BelloCell-500 USER MANUAL ver 2.0
Contents
1. CESCO Bioengineering User s Guide http www cescobio com tw 7 BelloCell 500 amp BelloStage 3000 DIMENSIONS Figure 6 BelloCell Bottle Dimension Hn Il Zenn CESCO Bioengineering User s Guide http www cescobio com tw 8 UNPACKING amp INSTALLATION 8 1 Unpacking After you have received your order inspect the boxes carefully for any damage that may have occurred during shipping Report any damage missing pieces immediately to the carrier and to your local CESCO distributor Save all packing materials and this User s Guide 8 2 Packing List Verification Verify on your packing list that you have received the correct materials Report any errors to the carrier or to your local CESCO distributor 8 3 Environment The BelloStage unit is designed to operate optimally in the following ambient conditions 20 to 42C 0 to 90 Relative Humidity inside a CO incubator 8 4 Electrical Requirements With the AC transformer provided the BelloStage system can run on 100 240 Volts 50 or 60 Hz You may require an additional adapter to mate the transformer plug to your electrical supply outlet a CAUTION A grounded electrical outlet is necessary for the safe operation of this instrument 8 5 Installation see Figure 8 1 Connect the controller signal cable on t2. Item Part number BelloStage system BS3000 BelloFeeder 1300 BF1300 BelloCell 500 bottle BC0500 BelloCell 500E bottle BE0500 BelloCell 500H bottle BH0500 BelloCell 500P bottle BC1000 BelloCell 500AP bottle BA1000 BelloCell 500EP bottle BE1000 CESCO Bioengineering User s Guide http www cescobio com tw 3 SYSTEM DESCRIPTION Burer High Density Cell Culture System provides a protected controlled and Figure 1 BelloCell Cell Culture System contained environment for the growth of cell cultures The BelloCell High Density Cell Culture System consists of three major components see Figure 1 a control box the BelloStage unit and ready to use disposable 500 ml bottles The unit fits easily dt Control box inside your incubator while the magnetic control box attaches conveniently to an BelloStage outside metal incubator wall for easy access Unit The BelloStage unit which holds up to four disposable cell culture bottles moves the bottles contents up and down according to your program using a platform to compress and expand the bellows built into each bottle to optimize oxygenation As the platform lifts it compresses the bellows sending the media into the chamber that contains the BioNOC II disks as the platform descends the media returns to the expanding bellows exposing the carrier disks to the atmospheric environment Figure 2 BelloCell 500 bottle
3. CAUTION The exterior surfaces of the BelloStage unit and controller are coated with an anti rust material Never use any abrasive materials and be careful not to scratch the exterior finish amp D sIoHAZARD WARNING The user is responsible for following local guidelines for handling hazardous waste and biohazardous materials that may be generated from the use of this equipment CESCO Bioengineering User s Guide http www cescobio com tw 10 OPERATION 10 1 General Instruction of BelloStage Take a moment to acquaint yourself with the use of the controller keypad to set up operating parameters before operation begins and to see how to change operating parameters during a run 10 1 1 Before operation 1 Press the STOP key to switch between the Up T_H and Down B_H in the display whre T_H means Top Holding time and B_H means Bottom Holding time see Figures 9 amp 10 Figure 9 Main Screen Rising Platform 2 Press the or part of the RATE key to change platform rising and lowering speed Eight moving speed are available in increments of 0 25 from 0 25 to 2 0 mm s 3 Press the a or part of the DELAY key to change the platform holding time at the top compressed bellows and bottom extended bellows positions Holding time can be set from 0 to 99 minutes and 59 seconds To set minutes press and hold either D
4. 2a and BelloCell 500P bottle 2b Be oce 500 see Figure 2a is made of disposable plastic materials BioNOC II carriers are packed in the upper chamber of a BelloCell 500 unit Animal cells are immobilized within carriers and the culture medium is moving up and down between the upper chamber and the bellows by BelloStage 3000 BelloCell 500P see Figure 2b not only has the same features of BelloCell 500 but can perform more advanced perfusion re circulation operation through its inlet and outlet ports to stabilize culture environment and reduce labor efforts CESCO Bioengineering User s Guide http www cescobio com tw 4 CONTROL BOX FEATURES Figure 3 BelloStage Control Box Display Moving direction A v Bellos tage 3000 START Controller cable Main power switch Button Functional Description To start operation 1 Stop operation 2 Switch the setup mode between Up T_H and Down B_H T_H top holding time B_H bottom holding time Alert mode Culture time recording View the BelloStage version Setup top T_H and bottom holding time B_H 0 99 mins 59 secs before or during operation Setup up and down speed 0 25 to 2 0 mm s before or during operation CESCO Bioengineering User s Guide http www cescobio com tw 5 BelloCell 500 BOTTLE FEATURES The following corss section of the BelloCell bottle see Figure 4 will acquaint
5. 3000 Control Box Dimension 137 x 226 x 40 m m L W D 5 4 x 8 9 x 1 6 inches Weight 1 16 kg 2 6 Ib Power 100 230 VOLTS AC 50 60 Hz Input 12 VOLTS DC Output Environment Room temperature outside CO incubator Materials Magnetic backplate to hold the controller to the side of the incubator BelloFeeder 1300 Pump Dimension 131 x 230 x 61 m m W L H 5 2 x9 1 x2 4 inches Weight 0 73 kg 1 6 Ib Power 85 250 VOLTS AC 50 60 Hz Input 5 A Environment 20 42 C 0 85 relative humidity in a CO incubator BelloCell 500 Bottle Dimension H 243 mm x Diameter 100 mm Working Volume 500 ml Carriers Volume 100 ml 5 5 g Vent Filter type 0 22 um PTFE membrane with PP support Carrier type BioNOC II carriers Material PETG LDPE EVA and PP BelloCell 500P Bottle Dimension H 243 mm x Diameter 100 mm Working Volume 500 ml Carriers Volume 100 ml 5 5 g Vent Filter type 0 22 um PTFE membrane with PP support Carrier type BioNOC II carriers CESCO Bioengineering User s Guide http www cescobio com tw Material PETG LDPE EVA and PP Inward flowing port 10 cm silicon tubing with PP female luer connector Outward flowing port 10 cm silicon tubing with PP male luer connector BelloCell System Part Numbers
6. Adding Media to BelloCell Bottle 500 ml 450 ml 400 ml 350 ml 00 ml 250 ml 00 mil 150 ml 100 ml 50 m Step 2 Aseptically open the cap and directly on top of the BioNOC II disks working in a spiral from the center dispense 30 ml cell suspension containing a total of 1 x 10 2 0 x 10 live cells Close and tighten the cap CESCO Bioengineering User s Guide 16 http www cescobio com tw Figure 13 Inoculation to BelloCell Bottle Step 3 Bring the bottle to BelloStage unit in the incubator making sure the blue ring locks in place Turn on the controller by pressing the red ON OFF button Set up the moving rate and holding time parameters for immobilization Push START button to progress inoculation procedure Figure 14 Loading BelloCell Bottle in Unit XA NOTE Do not delay start operation immediately after inoculation CESCO Bioengineering User s Guide http www cescobio com tw ZS NOTE pH is critical at this point After inoculation make sure that any added media has a pH of 7 0 7 2 if the media contains sodium bicarbonate CO concentration should be maintained between 5 10 in the incubator Abnormal pH range will retard the immobilization efficiency Step 4 After 2 5 hours reset the moving rate Rate and holding time T_H B_H parameters for culture ZS NOTE Increase top holding ti
7. BelloCell can sustain cell growth for months without causing distortion or contamination with an appropriate and aseptic operation 9 Q Can I use BelloCell for long term culture A BelloCell has passed the test of 200 000 times compression which is equal to 3 months at the highest speed or 33 month at the lowest speed Since the cells are immobilized in the carrier box user can culture the cells as long as providing them enough nutrients CESCO Bioengineering User s Guide 32 http www cescobio com tw 14 Reference Guide The patented BelloCell contains two chambers the upper chamber contains a porous fibrous matrix mounted inside where the cells are embedded the lower chamber contains a compressible bellows and has culture medium inside The BelloStage compresses the lower bellows in order to expel the culture medium out of the lower chamber and raise the culture medium level to submerge the matrix in the upper chamber Alternatively the BelloStage 3000 lowers compressible bellows in order to lower the culture medium level to expose the matrix to the air The matrix containing carriers packed in the culture vessel is thus exposed and submerged in the culture medium alternatively controlled by the moving rate of the platform of BelloStage see Figure 21 During the exposing phase see Figure 20 the cells embedded in the porous carriers will not expose directly to the gaseous environment but through a thin liq
8. but do not spray alcohol on the cap because it contains a filter 4 Aseptically open the cap tilt the bottle slightly and pipette out the desired amount of sample through the central hole in the BioNOC II chamber see Figure 4 5 Close the cap tightly and reinstall the bottle in the BelloStage unit locking it in place then press the START key again 10 2 4 Sampling Media For BelloCell 500P 1 Use sterilized syringe and withdraw samples through silicon plug of the sampling port Withdraw 1 0 ml medium or more for analysis CESCO Bioengineering User s Guide 20 http www cescobio com tw ZS NOTE Block the sampling tubing first by slide clamps before draw out needle to avoid back flow of the culture medium 10 2 5 Adjusting pH Level The CO concentration in the incubator may have to be adjusted manually to control the pH level of the BelloCell bottle s contents A good rule of thumb is that while the glucose concentration in the bottle is still sufficient and the media color has turned orange yellow it is time to adjust the CO concentration to a lower percentage 10 2 6 Media Replenishment For BelloCell 500 A general guideline is to replenish media once every day or two beginning on the third day following inoculation The frequency depends on residual glucose concentration and pH in the culture media Whenever there is a need to change the culture media follow the procedure in Sect
9. level gt pump head TT TTL a 2200 ml A BelloCell 500P Hook to BelloFeeder pump 2 L vessel CESCO Bioengineering User s Guide 19 http www cescobio com tw 1 Prior to connect to BelloCell 500P bottle fill the culture medium into the reservoir vessel and remain the headspace in the reservoir to be as little as possible Large headspace gt 500 ml will cause variation of the medium level in BelloCell e CAUTION No matter what s the volume of the vessel keep the headspace in the vessel below 500 ml after fill with culture medium 2 Make sure the cap in the reservoir vessel is firmly capped and air tight e CAUTION Any leakage from the cap will cause contamination and break the balance during medium recirculation 3 Make the connection as the suggested drawing 4 Plug the pump head onto BelloFeeder pump please refer to BelloFeeder 1300 Pump Quick Reference Sheet 5 Initial pumping rate should be around 500 ml to 1000 ml per day and gradually increases to 1999 ml day according to the glucose level 6 Take samples through the sample ports by syringe 10 2 3 Sampling Media for BelloCell 500 1 To take a sample if one is needed press the STOP key to stop the platform and by default send it back to its lowest position 2 Unlock the BelloCell bottle and remove it from the unit 3 Wipe the BelloCell bottle with a 70 alcohol solution
10. you with its features and components Figure 4 BelloCell Bottle Corss Section Fa Went Fitter 2 Carrier Support Plate Ta git Central Hole _ CLorriers og te LAE AE 2 26 Locking Ring Ex Carrier Support Box 7 Ba ff Pe SCH e Bellows WW o CESCO Bioengineering User s Guide http www cescobio com tw 6 BelloCell 500P BOTTLE FEATURES The cross section of a BelloCell 500P bottle see Figure 5 will acquaint you with its features and components Basic parts of BelloCell 500P are the same with those in BelloCell 500 besides inward flowing and outward flowing connectors Figure 5 BelloCell 500P Bottle Perspective Drawing 1 8 Female Luer Lock 1 87 Male plug Ki Se j E Inward flowing port A e N A lt Qutward flowing port SE SC 1 8 Male Luer Lock 1 8 Female plug
11. CESCO BelloCell High Density Disposable Cell Culture System MANUAL NO WISA0101 Revision 2 0 Feb 14 2008 TABLE OF CONTENTS p 1 SAFETY INFORMATION 44 58 4 54 0414 det 43n 508y en NEEN NEE de 1 2 PRODUCT SPECIFICATION 54 45 5a554442 40054 545404410442 54 20 5434e 135454 2 3 SYSTEM DESCRIPTION ee k k y P Wek y la he lat a kuy a 4 4 CONTROL BOX FEATURES keke keke keka eke k e kk k ek k ka ka k 5 5 BELLOCELL 500 BOTTLE FEATURES kk 6 6 BELLOCELL 500P BOTTLE FEATURES e eke 7 7 BELLOCELL 500 amp BELLOSTAGE 3000 DIMENSIONS 8 8 UNPACKING amp INSTALLATION Wke kk KK kk K k 9 9 IMPORTANT CAUTIONS amp WARNINGS ww kk k k k 11 TO OPERATION ee Made Eeer 12 10 1 General Instruction of BelloStage 0 00 cccccc cece eee e eee e eee eeenaes 12 LOD RUANING a EENS Ee EE 15 10 3 Cell Harvest and Sampling 0 ccc cece eee e EEE KEKE KRA 22 11 MAINTENANCE cic Eesen Ee EE EEN 28 12 TROUBLING SHOOTING 0c i p cence e ERa ene 29 13 QUESTION AND ANSWER gt i lt i i4 5444s ui bel d n Paa na ek QR g y pelan 30 14 REFERENCE GUIDE 444 5 ees gea k bed l 394405440250 h ku bez n 32 CESCO BIOENGINEERING CO LTD 36 20 Rd Taichung Industry Park Taichung Taiwan R O C Telephone 886 3 5910121 e Fax 886 3 591 0123 e Internet http www cescobio com e E mail info cesc
12. ELAY arrow key to set seconds press either DELAY CESCO Bioengineering User s Guide http www cescobio com tw arrow key in short bursts 4 Start and stop Press START to begin culture process Press STOP to terminate culture process 5 Press the TIME RESET key to change the display between Total Time Alert Mode and the current BelloStage system version In the Total Time display press the Time Reset key and hold it down for 3 seconds to reset the time record to zero In the Alert Mode display press the 4 or part of the DELAY key to change alarm mode In 30 seconds mode alarm will keep beeping for 30 seconds In Continuous mode the alarm will keep beeping until the STOP key is pressed 10 1 2 During operation To change the platform rising and lowering speed press the A or W part of the RATE key As the platform is rising you can change the rising rate and as the platform is descending you can change the lowering rate To change the platform holding time at the top compressed bellows and bottom extended bellows positions press the A or W part of the DELAY key As the platform is rising you can change the upper holding time T_H and as the platform is descending you can change the lower holding time B_H While the platform is holding you cannot change the hold time Press the TIME RESET key to check the culture time in t
13. HOOTING Cells could keep activity even have been shut down for over night Please try to turn on the machine or change the bottles to another equipment immediately Index Possible Cause Solutions Continuous alarm platform fail to move in the unit _L Rust on the ball screw cause resistance to the platform movement wech Remove the rust and grease the ball screw Be sure to grease the ball screw every 3 months 2 ee of the step Remove the BelloStage otor A unit from the incubator if 3 Loosen on signal cable not use for a while between control box and 2 Repl ie sieo mooi the BelloStage unit ee ier 3 Make sure the 9 pin signal cable between control box and platform unit is well connected UPPER LIMIT ERROR 1 Failure on the upper limit 1 Clean the upper limit optical Biati rm can n treaeh the optical sensor sensor Make sure no dirt on upper limit the sensor surface Controller looks normal but 1 Burn down of the 6V 1 Replace the 6V DC CD unit cease movement DC DC circuit circuit 2 Malfunction of the step 2 Replace the step motor motor Random codes on Display 1 Signal transfer between 1 Reset and restart but unit operates normal LCM and CPU abnormal 2 Try to use single power 2 Noise from other electrical source of the equipment signal Control box can not hold on 1 Dirt between the magnetic 1 Clean up the dirt the incubator backplate and the incubator 2 Fi
14. d possible re location This warranty shall be invalid due to damage caused by accident misuse theft neglect natural disaster use of non CESCO s authorized spare parts illegal disassembly etc relocation or deterioration caused by application which is not the original purpose to design this product e CAUTION This equipment must be operated as described in this manual If operational guidelines are not followed equipment damage and personal injury can occur Please read the entire User s Guide before attempting to use this unit Do not use this equipment in a hazardous atmosphere or with hazardous materials for which the equipment was not designed CESCO Bioengineering Co Ltd CESCO is not responsible for any damage to this equipment that may result from the use of an accessory not manufactured by CESCO CESCO Bioengineering User s Guide http www cescobio com tw 1 SAFETY INFORMATION Please read this entire instruction manual prior to use the system a The BelloStage 3000 system operates on 100 230 volts 50 60 Hz through a desktop AC transformer Note Ensure that the input voltage listed on the AC Transformer label is compatible with your line voltage Ze NOTE Ensure that the input voltage listed on the AC Transformer label is compatible with your line voltage a Use a properly grounded electrical outlet of correct voltage and current handling capacity a Do not disass
15. emble the BelloStage 3000 or AC Transformer a Do not submerge either the BelloStage 3000 system or BelloCell in water Do not place the control box inside the incubator The control box is designed to operate outside the incubator a The BelloCell culture system is for laboratory use only It is not intended for diagnostic or therapeutic use in humans or animals 53 One BelloCell is one time use only When the experiments are finished BelloCell should be decontaminated and discarded Consult local institution for detail WARNING Avoid inserting your hands inside the BelloStage unit during operation B Please feel free to contact CESCO Bioengineering Co Ltd at info cescobio com tw for technical assistance CESCO Bioengineering User s Guide http www cescobio com tw 2 PRODUCT SPECIFICATION BelloStage 3000 Console Dimension 264 x 359 x 170 m m W L H 10 4 x14 x6 7 inches Weight 7 0 kg 15 4 Ib Power 100 230 VOLTS AC 50 60 Hz Input 12 VOLTS DC Output _ Up Down Rate 0 25 to 2 0 mm sec Step of 0 25 mm sec Delay Time 0 to 99 min 59 sec Step of 1 0 sec or 1 0 min Driver Motor DC stepping motor Environment 20 42 C 0 90 relative humidity in a CO incubator Mechanical Hi Low Optical Sensor Protection Transmission Gear set ratio 1 1 2 and belt Materials Aluminum alloy chromic steel BelloStage
16. he Total Time or the Alert Mode display but you cannot change the values during operation Press the STOP key to stop platform movement the platform will by CESCO Bioengineering User s Guide http www cescobio com tw default return to its lowest position leaving the bottle bellows extended 10 1 3 Stopping a Run At any time during a run you can stop the BelloStage unit by pressing the STOP key on the controller The moving plate will go down to the bottom position A Return to Base Waiting will be displayed in the LCD screen see Figure 11 Two BEEP sound appeared indicate the termination of the operation Figure 11 Display after pressing STOP key BelloStage 3000 L TIME START RESET CESCO Bioengineering User s Guide http www cescobio com tw 10 2 Running a Culture 10 2 1 Running a Culture in BelloCell 500 CAUTION Be sure to read Sections 10 before taking any action to prepare for or to run a culture e Never spray alcohol on the BelloCell bottle cap because it contains a sterile filter e Never flame the neck of the BelloCell bottle Step 1 Remove a fresh BelloCell 500 or BelloCell 500P bottle from its bag It is already sterilized for use Place the BelloCell unit in a bio safety cabinet Aseptically remove the cap and fill in 470 ml pre warmed culture medium Tilt the bottle to send all the media down into the bellows chamber Figure 12
17. he left side of BelloStage unit to the control box at the 9 pin cable junction CESCO Bioengineering User s Guide 10 http www cescobio com tw 2 Place the BelloStage platform inside a humidified CO incubator and leave the control box outside the incubator 3 Thread the controller signal cable through an available port or between the door and door gasket 4 The control box is magnetized for easy attachment Position the control box on a convenient metallic outside wall of the incubator 5 After verifying that your unit s AC transformer plug mates to your outlet connect the power cable from the control box to your power supply outlet The BelloStage platform is ready to use now Figure 8 BelloStage Assembly ee Power adaptor lt ee ri M on off Control Box lt 3 Main body Signal cable e CAUTION Always turn off unplug and remove the BelloStage unit from the incubator if you do not plan to use the system for a while CESCO Bioengineering User s Guide http www cescobio com tw 9 IMPORTANT CAUTIONS amp WARNING WARNINGS Never place your hands inside the BelloStage unit during operation CAUTION Never spray alcohol on the BelloCell bottle cap because it contains a sterile filter Never flame the BelloCell bottle CAUTION A grounded electrical outlet is necessary for the safe operation of this instrument
18. ion 10 2 4 above for aseptically taking a sample but use the pipette to remove all of the media Then add 470 ml of fresh media 30 ml of conditioned media usually remains in the bottle Return the bottle to the BelloStage unit and press the START key to resume the run 10 2 7 Media Replenishment For BelloCell 500P 1 Turn off BelloFeeder pump and BelloStage 3000 3 Block medium flow by clamps in both in and out flow tubings Separate pump head from BelloFeeder pump module please check Step by Step Instruction for details Move and place the BelloCell 500P medium reservoir and the tubing set inside a biosafety cabinet 4 Prepare a reservoir containing fresh culture medium Open and move the cap set from the conditioned to the new reservoir and close it tight 5 Place the BelloCell 500P pump head and medium reservoir together inside the incubator again and insert the pump head into the rotary shaft of BelloFeeder pump Loosen the clamps on the tubing Turn on the BelloStage 3000 and BelloFeeder pump CESCO Bioengineering User s Guide 21 http www cescobio com tw WARNING Before turn on BelloFeeder pump module both the slide clamps on the in and out flow tubing must be loosened 10 2 8 Monitoring Glucose You can measure the glucose residue in a sample of the media from time to time to determine whether the media should be renewed You can also calculate the Glucose Uptake Rate GUR to mo
19. is to randomly pick six BioNOC II disks from the carrier disk chamber following the aseptic instructions in Section 10 3 1 above and to then follow this procedure Aseptically place the carriers two per vial into three 1 5 ml Eppendorf vials Add 1 ml CVD nucleus counting kit provided with your BelloStage system Vortex the vials for approximately one minute Incubate the vials in the CO incubator at 37 for two hours vortexing the vials every 30 minutes during the incubation period Count the nuclei in the solution using a hemocytometer see Figure 18 Average the cell count per carrier then multiply that average value times 860 the average number of BioNOC II disks in a BelloCell bottle to calculate the probable cell population OND 5 6 ZS NOTE This is not an accurate method to assess cell viability Figure 18 Nuclei of Vero Cells Stained with crystal violet dye Nuclei show bright purple color and oval shape in phase contract microscope 10 3 3 2 Counting Cells by Trypsinization Another method to count cells is to randomly pick six BioNOC II disks from the carrier disk chamber following the aseptic instructions in Section 10 3 1 above and to then follow this procedure CESCO Bioengineering User s Guide 25 http www cescobio com tw 1 Aseptically place the carriers two per vial into three 1 5 ml Eppendorf vials 2 Add 1 ml PBS EDTA 0 02 to the
20. me allows more nutrient exchange between bulk medium solution and medium carriers Increase top holding time can also reduce oxygen level if the condition prefers low dissolved oxygen Increase bottom holding time will increase the oxygen level during culture Increase bottom holding time can also help to stabilize the pH Recommended inoculation densities per bottle Cell Type Inoculation density cells bottle CHO VERO BHK C 127 RK 13 Hela 0 5 1 0x108 Hybridoma NSO Sf 9 Hi 5 Sf 21 HEK 293 1 5 2 0x108 Recommended Operating Parameters Rate mm sec Delay DEES UP DOWN ER B H Inoculation 2 0 2 0 20 secs 0 Cell Culture 1 0 1 5 1 0 1 5 10 secs 1 min 30 mins for the first 2 5 hours following inoculation CESCO Bioengineering User s Guide http www cescobio com tw 10 2 2 Running a Culture in BelloCell 500P XA NOTE Cell inoculation procedure is the same as Section 10 2 1 BelloCell 500 ZS NOTE Sterilize the tubing set and reservoir vessel by autoclaving at 121 C 30 mins prior for use XA NOTE Connect the tubing set with BelloCell 500P bottle and start pumping after 48 72 hours culture according to the figure 15 shown below Figure 15 BelloCell 500P Bottle Assembly ee AR ce ee Reduction Comte 1 8 lr Peristaltic Media
21. nd a smooth and ilat 2 Wall surface of the surface incubator is not flat 3 Find a surface made by 3 Wall surface is inert to iron or steel magnetic Keypad failure Keypad failure Change keypad If you are unable to solve the above problems please contact local CESCO distributor or e mail info cescobio com tw CESCO Bioengineering User s Guide 30 http www cescobio com tw 13 Question amp Answer 1 Q Can I harvest cells after culture A Yes you can harvest cells after culture terminated BelloCell 500 can be disassembled and the carrier box can be taken out for this purpose You can also harvest cells aseptically without disassemble the bottle Please refer to the instruction manual for detail instruction 2 Q How can I observe cell growth on the carriers A The carriers are made of porous nonwoven fabrics The common staining solution is hematoxylin coomassie brilliant blue G etc Please follow the instruction manual for each staining application Then you can see the cell morphology in carriers under a microscope 3 Q Can take out carriers for sampling during culture A Yes BelloCell 500 model is equipped with a sampling window on the carrier box With long arm forceps users can take out carriers through aseptic techniques during culture 4 Q Can I count the cell population during culture A Yes After take out carriers through aseptic technique
22. nitor the cell growth rate the GUR value should be proportional to the total cell volume To calculate GUR use this equation GUR Cau t2 Cau t1 X Vsolu t2 t1 where GUR is the Glucose Uptake Rate in mg hour Cau is the concentration of residual glucose t1 amp t2 are the sampling hours Vsou is the volume of media in liters Cesco Bioengineering also provides GlucCell glucose meter and kits for your convenience to measure glucose concentration during culture Please refer to Section 15 Options for BelloCell Cell Culture System 8 NOTE We recommend that you maintain the glucose concentration at more than 1000 mg L CESCO Bioengineering User s Guide 22 http www cescobio com tw 10 3 10 3 1 Cell Harvest and Sampling Sampling of BioNOC II Carriers The BelloCell bottle is equipped with a sampling port see Figure 16 The rotating BelloCell chamber can be turned to allow sampling of carriers anyplace in the chamber To sample BioNOC II disks 1 AUN 10 3 2 Press the STOP key on the controller to return the platform to its bottom position and to halt operation Unlock the BelloCell bottle from the unit and aseptically open the cap Tilt the bottle toward you until you can see the sampling window see Figure 16 Aseptically push against the sampling window with the tip of a sterilized forceps then pick the carriers Place the selected carriers in a
23. nts aseptically we recommend the following procedure 1 Discard the culture media from the BelloCell 500 bottle Observe proper handling and disposal procedures if the media contains potentially bionazardous material 2 Add 500 ml of pre warmed PBS EDTA to the BelloCell bottle Install the bottle in the BelloStage unit Program the Up Down Rate at 2 0 mm s and start the START key Stop the compression after 5 mins remove the bottle and asceptically open it to discard the washing solution 3 Repeat step 2 twice more at least 4 Add 400 ml of pre warmed trypsin EDTA solution 0 05 0 02 to the bottle Incubate for 10 15 mins CESCO provides CELL DISSOCIATION SUPPORTER to reduce trypsin requirement to 250 ml 5 Aseptically discard the enzymatic solution and incubate for 10 15 mins more 6 Tap the bottle sharply against your palm or a soft round counter edge at the matrix chamber level for 3 5 minutes to force cells to release from the carriers and the matrices they have created CESCO Bioengineering User s Guide 27 http www cescobio com tw 7 Add 500 ml Culture medium or PBS containing FBS or trypsin inhibitor to the bottle 8 Install the bottle on BelloStage in a CO incubator Run the unit for 2 3 mins at the Up Down rate at 2 0 mm s 9 Remove the bottle and aseptically harvest the cell laden solution and then harvest the cells by centrifuging the solution 10 Repeat step 6 9 at least
24. obio com tw http www cescobio com tw Copyright Notice CESCO Bioengineering Co Ltd 36 20 Rd Taichung Industry Park Taichung Taiwan R O C Copyright 2008 CESCO Bioengineering Co Ltd All Rights Reserved Reproduction adaptation or translation without prior written permission from CESCO Bioengineering is prohibited The information contained herein is designed only for use with this product CESCO Bioengineering is not responsible for any use of this information as applied to other products Neither CESCO Bioengineering nor its affiliates shall be liable to the purchaser of this product or third parties for damages losses costs or expenses incurred by the purchaser or third parties as a result of accident misuse or abuse of this product or unauthorized modifications repairs or alterations to this product or failure to strictly comply with maintenance instructions Disclaimer Notice CESCO Bioengineering Co Ltd reserves the right to change information in this document without notice Updates to information in this document reflect our commitment to continuing product development and improvement Manual Conventions Bold Text in bold face type emphasizes key words or phrases CS NOTE Notes contain essential information that deserves special attention Caution messages appear before procedures CAUTION which if caution is not observed could result in damage to the equipment WARNING Warning mes
25. s during culture users can measure the cell population through crystal violet dye nuclear count method Each BelloCell bottle contains 865 5 carriers User can estimate the total cell population by timing back the total carrier number Cesco also provide CVD nucleus count kit 5 Q Can I culture cells by using serum free medium in BelloCell CESCO Bioengineering User s Guide 31 http www cescobio com tw A Most serum free media can be applied to BelloCell culture If the medium allows cells to attach or partially attach 50 suspend 50 attach on typical T flasks then it should be able to be used in the BelloCell 500 system However for loosely attached cells we recommend to use BelloCell 500P instead of BelloCell 500 6 Q What is the BioNOC II carriers in BelloCell A BioNOC II carriers are made by FDA approved 100 PET fabrics They were processed through a special surface treatment to make it biocompatible The carriers provide around 2 400 cm g specific surface area for cell growth One BelloCell contains 5 5 g of the BioNOC II carriers 7 Q Can obtain BioNOC II carriers solely for other purposes A Yes you can purchase BioNOC II carriers They can be applied to our TideCell bioreactor or other packed bed type bioreactors 8 Q Can I reuse BelloCell 3 A BelloCell is designed for one time use only and it cannot be sterilized by autoclaving Reuse BelloCell is not recommended A
26. sages alert you to specific procedures or practices which if not followed correctly could result in serious personal injury d gt BIOHAZARD Biohazard Warnings alert you to user responsibilities WARNING in the handling and disposal of potentially hazardous biological material CESCO Bioengineering User s Guide http www cescobio com tw Warranty CESCO Bioengineering Co Ltd warrants that if a product manufactured by CESCO shall be free of defects in materials including mechanical parts and electronic parts and other workmanship for one 1 year from the first to occur of i the date the product is sold by CESCO directly or ii the date the product is purchased from authorized distributors by CESCO the Commencement date Except as expressly stated above CESCO makes no other warranty expressed or implied with respect to the products and expressly disclaims any and all warranties including but not limited to warranties of design merchant ability and fitness for a particular purpose An authorized distributor of CESCO must perform all warranty inspections In the event of defect covered by CESCO s warranty CESCO shall as its sole obligation and exclusive remedy provide free replacement parts to remedy the defective product This instrument warranty is not allowed to be transferable The warranty only applies to the original purchaser of this instrument and will be null and void in the case of resale an
27. sterilized vessel Use the tip of the same forceps to rotate the sampling window as necessary to obtain all the desired carriers Figure 16 BelloCell Sampling Window view from top of BelloCell bottle cap removed Observing Cells To observe a sample of cells under a light microscope 1 2 3 Aseptically remove a few BioNOC II disks from the carrier disk chamber following the aseptic instructions in Section 10 3 1 above Dehydrate and fix the cells in their carriers using EtOH 70 dehydration or 99 5 EtOH dehydration for 5 minutes Wash off the EtOH twice using either DI water or PBS CESCO Bioengineering User s Guide 23 http www cescobio com tw 4 Stain the cells with hematoxylin crystal violet or coomassie brilliant blue G dye for 5 10 minutes 5 Wash off the excess dye with DI water 6 Observe the cells in your light microscope see Figures 17a and 17b amp BIOHAZARD WARNING The user is responsible for following local guidelines for handling hazardous waste and biohazardous materials that may be generated from the use of this equipment Figure 17a Hi 5 Cells in BioNOC Il disks Figure 17b HEK293 Cells in BioNOC II disks Stained with hematoxylin Stained with hematoxylin CESCO Bioengineering User s Guide 24 http www cescobio com tw 10 3 3 Counting Cells 10 3 3 1 Crystal Violet Nucleus Count Method One method to count cells
28. three times until the cell residue harvested falls below the desired concentration Ce NOTE Because of the high cell density cell laden solution may contain many extra cellular matrices But it can be separated from cells by centrifugation CESCO Bioengineering User s Guide 28 11 MAINTENANCE Periodic maintenance is essential to the proper operation of your BelloStage system http www cescobio com tw 11 1 Cleaning Periodically clean the BelloStage unit and controller by wiping the outside surfaces with a soft cloth moistened with a 70 alcohol solution e CAUTION The exterior surfaces of the BelloStage unit and controller are coated with an anti rust material Never use any abrasive materials and be careful not to scratch the exterior finish 11 2 Maintenance We recommend that you grease the BelloStage unit s platform ball screw see Figure 19a once every three months to keep it operating smoothly 1 Turn off and unplug the BelloStage unit 2 Remove the unit from your incubator 3 Slowly and carefully apply approximately 0 5 ml of grease using a user supplied syringe to the platform ball screw from the bottom up see Figure 19a 4 Make note on the maintenance card see Figure 19b Figure 19a Greasing the Ball Screw Figure 19b Recording after Maintenance CESCO Bioengineering User s Guide 29 http www cescobio com tw S CAUTION 11 TROUBLE S
29. tracapillary EC space because of convective flow in EC may result in serious sedimentation and poor oxygen transfer Some commercial packed bed bioreactor system is designed on basis of airlifting principle combined with modified impeller to promote gentle liquid circulation through annular packed bed with low shear stress but good oxygen transfer However the height of packed bed limits the industrial scale up capability for this type of bioreactors In order to minimize the disadvantages and retain the advantages of the commercialized bioreactors aforementioned CESCO Bioengineering Co has developed BelloCell and TideCell bioreactors In BelloCell and TideCell a massive oxygenating surface area was created with simple medium movement relative to the cell embedded in matrix for highly efficient oxygen transfer without any agitation and air sparging BelloCell is a disposable bioreactor mainly designed for simple efficient and economical laboratory use while TideCell was for pilot and production plant application Figure 21 Diagram showing the nutrition and aeration phase during culture in BelloCell system Nutrition Aeration CESCO Bioengineering User s Guide http www cescobio com tw 34
30. uid film as shown in the following schematic diagram Not only this would not cause the damage of the culture cells but also facilitate the most efficient oxygen transfer from air to the cells During the submerging phase see Figure 20 the cells expose to the new liquid surface and facilitate the uptake of nutrients from the medium such as glucose glutamine and growth factors etc and removal of the wastes such as ammonium lactic acid etc from the cells Figure 20 Cross Section view of liquid left and gas right transfer across the carriers and cells Nutrition profile O2 profile Bulk solution 1 em em em em e pm xe em ze eee eee ooo af oo ao Cells in Cells in matrix matrix Gas phase Liquid film CESCO Bioengineering User s Guide 33 http www cescobio com tw Oxygen transfer limitation is always the threshold of achieving high cell density in cell culture In the stirring tank bioreactor system with or without use microcarriers it requires high air sparging and agitation rate to achieve high oxygen transfer but it also generates high shear stress and foaming problem which are detrimental to animal cells The hollow fiber bioreactor system is designed to generate lower shear stress and perform perfusion culture but it requires exterior oxygenation system and high circulation rate to provide sufficient oxygen supply Furthermore the axial non uniform distribution of cells and proteins along ex
31. vial and wash the carriers by inversion 2 3 times 3 Remove the PBS EDTA Repeat the wash at least twice 4 Add 1 ml trypsin 0 05 trypsin O 02 EDTA to the vial 5 Incubate at 37 for 10 mins 6 Flick the vial 10 more times by fingers until the solution becomes turbid 7 Count the cells and viability with trypan blue dye exclusive method in the solution using a hemocytometer 8 Average the cell count per carrier then multiply that average value times 860 the average number of BioNOC II disks in a BelloCell bottle to calculate the probable cell population XA NOTE Cell count might not be accurate if cells cannot release from the carrier efficiently CESCO Bioengineering User s Guide 26 http www cescobio com tw 10 3 4 Harvesting Cells Ka BIOHAZARD WARNING The user is responsible for following local guidelines for handling hazardous waste and biohazardous materials that may be generated from the use of this equipment When the culture run is terminated users may want to harvest cells or cell components for use elsewhere One solution is to disassemble BelloCell 500 bottle by BOTTLE OPENER to BioNOC II carriers and then harvest the cells or cell components either by adding lysis buffer or freeze thaw procedure Bottle Opener can be purchased from CESCO Bioengineering Ze NOTE The above harvesting procedure is not aseptic For aseptic cell harvest cells or cell compone
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