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CellCube Culture System User`s Manual

1. casses 8 amp 8 04A i mu QUT wee C ME ell JBN c x p aul E CIRCULATION PUMP HARVEST FRESH VESSE MEDIA EN l i Figure 1 Overview of the CellCube Svstem Circulation Loop GENERAL CELLCUBE SVSTEM OVERVIEW down the inside surface of the glass reservoir The media is continuously refreshed by the gas mixture supplied to the headspace of the Oxygenator by the System Controller The fluid flow and gas exchange The culture media within the system is removed from the Oxygenator by the Circulation Pump and is then pumped into and distributed through his out the CellCube Module The media flows from within the Oxygenator is carefully controlled to the outlet of the CellCube Module back to the help eliminate turbulence foaming and to prevent Oxygenator where the media is evenly distributed PPET degradation l CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CELLCUBE SVSTEM COMPONENTS The Standard CellCube Svstem is comprised ot the five basic components shown in Figure 2 from left to right Controller Circulation Pump b Oxygenator Media Pump CellCube Culture Module The Digital Controller Figure 3 is a fully integrated multichannel instrument which mon itors controls and records cell cultu
2. GASSES XE DATA DUT ii gH OXYGENATOR LINE POWER MEO A PUMP HARVEST FRESH VESSEL MED A Figure 103 CellCube System with 6L oxygenator Corning Cat No 3143 BAS IN PsA X FLTERS FL A WA im A m tl Ea SET uet 4 FOR i ie AR i Z MEU 2 FI MEDA FUME x CASSETTE INLET DATA DUT i 3 9 LITER i 0 EN meer QE OXYGEN FAGBL x 100 PRODUCTION GFLLCUBE SUP 4 x 700 CELLCUBE SHOWN IN CPEGATITINAI POSITHOM I CART REF 42100 PROOUCTION WANITOLE STUP FOR PROPER CONNECT ONS SEEING FORT TSRALES CFR WITH WIH d Figure 104 CellCube System with 36L oxygenator Corning Cat No 3400 44 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 45 contains detailed instructions on how to set up a CellCube System for your particular application with the lengths and diameters of tubing that may be required For instance a CellCube system that is completely in a warm room may have dif
3. Taaale INPUT f PUMP ALARH Figure 74 Output Configuration screen In the Output Configuration screen are the four current output displays Figure 74 Each of the outputs may be turned off or configured for either 0 to 20 milliamps ma or 4 to 20 ma The output is configured by pressing the appropriate key num ber Each time the key number is pressed the output is advanced to the next level The current loop outputs are accessible from the rear of the controller The outputs are isolated and provide an optional means to log data at a user selectable rate Corning recommends using the 4 to 20 ma output This is because using a 4 to 20 ma output will detect any wrong cables which will give a 0 ma output Your facility or IT engineer should be able to assist in this process Serial Configuration HO EREOE CONDITION Serial Configuration kes 1 Tossle Fort Tyre RS 232 PORT A B c Enter Controller ID number h 313 INPUT f PUMP ALARH Figure 75 Serial Configuration screen OUTPUT The Serial Configuration screen modifies com munication between the controller and a PC The serial port and or address configuration can be selected by pressing 1 or 2 Press 1 for Toggle Port Type to select the type of output port you are using for communication between the controller and the PC The typical configuration for most PC s is the RS232 setting For system wide monitoring with long serial cables the R
4. www watson marlow com Model 101 U R 07518 10 Pump Head CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 56 Luer Tubing Fittings Value Plastics Inc 3325 Timberline Rd Fort Collins CO 80525 Tel 970 267 5200 Fax 970 223 0953 www devicelink com Nylon Male luer with lock 3 16 hose barb VP Part No MI LL 250 1 Male luer with lock 18 hose barb VP Part No MT LL 230 1 Female luer 3 16 hose barb VP Part No FT LL250 1 GENERAL SPECIFICATIONS Polyproplyene Male luer with lock 3 16 hose barb VP Part No MT LL 250 6 Male luer with lock 1 8 hose barb VP Part No MI LL 230 6 Female luer 3 16 hose barb VP Part No FT LL250 6 Nvlon 3 4 Tubing Clamps MeMaster Carr P O Box 4355 Chicago Il 60680 4355 Tel 630 833 0300 Fax 630 834 9427 www mcmaster com M C Part No 5031 K 13 Digital Controller Corning Cat No 3220 Specs Environmental Temperature Max Humidity Static Discharge Radiated Electric Field Susceptibility Conducted Transient Susceptibility Power Supply AC Input DC Input Inputs Resolution Oxygen Channels 1 amp 2 Measurement Range Resolution Bias Voltage Input Current Range pH Channel Measurement Range Measurement Resolution Input Impedance Input Voltage Range RTD Measurement Range Sensor Current Sensor Type Sensor Resistance Sensor Curve 0 to 600 C 95 Noncondensing Meets IEC 801 2 Meets IEC 801 3 Meets IEC
5. 3221 is designed for continuous operation at 37 C The pump utilizes an autoclavable disposable tubing set Corning Cat No 3120 The tubing set is designed for continuous duty at the full rated speed of the Media Pump It is generally recommended that the tubing be replaced after approximately 2 000 hours of use or if noticeable scoring or deep gauges are observed on the tub ing To facilitate extended use of the tubing it is advantageous to move the tubing back and forth daily to minimize extended wear of the tubing at the same place on the tube UNPACKING The Media Pump is packaged with the following items Power cord Remote control cable 15 pin D Sub type connector male x female Data sheet Remote control interface and alarm connections Iwo female pin connectors for auxiliary analog external alarm connections The pump is equipped with an internal universal power supply that automatically adjusts to any AC power source between 90 and 250 VAC 1 Place the pump on a clean dry surface 2 Assure that the power selection knob on the back of the pump is in the OFF position 3 Assure that the power selection knob on the front of the pump is in the STOP position 4 Insert the power cord into the socket located at the rear of the pump 5 Assure that the safety shield is in position covering the pump mechanism To activate the pump the safety screw on the cover needs to be tightened w
6. If a CellCube System run is planned for the next day place the CellCube module and media in an incubator overnight in order to bring it up to temperature for the following day s run CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CellCube Svstem Operation Because of its unique design the CellCube Svstem requires minimal supervision during operation During the growth phase of the culture dailv adjustments to the media infusion rate and occasional adjustments to the gas flow mav be required GROWTH PHASE We reter the reader to the tutorial on the CD video tapes provided in the setup package that dis cusses various options for initial control strategv This tutorial details tvpical scenarios observed in CellCube System operation and how to what to do strategies for operation Generally speaking the first few days of culture are usually satisfied by the media contained within the system after the initial seeding The length of time between the initial setup and the time when media perfusion begins depends on the initial seed density and the metabolic rates of a particular cell line The meas urement of the residual glucose concentration in the circulating media and oxygen consumption are good indicators of the status of the culture Dur ing the process of establishing a standard operat ing procedure it may be beneficial to monitor glucose concentration several times daily at a vari ety of perfusion flow rates to det
7. The pump provides a remote alarm connection on the rear panel and a mating connector The alarm contacts are dry contacts rated at 30 VDC The contacts are normally open The contacts close under the following alarm conditions 1 Door open 2 Improper remote connection 3 Loss of rotation seizure in the run state 30 second delay This alarm connection may be used to signal your in house alarm system visual or aural alarm system CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 61 Notes CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Corning Incorporated Life Sciences Tower 2 4th Floor 900 Chelmsford St Lowell MA 01851 t 800 492 1110 t 978 442 2200 f 978 442 2476 www corning com lifesciences For additional product or technical information please visit www corning com lifesciences or call 1 800 492 1110 Customers outside the United States please call 1 978 442 2200 or contact your local Corning sales office listed below Worldwide Support Offices ASIA PACIFIC Australia New Zealand t 0402 794 347 China t 86 21 2215 2888 f 86 21 6215 2988 India t 91 124 4604000 f 91 124 4604099 Corning and CellCube are registered trademarks of Corning Incorporated Corning NY Japan t 81 3 3586 1996 f 81 3 3586 1291 Korea t 82 2 796 9500 f 82 2 796 9300 Singapore t 65 6733 6511 f 65 6861 2913 Taiwan t 886 2 2716 0338 f 886 2 2516 7500 EUROPE France t 0800 916 882 f 0800 918 636
8. probable and may have a deleterious effect on some cell lines The controller will then add O as nec essary to maintain the desired set point The flow rate should be increased when the set point cannot be achieved Note The recommendations and observations are guide lines to help you better understand the general operation of the system Each cell line and product may have special requirements not mentioned here CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Large Scale Operation Da THEORV OF LARGE SCALE CULTURE OPERATION Large scale operation of the CellCube System is similar to the operation of the standard CellCube system Special care should be taken when using the 4 x 100 assembly to make sure that each phase of operation from seeding to operation to produc tion can be accomplished with generally acceptable time limits for that particular phase of operation An example is the seeding of a 4 x 100 CellCube System assembly The 4 x 100 assembly requires approximately 26 liters of cell suspension to fill the modules The connectors connections and associated tubing should all be capable of deliver ing the cell suspension to the CellCube System at the low 2 to 3 psi pressures recommended in the setup procedure The delivery of the suspension should be completed in a period of time that assures that the cells remain in suspension and are not settling Air should be removed from the inlet and the outlet manifolds to
9. A button and the con troller will record the value as the zero point for this probe Note that the value is only slightly different from the value of the amplifier null test The difference helps the controller recog nize the difference between an error condition no electrolyte and low oxygen 3 Press 3 to change the target reference calibra tion point We typically use room temperature air as a reference point You may decide to cor rect this value to compensate for the effects of altitude depending on your location Enter the desired value and then press ENTER CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 4 Press 4 to display the calibrate slope menu Hold the probe in an oxygen environment until it reaches a stable reading This may take a few minutes The conditions surrounding the probe should remain constant during this portion of the test When the signal is stable press ENTER or the A button and the procedure is complete The procedures used to enter the probe s ID number will be discussed in the sec tion Configure Inputs on page 32 Calibration and Maintenance of pH probe HO ERROR CONDITION FH Calibration 2 ter 1 Calibrate tand Tossle Comrenzatian Tare Enter Fixed Ecl enero Malus Calibrate la Drift Come coe BB Ba BG ae Ge Figure 51 The pH Calibration Screen The controller is calibrated in a manner analogous to standard bench top pH meters using a two point calibrati
10. Consumption Max Normal Range Normal Operating Temperature Physical Dimensions Height Width Depth Weight Approvals 2 0 amp 250 V 5 x 20 mm 47 to 63 Hz Range 90 to 132 VAC 180 to 264 VAC 140 Watts 4 to 84 RPM 0 4 to 9 2 LPM 10 to 38 C 50 100 F 36 6 cm 14 47 42 0 cm 16 57 39 4 cm 15 57 30 5 kg 67 lbs ETL ETL C CE PUMP INTERFACE The pump interface connections provide access to the advance features of the pump this permits computer control and monitoring including the following 1 Current loop input remote speed control 2 Current loop output remote monitoring of speed Alarm circuit Frequencv output calibration Status indicators ON nn B Ww Pump Identification binary Caution A technician with electronic circuits experience should make the following connections Explanation of Remote Interface Pins 1 amp 2 4 20 mA Current Loop Input Pin 1 is positive The best results are achieved when pin 2 is grounded to pin 15 and the input current source is connected between pins 1 amp 2 The pump will run at the slowest speed with a 4 mA input and at the fastest speed with a 20 mA input The intermediate speeds may be calculated with the formula RPM MIN 1 4 x MAX MINY 16 where MIN and MAX are the minimum and maximum speed settings that may be achieved with the front speed pots The formula may be simplified to RP
11. Germany t 0800 101 1153 f 0800 101 2427 The Netherlands t 31 20 655 79 28 f 31 20 659 76 73 United Kingdom t 0800 376 8660 All other trademarks included in this document are the properties of their respective owners Corning Incorporated One Riverfront Plaza Corning NY 14831 0001 f 0800 279 1117 All Other European Countries t 31 0 20 659 60 51 f 31 0 20 659 76 73 LATIN AMERICA Brasil t 55 11 3089 7419 f 55 11 3167 0700 Mexico t 52 81 8158 8400 f 52 81 8313 8589 11 11 POD CLS BP 019 REVI 2011 Corning Incorporated Printed in U S A
12. OD tubing leading into the CellCube Note If the tubing length exceeds 1 meter 3 feet add approximately 2 mL of distilled water per meter 3 feet of tubing Position SINGLE SIDE ARM PORT Prepare the single side arm port assembly of the oxygenator for connection to the inlet bottom manifold of the Circulation Pump cassette Connect a piece of 12 ID x 34 OD silicone tubing from the single side arm port to the inlet manifold bottom of the Circulation Pump cassette Connect the outlet top mani fold of the Circulation Pump cassette by con necting a piece of 12 ID x74 OD silicone tubing of appropriate length with a clamp approximately 7 cm 2 5 in from the end of the tube terminating with a 1 2 male male female luer connector that will be connected to the CellCube module After attaching the tubing to the two female luer fit tings on the 2 male male female luer connec tor see below Step 4 prepare for sterilization by wrapping the exposed connector in autoclave paper Add approximately 5 mL of deionized water to the manifolds by holding the manifold in a horizontal position before connecting the tubing If using a bridge piece see Step 2 above then insert the male male female luer con nector into the other end of the bridge piece Position CELL SEEDING PORT AND MEDIUM ADDITION PORT Cut an appropriate length of 4 ID tubing to bridge between the male male female luer connector and t
13. This is the set point that will trigger a maximum alarm condition on channel 2 A typical value here would be 10 less than the set point Press 9 to set the MAX Auto Flow Rate which is used to limit the oxygen flow rate A maximum limit may be advantageous when utilizing a fast capture profile such as indicating low averaging numbers for the PID controller A typical value here would be a value equal to the air setting Correctly adjusting the maximum flow will help prevent overshoot exceeding the set point especially if higher O set points are desired pH Settings HO ERROR CONDITIONS FH Settings Tossle Trackina Tyre Set PH Setroint Set LIE Flow Fate Set FH Alarm MIH Set FH Alarm MARA Set NAS Auto Flow Fate Auto Track r 4 FH Figure 78 The pH Settings screen It is assumed that sodium bicarbonate buffered media will typically be used as the primary means of maintaining pH in the system Primary pH control is therefore provided through the introduction of CO into the gas inlet stream The pH control may be either Automatic or Manual Manual control provides a method to blend a known percentage of gasses to control pH in case the probe fails A typical value of 5 to 10 CO to the total gas flow will provide adequate control Press 1 to toggle between either Automatic or Manual control Automatic control is recom mended Press 2 you are able to input the set point and comple
14. after gamma irradiation Dye injection studies showing typical fluid flow distribution are shown on page 5 Figures 14 18 Figure 11 CellCube Culture Module with corner removed to display culture plates Figure 12 Flow Control Orifices Figure 13 CellCube Culture Module 100 Layer Corning Cat No 3264 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Table 1 Different CellCube9 Culture Modules Surface Area Equal to CellCube Cat No Description Surface Area Roller Bottle Flask Seeding Volume Qty Cs 3200 CellCube Modules 10 8 500 cm 10 114 0 6 L 2 3201 CellCube Modules 25 21 250 cm 25 284 LSL 1 3264 CellCube Modules 100 85 000 cm 100 1 134 6 0 L l Roller bottle from 850 cm roller bottles Cat No 3907 Flask 75 cm flasks Cat No 430198 Figure 14 18 CellCube Culture Module Flow Progression Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CellCube Oxvgenator The CellCube Oxvgenator is a thin tilm diffusion device Figures 19 and 20 The entire oxvgenator assembiv is autoclavable and incorporates a 100 mm headplate assembiv including an autoclavable gel tilled pH probe a stainless steel level sensor probe and various ports for media withdrawal and a gas exchange inlet tube A polarographic oxvgen probe is also located in the 100 mm headplate assembiv on the 6L Oxvgenator The Oxvgenator is designed to take advantage of
15. and arrows for x y cursor control Auxiliary Gas Flow Control timin O ce min co Uic A KE Elmin J4IFS a ha MANUAL GAS CONNECTIONS AE fax hae EHITPUT Figure 25 Gas Flow Control The Mass Flow Controllers MFC used to man age the input gasses O CO and air have a vari able independent manual control feature Figure 25 Each MEC includes a LCD readout showing the flow of gas in cc minute a selector switch to choose between automatic and manual gas flow control and a dial for manual gas flow adjust ment Gas Connections O CO and air are delivered to the MFC through a male luer fitting Figure 25 The gas inlet pressure should be regulated to 5 PSI 0 34 bar The blended gasses are delivered to the Mix Output fitting for delivery to the Oxygenator Process Inputs PROCESS INPUTS C NN L E T Figure 26 Process Connectors The front mounted process input fittings provide connections for a three wire RTD probe for tem perature monitoring Level sensor pH and two temperature compensating polarographic dis solved oxygen DO probes Figure 26 Connecting Probes to the Controller The Controller collects its information through the input cables from each of the probes and pumps The Controller utilizes a variety of con nectors with different pin styles and numbers The varied styles help assure that the proper probe is connected to the correct input It is important th
16. average and last Air Flow values for the time period shown on the graph are also displayed A more sensitively scaled Y axis can be viewed by pressing the ENTER key The bottom left portion of the display shows that the next possible selection is TEMP CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Temperature Temperature History ERROR DETECTED ETI History Figure 91 Temperature History ERROR DETECTED ETI History D A A ART rue LI STATUE PUMPS Figure 92 Temperature History sensitive OA GEN A typical RTD History Chart is displayed in Figure 91 Note that the graph history is labeled RTD History and the Y axis scale is 0 1000C The minimum maximum average and last RTD values for the time period shown on the graph are also displayed A more sensitively scaled Y axis Figure 92 can be viewed by pressing the ENTER key Pumps Media Pump History HO ERROR CONDITION Media Fume Flow History 333 H Figure 93 View Media Pump Flow History screen A typical Media Pump Flow history chart is dis played in Figure 93 Note that the graph history is labeled Media Pump Flow History and the Y axis scale is zero to the highest limit determined during the calibration routine The minimum maximum average and last L day values for the time period shown on the graph are also displayed A more sensitively scaled Y axis can be viewed by pressing the ENTER key The bottom left por tion
17. cali bration menu appears and the current value of the probe signal will be displayed When the current value is stable press ENTER or the A button A pH of 7 00 should yield a current value of 2048 plus or minus 100 units Values outside this limit may indicate a probe that is not clean or may have internal reference prob lems Rinse the probe with D I deionized water 2 Determine the proper compensation method to apply from the information listed above Each time you press 2 the compensation method Toggle Compensation Type will advance to the next type FIXED AUTOMATIC pre ferred method and NONE 3 Press 3 for the Enter Fixed Compensation Value step Enter the probe calibration temper ature e g 37 C and then press ENTER MON NU ERROR CONDITIONS Place electrode in 4 buffer solution of 4 BHBdrFH or 16 866FH When the value becomes stable press the ENTER key to accert or the CANCEL key to retain the current value Current Value 1898 Ho acceptable FH standard found Temrerature Compensation d Figure 53 The pH Calibration Slope Screen CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL 28 4 Press 4 to select the calibrate slope menu Figure 52 on previous page Place the probe after rinsing it with D I deionized water in a standard buffer e g pH 4 0 or 10 0 and stir with the electrode to ensure proper contact Allow the signal to stabilize and then press ENTER or the A but
18. during oper ation while not pinching so firmly that the tube is occluded 6 Fit one end of the tubing into the pinch clamp of the pump cavity 7 Check the desired flow of the fluid to assure the tubing is properly placed in the pump The tubing from the fresh media should be near the inlet of the pump and the tubing leaving the outlet of the pump should be directed towards the CellCube system There is a crank han dle that snaps in and out of the center of the pump head that can be used to help rotate the pump head while inserting the tubing 8 Feed the tubing between the rollers and the track aligning it with the rotor tube guides The tubing must lie naturally against the track without being twisted or stretched 9 Open the outlet pinch clamp and place the other end of the tubing in the pinch clamp and adjust the pinch clamp as detailed above 10 Close the crank handle by snapping it back into the lock position on the pump head and shut and lock the safety cover shield using the cover screw provided 11 When both of the covers are tight and the safety switches are activated the red alarm light will light and the green Jog Start light will flash The pump will not restart until both of the safety covers are properly posi tioned and the Jog Start switch is pressed to restart the pump Caution The area under the safety shield is a pinch point and extreme caution should be used while installing or removing tub
19. from a remote location Each selected alarm or set point will immediately activate the alarm switch that it is connected to whenever that limit is exceeded One example of a condition that would set off all the selected alarms would be a power failure When the power is restored the oxygen probes require a certain amount of time to stabilize extending the alarm period The pH levels may exceed the limits until the circulation has re established the set point which may require a number of volumetric turnovers depending on the length of time the pump was off Delay peri ods should be selected based on their ability to prevent spurious alarm activation without being so long that the process is compromised To set a delay period use the cursor to move to the selected Device e g DO 1 DO 2 and then over to the DELAY column Any number between 0 and 999 seconds is an acceptable entry Use the keypad to enter the numbers and then press ENTER to complete that selection You must either press ENTER or move out of the delay function area using the cursor control arrows before you are permitted to use the other main function buttons This does not affect the control functions or actual operation Configure Outputs HO EREOE CONDITION Qutrut Confisuratian eu i Toaale Ge 1 Qutrut Confia Toaale Of 2 utrut Confis 4 A a oe 1 7T rarono T Los Lew er Toaale FH utrFut Confia ETID Dutrut Confis
20. of the display shows that the next possible selection is CIRC Circulation Pump History HO ERROR CONDITION Circ Pume Flow History Figure 94 View Circulation Pump Flow History Screen A typical Circulation Pump Flow History Chart is displayed in Figure 94 Note that the graph his tory is labeled Circ Pump Flow History and the Y axis scale is zero to the highest limit determined during the calibration routine The minimum maximum average and last L day values for the time period shown on the graph are also displayed A more sensitively scaled Y axis can be viewed by pressing the ENTER key View Status ERROR DETECTED Mieg Device Status Information ta tus Ok kK OK A below min setting Dewi ce DO 1 PUMPS Figure 95 View Device Status Information screen The View Device Status Information menu dis plays the status of all the selected alarms A view device status menu is shown in Figure 95 When a device is operating outside its limits an error message is displayed The displayed menu depicts a low temperature condition This view facilitates quick identification of any error that is detected CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 41 AUXILIARV MENUS ERROR DETECTED Aux UuUMMarH4 creer BATCH IEF AULT Figure 96 Auxiliary Summary Screen FACTOR In the auxiliary menu you have access to start a new Batch to save the calibration values to change the internal cloc
21. processing handling use or repair by any party other than Corning The foregoing is the sole and exclusive representation and warranty made by Corning and all other representations and warranties express and implied including but not limited to the implied warranties of merchantability and fitness for purpose are expressly disclaimed The foregoing remedies constitute Corning s sole and exclusive liability for any alleged breach of any representation warranty or other defects whether a claim is asserted in contract tort or on any other basis Shown on front cover CellCube System Corning Cat No 3143 PLEASE COMPLETE THIS DOCUMENT WITH THE CELLCUBE SYSTEM INFORMATION TO REGISTER YOUR CELLCUBE EQUIPMENT FOR WARRANTY THIS FORM MAY BE FAXED TO 978 442 2492 CellCube System User s Manual Rev V1 02 Company Name Mailing Address 00000000 Thank you for your interest in CellCube System Applications If you have any questions please contact Corning s Technical Service department at 1 800 492 1110 within the United States Outside the United States please call 1 978 442 2200 CellCube System Information Document PLEASE FILL IN AND MAINTAIN THIS RECORD for warranty Receipt Date mo day yr Digital Controller Serial No Lot No Circulation Pump Serial No Lot No Media Pump Serial No Lot No Oxygenator Serial No Lot No Oxygen Probe Flow Cell Serial No Lot No Flow Cell No 2 If applicable Serial No Oxygen
22. production 1s usually discarded and a perfusion rate of 3 reactor volumes per day is being used the reduction in serum content is greater than 7526 per day The introduction of serum free medium one day prior to reaching confluency may help adapt the cells to the new environment while still providing some residual serum for the transition Another benefit of perfusion is that the transition from serum containing to serum free media is gentle CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL 52 The cells remain at the appropriate temperature and usually stay firmly attached in the CellCube Module This reduces lost time and the expense of wasted medium while the cells adapt to a rapid shift in the medium environment Alternatively the perfusion rate may be increased to a rate equal to 4 to 6 reactor volumes day to yield a similar result although at a potentially greater expense Once working parameters are established for a cell line that are reproducible it may be possible to eliminate perfusion by providing a single layer media bag or carboy which can hold an entire supply of medium for the duration of the run Please contact Corning Technical Service for additional information using this culture strategy pH and Oxygen Control The pH high limit control is coupled to the CO gas flow This is needed mainly during the initial growth phase of the culture The carrier gas air is provided by an external air pump capable of de
23. selector switch 1s turned on The green Jog Start light on the front panel should light and flash The green Remote light on the Figure 33 Dual Circulation Pump Corning Cat No 3223 10 l E front panel will light only when the remote control interface is properly connected and the safety switch has been activated by attach ing the safety shield To activate the safety switch push on the Jog Start button one time Turn the power switch on the front panel to the local setting on the pump The pump should begin to operate at a rate determined by the Rate knob on the pump The rate shown on the display is RPM Rotate the Rate knob counter clockwise until it stops The rate should stabilize at approximately 4 RPM Rotate the Rate knob clockwise until it stops The rate should stabilize approximately 84 RPM Reduce the pump Rate to 4 RPM 12 Slowly loosen all four of the knobs holding the safety shield in position When the safety shield cover becomes loose the red alarm indicator will light the pump should stop and the green Jog Start light will flash The pump will not restart until both the safety shield is properly positioned to depress the white safety switch on the left tube guide and the Jog Start switch is pressed to restart the pump 17 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CASSETTE INSTALLATION The Dual Circulation Pump Cassette Corning Cat No 3187 uti
24. to 10 volt output that may be read or recorded directly DO NOT APPLY ANY VOLTAGE TO THESE PINS Pin 10 Alarm Pin 10 is the alarm output connection This is a sinking current output that goes active when there is an alarm condition in the pump There are three conditions which can activate an alarm in the pump The pump is in the local or remote mode and the pump mechanism has been stopped mechanically seized or broken The pump is in the local or remote mode and the interlock mechanism has been activated or is still activated and the start switch has not been pressed The pump is in the remote mode and the remote available signal is not active pump runs at the local speed setting Pin 11 Remote Ready Indicator Pin 11 is the remote ready indicator This connection is a current sinking output that goes active when the pump is in the remote mode and ready to accept input from a remote source Pin 12 Pin 12 is not used at this time Pins 13 and 14 Pump ID Pins 13 and 14 are the pump ID pins These pins provide a binary code that defines the type of pump to a Controller Corning Cat No 3220 Pin 14 Pin 13 Not Used OPEN OPEN 3223 OPEN GROUND 3222 GROUND OPEN 3221 GROUND GROUND Pin 15 Ground Pin 15 is the common ground connection for the remote interface This is the ground reference for all of the input and output connections to and from the pump External Alarm Connections
25. 801 4 100 to 240 V 250 50 to 60 Hz 1 0A max 45 Watt 13 8VDC 10 2 5A max 12 Bits 0 0 to 200 0 DO up to 0 1 DO 0 685VDC typ 0 to 130nA typ 0 to 14pH 0 01pH 1012 ohm 450mV to 450mV 0 to 1000C Il mA 3 wire Platinum 100 ohm 0 0 00C European alpha 00385 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Level Sensor Nominal Frequencv High Level Threshold Outputs Analog Resolution Span Max Output Voltage Max Load Impedance Alarm Type Rating 1 1 Khz 50K ohm typ 12 Bit 0 20mA or 4 20mA IOVDC 500 ohm Drv Contact 30 V 1 Amp max Mass Flow Controllers Gas Connections Flow Range Accuracy amp Linearity Temperature Coefficient Repeatability Response lime Input Pressure Range Safety Emissions Meets Safety Requirements Emissions Serial Ports Port A Port B Physical Dimensions Height Width Depth Weight Approvals 0 and 10 to 500 cc min 1 of Full Scale 0 1 0C Within 0 2 Full Scale at any constant temperature 1 2 sec l to 10 psi EN 61010 1 UL 3103 1 CSA C222 EN 50081 1 FCC Part 15 Class A RS 232 DCE amp RS 485 RS 232 DIE amp RS 485 37 cm 14 47 18 cm 7 436 cm 14 15 5 kg 34 Ibs ETL ETL C CE Host Software Svstem Requirements optional Computer Operating Svstem RAM Hard Drive Space serial Port 486 33 Mhz min Windows 95 or more recent operating svstems r
26. CellCube Culture Svstem Users Manual Rev V1 02 CORNING Corning Incorporated Tower 2 4th Floor 900 Chelmsford St Lowell MA USA t 1 800 492 1110 1 978 442 2200 f 1 987 442 2476 www corning com lifesciences Copyright 2011 CORNING INCORPORATED All rights reserved No part of this document may be reproduced in any form without the written permission of CORNING INCORPORATED CORNING has taken every reasonable precaution to ensure the accuracy of the information descriptions and instructions in this manual However Corning assumes no responsibility for errors omissions or possible misinterpretations in addition to conse quences resulting from the operation of the equipment based on these instructions CORNING Limited Warranty Corning warrants that at the time of delivery the product will conform to the physical characteristics noted in the drawings and specifications referenced herein in this instruction manual If the product does not so comply Corning will at its discretion either repair the product or provide a replacement so long as Corning receives written notice of a claim asserting noncompliance prior to the earlier of 12 months after the time of delivery Thereafter Corning will have no obligation to perform any repair or replacement services Corning is not responsible for any nonconformance due to wear and tear negligence on the part of any party other than Corning inappropriate use of the product or
27. DITION Connect probe for this channel and immerse in a BE oxygen enviornment Press the to retain the current values Current Value Figure 48 Calibration in an oxygen free environment 3 Press 3 to display the Enter Target DO screen in order to change the target reference calibration point Figure 49 This controller is preset at the factory to read O saturated liquid as 100 You may decide to correct this value to compensate for the effects of density altitude depending on your location Enter the desired value and then press ENTER Unless the CellCube System is used in a super saturated environment it is best to leave the value at 100 MU ELI LUMLLTLU S Oxqaen 1 Calibration mt EF Amelifier Hull J Calibrate era 16 Enter Tarset 400 Calibrate lore M e D 4 B mad Figure 49 Enter target DO 4 Press 4 to display the Calibrate Slope menu Figure 49 Place the probe in an environment saturated with O until it reaches a stable read ing This may take a few minutes The conditions surrounding the probe should remain constant during this portion of the test An acceptable reference is a liquid in a contain er open to ambient air a cylinder of com pressed air or a small pump such as an aquari um pump When the signal is stable press ENTER or the A button and the procedure is complete The procedures used to enter the ID number will be discussed in the section Con
28. G CELLCUBE CULTURE SYSTEM USER s MANUAL 47 2 Position MEDIA RETURN The connector kit Corning Cat No 9267 contains a pre assembled tubing set that includes two 4 x 7 16 silicone tubes on a Y connector and terminating in a 1 4 x 1 2 male adapter This portion of the connector kit is used between the two media inlet tubes of the oxygenator headplate and the 25 mm oxy gen probe holder Place one each of the shorter tubing pieces on the Y connector over each of the media inlet tubes on the oxygenator head plate Connect a piece of 12 ID x 34 OD sili cone tubing of appropriate length between the media inlet adapter and the 25mm probe hold er Connect the same length of silicone tubing between the other side of the 25mm probe holder and the outlet tube of the CellCube System Install a clamp midway on this piece of tubing and terminate with a male male female luer connector Prepare for sterilization by wrapping the outlet adapter and loosely inserting male plugs in the luer fittings Alter natively a bridge piece 1 foot 30 cm length of 12 ID x 4 OD silicone tubing can be used to connect between this 12 male male female luer connector and its equivalent on the Single Side arm Port during autoclaving acting in essence as a substitute for the CellCube during the autoclave process to complete a loop Each connector would then be removed after auto claving and reinserted into the inlet outlet 1 2 ID x 4
29. M 4 I 4 x 5 for the Corning Cat No 3222 Circulation Pump There is a resistance of 250 ohms between pins 1 and 2 A 20 mA current will be developed with an applied voltage of 5 VDC to pin 1 and common to pin 2 The best results are achieved when pin 2 is connected to ground Digital to analog outputs 0 to 5 VDC may be connected using this method You may use other voltage inputs with the addition of the proper current limiting resistor It is important NOT TO EXCEED 20 VDC or 20 mA on these INPUT CONNECTIONS The remote interface is buffered from the actual control circuit The accidental destruction of the remote interface should not have any effect on the operation of the pump while in the local control mode One safety feature of the pump is that the actual speed control of the pump will default to the setting of the rate control on the front panel if all the remote connections are not complete CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Pin 3 Pin 3 is not used at this time Pin f4 Remote Enable Connection Pin 4 is the remote enable connection When pin 4 is connected to ground Pin 15 the pump will run in the remote mode see front panel if the remote available pin is also active see Below Do not apply voltages above 12 V to this pin The current sink capability must be less than 5 mA Pin 5 Remote Available Connection Pin 5 is the remote available connection This pin must
30. PC Host Software is a program that is designed to run in a Windows environment and is operational in all of the Windows operating svstems from Windows 95 through Windows XP If the PC is on a network and can be accessed via pcAnywhere 1 0 then the program can even ofter off site remote access capabilitv The functions of the PC Host software include communication via serial cable and displav of the LCD screen from the CellCube Controller Unit Corning Cat No 3220 A graphical view of the last 24 hours settings and calibrations of virtually all the different views can also be controlled by the Host Software The software is required to download the data from the controller itself in a spreadsheet compat ible history file This data is in a raw text format and can be imported in to a variety of spreadsheet programs for analysis Transferring the capture screens from the display on the controller in a bit map format is also an option of the Host Software Thirty two controllers can be monitored with the Host Software on a PC when the controllers are serial connected to each other Each controller can be viewed on the PC screen by selecting the corresponding controller designation number from 0 to 31 These controller designation num bers are manually entered on the controller itself pcAnywhere is a registered software with Symantec Corporation Similar software applications on the market may also act in this capac ity b
31. PS STATUS Figure 82 View Summary Screen The menus inside VIEW provide rapid access to all the summary menus that display the operating conditions of the system for the last 24 hours The View Summary screen provides a current display of the most critical operating parameters There are five sub menus OXYGEN pH AIR TEMP PUMPS and STATUS Oxygen Oxygen First View DO 1 HO ERROR CONDITION Oxygen History CH CHA d LA T MAKE MIH nv 114 7 lee 114 4 117 5 Figure 83 View Oxygen History Channel 1 Screen Figure 83 is a typical Oxygen History chart for the last 24 hours Note that the graph history is labeled CH1 and the Y axis scale is 0 to 200 DO maximum setting in this case The mini mum maximum average and last DO values for the time period shown on the graph are also displayed When a run is longer than a 24 hour period a more sensitively scaled Y axis can be viewed by pressing the ENTER key that will auto scale to the minimum and maximum meas ured value for the last 24 hours The bottom left portion of the display shows that the next possible selection is DO 2 Oxygen Second View DO 2 EFROR DETECTED Oxygen History LHE CHA A J STATUS DEF LUN PUMPS Figure 84 View Oxygen History Channel 2 Screen Figure 84 is a typical Oxygen History chart for the last 24 hours Note that the graph history is labeled CH2 and the Y axis scale is 0 to 200 DO maxi
32. Probe Serial No Lot No Oxygen Probe Serial No Lot No pH Probe Serial No Lot No Cart 4 X 100 STACK Serial No CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Table of Contents Before YOu Starts zo re dei walk nt Y erts iv Unpacking the CellCube Svstem iv Check Inventory 1 o64424 0800 Tv iue deese iv Introduction xiii u bb ctten b Ebda Chats erae 1 General CellCube Svstem Overview 1 CellCube System Components 2 CellCube Culture Module 4 CellCube Oxygenator 04 6 Glass Reservoir Cleaning cias err ds CellCube Digital Controller 8 Unpacking and Setup ias mac pem tad ans 8 DVSLEI CONGO 2144 24 cade cates enon 8 Working With Me ts sx0sssaotosazajnzba 8 Perino Dalis ono 59 cu Ree y PPP EN UE 9 Auxiliary Gas Flow Controls ssti isestonte g Cias COMMECHONS ss sa ni t iz E PES 9 Process INDUS issir nie b ka baja weeded 9 Connecting Probes to the Controller 9 Coming Pumps 222 0x0 E EIPRVRE Ex 10 Background Information s ouis 44504 cease 10 CellCube Media Pump 11 WAC acd 11 Lupine Installation se dcus oc hte ted pobre dens 12 Pump Calibration Flow Charts 12 CellCube Single Circulation Pump 14 MP vxo oxide en Leuk eee needles 14 Cassette Inst DS BIO DES eng deep es ane ens 15 Pump Calibration Flow Charts 15 CellCube Dual Circulation P
33. ROR CONDITIONS PAS 1 Place EHFT measurement container under Fume outlet and rlace rumr inlet in a The Fume should be fully Primed before this calibration iz The FUME will run For 2 min started Fume is ready to calibrate PRIME Figure 57 Calibration Pump Screen High Speed ABORT Place a receiving vessel of sufficient size to retain the sample under the output tube and press START The calibration will begin and the pump will run for two minutes Figure 58 HO ERROR CONDITIONS The total test time was amp Hin B H Sec Average Speed 326383 Enter the volume of Fluid FUMFE during this test in m Figure 58 Calibration Media Pump High Speed Enter Volume The pump will stop automatically Measure the sample collected volume or weight The screen displayed in Figure 58 will prompt you for the volume collected during the first test period Enter the volume in mL in the space as requested and press ENTER HO ERROR CONDITIONS The total test time was amp Hin B 1 Sec Average Speed 1695 Enter the volume of Fluid FUMFE during this test in m pu ESS EES uu pu Figure 59 Calibration Media Pump Enter Volume Low Speed The screen displayed in Figure 59 will prompt you to prepare and to collect the second cali bration sample Repeat if necessary Empty the receiving vessel replace it back under the output tube and press START The calibration will begin and the pump will run for two mi
34. S485 is more appropriate Your IT department should be able to assist with deter mining the appropriate course of action CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Press 2 to enter the controller ID The controller ID is a number generated by the user where the acceptable range is between 0 and 31 A maximum of 32 Controllers can be daisy chained together through the RS232 outputs to a single PC This is done by routing the output of the 1st Controller Part B on the back of the Controller to the input of the second Controller Part A and so forth Controller ID numbers are only changeable on the controller itself THE SETTINGS FUNCTIONS HO ERROR CONDITIONS Settinas Summar Screen Oxysen Mangement Autotrackina Probe 2 at 68 8 SLD EFH C z Management Autotracking at r BH FH Airflow SetPoint 166 86 ccemin Media Pume xetrolmt 49 9 leday Status RUNNING Figure 76 Settings Summary Screen The settings function provides access to the menus where the set points and alarm values minimum and maximum are entered into the controller The Settings Summary Screen displays the currently active set points and the probe selection Figure 76 The default values of the Controller are Oxygen is set to 80 0 and the controller tracks DO Probe 2 pH CO Management is set for Autotracking at 7 00 pH and the Airflow Setpoint is defaulted to 200 cc min The pumps in this example are connected an
35. alibration point is just a few degrees above operation temperature Place the thermistor in the second temperature standard and stir the probe momentarily to ensure a proper contact 4 Press 4 for Calibrate Target 2 Value to select the second calibration menu The signal will stabilize as indicated by a stable reading of the current value Press ENTER to record the second standard temperature Calibration of Corning Pumps Background Information The CellCube Controller is capable of recogniz ing any of the three pumps that are designed to integrate with the controller The following three pumps are automatically recognized when con nected to the controller with the pump communi cations cable Media Pump Corning Cat No 3221 Circulation Pump Corning Cat No 3222 and the Dual Circulation Pump Corning Cat No 3223 The controller is equipped with a cali bration routine to convert RPM reciprocations or revolutions per minute depending on the pump The three pumps are driven with the same servo type brushless DC motor to provide accurate CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL low maintenance pump operation The calibration Media Pump Calibration procedure is the same for each of the pumps how ever the volume collected during the procedure NO ERROR CONDITIONS will vary depending on the pump used As with O Pume Calibration screen e e e e key and pH probes a two point calibration method is Ar eirate M
36. an be found in the files found on the CD video tapes provided in your installation package The schemes shown are for a standard CellCube system using a 100 Layer CellCube Module Table 4 summarizes the volumes that would be appropriate for the smaller CellCube modules It 1s best to consider the circulation loop initially Note It is advised to not save cost on tubing and connectors when you do not have a lot of experi ence with scale up processes However when think ing through your process flow try to minimize redundancy in tubing especially in the assembly of the circulation loop and perfusion circuit For an in depth look at how to start up a CellCube system please refer to the Power Point presenta tion in the CellCube system Video CD 1 If you do not have a set of videos or CDs please contact your local sales representative This Video CD Table 4 Fluid Capacities for CellCube Modules Approximate Fluid Capacities of CellCube Systems Total RB Sk Approx Oxygenator Total Equivalent Aea Module Dead and Circ Volume 10 Layer 10 8 500 600 1 5 2 1 25 Layer 29 21 250 1900 15 3 50 Layer 50 42 500 3000 1 5 45 l i I l 100 Layer 100 85 000 8000 5 15 75 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Q P H amp LEVEL INFORMATION X KA GAS IN 1 4a KON mi Fil lk AUT
37. at you never force a connector into one of the inputs as most of the connectors are keyed so that they will only fit in one orien tation The Controller contains very sensitive amplifiers for pH and DO measurement It is beneficial whenever working with electronic instruments to be grounded prior to touching an input connection Poo Ia gf Papst ant Figure 27 Smart ports of the controller The rear of the Controller contains two pump interface ports These ports are smart ports Figure 27 that are capable of recognizing any of the Corning Media or Circulation Pumps Corning Cat No 3221 3222 or 3223 The data cables must be connected to the Controller for remote control of the Media Pump and to record pump data CORNING CELLCUBE CULTURE SVSTEM USER s MANUAL Corning Pumps 10 BACKGROUND INFORMATION The Controller is capable of recognizing anv ot the three pumps that are designed to integrate with the controller The following pumps will be automaticallv recognized when connected to the controller with the pump communications cable Media Pump Corning Cat No 3221 Single Circulation Pump Corning Cat No 3222 and Dual Circulation Pump Corning Cat No 3223 The controller is equipped with a calibration routine to convert the pump speed into tvpical engineering units Table 2 Volume Collected During 24 Hour Test Period The three pumps are driven with the same servo tvpe brushless DC motor to p
38. be tied to ground Pin 15 before the pump will run in the remote mode It may be connected directly to ground or it may be con nected to a watchdog circuit of the controlling device If the connection is open the pump will display an alarm condition and it will run at a speed determined by the speed pot on the front panel Do not apply voltages above 12 V to this pin The current sink capability must be less than 5 mA Pin 6 Is not used at this time Pin 7 Frequency Output Pin 7 is the frequency out connection The frequencv out connection is a current sinking output that provides a frequency that is directly proportional to the pump speed The formula to calculate the speed for the Circulation Pump Corning cat No 3222 is F RPM x 42 47 15 or F RPM x 2 831333 Pins 8 and 9 4 20 mA Current Loop Output Pins 8 and 9 are the 4 to 20 mA current loop output of the Circulation Pump Corning Cat No 3222 Pin 8 is positive These pins provide a current output that is proportional to the pump speed The speed of the output can be determined by using the following formula T 4 RPM 16 MAX where MAX is Maximum speed available from the speed pot on the front panel of the pump The formula can be simplified to T 4 RPM x 0 1904761 The maximum voltage that can be produced at pin 8 is 20 VDC The maximum load between pins 8 and 9 is 1000 ohms The use of a 500 ohms resistor would provide a 2
39. been entered This feature allows the user to restore the preferred calibration values to the controller if the processor resets for any reason Time Menu ERRORS DETECTED Set Time KEY 1 Enter Hours e Enter Minutes 2 Enter Seconds d Enter Honth 2 Enter Day 6 Enter Year ru banco Ren Current Time 8r 29 64 HHB e3 83 Figure 100 Auxiliary Time Screen The Set Time menu is used to enter the current time and date into the controller There are six selections and the values are entered using the numeric keypad CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL 42 Factorv Menu ERRORS DETECTED Enter Factory Password and Press ENTER to Access Factory Screens Figure 101 Auxiliary Factory Screen ERROR DETECTED Factory Settings Screen Figure 102 Auxiliary Factory Settings Screen The Auxiliary Factory Settings Screens Figures 101 and 102 are to be accessed by Corning Technical Applications Support only Please contact your local representative for help with these functions if necessary CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL OVERVIEW OF CELLCUBE FLUID PATHS AND PHILOSOPHV OF OPERATION The CellCube Svstem tvpicallv consists of two intersecting flow paths Figures 103 and 104 on page 44 The primarv flow path that supplies the usual nutrients gases and controls pH is the cir culation loop It is perfectly feasible to operate the CellCube System with onl
40. ble Level sensor probe Level sensor cable 100 mm Head plate 45 mm One port sidearm assembly 45 mm Two port sidearm assembly 12 mm Oxygen probe replacement kit 25 mm Oxygen probe replacement kit Secondary O probe flow cell assembly 25mm Oxygen probe 25mm Oxygen probe flow cell 25mm Oxygen probe cable 36L Oxygenator Complete Corning Cat No 3188 Oxygenator glass 36L pH Probe pH Probe cable 25 mm Oxygen probe 2x Oxygen probe flow cell 2x Oxygen probe cable 2x Level sensor probe Level sensor cable 100 mm Head plate 45 mm One port sidearm assembly 45 mm Two port sidearm assembly 25 mm Oxygen probe replacement kit Setup Kit for the 6L Oxygenator System Corning Cat No 3135 Single Circulation Pump cassette Media Pump tubing set Various tubing connectors Silicone tubing Setup Kit for the 36L Oxygenator System Corning Cat No 3115 Media extension set 10 Collection Set 25 Pressure relief valve assay Various tubing and connectors Dual Circulation Pump Cassette Corning Cat No 3187 Ouad Incubator Cart Corning Cat No 9234 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Introduction O P H amp LEVEL INFORMATION GAS IN
41. d running at their respective flow rates Note The values that are expressed above are for reference only Actual values will no doubt be developed depending on the cell lines media and products expressed Those parameters as developed empirically are There are five sub menus OXYGEN pH AIR TEMP and PUMPS Oxygen Settings The oxygen set point and alarm values are entered here There are nine different options displayed in the oxygen settings menu Figure 77 HO ERROR CONDITIONS Oxygen Settings ke Toaale Tracking Tyre Auto CH 2 Toggle System Size j Medium A l e 3 4 Sis 6 ng a a CE min Figure 77 Oxygen Settings Screen Press 1 to change the control method The control of the oxygen in the system may be con trolled by O Probe 1 O Probe 2 or manually There are three selections Auto CH 1 auto tracking Auto CH 2 auto tracking and Manual that can be toggled respectively Each time that you press 1 the selection is advanced one posi tion The default setting is Auto CH 2 auto tracking Press 2 to select the system size and choose one of three values Small Medium or Large The size selection loads a set of algorithms that were developed to match the response of the system to the appropriate oxygenator vessel A standard system that has the 6L Oxygenator Corning Cat No 3101 with one 100 Layer CellCube Module Corning Cat No 3201 or Corning Cat No 3264 wo
42. dling Considerations for CellCube System Module Size 25 Layer 50 Layer 100 Laver system Volume L 3 45 7 5 Flow Rabe Tubing LD RPM mimin Liday VolExchangesidaw at Flow Rate 1167 Tubing 10 43 62 21 1 4 08 20 86 124 41 28 17 30 129 186 62 41 25 40 172 248 83 55 33 50 215 no 103 69 l 41 1 8 Tubing 10 179 258 86 57 34 20 372 536 179 119 71 30 558 804 28 179 107 40 44 1071 2357 238 143 i 50 930 1339 446 298 179 1167 Tubing 10 405 583 194 130 78 20 810 1166 389 259 155 30 1215 170 583 389 233 40 162 0 2333 718 518 314 50 2025 2916 972 048 389 Figure 107 Flask with Portcap CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Autoclave Preparation The recommended autoclave cycle is between 45 and 90 minutes on slow exhaust at 121 C It is simplest to assemble the System in the morning or early afternoon autoclave it and then let the system cool down over night in a hood or a bio logical safety cabinet The use of a plastic or metal container sufficiently large enough to hold the oxygenator and tubing while in the autoclave is recommended It is also a good idea to slide the entire assembly into an autoclave bag that is loosely closed This provides additional safety after autoclaving while transporting the unit to the biohood for coolin
43. e found later in this manual See section on Media Pump Calibration on page 29 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 13 400 390 300 250 200 150 Liters dav mall c o O O Tubing Size RPM 1 16 1 8 3 16 2 1 2 5 4 11 7 5 3 1 13 4 29 2 10 6 2 25 8 58 3 15 9 3 40 2 87 5 20 12 4 53 6 116 6 25 15 5 67 145 8 30 18 6 80 4 175 35 21 7 93 7 204 1 40 24 8 107 1 233 3 45 27 9 120 5 262 4 50 31 133 9 291 6 55 34 1 147 3 320 8 60 37 2 160 7 349 9 64 3 6 171 4 2722 1 16 A 1 8 lt 3 16 Figure 29 An example of a flow chart for the Media Pump CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL The Circulation Pump Corning Cat No 3222 is designed for continuous operation at 37 C The pump utilizes a single use Disposable Cassette Corning Cat No 3121 The cassette is designed for continuous duty at full rated speed of the Circulation Pump UNPACKING The Circulation Pump is packaged with the following items Power cord Remote control cable 15 pin D Sub type connector male x female Data sheet remote control interface and alarm connections Two pin female connectors for auxiliary analog external alarm connections The pump is equipped with an internal universal power supply that automatically adjusts to any AC power source between 90 and 250 VAC 1 Place the pump on a clean dry surface 2 Assure that the power selection knob on t
44. e is very useful for tracking the actual operation temperature of the system Although the CellCube System is typically operated at 37 C in an incubator warm room during long perfusion runs the medium added may be from a refrigerated environment to preserve labile components in the medium One can use a nonsterile thermistor placed under the oxygenator to measure the circulating tempera ture of the media in the system Tracking the tem perature in the system will then provide valuable information for the future operation Calibrate the temperature probe with a calibrated thermometer ee HD ERROR CONDITION Temrerature Calibration Screen ter 1 oe Target 1 H A FL e Calibrate Taraet 1 Value 668 3 Enter Target 2 46 6 C d Calibrate Target 2 Value 18828 On TIEN FH FLOM F LIMF 2 Figure 55 Temperature Calibration Screen 1 Place the thermistor in an ice bath and stir the probe momentarily to ensure proper contact Press 1 to Enter Target 1 Enter the target temperature e g 40 C or the temperature from the reference thermometer then press ENTER 2 Press 2 for Calibrate Target 1 Value to select the first calibration menu The signal will sta bilize as indicated by a stabile reading of the current value Press ENTER to record the first standard temperature 3 Press 3 to Enter Target 2 Enter the value for the second calibration point The most accurate result will be ensured when the second c
45. ec Average Speed 33168 Enter the volume of Fluid rumred during this test in liters Figure 63 Calibrate Circulation Pump Screen High Speed_ Enter Volume The pump will stop automatically after two min utes and the sample may be measured Figure 63 The screen displayed will prompt you for the vol ume collected during the first test period Enter the volume in liters in the space as requested and press ENTER HO ERROR CONDITIONS The total test time was amp Hin B 2 Sec Average Speed Irdr Enter the volume of fluid rumred during this test in liters The calculated flow rate is B ebbt l mini Figure 64 Calibration Pump Screen Low speed Figure 64 will prompt you to prepare to collect the second calibration sample Empty the receiving vessel and replace it back under the output tube and press START The calibration will begin and the pump will run for two minutes HO ERROR CONDITIONS The total test time was amp Hin B 2 Sec Average Speed 1747 Enter the volume of fluid FUMFE during this test in liters Figure 65 Calibration Circulation Pump Low Speed_ Enter Volume The pump will stop automatically after two min utes and the sample may be measured The screen displayed in Figure 65 will prompt you for the volume collected during the second test period Enter the volume in liters in the space as requested and press ENTER IE ERROR CONDITIONS Pass 1 Fesul Flow Rate 3 ja
46. ectors fluid viscositv suction and head pres sure components and operating temperature that will be used in the process It is advantageous to break in a new pump tube cassette prior to cali bration by installing it in the pump head and operating it in the pump head at a moderate speed for several hours prior to calibration Figure 32 indicates the typical delivery results after calibration of the Single Circulation Pump Manual calibrations are performed by measuring the volume collected over a fixed time period for a range of speeds that would cover your typical process The specific details and procedures for calibrating the Circulation Pump are found in a later portion of this manual See Calibrating the Circulation Pump page 30 4500 4000 3500 3000 2500 2000 1500 1000 900 ml min 44 16 20 22 64 95 20 ml min 156 50 296 08 577 03 717 61 1134 17 2385 00 3943 33 40 RPM ml rev 35 57 37 01 36 06 35 88 35 44 327 46 39 60 L day 225 36 426 36 830 92 1033 36 1633 20 3434 40 5678 40 ii ml min 80 100 Figure 32 An example of a flow chart of the Single Circulation Pump Corning cat No 3222 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CellCube Dual Circulation Pump The Dual Circulation Pump Corning Cat No 3223 is designed for continuous dutv operation at 37 C The pump utilizes a single use disposable cassette Corning Cat No 3187 The ca
47. edis Pun used The calibration routine automatically per slore 3 5896e 85 Offset 2 9377e 82 forms a timed two minute interval as the collection 2 Calibrate Circulation Pum period calibration This is repeated twice The first with the pump operating at approximately 7 4 of full speed and the second at approximately 1 4 speed The approximate volume collected during the test period would be as shown in Table 3 lorPe 1 2884e BH3 ffzet d eBB1enn Fume i TurFe nne FLIMP Fume amp TsrFe EC PUMP Figure 56 Pump Calibration Screen The accuracy of volumetric delivery during actual operation and reproducibility of delivery from run to run is directly proportional to the accuracy of the measurement during the calibration procedure It is important therefore to accurately calibrate all pumps by weighing measuring the output vol ume The Circulation Pumps require the collec tion of larger volumes and will require either a large graduated cylinder or large empty containers to collect the volume of fluid during calibration Much less volume is used during Media Pump calibration but it is important that the same diam eter of tubing 1s used in the pump head during calibration as will be used in the production run By selecting the category Pumps from the main calibration menu the Pump Calibration Screen is displayed Figure 55 Press 1 to select the Calibrate Media Pump menu Figure 56 on page 30 The pump should be equip
48. ensure better distribution The vessels or reservoirs for media and product waste should also be sized to accommodate the operation of the system in its most active state 30 to 100 LPD Flow rates of all supplies air CO oxygen as well as media should be set to control the system during unsupervised periods while accommodating the most active needs of the sys tem Failure to control the system with either enough media or gasses may cause the loss of Figure 108 Production Cart with 4 X 3264 Corning Cat No 9234 Figure 109 Seeding Position of Cart Corning Cat No 9234 pH control or the premature reduction in growth rate within the entire system DESCRIPTION OF THE CART The CellCube Quad Production Cart Figure 108 is a device designed for simplified installation fill ing seeding operation and harvesting of up to four 100 Layer CellCube Modules Corning Cat No 3264 The quad production cart is a stainless steel autoclavable mobile rack that holds four 100 Layer CellCube modules or 340 000 cm of culture surface in a small footprint All opera tional procedures may be performed in this device because the CellCube modules can be fully rotat ed 360 degrees The cage in the center of the unit can rotate freely facilitating the attachment process of cells in the CellCube modules To seed the module rotate the cage first 90 counter clockwise Figure 109 from the operational hori zontal position Fi
49. equired Not compatible with Mac operating svstems 8 Meg min 5 Meg min 16550 UART recommended CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Circulation Pump Corning Cat No 3222 Specs Specifications Fuses 2 ea Universal Power Supply Frequency Auto switching Voltage Ist Stage 2nd Stage Power Consumption max Normal Range Normal Operating Temperature Physical Dimensions Height Width Depth Weight Approvals 2 0 amp 250V 5 x 20 mm 47 to 63 Hz Range 90 to 132 VAC 180 to 264 VAC 140 Watts 4 to 84 RPM 0 5 to 3 9 LPM 10 to 40 C 50 104 F 36 6 cm 14 47 17 6 cm 6 97 40 0 cm 15 87 21 kg 46 Ibs ETL ETL C CE Media Pump Corning Cat No 3221 Specs Specifications Fuses 2 ea Universal Power Supply Frequency Auto switching Voltage Ist Stage 2nd Stage Power Consumption Max Normal Range Normal Operating Temperature Physical Dimensions Height Width Depth Weight Approvals 2 0 amp 250 V 5 x 20 mm 47 to 63 Hz Range 90 to 132 VAC 180 to 264 VAC 40 Watts 172 VA 2 to 64 RPM 3 354 Liters dav 10 to 40 C 50 104 F 36 6 cm 14 4 17 6 cm 6 97 40 0 cm 15 87 26 kg 57 lbs ETL ETL C CE CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 59 Dual Circulation Pump Corning Cat No 3223 Specs Specifications Fuses 2 ea Universal Power Supplv Frequencv Auto switching Voltage Ist Stage 2nd Stage Power
50. ermine the most economical and productive operating parameters for the system through the growth and produc tion phase of culture In most cases glucose values should not fall below 50 If this is observed adding fresh medium or increasing oxygen to the system may be warranted Similarly a drop in the O value at the secondary oxygen probe is typical ly observed The difference between the O values in the primary probe and secondary probe the delta O should become more pronounced indicating that the cell population in the CellCube is in an active metabolic state Remember that the cells within the system rapidly reach a higher number of cells mL than most traditional culture systems Many of the typically used basal media such as MEM are designed to support only 1x10 cells mL per day A typical CellCube System using four 100 Layer modules Corning Cat No 3264 runs with approximately 85 000 cm surface area per module and contains approximately six liters of media within the mod ule The ultimate cell density depending on cell line exceeds 1x10 cells mL in the culture vessel At confluency 2 to 4 reactor volumes of media are typically required per day to meet the demands of the increased cell number conservative approach if perfusion is to be used might be to begin perfusion on day two to day four with one eighth the total system volume then double the volume perfuse each day until day 7 or 8 Conf
51. ervoir of the oxygenator a Inspect the sealing ring on the headplate to be sure it is not damaged b Remove and store as necessary c Go to the Glass Reservoir Cleaning Protocol described in the CellCube Oxygenator Section on page 7 if the vessel displays significant proteinaceous buildup after a lengthy operation ASSEMBLY OF THE CIRCULATION LOOP 1 Position HARVESTING PORT Add an appropriate length of tubing use 7 16 ID x As OD silicone tubing for use between the long harvest tube on the stainless steel combo side arm fitting of the oxygenator and the export pump tube set harvest tube 16 by j is Pharmed or Bioprene for the Watson Marlow pump head alternate sizes may result in improper operation Choose a pump tubing diameter size that 1s the same or one size larger than will be used on the import pump i e if using the 16 tubing on the import pump then use the 1s tubing on the export pump At the other end of the harvest tube attach some sili cone tubing with a female luer on the end This can be of sufficient length to connect directly to a harvest bottle bag or a shorter piece that will act as a bridge between the pump and a harvest container Place a clamp 7 cm 2 5 before the female luer fitting Wrap the end in autoclave paper A male luer plug can be wrapped separately and autoclaved to plug the opening on the female luer after autoclaving Secure all connections with a cable tie CORNIN
52. ess control data espe cially when perfusing at high flow rates with refrigerated medium HO ERROR CONDITIONS Temrerature Settings Key i Set Temrerature Alarm HI c Set TemrPerature Alarm H M JB E P 4 C Ds GEN AIF PLIMP Figure 80 Temperature Settings screen Press 1 and the desired temperature for the minimum alarm set point can be entered and the selection will be completed by pressing ENTER A typical value used here would be two degrees below the working temperature 35 C Press 2 and the desired temperature for the maximum alarm set point can be entered and by pressing ENTER the selection will be com pleted A typical value used here would be two degrees higher than the working temperature 39 C Pump Settings Screen The pump setting screen has nine options Figure 81 Selections 1 4 are for regulating the Media Pump and selections 5 9 are to control the Circu lation Pump Be sure to calibrate all pumps prior to adjusting the settings HO ERROR CONDITIONS Fume Settinas Screen Ke ia Flow Media Flow Rate Media Flow Rate ia Delay Time tor Media Pumr culation Flow Circ Flow Rate Circ Flow Rate tor Circ Pumr l gs 3 4 d 6 ia a a AAA L L LP L E cri DD i T T ID tu oro mi ag ey a a Drs GEN Figure 81 Pump Settings Screen Press 1 for Set Media Flow in order to select the Media Pump flow rate Input the desired value and then press ENTER to comple
53. ferent tubing requirements than a System that is set up in a roll in incubator and utilizes media feed and harvesting bags in another location ASSEMBLY OF THE OXYGENATOR HEADPLATE The assembly of both headplates is essentially the same The exception is that the 36L Oxygenator has two 25 mm oxvgen probes in line while the 6L Oxvgenator has a 12 mm oxvgen probe in the headplate and one 25 mm oxygen probe in line Because of the liquid flow the tubing lengths will Unrolled View clockwise from position 1 be different for both systems Be sure that the glass vessel has been treated with sodium hydroxide as was described earlier see page 7 so that proper fluid flow of the medium over the glass is maintained The pH and Oxygen probes should be calibrated and polarized prior to assembly 1 HEADPLATE Install the 100 mm headplate assembly onto the oxygenator bottle It may be necessary to slightly tip the headplate so that the inlet tubes fit into the vessel neck Be sure to inspect the sealing ring on the headplate so that it is cor rectly positioned the concave portion should rest on the top of the glass rim and that it is not nicked or distorted if the System is being Top View Position 1 12X420mm DO probe Media inlet tube Level sensor probe pH probe Media inlet tube Gas inlet Figure 105 Drawing of a 6L Oxygenator Headplate Unrolled View clockwise from position 1 Top View Posi
54. figure Inputs on page 32 Calibration of Oxygen Channel Two HO ERROR CONDITIONS Oxygen 42 Calibration st EF Amprlifier Hull Calibrate era E E Taraet 300 a l e 3 d librate lore O2CH1 EH FLOW TEMP PUMP Figure 50 Calibration summary screen for Oxygen Channel Two It is important that the oxygen probe has been connected to the controller and the controller has been turned on for at least six hours or the probes should be maintained in a polarized state prior to calibration Press the A button to select Oxygen channel two 1 Press 1 to display the Amplifier Null screen There are two cable connections for each of the oxygen probes The oxygen probe signal is transmitted through the BNC connector The three pin connector is connected to the thermistor that is built into each of the oxygen probes The thermistor connector should be connected to the controller and the BNC connector should be disconnected for the Amplifier Null procedure Figure 45 When the reading is stable press ENTER or the A button Reconnect the BNC connector and proceed to step two 2 Press 2 to display the Calibrate Zero screen It is important that the oxygen probe is in an oxygen free environment This could be a solu tion of sodium beta bisulfate or gas such as nitrogen that is near zero in oxygen content prior to executing this function When the probe signal has stabilized at the lowest point press ENTER or the
55. fill and installation procedures The electrode is mildly caustic and should be rinsed off of any contact with your skin The membrane of all O probes is very delicate and care must be taken to avoid damaging its integrity Even the slightest puncture of the membrane will result in faulty readings 1 Unscrew the cap sleeve from the electrode shaft and carefully slide it off the electrode If the membrane cartridge remains in the end cap dislodge it by gently pressing on the membrane end 2 Pull the membrane cartridge from the interior of the electrode body Discard the old mem brane cartridge if it is faulty or has been auto claved three times 3 Rinse the anode cathode assembly with tap water and dry it with a piece of lint free tissue paper Inspect the glass tip to ensure that it has no hairline cracks Never polish or sand it with an abrasive and avoid touching it with your fin gers Inspect the anode or brass colored area above the glass tip It should have a bright fin ish although an irregular or blotched finish is acceptable If a deposit is noted use a toothbrush with soft bristles to clean the anode 4 Check all O rings visually for mechanical defects and replace them if necessary 5 Completely fill the membrane cartridge with O electrolyte remove any air bubbles from the membrane cartridge by gently tapping on the side of the membrane cartridge 6 Position the probe so that the fill groove in the elec
56. g and final assembly Note The above is a conservative general recommendation and should be replaced by your validated procedure 1 Secure wire ties around each luer and plastic connector and clip off the excess from each wire tie 2 Securely tighten any luer to luer connections and loosen any clamps on the tubing to ensure free movement of steam through the fluid paths 3 Loosely gather the longer lengths of tubing into large loops that have no constrictions and loosely secure with a large wire tie or lab tape It is a good idea to drape the large loops over components of the oxygenator that can act as a hanger such as the side arms on the oxygenator or a probe on the headplate 4 Ensure that any exposed ends of tubing are wrapped with autoclave paper STERILE SET UP AFTER AUTOCLAVING The following procedures require standard aseptic procedures for assembly 1 Check all the connections to be sure that everything is still securely tightened 2 Remove the oxygenator tubing Circulation Pump cassette assemblies from the autoclave and place into a biohood or a biological safety cabinet 3 Unravel the tubing and lay it out in a hood or safety cabinet in a logical flow path orientation 4 Place the CellCube module sample septum media in cell seeding and harvest tubing in a sterile biohood 5 Connect the CellCube module outlet tube to the 12 male male female luer connector near the secondary oxygen p
57. ger tightened Note Do not twist the DO probe counter clockwise during insertion because the DO probe membrane is held in position with a clockwise thread Position GAS INLET Prepare two 0 20 pm hydrophobic vent filters for sterilization by connecting in series utilizing a short length of 7 16 x 3s silicone tubing Insert the barb end on one of the filters onto a 7 cm 2 5 in 16 ID x 3s OD silicone tubing and place the other end of the tubing on the gas inlet of the oxygenator headplate Position GAS VENT Use 316 ID x Ys OD silicone tubing for con nection in order to support the weight of the filter Cut a tubing length of 7 cm 2 5 in and place this on the short stainless steel tube of the combo side arm fitting Insert a single 0 20 pm hydrophobic vent filter for steriliza tion on the other end of the tubing Note Be sure to place the appropriate protective cap for autoclaving over the attachment point for the electronic wiring on both the pH and oxygen probe prior to autoclaving BREAKDOWN OF THE OXYGENATOR HEADPLATE AFTER OPERATION 1 Decompress remove and store the level sensor cinch bushings remove and store the level sensor pH and DO probes a Inspect the O rings on the level sensor b Make sure to protect the end of the DO probe on the 6L vessel by replacing the black vinyl cap Loosen the orange headplate securing ring and remove the 100 mm headplate assembly from the glass res
58. gure 110 After attachment on the first side the cage is turned 180 in the clock wise direction to allow the cells to attach to the second side of the CellCube module After attach ment the cage is turned back 90 counter clockwise back to the horizontal operating position The quad production cart has an adjustable wheelbase and can be sized to fit in a wide selec tion of tracks founds within a full size standard roll in incubation or environmental chamber during growth periods Figure 110 Operation Position of Cart Corning Cat No 9234 CORNING CELLCUBE CULTURE SVSTEM USER s MANUAL General Material Advisorv 54 Below are some general guidelines for GLP GMP environments Corning recommends replacing all the disposables each batch including the vent filters and silicone tubing All CellCube Modules are pre sterilized and single use disposable items DO NOT auto clave a CellCube module at the end of a run because it may compromise the integrity of the CellCube module The Circulation Pump cassette and the dual Circulation pump cassette are one time auto clavable single use items These items should be discarded after each CellCube system run The pH and dissolved oxygen probes from either Broadly James or Ingold are supplied along with the manufacturer s recommended procedures for cleaning and maintenance These instructions are included with each new probe and should be filed for your reference For fu
59. h all of the I O devices necessary to operate the CellCube System including calibration set point assignments of a CellCube system gas flow con trol and the measurements of the different probes The LCD screen will show which version of Firmware is installed on the machine the moment the controller is turned on If upgrading the Firmware is necessary the con troller must be connected to a PC with operational Host Software It is imperative that these up grades are performed by trained Corning9 CellCube System personnel or in conjunction with guidance from a Corning CellCube System technical spe cialist to ensure that the Firmware and Host Soft ware are correctly matched After uploading on the host software select the control bin file The Host Software will ask to confirm the update A window will display the status of the firmware update on the PC screen Once the firmware is updated the controller will automatically restart itself There is no need to restart the PC Detailed Open File File name i Folders Open file of type Drives Controller Firmware bin c s Figure 40 Starting the host software program instructions are provided from a Corning technical specialist together with the software upgrade instructions in the next section UPGRADING FIRMWARE Start the Host software program See Figure 40 On the menu is the option Transfer which will give vou two options after clicking on it The fir
60. he back of the pump is in the OFF position 3 Assure that the power selection knob on the front of the pump is in the STOP position 4 Insert the power cord into the socket located at the rear of the pump a Assure that the safety shield is in position covering the pump mechanism The safety shield 1s held in place by four thumb screws which must be tightened sufficiently to depress the white safety switch found on the left tube guide of the pump 6 Insert the power plug into the wall outlet Insert one part of the remote control cable in the back of the Dual Circulation Pump and the other end in one of the pump interface ports at the back of the Controller Corning Cat No 3131 7 Activate the power switch located above the power cord on the rear panel The red indica tor on the front panel should light when the power selector switch 1s turned on The green Jog Start light on the front panel should light and flash The green Remote light on the front panel will light only when the remote control CellCube Single Circulation Pump Figure 30 Single Circulation pump 3222 interface is properly connected and the safety switch has been activated by attaching the safety shield Io activate the safety switch push on the Jog Start button one time 8 Turn the power switch on the front panel to the local setting on the pump The pump should begin to operate at a rate determined by the Rate knob on the pump The
61. he input pump on the Media Pump Unit Position a clamp on the tubing approximately 7 cm 2 5 from one end Place a male luer connector on the same end and connect it to the female luer fitting on the 2 male male female luer connector that will be closest to the CellCube inlet tube This will become the cell seeding tubing and its connec tion point will be the CELL SEEDING PORT At the other end of this tubing attach a luer fit ting to mate with an appropriate luer fitting on the Cell Seeding Reservoir Repeat the process above with another similar length of tubing that you connect to the other female luer fitting with a male luer fitting This will become the medi um addition tubing and its connection point will be the MEDIUM ADDITION PORT At the other end of this tubing insert a male luer fitting and a clamp as was done previously for the CELL SEEDING PORT Attach this to one of the 16 Va or 16 Pharmed tubings provided in the Inlet Pump Tubing Set The selection of tubing is dependent on the amount of medium to be perfused into the CellCube System Table 5 on page 48 will provide you with a guide to the range of volumes that can be delivered If you are unsure of the amounts that will be used then connect all three tub ings in the Inlet Pump Tubing Set in a line of increasing size and connect the smallest tubing to the line above At the other end of the Inlet Pump Tubing attach a length of 1 4 ID tubing with a male l
62. hould flash 4 The upper tube guides contain four slotted guides to locate the outlet check valves The tubes may be inserted into the guides by pinching the tube approximately 3 cm below the valve assembly and sliding the tube into the slot 5 Insert the four Dual Circulation Pump tubing into each of the top tube guides 6 Center each tube in the guide and push down on each of the valve assemblies 7 Press down on the bottom cassette manifold to gently stretch the cassette over the bottom locating pin This may take some physical exertion especially with a new cassette that is a bit stiff a Figure 34 Drawing of the Dual Circulation Pump Cassette Corning Cat No 3187 Figure 35 Installing the Dual Circulation Pump Cassette Top View Figure 36 Installing the Dual Circulation Pump Cassette Total View 8 Press down and toward the faceplate on the bottom manifold until it slides over the bot tom locating pin Generallv speaking vou will hear a snap as the bottom manifold CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 19 snaps into the mouth on the bottom locating pins Figure 37 9 Slide the tube between the tube guide and the piston the open position from step two 10 Press the Jog Start switch intermittently to move the piston over to the other side allowing the second of the silicone tubular diaphragms to move into position Repeat the process by pressing the J
63. ientation It is a good idea to use a marking pen and write top and bottom on the front of the cassette manifolds and also indicate the direction of flow with an arrow to avoid confusion later Caution The area under the safety shield is a pinch point and extreme caution should be used while installing or removing the cassette 1 Place the Power Selector switch in the Stop position 2 Using the Jog Start switch place one of the pistons in the full right left position Note The Jog Start switch needs to be continually pressed to operate the pump 3 Remove the safety shield The red Alarm light will light and the Jog Start light will flash 4 Place the cassette loosely into the pump cavity 5 Slide the top manifold onto the top locating pin until it settles into the notch in the pin 6 Press down on the bottom cassette manifold to gently stretch the cassette over the bottom locating pin This may take some physical exertion especially with a new cassette that is a bit stiff Generally speaking you will hear a sharp snap as the bottom manifold snaps into the notch on the bottom locating pin Repeat with the top of the cassette if it has shifted during positioning of the lower manifold 7 Press the Jog Start switch intermittently to move the piston over to the other side allow ing one of the silicone tubular diaphragms to move into position Repeat the process by pressing the Jog Start button d
64. ing PUMP CALIBRATION FLOW CHARTS The flow charts Figure 29 page 13 depict the typical output of the Media Pump Corning Cat No 3221 The volume range that can be delivered is completely dependent on the internal diameter I D of the pump tubing used in the pump head and is independent of the size of the tubing out side of the pump head The display indicates the rate in RPM and it is typically linear 0 5 FS full scale of the set point for the usable speed range of the pump Mechanical variations back pressure tubing dimensions and actual tube loca tion can all affect the performance of the pump The most accurate delivery will occur when the calibration of the pump is performed prior to sterilization in conjunction with the same size tubing connectors fluid viscosity suction and head pressure components and operating temper ature that will be used during actual operation It is advantageous to break in a new segment of pump tubing prior to calibration by installing it into the pump head and operating it in the pump head at a moderate speed for several hours prior to calibration Figure 29 on page 13 indicates the typical delivery results after calibration of the Media Pump for certain ID tubing Manual calibrations are pre formed by measuring the volume collected over a fixed time period for a range of speeds that would cover the process The specific details and proce dures for calibrating the Media Pumps ar
65. irculation loop manually at given intervals can be both beneficial and sup ply a wealth of information on how to proceed 4 Take small steps and avoid multiple variable changes in conditions that can confound an explanation of a particular effect 5 Lastly especially as cell populations increase in the CellCube Modules it is very easy for things to get out of hand in the process Factors such as pH or available nutrients can change very rapidly when populations exceed 1x10 CellCube System Setup Protocols cells in a confined area Try and stay ahead of the process whenever possible As you expect as you grow in your expertise you should become more adept at noticing a condition and dealing with its appropriately BUILDING UP CELLCUBE FLUID PATHS The underlying flexibility of the CellCube system allows a wide variety of configurations and envi ronments to be adopted for cell culture While the basic pathways of fluid movements are essen tially the same minor variations may be necessary due to decisions on where the equipment will be positioned warm room versus roll in incubator whether the harvest stream is heat labile and must be refrigerated in an adjacent area or removed from the culture area or the upstream feed stream have temperature or light constrains We have pro vided you a few variations that we have compiled from a variety of sources to assist you in planning an optimal flow path These schemes c
66. ith a screwdriver by turning it approximately 90 in a clockwise direction For safety reasons the pump will not activate until this sequence has been completed 6 Insert the plug into the wall connector Insert end of the remote control cable into the back of the Media Pump and the other end into one of the pump interface ports at the back of the controller Corning Cat No 3220 Figure 28 Media pump 3221 7 Activate the power switch located above the power cord on the rear panel The red indica tor on the front panel should light when the power selector switch is turned on The green Jog Start light on the front panel should light and flash The green Remote light on the front panel will light only when the remote control interface is properly connected and the safety switch has been activated To acti vate the safety switch push on the Jog Start button one time 8 Turn the power switch on the front panel to the local setting on the pump The pump should begin to operate at a rate determined by the Rate knob on the pump The RPM rate 1s shown on the display 9 Rotate the Rate knob counter clock wise until it stops The rate should stabilize at approximately 2 RPM 10 Rotate the Rate knob clock wise until it stops The rate should stabilize at approxi mately 64 RPM 11 Reduce the pump Rate to 2 RPM CORNING CELLCUBE CULTURE SVSTEM USER s MANUAL 12 Safety check Slowly loosen either
67. k and factory history data The Auxiliary Summary Screen shown in Figure 96 displays the functions that are available for BATCH DEFAULT TIME and FACTORY Batch Menu ERROR DETECTED Batch Processing Screen Figure 97 Auxiliary Batch Processing Screen In the Batch menu you can start a Batch By entering this START Batch menu you clear the entire history of the controller Before you do this Corning recommends saving the current data before starting the run batch It will only clean the history data The settings calibration and configuration data will be unchanged ERRORS DETECTED TAET BATCH l history will be CLEARED Are You sure this is what you want to do Figure 98 Auxiliary Batch Start Screen Defaults Menu ERRORS DETECTED Defaults Nenu ke FI 1 Load Factory Defaults E Load User Confisuration Save Current User Confisuration Figure 99 Auxiliary Default Screen The Defaults Menu provides three selections Load Factory Defaults Load User Configuration and Save Current User Configuration Figure 99 Load Factory Defaults restores the values that were entered at the factory Load User Configuration loads the values that were saved by the user selec tion 3 Save Current User Configuration provides a means to save the preferred set up parameters of the user It is advisable to utilize this feature after all the probes have been calibrated and the process parameters have
68. l Note The above method assures that there is a small bubble of air inside the probe to provide room for expansion during sterilization Calibration of Oxygen Channel One A good laboratory practice is to record all of the values achieved during the calibration procedures in a notebook to ensure that a history of probe response is accumulated for the life of the probe Selecting the OXYGEN Calibration screen by pressing the A button displays the calibration menu for oxygen channel one Oxygen 1 Cali bration The functions that may be calibrated are displayed and are listed as items 1 4 You may also note that when the first oxygen screen is selected that the variable function button is O2CH2 or oxygen channel two By pressing the A button again at this point you switch and calibrate channel two As mentioned above it is important that the probes are connected to the controller and the controller is turned on for at least six hours or the probes should be maintained in a polarized state prior to calibration The following proce dures refer to the current value of the probe signal during the calibration procedures The CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL numbers displaved are digital values of the amp lifier The amplifier is 12 bit and the full scale is represented bv 4096 digital levels The full scale is 0 to 4095 units with 0 units equivalent to 0 input and 4095 the maximum input This pr
69. l of the desired set points HO ERROR CONDITIONS Airflow Settings Ke i j ate H cermin H ccz min cc mir Figure 79 Airflow Settings screen Press 1 to enter the desired Air Flow Rate and complete the selection by pressing the ENTER key A typical value used here would be 200 cc min for the 6L oxygenator and 400 cc min for the 36L oxygenator Avoid excessively high values which can displace other desirable control gasses such as oxygen or CO which are less soluble in the combined carrier gas stream Press 2 for the Set Air Flow Alarm MIN in order to enter the desired Air Flow rate for the minimum alarm set point and complete the selec tion by pressing ENTER A typical value used here would be 100 cc min less then the Set Air Flow Rate CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL 37 Press 3 for the Set Air Flow Alarm MAX in order to enter the desired air flow rate for the maximum alarm set point and complete the selection bv press ing ENTER A typical value used here would be a 100 cc min more then the Set Air Flow Rate Temperature Settings The alarm temperature settings should be set within a range that will not generate numerous false alarms Tests have shown that the tempera ture probe placed under the oxygenator and insu lated from the surface under the oxygenator very closely simulates a probe immersed in the media When properly calibrated against a reference it can provide valuable proc
70. le minimum and maximum set points alarms and error condi tion alarms for DO 1 DO 2 pH RTD Media Pump flow rate Circulation Pump flow rate and the Air flow rate The controller provides selectable error condition alarms for CO O and the level sensor The error conditions for the probes are as follows DO 1 and 2 Probe shorted pH Probe shorted RTD Probe not connected or shorted Pumps Interface error or not connected Mass Flow Control Output less than requested loss of pressure Level Sense Not connected or High Level Status Alarms ERRORS DETECTED ieu Device Status Information Tew ce Status DO 1 Ok OIE Ok zu below min zettina PUMP Figure 71 View Device Status Information screen The View Device Status Information menu Figure 71 provides a current look at all the alarm conditions showing which alarms are active and what type of activity caused the alarm Alarm Categories ERROR DETECTED Alarm Confisuration E T T ka ka ka pm s n n n n n fa Lt Los Duc Din Lc eT Et Lus Du Lic Lc Du Dt Led in Dict Et Lc Duc Dict Lac Dac D icn Lc Tics Dict INPUT 1 PUMP OUTPUT Figure 72 Alarm Configuration Screen Active The alarm configuration screen displays the dif ferent categories visible with their alarm function device minimum maximum error and delay Figure 72 By default all alarms are set in the off position at the factory This is to
71. livering up to 1 0 LPM of air through a 0 2 pm filter As the airflow rate is increased the CO flow should be increased to maintain the same proportional mix of air and CO during the initial stages of culture Often when cell densities reach sufficient levels the CO may be lowered since the cells will provide sufficient CO from metabolism to maintain pH set points The Oxygenator affords excellent O transfer and CO removal Generally increased gas flow and increased perfusion are all that are required to maintain the culture at the desired state Oxygen Addition The addition of oxygen to the headspace gases increases the transfer of oxygen to the media returning to the oxygenator Oxygen addition may be required for some cell lines depending on the size of the culture module used the metabolic activity of the cells cultured and the minimum dissolved oxygen level desired Oxygenation can be controlled in two ways If the 1 primary probe is selected then additions of O will occur based on the readings at the upstream oxygenator end of the process If the 2 secondary probe is selected then additions of O will occur based on the readings at the outlet of the CellCube Either strategy is acceptable Typical set points for oxygen control are 60 on the 1 primary probe or 40 on the 2 secondary probe Higher initial O settings on the primary probe should be avoided because over shoot of the desired set point is more
72. lizes eight stainless steel ball check valves located in the top valve assemblies and bottom manifold assemblies The bottom manifold is labeled in the area between the sili cone tubular diaphragms The bottom manifold is marked faintly with bottom and represents the inlet section of the cassette and the pressure relief valves The top valve assemblies represent the outlet section of the cassette The cassette will operate in one position only It is important to locate the cassette in the pump with the bottom manifold over the location pin near the lower por tion of the arrows that indicate the direction of flow in the pump It is a good idea to use a mark ing pen and write bottom on the front of the cassette manifolds and indicate the direction of flow with an arrow to avoid confusion in the future Caution The area under the safety shield is a pinch point and extreme caution should be used while installing or removing the cassette There are a variety of methods used to install the cassette in the pump The following is one method that permits the installation of the cassette with only one operator 1 Place the Power Selector switch in the Stop position 2 Using the Jog Start switch place one of the pistons in the full right left position Note The Jog Start switch needs to be continually pressed to operate the pump 3 Remove both of the safety shields The red Alarm light will light and the Jog Start light s
73. luency would ordinarily be reached on day seven depending on the doubling times of the cell line and the initial seeding density PRODUCTION PHASE The timing and parameters of the production phase of the culture depend on the type and ulti mate use of the particular cell line Many cultures require a medium for production that is different from that used in the growth phase of the culture The transition from growth phase to production in traditional cell culture technology generally involves various washing and rinsing steps A wash process might require as little as 1090 wash vol ume for roller bottles up to as much as 4 reactor volumes for a stirred tank reactor The media and buffer used during these procedures are not gen erally taken up by the cells because of the short contact time involved The CellCube System can employ a perfusion methodology to accomplish this goal The benefit of perfusion is the ability to provide a gentle tran sition between various operating conditions as opposed to fed batch arrangements where fresh media can shock the cells and introduce a lag phase and a delay in productivity Traditional washing steps may be unnecessary with the perfusion system One object of a wash step might be to reduce or remove the serum component of the growth media The use of a perfusion system allows the user to easily pick the rate of change and the time of completion For example if the media from the first day of
74. lvcarbonate Connectors Qosina Corp 150 Q Executive Drive Edgewood NY 11717 Tel 631 242 3000 Fax 631 242 3230 www qosina com 12 x 1 2 male male with two female luer ports Qosina Cat No 27210 12 x 1 2 male male with two female luer ports Qosina Cat No 27226 12 x 12 X 1 2 male male male 45 connector Qosina Cat No 60127 Va x 1 2 reducer connector with female luer port Qosina Cat No 27224 14 x V2 reducer connector no female luer port Qosina Cat No 27215 Or Alternatively from Corning directly in the start up kit Vent Filters Corning Incorporated Life Sciences 45 Nagog Park Acton MA 01720 Tel 978 442 2200 Fax 978 442 2476 www corning com lifesciences 50 mm 0 22 um vent filter 12 pk Cat No 131261 Syrfil FN Miscellaneous Cole Parmer Instrument Co 7425 North Oak Park Avenue Chicago Il 60648 Tel 800 323 4340 Tel 847 549 7600 Fax 847 247 2929 www coleparmer com Nylon Cable Ties Nylon 3 16 tubing connectors Nylon 1 2 tubing connectors Nylon 16 tubing clamps Quick tie tensioning tool C P Cat No L 06830 54 6463 10 6463 25 L 06833 00 G 06830 00 Alternatively the cable ties may be purchased at a local hardware stor or as part of the Corning CellCube System Start up Kit Buffer Addition Pumps New Digital System Watson Marlow Inc 220 Ballardvale St Wilmington MA 01887 Tel 800 282 8823 978 658 6168 Fax 978 658 0041
75. m Requirements Optlotab peesaa derit d a7 Circulation Pump Corning Cac ING 3277 SPECS occacet ba d har 58 Media Pump Corning Cat ING2 O22 Speer a ces wid i ebebd REP UPS 58 Dual Circulation Pump Corning Cat ING 3273 B DGES o cavet vues bib wees 59 Pump DOLOS niseaencteae toe beset re bene 59 Explanation of Remote Interface 59 External Alarm Connections 60 iii CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL Before Vou Start UNPACKING THE CELLCUBE SYSTEM 1 Using a sharp tool carefully cut the tape on the large outer box to open 2 Remove all inner boxes and open each 3 Remove components from each inner box and place on a stable bench top 4 Important Save all packaging materials CHECK INVENTORY Each system component box should include the following Digital Controller Corning Cat No 3220 Digital Controller Digital Controller power cable with power supply Bag with different cables Media Pump Corning Cat No 3221 Media Pump Power cable Pump Controller interface cable Single Circulation Pump Corning Cat No 3222 Circulation Pump Power cable Pump Controller interface cable Dual Circulation Pump Corning Cat No 3223 Dual Circulation Pump Power cable Pump Controller interface cable 6L Oxygenator Complete Corning Cat No 3101 Oxygenator glass 6L pH Probe pH Probe cable 12 mm Oxygen probe 12 mm Oxygen probe ca
76. minimize the annoyance of loud alarm noices during initial setup of the CellCube System when a large number of process parameters would be out of limit Minimum maximum and error has its own group of alarm functions and they are labeled SI2D where S Sound 1 Switch 1 2 Switch 2 and D Delay Any combination of selections is acceptable Selecting sound for each alarm could be annoying each active alarm must be resolved before the aural alarm stops and that could take a few minutes It is best to activate the second function only after a run has started and culture parameters have stabilized Selecting an Alarm Function Each alarm function is selected by moving the cursor an underscore symbol to the desired CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL alarm condition utilizing the left right up and down arrows Figure 73 Each time one of the arrows is pressed a beep tone is generated and the cursor moves one position in the direction of the arrow selected Alarm functions that are not selected are displayed as a black character on a blank background Alarm functions that have been selected are displayed as a white character on a black background Press ENTER to either select or deselect the function Figure 73 Arrow Cursor Delay Function The delay function helps prevent unnecessary activation of the connected alarm system This is especially useful if the Controller is being oper ated
77. mum setting in this case The mini mum maximum average and last DO values for the time period shown on the graph are also displayed EFROR DETECTED Oxygen History CHe Figure 85 View Oxygen History Channel 2 Sensitive Screen When a run is longer than a 24 hour period a more sensitively scaled Y axis can be viewed by pressing the ENTER key which will change the scale to the minimum and maximum measured value for the last 24 hours A typical oxygen his tory chart with the sensitive scale is displayed in Figure 85 The bottom left portion of the display shows that the next possible selection is O2FLOW Oxygen Flow History HO ERROR CONDITION Oxygen Flow History Hage LIN STATUE PUMPS Figure 86 View Oxygen Flow History Sensitive Screen a LICHE 1 CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL The third oxvgen view displaved is the Oxvgen gas Flow Historv A tvpical Oxvgen Flow Hist orv chart in auto scale mode will have the graph historv labeled Oxvgen Flow Historv and the Y axis scale will be 0 to 500 cc min 500 cc is the maximum Air Flow setting in this case The minimum maximum average and last oxygen air flow values for the time period shown on the graph are also displayed A more sensitively scaled Y axis can be viewed by pressing the ENTER key Figure 86 The bottom left portion of the display shows that the next possible selection is DO 1 pH Air pH History HO ru ptas FH Histor
78. nutes E ERROR CONDITIONS Pass 1 Fesul Flow Rate a 7415 l mini regy JE S Pass Results Flow Rate B OHHIB l mini Feed 1695 poA rezultz xSlore d d rHr 56e Hj Ffzet 7 9693e HBhl LI slorPe 2 amp 6H8re Hj Offset 3 681re 61 Use These Values Figure 60 Summary Media Pump Calibration The pump will automatically stop after 2 minutes The screen displayed in Figure 59 will prompt you for the volume collected during the second test period Enter the volume in mL in the space as requested and press ENTER The calibration summary screen will display the results of the two tests Figure 60 The results may be either accepted or rejected Press YES to accept the values If results are rejected press NO and the prior calibration values will be used The Media Pump is now calibrated Note It is a good laboratory practice to enter results of the calibration into a lab notebook in order to compare them to past or future results Warning Each time you access the pump cali bration routine the pumps will be paused Calibrating the Circulation Pump HO ERROR CONDITIONS Fume Calibration creern Key 1 sTrbsste Media Pum Slope 3 5896e 85 Of set eg a3rre BWe e Calibrate Circulation FumF s lore l 2884e H3 FFzet 1 25B8let 8 Fume i Tyre MEDIA PLIMP Fume 2e Tyre CIRC PLIHP Figure 61 Pump Calibration Screen The calibration of the Circulation Pump is a two point calibra
79. ocedure is generally performed prior to sterilization although it can be done after sterilizing however the meth odology for performing this step aseptically is a bit cumbersome 1 Press 1 on the control pad to display the Amplifier Null screen with the current value of the probe signal Figure 44 There are two cable connections for each of the oxygen probes The oxygen probe signal is transmitted through the BNC connector HO ERROR CONDITIONS Oxygen 1 Calibration Flifier Mull librate era Enter Tarset 00 librate lore FLOW PUMPS Figure 44 Calibration screen of Oxygen Channel One ecHe The three pin connector is connected to the thermistor that is built in to each of the oxygen probes Disconnect the BNC connector from the controller for the amplifier null procedure Figure 45 The value on the display should rapidly decrease to between 4 and 10 units Figure 46 When the reading is stable press ENTER or the A button Reconnect the BNC connector and proceed to step two Figure 45 Controller inputs showing BNC connector disconnected MORE HOM ERROR CONDITION Disconnect only the Coaxial BMC cable from the oxygen Probe for this channel When the value stabilizes Press the ENTER key to accert or the CANCEL kes to retain the current values Current Value J Figure 46 BNC connector disconnected screen 2 Press 2 to display the Calibrate Zero screen Place the oxygen probe in
80. of the screws holding the safety shield of either pump in position by turning counterclockwise 90 When the cover becomes loose the red alarm light will light the pump should stop and the green Jog Start light will flash The pumps will not restart until the safety cover is properly positioned the screws tightened again and the Jog Start switch is pressed to restart the pump TUBING INSTALLATION The Media Pump incorporates two Watson Marlow pump heads The direction of rotation clockwise is the same for each pump The lower export pump rotates at a rate of 1 2 times the speed of the upper import pump The tubing set Corning Cat No 3120 includes three different size import tubes that include luer connectors and one tube that includes barbed hose connec tors Arrows on the face of the pump show the direction of the flow for each of the pump heads 1 Turn the rate control knob counterclockwise until it stops 2 Place the Power Selector switch in the Stop position 3 Place the rotor in the horizontal position using the Jog Start switch 4 Unlock and open the Safety Cover and swing out the rotor crank handle 5 Lift up the tubing pinch clamp and take notice that the closed position is adjustable The adjusting tab slides out away from the face of the pump The tab should be out for the smallest tube and in for the larger tubes The object of the pinch clamp is to stop the tubing from being drawn into the pump
81. og Start button until all four silicone tubular diaphragms move into position When the Jog Start Button is released the pistons tend to move back toward the center point between the two diaphragm tubes 11 Check the location of the silicone tubular diaphragms The front of the tube should be flush with the face of the tube guide 12 Install the safety shield using the four cover screws provided for each plate Note When both covers are tight and the white safety switches are activated the red alarm light will light the pump should stop and the green Jog Start light will flash The pump will not restart until the safety shield is properly posi tioned and the Jog Start switch is pressed to restart the pump 13 Repeat steps 1 through 12 for the opposite side of the pump PUMP CALIBRATION FLOW CHARTS The flow chart Figure 39 page 20 depicts the typical output of the Dual Circulation Pump Corning Cat No 3223 The display indicates the rate in RPM and it is typically linear 0 5 FS full scale of the set point for the usable speed range of the pump Mechanical variations back pressure tubing dimensions and actual tube loca tion can affect the performance of the pump The most accurate delivery will occur when the cali bration of the pump is performed prior to sterili zation in conjunction with the same size tubing connectors fluid viscosity suction and head pres sure components and operating temperature A
82. on with pH 7 0 and a second refer ence Since bioreactors using mammalian cells typically progress into the acidic range during scale up we recommend a pH 4 0 buffer as the second standard A pH 10 standard can be used but the values collected will be less accurate than calibrating at pH 4 0 One major difference from benchtop pH meter calibration is temperature compensation Since the CellCube System is usually operated at 37 C there are three possible compensation methods NONE FIXED and AUTOMATIC Select NONE if you are cali brating the probe at the actual use temperature and the probe and solution are at the same tem perature Select FIXED if you are calibrating at room temperature and the system is brought up to temperature later Select AUTOMATIC if you have the temperature probe calibrated and con nected and you are measuring the pH in a solu tion at the same temperature as the temperature probe Note For the best results always use fresh buffer solutions pH buffer solutions higher than 7 0 are less stable and have a shorter lifetime MO ERROR CONDITION Place electrode in 4 buffer solution of BHBdEH When the value becomes stable press the ENTER key to accert p CANCEL key to retain the current Walue Current Value 15559 Figure 52 The Calibration at pH 7 0 1 Press 1 and immerse the probe in a standard solution e g pH 7 0 Stir the probe momen tarily to ensure proper contact The first
83. onized water as required Unrolled View clockwise from position 1 Top View Position 1 1 Media inlet tube 2 Level sensor probe 3 Infection port 4 Media inlet tube 5 pH probe port 6 Gas inlet Figure 22 Schematic of a 36L Oxygenator Headplate CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CellCube Digital Controller UNPACKING AND SETUP The controller is equipped with a universal power Note The CellCube Controller Figure 23 Suey enon l eC pews utilizes a variety of different connectors for power source between 90 and 250 VAC and signal inputs Each connector is different to 1 Place the controller on a clean dry surface help prevent improper connections It is recom 5 mended that you become familiar with each con nector and its purpose prior to beginning the Assure that the power selection knob on the back of the controller is in the OFF position initial testing of the controller It is important 3 Insert the power connector from the power that you never force a connector into one of supply into the socket located at the rear of the inputs the controller The CellCube Controller is packaged with the 4 Insert the female end of the power cord into following components the power supply and the male end into the 1 Controller power receptacle 5 Activate the power switch located above the power cord back rear panel The controller 3 Support equipment kit will begin an initialization ro
84. own until the second tubular diaphragm moves into posi tion When the Jog Start button is released the piston will tend to move back toward the center point between two diaphragm tubes 8 Check the top and bottom manifolds to assure that they are properly located in the notch of the locating pin 9 Check the location of the tubular diaphragms The front of the tube should be flush with the face of the tube guide 10 Install the safety shield using the four cover screws provided Note When the safety shield cover is tight and the white safety switches are activated the red alarm light will light the pump should stop and the green Jog Start light will flash The pump will not restart until the safety shield is properly posi tioned and the Jog Start switch is pressed PUMP CALIBRATION FLOW CHARTS The following chart Figure 32 on page 16 depicts the typical output of the Single Circulation Pump Corning Cat No 3222 The display indi cates the rate in RPM reciprocations per minute and it is typically linear 0 5 FS full scale of the set point for the usable speed range of the pump Mechanical variations back pressure tub ing dimensions and actual tube location can all affect the performance of the pump The most accurate delivery will occur when the calibration on the pump is performed prior to sterilization CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 16 making sure to use the same size tubing conn
85. ped with the same ID tubing that will be used during operation There are three choices START PRIME or ABORT The inlet section of the tube should be immersed in water and the air should be purged from the system This can be achieved by placing the outlet tubing in the same vessel as the inlet tubing and pressing down on the PRIME button to purge all of the air in the tubing When you As mentioned in the section Cassette Installation release the PRIME button the pump stops on page 15 more accurate calibration is obtained priming immediately when the tubing has been broken in by operation for a period prior to calibration Table 3 Volume Collected During a Test Period Module Size 25 Layer 50 Layer 100 Layer System Volume L 3 4 5 7 5 Flow Rate Tubing LD RPM mimin Lidav Vol Exchanges day at Flow Rate 1H6 Tubing 10 43 6 21 1 4 08 20 86 124 41 28 17 30 129 186 82 41 25 40 172 248 83 55 33 50 215 310 1703 69 41 1 87 Tubing 10 179 258 86 57 3 4 20 372 536 179 119 71 30 558 804 268 179 107 40 44 1071 2357 238 143 50 930 1339 446 298 179 3167 Tubing 10 405 583 194 130 78 20 810 1166 389 259 155 30 1215 1750 583 389 233 40 162 0 2333 718 518 311 50 2025 2916 972 648 389 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL NU ER
86. qa l mini FEE 3312 Pass Results Flow Rate 8 199 l mini Feed lr4v n DEM rezultz Elore l 1 8532e 83 Offset 3 838Heths LI slore l1 4134de H3 Of Fset 1 2463e 68 Use These Values Figure 66 Summary of the Circulation Pump Calibration The calibration summary screen will display the results of the two tests Figure 66 The results may be either accepted or rejected Press YES to accept the values The Circulation Pump is now calibrated Note It is a good laboratory practice to enter results of the calibration into a lab notebook in order to compare them to past or future results CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL SYSTEM CONFIGURATION The Configure menus are accessed by pressing the main selection button CONFIGURE on the Digital Controller Faceplate see Figure 24 on page 8 The first menu that appears is the Configuration Summary screen HO ERROR CONDITIONS Conf isuration xummar4 utz D2 1 88538 Oe 2 seeroe FH er ETI im Pume 1 MEDIA Fume ce CIRCULATION Alarms 1 DISABLED INACTIVE 2 D01SABLED INACTIVE CHE dea 20 Oe 2 4 28 FH d ZH ETI d b Serial Port Ri 232 PORT HI IHFUT OUTPUT Figure 67 Configure Summary Screen There are five selection categories under the main configure menu INPUT PUMPS ALARMS OUTPUT and SERIAL ports Configure Inputs HO ERROR CONDITIONS Er Canfisuratian I E Toggle Averaging E Enter Humbe
87. r ODSrdtloft 222a eR PI IRE ER 46 Assembly of Circulation Loop 46 Autoclave Preparation Lulli 49 Sterile Set Up After Autoclaving 49 CellCube System Operation 51 Growth Phase 4a oce doe Rob Hew had iais 51 Production PIS6 tanker dodo Ke ed Ae 51 pH and Oxygen Control 52 Oxygen JOE OBbass ua docuerit ras 52 Large Scale Operation 53 Theory of Large Scale Culture Operation 53 Desenption Of tie Cart osos see sie be Ep 53 General Material Advisory 54 Appendics 4 10353 iens rape REP dame E PES 29 Vendor LEE oa sese booa Pad ertoe saint 55 Lir m e eee neo ame 55 Polycarbonate Connectors 25 Vent B NET Sa sowas ee eo A 55 Miscellaneous osese wie ok Ra Ede risen 55 butter Addition Pumps iux ox arra om RR am 93 Loer Tubing Fittings 2224 x axo een 56 Nylon 5 4 Tubing Clamps 56 General SpeciiCatiOns es dud i wg be duce debi 56 Digital Controller Corning C36 ING 3220 po ibus a acri eso ache dicen d 56 Envitoninental aw eae e Se ae ere 56 Power Supply 6240003 6200434000 40083 56 ID aie cece ecaceteeses een ene tees 56 Level Sens r 224p eR E rene b bed 57 OU serer der PREREEXTEPA E E ERES 57 Mass Flow Controllers Gas Gonneetlollse es veese pes PEDE XS X 5alety E1108810B8 4 2 rr rtr dam ds 57 belg PORS oes ar 57 Physical Dimensions Lua uu deua s 57 Host Software Syste
88. r OF Averages 1 Enter aen 1 ID Humber 6885 Enter Oxygen 42 ID Humber 3880 5 Enter FH ID Humber ei Enter RTD ID Humber hu Enter Oxygen 1 Tyre Enter Oxygen 2 Tyre Enter FH Tare Enter ETD Tare TD boa oem GoGo Oa Figure 68 Configuration Input Screen There are ten selections available in this category indicated by the numbers 1 to 0 Figure 68 Averaging 1 Press 1 to select Toggle Averaging the aver aging function to be ON or OFF The micro processor continuously scans each of the input channels The probe output signal may demon strate some minor bounce that when digitized would be displayed as a rapidly changing digit representing the most sensitive level of the signal a tenth of 80 0 DO etc Selecting the Toggle Averaging to be ON results in a displayed value that is the average of a number of samples Select ing the Toggle Averaging to be OFF sends the digitized value directly to the display Number of Averages 2 Press 2 to select Enter Number of Averages and input your desired number of averages The number of averages may vary from 5 to 250 aver ages The response time of the displayed value will be dampened by very high averaging numbers and that may interfere with the ability to accurately view signals that are changing rapidly A fast capture profile would use low averaging numbers or no averaging at all and would be more responsive to changing conditions perhaps exce
89. rate shown on the display is in RPM 9 Rotate the Rate knob counter clock wise until it stops The rate should stabilize at approximately 4 RPM 10 Rotate the Rate knob clockwise until it stops The rate should stabilize at approxi mately 84 RPM 11 Reduce the pump rate to 4 RPM 12 Slowly loosen all four of the knobs holding the safety shield in position When the cover becomes loose the red Alarm light should light the pump should stop and the green Jog Start light will flash The pump will not restart until both the safety cover 1s properly posi tioned to depress the white safety switch on the left tube guide and the Jog Start switch is pressed to restart the pump CORNING CELLCUBE CULTURE SVSTEM USER s MANUAL CASSETTE INSTALLATION Figure 31 Drawing of the Single Circulation Pump Cassette Corning Cat No 3121 The Single Circulation Pump Cassette Corning Cat No 3121 utilizes four stainless steel ball check valves located in the top and bottom man ifold assemblies Each manifold is labeled in the area between the silicone tubular diaphragms The bottom manifold is marked faintly with bottom and represents the inlet section of the cassette The top manifold represents the outlet section of the cassette and is marked faintly with top The cassette will operate in one position only It is important to locate the cassette in the pump with the top manifold in the right side up or
90. re parameters including pH dissolved oxygen gas flow and pump rates All functions are accessible through a menu driven user interface on the faceplate It also allows monitoring and control of the system from the user s PC The software is fully compati ble with WIN 95 98 ME NT4 2000 and XP Figure 2 Corning Standard CellCube System Corning Cat No 3143 operating systems In addition it is strongly suggested that a spreadsheet such as Excel QuatroPro or Lotus is also installed to facilitate analysis of the data collected and downloaded from the controller through the course of a run The Circulation Pump is a variable rate pump that utilizes a removable disposable elastomeric cassette that can be autoclaved one time The Circulation Pump provides a continuous supply of gas conditioned media and nutrients to the CellCube Module There are two Corning Circulation Pumps a Single Circulation Pump Figure 4 and a Dual Circulation Pump Figure 5 The Oxygenator is designed to provide a constant supply of gas conditioned media to the CellCube Culture module and operates using a thin film gas exchange process There are two sizes of Corning Oxygenators a 6L Oxygenator Figure 6 page 3 and a 36L Oxygenator Figure 7 page 3 XN Figure 3 Digital Controller Figure 4 Single Circulation Pump Figure 5 Dual Circulation Pump CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL The Media Pump Figure 8 i
91. robe and the inlet tube to the 12 male male female luer connector near the pump cassette This is done by unwrapping any autoclave paper or removing the connectors at the bridge pieces and insert ing them aseptically into the inlet and outlet tubes of the CellCube module If making all of the connections to Media In Seeding Reservoir and Harvest Reservoir in the hood complete those connections to the appropriate reservoirs before carefully moving the completed assembly out of the biohood and into the incubator or to a cart holding the CellCube Circulatory Pump Media Pumps and Oxygenator that will be moved into a warm room Alternatively if a biohood safety cabinet is positioned next to the incubator or if a Sterile Connect Device SCD is to be used to make future connections the Circula tion Loop and Oxygenator can be moved into the warm room incubator and connections can be done by moving the long leads to the reservoirs into the biohood safety cabinet and making the connections in the biohood at those specific locations A similar arrangement would occur with the use of the SCD except that the lengths of tubing to the reservoirs could be considerably shorter Attach the sample septum to the appropriate location If it was not done so prior to autoclaving place quick ties on every point of tubing con nection both to the Oxygenator and Pump Cassette and to all luer fittings barb fittings or quick connects that
92. rovide accurate low maintenance pump operation Although the cali bration procedure is the same for each pump the volume collected will depend on the ID of the tubing positioned in the pump head The calibra tion of the pumps is accomplished bv an automat ic sequence that measures the volumes collected during two minute intervals at two different pump speeds Typical volumes collected during a 24 hour period of operation of the standard CellCube System are shown in Table 2 The amounts col lected will depend on the size of the CellCube System and the system volume exchanges that are desired per 24 hour period Module Size 25 Layer 50 Layer 100 Layer 4 5 7 5 Vol Exchangesiday at Flow Rate System Volume I 3 Fiona Rate Tubing LD RPM mifmin Liday 1H6 Tubing 10 43 6 27 21 20 86 124 1 41 30 129 186 62 40 172 248 l 83 50 215 no 103 187 Tubing 10 179 258 86 20 32 536 179 30 558 804 268 40 744 1071 357 50 930 1339 446 3467 Tubing 10 405 583 194 20 810 1166 389 30 1215 1750 583 40 162 0 2333 718 50 2025 2916 972 14 i 08 28 17 41 25 55 33 69 41 Di 34 19 71 179 107 238 143 298 179 130 78 259 155 389 233 518 311 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CellCube Media Pump The Media Pump Corning Cat No
93. rovides a com prehensive array of selections that may be routed to remote switches to signal undesirable operating conditions Caution Do not connect the alarms connector to your central alarm system until the system has reached a stable operating condition The three lower green connectors on the back of the controller are fused alarm switches The switches may be used for a variety of functions Switch 1 and 2 are normally open and close on activation In the open position default there is no power to the circuit Switches 1 and 2 are addressable from the configure alarms menu The switches may be activated by any of the minimum maximum or error alarm functions When closed power is routed through the circuit and thus the switches may also be used to activate a buffer pump if equipped with a suitable remote switch interface While use of a buffer pump is typically not necessary when the system is sufficiently per fused with properly buffered media the pump could be used to deliver a basic solution as a fail safe if the pH drops to a level below a set point at which perfusion is no longer capable of control ling the pH The third switch is set up in the opposite orientation Switch 3 is normally closed i e always on during normal operation and is not addressable Therefore if wired to an external alarm it could be used to indicate power failure or failure of the microprocessor The controller provides user selectab
94. rther explanation of this maintenance refer to the instruction video tape CD presen tation and the information in the Appendix Vent filters should be replaced after four auto clave cycles by proper use For GMP and GLP batches we recommend single use Silicone tubing should be replaced after each CellCube system run All O rings should be replaced after 10 auto clave cycles or earlier if noticeable fraying or cracking of the O rings is observed Nylon ties are considered nonfunctional after autoclaving The sample septum Corning supplies is pre sterilized and a single use disposable Under no circumstances should the sample septum be autoclaved since it may melt during the autoclave process CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Appendices VENDOR LIST Tubing Platinum Cured Silicone 12 ID X 3 4 OD Silicone Tubing Durometer 50 3A6 ID X 16 OD Silicone Tubing Durometer 50 3 16 ID X 7 8 OD Silicone Tubing Durometer 50 PharMed 1 8 ID x 1 4 OD PharMed Tubing Norprene For the tubing in pumps and the size will be dependent on pump head size pH and Oxygen Probes including membranes replacement kits are available from the following vendors Ingold Electrodes Inc 261 Ballardvale Street Wilmington MA 01887 Broadlev James 1714 S Lvon Street Santa Ana CA 92705 Tel 714 547 8061 Tel 508 658 7615 Fax 714 547 8988 Fax 508 658 6973 www broadleyjames com www elscolab nl com Po
95. rvoir will promote the formation of a wide uniform liquid film when a stream of water is projected onto the surface In the case of build up of organic material over time it may be necessary to use sodium hydroxide as outlined below to ensure the flow of a uniform fluid film 1 Fill the reservoir to the base of the neck with a 10 mM solution of sodium hydroxide 400 mg L and autoclave for 45 minutes at 121 C and 1 psi slow exhaust Alternatively some people prefer to use a Normal sodium hydroxide soak at room temperature overnight as a cleaning method 2 Remove the glass reservoir from the sodium hydroxide solution and rinse it well with deion ized water Confirm that the O ring contact sites are free of the cleaning solution Note Organic materials may be reduced to ash by cooking the glass at 450 C for at least 3 hours The seal rings on the glass side arms should be removed and stored prior to use of this technique 1 Use a detergent such as 7 x or equivalent Avoid detergents commonly used for chemistry applications such as Alconox 2 Rinse as soon as practical after use Do not allow detergents to dry on any components during washing 3 Place the clean etched glass reservoir on a l l lint free absorbent surface 3 Store items to be cleaned in a cleanser or in A E E ee 4 Do not touch the interior of the glass reservoir 4 Thoroughly rinse all items in tap water with a final rinse of distilled or dei
96. s Circulation and Media The calibration menus are accessed by pressing the main selection button Calibration on the right side of the display Figure 24 page 8 The first menu that appears 1s the Calibration summary Screen Figure 42 HO EEEOE CONDITIONS Calibration Summar Screen CALIBRATED H4 11 835 Circ CALIBRATED dd 11 03 Mass Flow Control Factory Calibration In Use On GE FLOW FLIMP x Figure 42 Calibration Summary Screen Before ID numbers are entered HO ERROR CONDITION Calibration Umman Creer De 1 ID 42261 CALIERATED He ir 83 e ID 23188 7 CALIBRATED Be ir Bz 965 CALIBRATED 05 26 63 2096 CALIBRATED do az as CALIBRATED Circ CALIBRATED Ba c6 83 a i7 ds Hass Flow Control Factory Calibration In Use Oe GEN FLOW PLUMP Figure 43 Calibration Summary Screen After ID numbers are added The Calibration Summary Screen provides a list ing of each probe s identities and calibration status Each probe is identified by the input channel an optional ID number e g serial number status calibrated or uncalibrated and the date calibrated The calibration status of the Corning pumps and date of the calibration are also listed on this screen The various sub menus are displayed at the bottom of each screen and above a corresponding function button The functions at the bottom of the Cali bration Summary Screen are OXYGEN pH FLOW TEMP and PUMPS After you ha
97. s with the Media Pump it is an advantage to break in a new pump cassette prior to calibration by installing it in the pump head and operating it in the pump head at a moderate speed for several hours prior to calibration Figure 37 Installing the Dual Circulation Pump Cassette Bottom View Figure 38 Installing the Dual Circulation Pump Cassette Complete Figure 39 on the next page indicates the typical delivery results after calibration of the Dual Circulation Pump Manual calibrations are per formed by measuring the volume collected over a fixed time period for a range of speeds that would cover the process The specific details and proce dures for calibrating the Dual Circulation Pump is found in a later portion of this manual See Calibrating the Circulation Pump page 30 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 20 ml min Oa NUA 010 OO OQ O a L min 0 20 60 80 100 RPM RPM L min ml rev L day 4 0 4 0 10 576 2 0 5 0 10 720 10 1 1 0 11 1584 15 1 6 0 11 2304 20 22 0 11 3168 25 2 7 0 11 3888 30 3 2 0 11 4608 35 3 8 0 11 5472 40 43 0 11 6192 45 4 9 0 11 7056 50 5 4 0 11 7776 55 6 0 0 11 8640 60 6 5 0 11 9360 65 7 0 0 11 10080 70 7 6 0 11 10944 75 8 1 0 11 11664 80 8 7 0 11 12528 85 9 2 0 11 13248 Figure 39 Flow chart Dual Circulation Pump Corning Cat No 3223 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Software 21 The
98. s a variable speed dual head peristaltic pump for the continuous infusion and removal of medium It utilizes tubing sets of different diameters to span the 3 to 354 liters per dav LPD pertusion range of the svs tem The output pump operates at or 1 2 times the rate of the input pump to continually remove spent medium from the Oxvgenator and to main tain the liquid level of the Oxvgenator The CellCube Culture Module Figure 9 pro vides a large stable styrenic tissue culture treated surface area for the immobilization and growth of attachment dependent cells The CellCube module provides an environment which closely simulates in vivo conditions and reliably distrib utes nutrients and oxygen across all cells within the modules with low differential gradients Figure 6 6L Oxygenator Corning Cat No 9983 Figure 8 Media Pump Corning Cat No 3221 Corning Cat No 3101 Figure 7 36L Oxygenator Corning Cat No 9979 Figure 9 CellCube Culture Module 25 Layer CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL CellCube Culture Module The CellCube Module is an integrallv encapsulated sterile single use device and is 100 pressure hold tested before shipment Figures 10 and 11 It is comprised of a series of parallel culture plates joined to create thin sealed laminar flow spaces between adjacent plates Each culture plate receives proprietary tissue culture treatment prior to assembly to ensure den
99. se uniform cell growth The encapsulation and end plates are made from polycarbonate for strength and long term reliability The outlet port is located on the upper rear of the CellCube module diagonally opposite the inlet port The outlet port interconnects all the parallel laminar flow spaces of the module Each port inlet and outlet is supplied with a 25 cm 10 length of 1 2 ID x 4 OD silicone tubing that terminates in a sterile wrap only for Cat No 3201 3202 3264 Even distribution of medium to each of the channels between the plates is provided by the Flow Control Orifices Figure 12 through a 25 cm 10 length 12 ID x 3 4 OD silicon tubing located at the lower front of the module The CellCube module is available in three basic sizes Table 1 The 25 Layer module is com prised of 25 culture plates while the 100 Layer module Figure 13 is made up of four 25 Layer modules in parallel External clip arrangements on all four corners of the 100 Layer modules ensure that the assembly runs intact and provides a robust leak proof environment for operationng All individual CellCube modules are separately Figure 10 CellCube Culture Module 25 Layer Corning Cat No 3101 leak tested prior to assembly and then further tested after combined assembly of the 100 Layer module Each lot is tested on surface treatment and sterility what is gamma radiated The surface treatment and sterility of each lot is tested
100. ssette is designed for continuous duty at the full rated speed of the Dual Circulation Pump UNPACKING The Dual Circulation Pump is packaged with the following items Power cord Remote control cable 15 pin D Sub type connector male x female Data sheet remote control interface and alarm connections Two pin female connector for auxiliary external alarm connections The pump is equipped with a universal power supply that automatically adjusts to any AC power source between 90 and 250 VAC 1 Place the pump on a clean dry surface 2 Assure that the power selection knob on the back of the pump is in the OFF position 3 Assure that the power selection knob on the front of the pump is in the STOP position 4 Insert the power cord into the socket located at the rear of the pump a Assure that the safety shields are in position covering the pump mechanism Each safety shield 1s held in place by four thumb screws which need to be tightened sufficiently to depress the white safety switch found on the left tube guide of the pump 6 Insert the power plug into the wall outlet Insert one part of the remote control cable in the back of the Dual Circulation Pump and the other end in one of the pump interface ports at the back of the Controller Corning cat No 3220 7 Activate the power switch located above the power cord on the rear panel The red indica tor on the front panel should light when the power
101. ssively so ID Numbers 3 Press 3 6 to enter ID numbers for the probes used during a run The ID feature is useful for batch records probe calibration maintenance and repairs Many of the probes exhibit their own personal characteristics It is important that the correct probe is connected to the same input that was used during the calibration procedure Type Selection 4 Press 7 0 to further identify the probes in facilities that may have probes from different vendors that perform the same function Pump Configuration HO ERROR CONDITION Pume Confisuration Kew Toaale Fume 1 Mode Fumr 1 Tyre Taaale Fume 2 Mode umr z Tyre Taaale Media Fume tatusz Taaale Circ Pume Statue INPUT ALARM POLITPLIT Figure 69 Pump Configuration Screen The pump configuration section provides access menus to disable the output to the displav if vou are not using the Corning pumps The ability to receive data from non Corning tvpe pumps will be discussed in the Appendix on page 59 The use of the inputs for non Corning pumps should only be attempted bv skilled electronics technicians Alarm Configuration HO ERROR CONDITIONS Alarm Confisuratian Dew ce D 1 IU 2 FH SSS SSeS ERR sizi sien sieb slao sieb slao leD slo siel si l INPUT 1 PUMP Figure 70 Alarm Configuration screen OUTPUT CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 33 The Alarm Configuration section p
102. st option is Receive Historv and the second option is Update Firmware 1 Using the left mouse button click on Update Firmware 2 In the middle of the screen a prompt will ask Are you sure you want to update the Controller Firmware 3 Press Yes 4 Put the Firmware disk in drive A 5 Go to drives and select driver A Using the left mouse button select the control bin file on the left side of the screen and press OK Now the software will take over the controller and upload the firmware After the Firmware is uploaded the Controller will reset itself auto matically You can run the menus from the controller on the PC dd CORNING CELLCUBE CULTURE SVSTEM USER s MANUAL WORKING WITH MENUS Start up Screen HO ERROR CONDITION amp CORNING Discovering Beyond Imagination CONTROLLER a Current Time 18 55 59 B4 25 03 FFlication Firmware Verzion Z B Figure 41 Start up Screen The start up screen 1s displayed each time the controller is either powered up or a re boot is activated The start up screen displays the current time date month day year Firmware version error conditions and the Corning logo CALIBRATION MENUS The calibration menus are self prompting menus to assist in the process of calibrating the probes to the system controller Sub menus are provided for the calibration of the two oxygen inputs the pH probe the temperature probe and the Corning Pump
103. te the selec tion Press 2 for Set MIN Media Flow Rate in order to select the Media Pump flow rate for the mini mum flow alarm Input the desired value and then press ENTER to complete the selection Press 3 for Set MAX Media Flow Rate in order to select the Media Pump flow rate for the maxi mum flow alarm Input the value and then press ENTER to complete the selection Press 4 for Set Media Delay Time in order to set the delayed start time of the Media Pump in hours This is especially useful once process parameters have been established and the need to introduce fresh nutrients is not immediately necessary Press 5 for Start Stop Media Pump in order to start or stop the Media Pump Press 6 for Set Circulation Flow in order to set the Circulation Pump flow rate Input the values and press ENTER to complete the selection Press 7 for Set MIN Circ Flow Rate in order to select the Circulation Pump flow rate for the minimum flow alarm Input the value and then press ENTER to complete the selection Press 8 for Set MAX Circ Flow Rate in order to select the Circulation Pump flow rate for the minimum flow alarm Input the value and then press ENTER to complete the selection Press 9 for Start Stop Circ Pump in order to start or stop the Circulation Pump CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL VIEW MENUS HO ERROR CONDITION Mo eo Gb 00000 eso PUM
104. te the selection by pressing ENTER A typical value would be pH 7 10 to 7 2 Press 3 to select the CO flow rate that would be provided if the manual mode option in 1 above is selected Press 4 to enter a minimum pH alarm value press ENTER to complete the selection A typ ical value is 0 50 pH lower then the set point Press 5 to enter a maximum pH alarm value press 3 to complete the selection A typical value is 0 50 pH higher then the set point Press 6 to limit the maximum automatic CO flow rate Enter the value and press ENTER to complete the selection A maximum limit may be advantageous when utilizing a fast capture profile for the PID controller A typical value here would be 5 to 20 of the total gas flow see Air Flow Settings below Correctly adjusting the maximum flow will help prevent overshoot exceeding the set point especially as the cell culture process and CO begins to build in the system as the result of metabolic activity of the cell population Air Flow Settings The air flow settings are used to control the carrier gas flow of air into the oxygenator and the alarm settings Although it is possible to choose another carrier gas such as nitrogen air is the typical car rier gas into which CO and oxygen are blended It is important to maintain a continuous flow of a gas that is not being controlled The carrier gas dilutes out the controlled gasses providing smooth capture and contro
105. the natural tendencv of fluid to spread evenlv across a large hvdrophilic surface Fluid is deliv ered to the neck of the Oxvgenator where it spreads out and flows down the interior wall of the glass reservoir The media forms a thin film that facilitates rapid exchange of gases between the gas and media interface This process mini mizes the generation of foaming typically seen in directly sparged systems and eliminates the need for pluronic or other anti foaming agents The falling thin film of media is constantly maintained by an adjustable rate Circulation Pump This pro vides a large surface area to volume ratio for effi cient diffusive exchange between the circulating media and the gas mixture in the Oxygenator The media level is maintained by the continuous withdrawal of media through a stainless steel side arm of the oxygenator The pH and dissolved Unrolled View clockwise from position 1 Figure 19 6L Oxygenator Figure 20 36L Oxygenator oxygen levels are monitored by probes that are in constant contact with the media An adjusted gas mixture is continuously added to the Oxygenator headspace under the direction of the Digital Controller system This provides excellent dif fusion of CO into and out of the media for pH control and addition of oxygen to the media nec essary for maintaining the large cell density typi cally held by a CellCube Module The standard 6L Oxygenator Corning Cat No 3101 is designed
106. tion This means it will be run first for two minutes at high speed followed by two minutes low speed By selecting the category PUMPS from the main calibration menu the Pump Calibration Screen will be displayed Press 2 to select the Calibrate Circulation Pump menu The pump should be equipped with a pump cas sette similar to the one that will be used during operation See Figure 63 for the menu displayed There are three choices START PRIME or ABORT CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL 31 The inlet section of the tube should be immersed in water and the air should be purged from the system This can be achieved by placing the outlet tubing in the same vessel as the inlet tubing and pressing the PRIME button to remove of all the air in the tubing When you release the prime button the pump stops priming immediately HO ERROR CONDITION PAS 1 Place ENPTY measurement container under FUMF outlet and Place Fume inlet in a resevoir The Fume should be fulls Primed before this calibration is started The Fume will rum for Z min Pume iz ready to calibrate PRIME ABORT Figure 62 Calibrate Circulation Pump Screen High speed Once all the air is purged from the system place a receiving vessel of sufficient size to retain the sample under the output tube and press START The calibration will begin and the pump will run for two minutes Figure 62 HO ERROR CONDITIONS The total test time was amp Hin B H S
107. tion 1 1 Media inlet tube 2 Level sensor probe 3 Infection port 4 Media inlet tube 5 pH probe port 6 Gas inlet Figure 106 Drawing of a 36L Oxygenator Headplate CORNING CELLCUBE CULTURE SVSTEM USER s MANUAL 46 used after initial operation Secure using the orange headplate securing ring Position LEVEL SENSOR Insert the level sensor and cinch bushings into the appropriate opening Finger tighten the level sensor cinch bushings Position pH PROBE Wet the probe with distilled water to facilitate installation Insert the pH probe in the pH probe fitting by gently twisting into place The pH sensor should be threaded installed into position and finger tightened To relieve stress on the pH probe during autoclaving loosen by turning counter clockwise a half turn Refer to the subsection Calibration and Maintenance of pH probe in the Calibration Menus section page 27 Position OXYGEN PROBE The 6L Oxygenator Corning Cat No 3101 has one oxygen probe in the headplate and one in line The 36L Oxygenator has both probes in line Remove the DO probe membrane protection black vinyl cap on the end of the probe Wrap the threads on the probe with a small piece of polytetrafluorethylene PTFE tape Wet the probe with distilled water to facilitate installation Insert the probe in the probe fitting by gently twisting into place The oxygen probe should be threaded installed into position and fin
108. to an oxygen free environment This could be a solution of sodi um beta bisulfate or gas such as nitrogen that is near zero in oxygen content prior to executing this function The method used to reach the zero value will effect the time necessary to perform this test Typically a probe should reach a stable zero point after just a few min utes in the presence of a saturated continuous flow of nitrogen gas The same procedure performed in the liquid phase however may require a very long stabilization period A graph depicting the time required 60 minutes to reach 3 5 DO using a small flask and 200 cc s of water is shown as a general indicator of the time that might be required using the liq uid phase test system The time required may be much greater if the volume of fluid gas headspace or the gas flow rate are changed 8 Time to Zero in Water 5883588388 Figure 47 DO in water during a time frame When the probe signal has stabilized at the lowest point press ENTER or the A but ton and the controller will record the value as the zero point for this probe Typically the value obtained in the O free environment is only slightly different than the value of the amplifier null test The difference helps the controller recognize the difference between an error condition no electrolyte and low oxygen See Figure 48 page 26 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL HO ERROF CON
109. to maintain a media volume of approxi mately 1 2 to 2 0 liters that is sufficient to operate the 10 25 and 100 Layer CellCube modules Top View Position 1 1 12X420mm DO probe 2 Media inlet tube 3 Level sensor probe 4 pH probe 5 Media inlet tube 6 Gas inlet Figure 21 Schematic of a 6L Oxvgenator Headplate CORNING CELLCUBE CULTURE SVSTEM USER s MANUAL The 36L Oxvgenator Corning Cat No 3188 is designed to maintain a media volume of approxi mately 9 0 to 10 0 liters for operating two to four 100 Layer CellCube Modules An important component in the headplate is the level sensor In the unlikely event that the fluid level rises above a predetermined fixed volume contact of the media with the level sensor acti vates an alarm circuit which interrupts power to the Media Pump thereby eliminating the possibility of system overflow Note This level sensing function is extremely important Do not disable this feature GLASS RESERVOIR CLEANING There are a variety of cleaning methods utilized for glassware This section discusses some of the methods used successfully to clean the glassware used in the CellCube System The use of a labora tory glassware washer with standard protocols is usually all that is required to clean the reservoir Note Be careful when handling glassware The Oxygenator should be decontaminated before cleaning Avoid heat sterilization before cleaning A properly cleaned rese
110. ton The controller will warn you if it determines that the probe response is not typical A typical response to a change from pH 7 0 to either 4 0 or 10 0 would generate a shift of approximately 870 units plus or minus depending on the buffer from the current value determined at pH 7 0 e g 2048 IE HO EEEOE CONDITION FH Calibration ter 1 Calibrate tand 1986 e Toggle Compensation Tyre AUTOMATIC a Enter Fixed ComrFenzation Value 37 8 d Calibrate lore A BAA5165 Enter ACTUAL FH VALUE eee Ffzet will be CALCULATED O2CH1 FLOW TE P PUMP Figure 54 The pH Calibration Drift Step During repeated autoclaving and long production runs the probe may display drift or movement away from the set points This will result in a steady increase in inaccurate values It is good practice to routinely check the pH of the medium during the course of a run using another calibrated pH meter on freshly drawn samples as close to the CellCube System operating temperatures as pos sible If drift is observed it can be compensated for by the following procedure Take a sample and measure it on another calibrated pH electrode It is possible to enter this value in your run Figure 54 During the calibration of the pH electrode the drift will be zero and after the correction it will calculate the drift The time and date of this correction will be monitored Temperature Probe Calibration The thermistor temperature prob
111. trode faces you and the probe is in a verti cal position Slowly and gently slide the mem brane onto the top of the assembly When slid ing the membrane onto the cathode some of the electrolyte will be displaced The electrode is mildly caustic and should be rinsed off of any contact with your skin When the membrane is in position rinse the outside of the membrane with clean tap water and pat dry with a clean tissue to remove any residual electrolyte 7 Replace the cap sleeve of the probe and gently screw it on the housing The probe is now ready to be polarized Changing the Membrane Cap 12 mm DO Probes The membrane for the 12 mm probes is an inte gral part of the probe The entire tip stainless steel sleeve and membrane can be unscrewed from the probe There is only a small amount of electrolyte in the probe 1 Refer to points 3 and 4 above regarding probe inspection and cleaning 2 The membrane cap should be filled with elec trolyte to the first graduated marking line ust below the bottom of the threads with fresh electrolyte 3 The cap should then be carefully reinserted onto the probe 4 While slowly turning the threaded cap onto the probe you should look for electrolyte being displaced It is preferable that the cap reach the O ring without displacing the electrolyte If electrolyte is displaced prior to reaching the O ring remove the cap and wipe the cathode dry with a lint free towel and reinstal
112. u dia dd ae 33 Selecting an Alarm Function 33 Delay Punch Ot errete ent dn men 34 Congre Outputs ses drea eri e Enc 34 Serial Configurations aa de ies ace Qaia 34 The Settings BUBcHONS s se ereterso px ba it 35 OW ce SEE sat etek ie sibi fi nee noes 35 DEL SSLUDUO st etedes secon ta USES E PES 36 Ait Blow SettllibSi 1d eactacceradc eR t bad 36 Temperature Settings s ssssiiesjawte e a 37 Pump Settings DOPOE Uus eode 1E eodd db ene 3 li CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL boo ia M Pm 38 B C ee re re rae eee rer 38 Oxygen First View 7 DO 1 38 Oxygen Second View DO 2 38 Oxycen Flow History pa i legend bes 38 Ui ne oe M 39 pil USTO edena ina Pad ies bmi 39 CO Fow TISON ta eere no ayes 39 Air Flow Historv 000000 39 d nn MET 40 Temperature Histo saw d urat eas 40 PPS Ludos etude eee FRE LEOTE REDE 40 Media Pump History ii xikerrjszaatei 40 Circulation Pump History 40 VIEW S Pr 40 AUI MONDO ore SS saute bees RPPERT 4 Batch Menit 6506 ed etx EP RE S ee 4 Denu Mon 4544 eer tbe SLEEVE TS 4 Time ICAU usta eost an de dor dod a EE ani 4 Factory Menu 22 4 iu 23 area bad i parare dd 42 CellCube System Setup Protocols 43 Overview of CellCube Fluid Paths and Philosophy of Operation 43 Building Up CellCube Fluid Paths 43 Assembly of the Oxygenator Head Plate 45 Breakdown of the Oxygenator Head Plate Afte
113. uer fitting sufficiently long to reach the Medium In reservoir or bag Wrap the ends of the Cell Seeding Tubing the Medium Addition Tubing and the 2 male male female luer connector in autoclave paper The appro priate luer plugs can be wrapped separately and autoclaved to plug the opening on the luer fit tings on the tubing after autoclaving Secure all connections with a cable tie If using a bridge piece see Steps 2 and 3 then insert the 2 male male female luer connector into the other end of the bridge piece T CORNING CELLCUBE CULTURE SYSTEM USER s MANUAL 48 5 Place the oxvgenator tubing Circulation Pump zation Detailed information on autoclaving is assembly and cassette into an autoclavable con provided in the next section tainer The entire container can then be cov 6 ered by the perforated autoclave bag Check to see that all the connections have a cable tie and that the sterile wrappings will not rest in any Collect and autoclave the appropriately sized vessels to seed use for media and harvest Vessels should be autoclavable and assembled with 0 20 pm air vents and full length dip tubes with potential condensate puddles while the assembly a a fitting on the end this arie Draai is cooling down Invert or lay the Circulation l l tubing connection Example is shown in Pump cassette down during the autoclave cycle Figure 107 in order to disable the check valves for sterili Table 5 Fluid Han
114. uld be set to Medium The Large setting would incorporate the 36L Oxygenator Corning Cat No 3188 which can handle four modules Corning Cat No 3264 The default setting is set to Medium Press 3 to set the DO level which is used to enter the desired set point for the probe selected in the control method A set point of 80 for Channel 1 and 60 for Channel 2 are the default settings Typical starting set points for operations are 60 on Channel and 40 on Channel 2 Press 4 to set the O Flow Rate This is the flow that will be provided when the manual mode is selected as the control strategy in option 1 above Press 5 to set the CH 1 Alarm Min This is the set point that will trigger a low alarm condi tion on channel l A typical value here would be 10 less than the desired 0 set point Press 6 to set the CH 1 Alarm Max This is the set point that will trigger a maximum alarm condition on channel 1 A typical value here would be 20 to 40 higher than the desired O set point The Max set point should be increased proportionally as the O set point is increased to minimize overshoot that typically occurs as the O set point increases CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 36 Press 7 to set the CH 2 Alarm Min This is the set point that will trigger a low alarm condi tion on channel 2 A typical value here would be 10 less than the desired O set point Press 8 to set the CH 2 Alarm Max
115. ump 17 Uopac kini sudes ac der sor SEER bat ng ewe bee 17 Cassette Installato eces 4 4d oret onii 18 Pump Calibration Flow Charts 19 SONA eE EAE ib due EE dated 21 Installing the PC Host software 21 Firmware Operating System 22 Upgrading BIERINVAFG 2 22 21 w Ph dried drhaa 22 Working with Menus sia esubirsebatieg na 23 Startup SCE 1 o3 944392 ORNARE eras 23 Galibrition AIGmus iua seres ere ba ieegb o 23 Calibration of DO Probes 23 Maintenance of O Probes reet 23 Changing the Membrane Cartridge 25 mm DO Probes 23 Changing the Membrane Cap 12 tm DO Proves x ove etg E eRrns 24 Calibration of Oxygen Channel One 24 Calibration of Oxygen Channel Two 26 Calibration and Maintenance of Del tole esr Po EE sa PPP UNT MPO PAESE 27 Temperature Probe Cabhbration os 28 Calibration of Corning Pumps 28 Background Information 2 e 28 Media Pump Calibration 20 Calibrating the Circulation Pump 30 System Configuration au yc d ke eden coe ee 32 Conhioute Inputs 5 ince rea retrete 2d OO oa edP ud ERO cie UU de 32 Number OL AVERAGES es ve preter nee es 32 ID Nambers sarsn izzzsg inuntebazi 32 Type SeleCHONs eresi qu ctectnetsese4e 32 Pomp Configuration 424309 o br e be tema 32 Alarm Configuration aa sd e ed deris 22 DES AT Sae citur ater mde rara eura 33 Alarm Categories saliba tipik
116. ut have not been tested to date via the configuration tools which are accessible on the controller In addition any upgrades to the Firmware have to be done through the Host Soft ware on the PC by transferring the new Firmware file from the PC to the Controller itself INSTALLING THE PC HOST SOFTWARE Installing Version 2 00 1 Start the installation of the new PC Host Software version 2 00 by inserting Disk 1 in the floppy drive 2 Click Start select Run and then type in A Setup exe substitute A for the correspon ding Floppy drive letter and hit OK This will begin the Controller software installation 3 Make sure all other applications are closed before going to the next step of the installation then click OK 4 Leave the installation directory as the default directory C NCorningNHost and press the Installation button to continue inserting Disk 2 and 3 when prompted to When the installation has been completed there will be a shortcut to the PC Host software pro gram group within Program Files CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL Firmware Operating Svstem 22 Firmware is the working on board program for the CellCube Controller itself With this soft ware it is possible to view the last 24 hours change settings and alarms calibrate and identify the equipment The firmware is responsible for the local interaction of the digital controller wit
117. utine and after 10 to 20 seconds you will hear a beep and the startup screen with the time and date will be displayed 2 Multinational power supply a U S power cord b Universal power cord c Serial port terminator d Connector 12 pos e Connector 8 pos f Level sensor cable g Level sensor adapter h DB25 M F serial cable i lemperature sensor Figure 24 CellCube Digital Controller Faceplate 3220 SYSTEM CONTROLS Working With Menus As shown above Figure 24 the Digital Controller incorporates a menu driven faceplate Each func tion for the controller may be addressed by select ing the proper menu screen Main menu screens are accessed through the vertical row of buttons to the right of the screen labeled View Settings Calibrate Configure and Auxiliary Sub Menus to the Main Menu screens are selected by choosing the appropriate function buttons labeled A through E located below the screen The Digital Controller Figure 23 Digital Controller Corning Cat No 3220 8 CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL when delivered is configured with factory default settings when used with probes that have charac teristics similar to a standard probe shipped with the unit It is also possible to control the Digital Controller on a PC using the Corning PC Host Software Entering Data Below the Sub Menu buttons is a numeric keypad for entering data into the Digital Controller
118. ve entered all of the ID numbers for each probe the Calibration should appear similar to Figure 43 Calibration of DO Probes Maintenance of O Probes Prior to calibration fresh electrolyte and or a new membrane should be installed on the probe After this procedure the probe will need to be connected to the controller by the probe cable for a MINIMUM of 6 hours preferably overnight Calibration of a nonpolarized probe is a useless endeavor which will only result in faulty operation Changing the Membrane Cartridge 25 mm DO Probes The membrane cartridge in the electrode should be renewed if the electrode has been stored for several months The manufacturer of the probes also recommends that the membrane is replaced every third autoclaving Replacement of the elec trolyte should be done prior to each autoclaving CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 24 Further detailed information is provided bv the manufacturer in the brochure accompanving each probe To verify that the membrane cartridge is functioning properlv check the electrode zero point and calibration in room temperature air If the membrane fails to operate flawlessly for instance the response is sluggish or mechanical damage is detected it must be replaced When replacing the membrane cartridge strictly observe the following instructions Caution The use of protective gloves and eye wear is recommended throughout the membrane cartridge re
119. will experience any pressurization during operation Place the Circulation Pump cassette in the Circulation Pump Note Ensure the orienta tion of the cassette is correct Insert the Import and Export tubing seg ments into the appropriate pump heads on the CellCube Media Pump Unit If using the Harvest Pump to introduce medium into the Oxygenator be sure to orient the tubing in the pump head in a reverse orientation to be able to pump into the Oxygenator Alternatively an external peristaltic pump with a greater pumping action can be used for this purpose CORNING CELLCUBE CULTURE SYSTEM USER S MANUAL 50 10 The svstem is readv for use when all the prop 11 12 er connections have been made ensuring there are no loose or open connections It can now be transported to an environmental chamber if that has not already been done Close or open the appropriate clamps on any lines that are necessary to fill the system prior to inoculation With a reservoir of fresh medium attached turn on the appropriate pump and begin to fill up the system with medium Use the slowest speeds initially to ensure that there are no con strictions in the fluid paths or that a clamp has not been accidentally left open 13 We recommend that the system is operated at least overnight at the appropriate temperature to ensure that there are no leaks and as a sterility check prior to introducing the cells for attachment Note
120. y Figure 87 View pH History Screen A typical pH History chart is displayed in Figure 87 Note that the graph history is labeled pH History and the Y axis scale is 0 to 14 pH The minimum maximum average and last pH values for the time period shown on the graph are also displayed HO ERROR CONDITION FH History Figure 88 View pH History Screen Auto Scale A more sensitively scale Y axis can be viewed by pressing the ENTER key A typical auto pH History scale chart is displayed in Figure 88 The bottom left portion of the display shows that the next possible selection is CO CO Flow History HO ERROR CONDITION Coe Flow History LA T 2 8 AX 22 5 IN a AMS 11 3 Figure 89 View CO Flow History Screen A typical CO Flow History chart is displayed in Figure 89 Note that the graph history 1s labeled CO Flow History and the Y axis scale is 0 to 500 cc min The minimum maximum average and last CO Flow values for the time period shown on the graph are also displaved A more sensitivelv scaled Y axis can be viewed by pressing the ENTER key The bottom left portion of the display shows that the next possible selection is AIR Air Flow History HO ERROR CONDITION AIF Flow History Figure go View Air Flow History screen A typical air flow history chart is shown in Figure 90 Note that the graph history is labeled Air Flow History and the Y axis scale is 0 500 cc min The minimum maximum
121. y this flow path in operation This has been done in some facilities especially once the nutritional requirements and buffering capacity of a fixed volume necessary to bring up and support a specific population of cells has been determined Certainly in situations where the cell themselves are the product or where a more concentrated product is desired this mode of operation is more practical less expensive and less technically challenging Our experience how ever from both in house and customer application input suggest that the addition of the perfusion circuit in many cases dramatically enhances the productivity of the system both from the man hours for operation and productivity Your deci sion to use one or both of these pathways can only be determined empirically We typically recommend the following sequence of events in the selection process 1 Seed and establish a viable cell population in the CellCube System 2 Carefully monitor as many growth parameters as are feasible such as glucose consumption oxygen utilization pH lactate production etc to create a baseline set of data from which sin gle variable experiments can be created While this would seem to be intuitive we are often perplexed by investigators reluctance to gather this information 3 If costs of media or volume collected become a limiting factor fed batch strategies where only a partial volume of medium is removed and replaced from the c

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