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Flexcell® Tissue Train® Culture System

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1. Hi Ha 10 ft 3m 3 8 9 53mm 10 ft 3m 1 4 6 35mm tubing to Flex Out tubing to Flex In Figure 8 Single baseplate connection Ea ie n E t 10 ft 3m 1 4 6 35mm tubing to Flex In Figure 9 T connection for two baseplates E FLEXCELL INTERNATIONAL CORPORATION 2 5 SETUP OF BIOFLEX BASEPLATE S WITH LOADING STATIONS AND CULTURE PLATES Place the BioFlex baseplate onto one of the incubator shelves Be sure the shelf is level front to back and side to side in the incubator 1 Attach the FLEX IN and FLEX OUT quick disconnect fittings to the respective fittings on the baseplate 2 If using Loading Stations place the four sets of Loading Stations into the baseplate wells and apply silicone grease to the tops of the posts see the Culture Plate and Loading Stations user s manual for detailed instructions 3 Place the 4 red gaskets onto the bases of the four Tissue Train culture plates Be sure that the gaskets are fully pressed onto the culture plates and that no parts are folded under 4 Place the culture plates and gaskets into the baseplate wells Check to be sure that the gaskets are not folded up at any point of contact with the baseplate If the four gaskets feel loose within the plane of the baseplate can be easily moved right or left forward or backward without liftin
2. Mn Max Freq DC Cydes Duration Back To Repeat 0 0 5 0 L0 50 0 30 0 00 00 30 00 50 10 S00 3 0 00 00 30 00 00 1 0 500 3600 0 01 00 00 AddStep Remove Step Save Regimen Delete Regimen Help dse 2 3 4 5 6 Figure 15 FX 5000 regimen editor Choose the desired waveform shape under Shape by clicking on the downward arrow and selecting an entry from the drop down list Enter the desired minimum and maximum elongation for the plate membranes 0 30 in the boxes under Min and Max Enter the frequency for the waveform 0 01 5 Hz in the box under Freq If a static regimen is required enter the desired elongation at which the membrane is to be held in the Max box Enter 0 in the Min box and 1 0 in the Freq box Min and Freq are irrelevant for static regimens If a duty cycle for the waveform is to be defined enter the desired value in the box under DC The duty cycle is the percent of time that the waveform shape remains on at the high square wave or rising triangle part of the waveform Duty cycle only affects the triangle and square waves 17 7 Enter the number of times for the regimen to repeat 0 65 535 in the box under Cycles When moving out this box the box underneath dd hh mm ss will update to the calculated regimen time according to the set parameters The format for this entry is days hours minutes seconds However if the time is entered first the p
3. LEL DB Lal Triangle Wave Square Wave This waveform provides a linear ramped rise This waveform provides the fastest possible to the max and a linear ramped fall back to rise to max followed by the fastest possible the min fall to min l lll l lll SEWA SS g di di PUTT Lud BEHBHEEHEHEHEEEEERREN Lud 29 E FLEXCELL INTERNATIONAL CORPORATION Heart E Wave Heart P Wave This waveform applies a pressure pattern that This waveform applies a pressure pattern that mimics the shape as seen on an mimics the actual pressure wave generated by electrocardiogram a heart beat Lx Eren Nm imde w 33 j 25 Tee TTT Th Ju Static Wave This waveform applies a constant state of pressure ELLE 3231 ARI T 30 trt 3231 du Il FLEXCELL INTERNATIONAL CORPORATION PART C FLEXCELL FX 5000 TENSION SYSTEM SPECIFICATIONS E FLEXCELL INTERNATIONAL CORPORATION FX 5000 TENSION SYSTEM FEATURES Uses vacuum to deform a flexible bottom culture plate yielding up to 33 substrate elongation Minimum strain resolution capability is 0 1 elongation Applies a defined controlled static or cyclic deformation to growing cells in vitro Housed valving mechanism automatically regulates and maintains pressure to provide the specified strain regimen Capable of driving up to 4 independent FlexLink remote
4. excell Tissue Train Culture System Tissue Train 3 D Bioartificial Tissue TOP VIEW FLEXCELL SIDE VIEW Anchor stem Anchor Trough bay B Rubber membrane Trough Loader Vacuum hole Add cells in a gel matrix to trough bay f Rubber membrane deflected into trou 2 Vacuum Gel Cells Tr 12 15 14 Rev 4 4 Culturing Cells in a Mechanically Active Environment Flexcell International Corporation e 2730 Tucker Street Suite 200 e Burlington NC 27215 800 728 3714 919 732 1591 FAX 919 732 5196 www flexcellint com COPYRIGHT 2009 FLEXCELL INTERNATIONAL CORPORATION g FLEXCELL INTERNATIONAL CORPORATION TABLE OF CONTENTS One Year Limited Parts Warranty i5i exiit o Db VEI PUR ME sk HAN RIN N a n y HANA Wa vu n H d Hek E ill Dell Computer Warranty Information eee e Roca deer keke kek ke kl ke kk Kek iv Federal Communications Commission Radio Frequency Interference Statement V Tissue Train Quick Start Instructions tette tette teen nt vi PART A Creating 3D Cell Seeded Hydrogele 1 1 Intro HO cocido RD U Oxo ase Ope cari ease ae om 2 2 Method to Create 3D Cell Seeded Hydrogel eeeeee 2 2 1 Preparation of Cells in 3D Linear or Trapezoidal Gels in a Tissue Train Culture Plate 2 2 2 Preparation of Cells in 3D Circular Gels in
5. 18 0 BioFlex 28 mm 6 5 11 0 BioFlex 31 mm 2 0 33 V0 Tissue Train Arctangle 10 0 16 0 UniFlex Arctangle 5 0 8 5 HT BioFlex 7 0 11 5 36 xm ga FLEXCELL INTERNATIONAL CORPORATION NOTICE The information in this document is subject to change without notice Flexcell International assumes no responsibility for any errors that may appear in this guide This manual is believed to be complete and accurate at the time of publication In no event shall Flexcell International Corporation be liable for incidental or consequential damages in connection with or arising from the use of the manual Opening the case of the FX5K Tension FlexLink voids the warranty Customers must contact Flexcell International Corporation or an authorized distributor before opening the case on the FX5K Tension FlexLink 37
6. E FLEXCELL INTERNATIONAL CORPORATION not it is possible that the valves are locked due to residue buildup Try applying positive pressure to the VENT port to free the valves If the regimen wave is flatlining but the plates are still cycling air pressure try the following Disconnect the blue 1 4 6 4mm FLEX IN tubing from the back of the unit and place a finger over the end of the tubing while a regimen is running If air is cycling at the end of the tubing then the problem is with the FX5K Tension FlexLink Contact Flexcell International for further instructions Problem 3 Regimen wave is fluttering or not smooth Solution 3 This problem could be due to water in the valves or the system being out of calibration Run the drying regimen with the Drying Filter as described in the manual to dry the system and valves If this does not resolve the problem try running the drying regimen once more If the problem continues contact Flexcell for further instructions Problem 4 Programming a long regimen 1 day or more but the software will not allow the regimen to be saved Solution 4 The software has a limit on the number of cycles that can be entered into any given step 65 535 If a regimen is programmed with a step that requires more than 65 535 cycles the software will give an invalid entry error To run the regimen for the time required additional steps will need to be programmed This wil
7. Using additional FX5K Tension FlexLinks will allow two or more baseplates platforms with different regimens to run simultaneously All tubing connections made to the first FlexLink should be made exactly the same with any additional FlexLinks Each FlexLink will have its own separate FLEX IN and FLEX OUT tubing for each baseplate However the SYSTEM tubing can be shared between FlexLinks with T fittings as long as the vacuum source is capable of running two or more FlexLinks 13 with the programmed regimens Regimens with a combination of high frequencies and elongation levels will require a more powerful vacuum source The capability of the vacuum source with more than one FlexLink will need to be tested experimentally with the users particular setup FlexLinks must be placed side by side during use If the user desires to use the vacuum pump auto control feature with each FlexLink then separate vacuum pumps will need to be used with each FlexLink To connect an additional FlexLink s to the computer connect the Ethernet cable from each FlexLink to an Ethernet hub Then connect the hub to the Ethernet port on the back of the computer using the Ethernet communications cable provided The LCD on the front of the FlexLink will display the number 1 4 that the FX5K Tension FlexLink has been assigned at the time of production NOTE The numbering of each FX5K Tension FlexLink will not de
8. ear eeu 28 Baseplate and Gasket Cleaning and Disinfecting eke keke 28 Running Low Frequency Regimens with BioFlex Culture Plates 28 Waveform Shape Definitions e tee ce rre ver ss ea v i REP abe E ec e 29 PART C Flexcell FX 5000 Tension System Specificationes 31 Appendix A FX 5000 Tension System Dual Baseplate Operation 35 I i FLEXCELL INTERNATIONAL CORPORATION ONE YEAR LIMITED PARTS WARRANTY Flexcell Tissue Train System 1 FLEXCELL INTERNATIONAL CORPORATION warrants to the original purchaser customer all hardware components of the Flexcell Tissue Train System serial for a period of one year from the date of delivery to the purchaser customer to be free from manufacturing defects in workmanship or materials with the following exceptions terms and conditions a ITEMS EXCLUDED FROM THE WARRANTY ARE Dell OptiPlex desktop computer and monitor software disks manuals and external peripherals such as printers mouse or track ball units imaging devices vacuum pumps air tanks electric voltage converters compressors surge suppressers and all other accessory equipment b DURING THE WARRANTY PERIOD the purchaser customer must notify Flexcell of any warranty claim in writing by telephone fax transmission or email identifying each defective part or specifically describe the exact problem no later than the last day the warranty is in effect c FLE
9. 1 e 500 points sec or 1 800 000 points hour Therefore a one hour regimen will have approximately a 4 0 Mb data file To turn this feature OFF please contact Flexcell To see the stored data in the Data Analysis menu select Data Viewer Open the appropriate dat file from the C FlexSyst directory Two new windows will appear Fig 17 In the window labeled filename 1 the data presented defaults to the raw data In the window labeled filename 2 the data presented defaults to a moving average of the raw data with data averaged every 10 seconds i FLEXCELL INTERNATIONAL CORPORATION Figure 17 FX 5000 data display The y Axis control box allows for automatic or manual control of the scale on the y axis If Auto is selected the y axis scale can be changed by clicking on one of the two small double arrow buttons at either end of the axis The button with arrows pointing away from each other increases the axis scale while the button with arrows pointing toward each other decreases the scale The minimum value is 2 kPa and the maximum is 100 kPa If Manual is selected the user can enter the upper and lower limits of the y axis scale in the white boxes to the left side of the axis The small double arrows will not function if Manual is selected The x axis scale allows for a time interval to be set between 0 1 and 1000 seconds Click on one of the two small double arrow buttons at either end of the axis to change
10. be sure the voltage selection switch ON the computer Fig 7 is set to 115 V or 230 V respectively When the instruments are completely set up the power cords should be connected to a surge protected power strip and then to a 115 VAC or 230 VAC 60 Hz power outlet 2 4 INCUBATOR ACCESS PORTS Consult the operations manual of the CO incubator for the location of the incubator access ports Most incubators have an access port at the top or the rear of the incubator If this opening is not immediately apparent consult the incubator manufacturer about the location of an access port To prevent voiding the incubator warranty or 10 encountering an electrical hazard a trained incubator representative should open the incubator access ports Once the access port has been located insert the FLEX IN and FLEX OUT tubing through the access port into the incubator Attach the 4 6 4 mm blue tubing to the Ya 6 4 mm quick disconnect on the baseplate Attach the 3 9 5 mm clear tubing to the 3 9 5 mm quick disconnect on the baseplate Fig 8 If two baseplates are to be used simultaneously connect the baseplates using the configuration as shown in Figure 9 NOTE Please see Appendix A pg 35 for the maximum allowable 96 elongations when using two baseplates simultaneously 1 4 6 385mmm tubing he She HE HET ds wu d ir ia Quick disconnects a H
11. strain and or compression controllers Capable of delivering frequency ranges from 0 0135 Hz Works with BioFlex Tissue Train and UniFlex culture plates Ability to program multiple frequency amplitude and wave changes in one regimen Waveforms available Static wave Sinusoidal wave Heart wave Electronic and Pressure Triangular wave Square wave and custom waveforms Provides equibiaxial strain or unconstrained distension to the BioFlex culture plate membrane Optional Tissue Train and UniFlex plates can be used in conjunction with Arctangle Loading Stations to achieve uniaxial strain Optional StageFlexer and StageFlexer Jr devices allow strain application to cells while viewing in real time under an upright microscope Optional FlexStop provides a way to prevent pressure induced flexing of any number of BioFlex wells FX 5000 TENSION COMPONENTS AND SPECIFICATIONS Dell OptiPlex Desktop Computer USB Keyboard USB Mouse 17 Flat panel Monitor FlexSoft FX 5000 V1 0 Flexcell FX 5000 Software and Microsoft Windows 7 FX5K Tension FlexLink controller Accessory Pack nuts ferrules Flexcell vacuum pressure baseplate gaskets and acrylic window for BioFlex culture plates BioFlex 25mm diameter Loading Stations and Lubricant Water Traps System Drying Filter 25 ft small blue tubing 4 6 4 mm O D for FLEX IN connection 25 ft clear tubing 9 5 mm O D for FLEX OUT connection 25
12. that it is highlighted in blue and then click on Delete User The program will ask for a confirmation and issue a warning click Yes to confirm deletion The user and any of the user s defined regimens will be deleted To exit the Users window click on Close 3 5 CREATING REGIMENS NOTE Please see Appendix A pg 35 for the maximum allowable 96 elongations when using two baseplates simultaneously Before beginning a regimen must be created for downloading In the Regimen menu select Create new A new window will appear entitled Regimens with a list of the existing users and their regimens Fig 15 In the Existing Users box click on the user name under which the regimen is to be created A list of current regimens created under this user will appear in the Regimens for Selected User box unless no regimens have been saved under this user in which case this box will be empty To create a new regimen click on Run Wizard in the bottom left hand corner This option will bring up a step by step process of creating a new regimen Alternatively follow the sequence of steps below 1 Regimen Name Test Shape Min sw oo 5 0 1 0 E FLEXCELL INTERNATIONAL CORPORATION Type a regimen name in the box labeled Regimen Name Regimens for Selected User Step 1 Duration 0 01 01 00 Max Freq DC Cydes dd hh mm ss Back To Repeat so js 0200030 je fo Regimen Steps Step Shape Run Wizard
13. the total duration time 7 Click on Save Regimen 8 When finished with regimen editing click on Close 3 7 ASSIGNING REGIMENS In the Controllers menu select the appropriate FlexLink n where n is the FlexLink number to which the regimen will be downloaded In the Regimen menu select Assign then choose the Platform User and Regimen from each drop down menu Fig 16 A message box will appear to confirm regimen assignment Click Yes 19 NOTE Be sure to choose the platform that properly corresponds to the setup being used otherwise the elongation values will be incorrect For example If using the Arctangle Loading Stations with linear or trapezoidal Tissue Train plates click on Tissue Train Plate 24mm Arctangle LS for the choice of platform If using the 25 mm BioFlex Loading Stations with Tissue Train Circular Foam plates click on BFlx Loading Station 25mm for the choice of platform Figure 16 Assigning regimens for downloading to the FX5K Tension FlexLink If the download progresses no further messages will appear and the Start and Simulate buttons will be enabled If an error occurred in downloading the regimen to the FlexLink a message will appear alerting the user of a problem and the regimen will not be able to run In which case click Reset in the FlexLink window and then assign the desired regimen again 3 8 STARTING REGIMENS To run an assi
14. this scale The button with arrows pointing away from each other increases the axis scale while the button with arrows pointing toward each other decreases the scale The Input data box allows the user to switch the data in the graph from raw data to averaged data If the Raw data option is selected the graph will plot the pressure in kPa over time in seconds for the selected regimen If the Moving average option is selected the graph will plot the average pressure over the designated time period in 22 seconds range of 0 1 1000 seconds shown in the Time s box The Start time box can be set so that the data viewed in the plot begin at the specified time entered here Use the timeline at the top of the graph to navigate through the regimen Users can move the slider function within the timeline to manually progress through the regimen plot If the user clicks on the arrows at either end of the timeline the plot will progress to the next time period as set by the x axis scale For instance if the scale is set to show 20 seconds of data and the user clicks on the right arrow button the plot will progress to the next 20 seconds of data 3 17 SHUTTING DOWN THE SYSTEM A system Drying Filter Fig 18 has been supplied with the FX 5000 This device must be used whenever the system has been operated for more than one day and will not be operated again for more than one day If the system will be operated again immediately a
15. 000 15 9 31mm Diameter Loading Stations equibiaxial strain Minimum achievable elongation with the FX 5000 0 8 Maximum achievable elongation with the FX 5000 6 0 Tissue Train with Arctangle Loading Stations uniaxial strain Minimum achievable elongation with the FX 5000 1 6 Maximum achievable elongation with the FX 5000 20 8 33 E FLEXCELL INTERNATIONAL CORPORATION UniFlex with Arctangle Loading Stations uniaxial strain Minimum achievable elongation with the FX 5000 1 1 Maximum achievable elongation with the FX 5000 12 2 HT 24 Well Plate with Cylindrical Loading Stations equibiaxial strain Minimum achievable elongation with the FX 5000 1 2 Maximum achievable elongation with the FX 5000 15 0 FX 5000 TENSION PHYSICAL SPECIFICATIONS FX 5000 Computer Size W x Hx D Power Requirements Weight unit only FX5K Tension FlexLink Size W x Hx D Power Requirements Weight unit only Monitor 17 LCD Flat Panel Size W x Hx D Power Requirements Weight unit only 3 65 x 12 45 x 13 40 9 3 x 31 6 x 34 0 cm 115 230V 8 8 4 3A or 4 3 2 2A 60 50 Hz 15 0 Ibs 6 8 kg 10 5 x 7 0 x 18 5 26 7 x 17 8 x 46 9 cm 115 230V 6 3A 60 50 Hz 22 15 Ibs 10 06 kg 14 6 x 15 7 x 7 1 37 1 x 39 7 x 18 0 cm 115V 60 Hz Auto switch to 230 V 11 22 Ibs 5 1kg FX 5000 TENSION VACUUM REQUIREMENTS
16. 5000 software Preinstalled software FlexSoft FX 5000 Microsoft Windows 7 Microsoft Office 2010 Adobe Acrobat FX5K Tension FlexLink USB keyboard USB 2 button entry mouse 17 Flat panel monitor Accessory pack of nuts and ferrules Flexcell BioFlex baseplate Sey Pie SP be 8 Four gaskets 9 Four Arctangle Loading Stations 10 Four Trough Loaders 11 Silicone lubricant 12 Plexiglas cover 13 System Drying Filter device 14 25 ft small blue tubing 6 4 mm O D for FLEX IN connection 15 25 ft clear tubing 9 5 mm O D for FLEX OUT connection 16 25 ft large blue tubing 9 5 mm O D for vacuum source connection 17 Flexcell Tissue Train Culture System users manual and other ancillary manuals for computer and monitor 18 Surge protected outlet strip 19 Water Trap x2 20 Vacuum source optional 2 SETUP OF THE FLEXCELL TISSUE TRAIN SYSTEM Do not power up the Flexcell Tissue Train system until all connections among components have been properly made and checked Never power up the computer or the FX5K Tension FlexLink with the cover removed an electrical shock hazard exists Operation of the computer without the cover is in violation of FCC regulations and will void the warranty 2 1 PACKING LIST Remove the packing list and check off the instrument components as they are removed from the shipping containers If any parts are missing contac
17. NT This computing equipment has been type tested and found to comply with the limits for a Class B digital device in accordance with the specifications for Subpart J of Part 15 of FCC Rules These limits are designed to provide reasonable protection against harmful radio and TV interference in a residential installation There is no guarantee that interference will not occur in a particular installation The following instructions and precautions must be observed by the user when installing and operating this device 1 The equipment must be operated in strict accordance with the manufacturer s instructions 2 The unit s must be plugged into a properly grounded power outlet and used with the power cord supplied with the unit s unmodified The unit must be operated with the factory installed cover in place 4 No modifications of the equipment can be made which may result in the equipment failing to meet the limits of the FCC Rules 5 The equipment must be maintained in a satisfactory state of repair 6 Shielded cables must be used with this equipment to ensure compliance with the FCC Rules If the unit interferes with radio or television reception which can be tested by turning the unit off and on the user is encouraged to try the following procedure to correct the interference 1 Reorient the antenna of the receiver Relocate the equipment to increase the distance between the unit and the radio TV Connect the unit to an out
18. ON If Off is selected the FlexLink will turn the pump OFF For automatic control of power to the pump while regimens are running select Auto prior to downloading the regimen Once Auto is selected the FlexLink will turn the pump ON The software will turn the pump OFF when a regimen is complete This option also allows the pump to be turned OFF during a regimen step when a static waveform regimen is selected that has a 0 maximum elongation longer than 60 minutes The pump will power up 10 minutes before the next regimen step begins Users will be prompted 21 to select this option or not when creating a regimen The status of the pump is indicated by a light box to the right side of the On Off Auto options A green light indicates that the pump is ON and a red light indicates that the pump is OFF 3 16 DATA ANALYSIS The FX 5000 software has the capability of storing data a set of waveform points from a regimen run to a file on the hard drive The filename consists of the FlexLink number a V or C code letter V for vacuum or C for compression the regimen name and a sequence number If a tension regimen is run three times on FlexLink 1 then there will be three data files on the hard drive with numbers 000 001 and 002 i e 1_V_regimenname_001 dat 1 V regimenname 002 dat etc The file size depends on the regimen duration The current settings take pressure data every 2 milliseconds
19. Then click on Remove Step at the bottom of the window Confirm the deletion by clicking on Yes when prompted 12 After all steps and parameters have been entered click on Save Regimen 13 To delete a regimen simply double click on the desired regimen name in the Regimens for Selected User box and then click on Delete Regimen Confirm the deletion by clicking on Yes NOTE If the regimen to be deleted is currently assigned the regimen must be removed from assignment before deleting 14 When finished with regimen editing click on Close 3 6 CREATING A REGIMEN WITH A CUSTOM WAVEFORM SHAPE In the top row of an Excel spreadsheet enter the elongation values for the desired waveform shape for one cycle Enter one value per cell Values should be chosen along a curve at even time intervals i e every 100 msec milliseconds Save this spreadsheet as a csv file comma delimited file format in the C FlexSyst directory Elongation 0 100 200 300 400 500 600 700 800 900 1000 Time msec Example Custom waveform shape Ramp waveform shape Example Excel file Ramp waveform shape 1 1 2 2 3 3 4 4 1515 10 3 3 5 5 5 Example CSV file viewed in Notepad Ramp waveform shape 1 1 5 2 2 5 3 3 5 4 4 5 5 5 5 0 In the Regimen menu of the FX 5000 software select Create new A new window will appear entitled Regimens with a list of the existing users and the
20. To achieve the maximum system capability the minimum vacuum source requirements are e Maximum Vacuum 100 kPa e Free Airflow Rate 5 7 cfm 161 L min 34 Il FLEXCELL INTERNATIONAL CORPORATION Appendix A FX 5000 TENSION SYSTEM DUAL BASEPLATE OPERATION A FLEXCELL INTERNATIONAL CORPORATION When using the FX 5000 Tension System in dual baseplate operation the user may notice a drop in the overall programmed elongation depending upon the desired elongation and frequency of the regimen This drop is due to the vacuum source being shared between both baseplates It is therefore recommended that users reduce the frequency of such a regimen to 0 5 Hz or lower A lower frequency will allow the FX 5000 Tension System the correct amount of time to intake and exhaust the vacuum which in turn gives the system enough time to reach the desired elongation For example if an FX 5000 Tension System is connected in the dual baseplate configuration using the BioFlex 25 mm Loading Stations and a regimen is programmed with the following variables Waveform Sine Elongation 20 Frequency 1 Hz The maximum elongation achieved might only be 10 Reducing the frequency of the regimen to 0 5 Hz will produce an achievable elongation of 18 The maximum allowable elongation for dual baseplate operation with the FX 5000 Tension System is as follows Platform Frequency 1 0 Hz 0 5 Hz BioFlex 25 mm 10 0
21. XCELL AGREES to correct any defect in workmanship or material and supply new or rebuilt parts in exchange for defective parts upon completion and submission by purchaser customer of a printed Parts Return Authorization form furnished by Flexcell Parts must be properly packed in original container and shipped to our factory service center or distributor with all shipping costs prepaid if the unit is out of warranty coverage If the original shipping box is not available Flexcell will send the required protective shipping container Flexcell will recommend the insurance value for parts or equipment to be shipped Return carrier shipping costs will be paid by Flexcell from the service center The purchaser customer is solely responsible for payment of custom fees taxes holding fees or value added taxes d THIS LIMITED WARRANTY only covers failures due to defects in materials or workmanship which occur during normal use It does not cover damage which occurs in shipment or failures of original equipment due to products identified as add ons not manufactured by Flexcell International Corporation or its distributors nor does this limited warranty cover damages or failures which result from accident or disaster such as fire explosion flood wind lightning or earthquake or misuse abuse neglect mishandling misapplication alteration faulty installation modification or service by anyone other than our factory or distributor Breaking the seal and opening
22. a Tissue Train Culture Plate 5 3 Application of Regulated Strain to Cells in a 3D Gel Using the Tissue Train C lt re VS ORS OO cso 1 xiya an THERE IND el ren e ke IUD EUREN IEEE END NH ees 6 PART B Set Up of the Tissue Train System Tension Equipment 7 1 Flexcell Tissue Train System Components es 8 2 Setup of the Flexcell Tissue Train System 8 2A Packing JR c c 8 2 2 Location of the Tissue Train System ssssete ttt tente 8 2 3 Setup of the Computer and FX5K Tension FlexLink e 9 2 4 Incubator Access POTIS osea ito ENSE IN CR DENM aO ua PI RR QURE ANNUM MEUSE 10 2 5 Setup of the BioFlex Baseplate s with Loading Stations and Culture Plates 11 2 6 Connecting the System Vacuum LineS E ekere eke kek ee keke kek ke k k ata 11 2 7 FlexSoft FX 5000 Software and System Power up cescsscssssssssssessessesseesestsestesteseen 12 2 8 LCD Display on the FX5K Tension FlexLink c cccsccesseessessseesesseesseessesseesseesseessen 13 2 9 Using Additional FX5K Tension FlexLinks ee 13 3 Operation InStrUCtiOTS L feni ete eate a ee 14 3 1 Starting the FX 5000 Program ettet tette teen 14 3 2 Using the FX 5000 Tutorial eet tette tentent 14 3 5 Mail Display Win dO W gt oa ss trn diat 1590455544150450 da akan 1a kn ufo
23. ain well attached to the anchor stem The vacuum holes deform the rubber membrane downward to create a space for the gel The side view shows the membrane conforming to the trough and the loading of a gel matrix in the trough 11 Place the baseplate with culture plates in a 37 9C incubator and allow the solution to gel After the gel has set release the vacuum and add 3 mL of serum containing media to each well The gels should appear as a band of gel attached at each anchor end in the Tissue Train well Fig 3 Remove culture plates from the BioFlex baseplate if needed Figure 3 Tissue constructs in A a linear Tissue Train plate atop Trough Loaders and B a Trapezoidal Tissue Train culture plate 12 Culture constructs according to the laboratories established protocol Cultures may be observed or assayed for various functions The user can monitor cell shape organization migration division gene expression protein expression secretion mediator secretion DNA and protein synthesis in the gels to name a few Cultures may also be mechanically loaded see Part B at any time after gelation or after cells have self assembled their own matrix The elongation frequency and duration of the applied strain to the construct must be determined by the investigator for his her specific use Typically spherical shaped cells can be observed in the gel using an inverted phase contrast microscope directly afte
24. boratories established protocol Cultures may be observed or assayed for various functions The user can monitor cell shape organization migration division gene expression protein expression secretion mediator secretion DNA and protein synthesis in the gels to name a few Cultures may also be mechanically loaded see Part B at any time after gelation or after cells have self assembled their own matrix The elongation frequency and duration of the applied strain to the construct must be determined by the investigator for his her specific use Typically spherical shaped cells can be observed in the gel using an inverted phase contrast microscope directly after plating By day one the cells will begin to attach to and spread into the matrix Cells will then form attachments to each other and intercommunicate By days 3 5 cells will reorganize and contract the matrix 3 APPLICATION OF REGULATED STRAIN TO CELLS IN A 3D GEL USING THE TISSUE TRAIN CULTURE SYSTEM Cell matrix constructs can be mechanically loaded in the 3D matrix by using a Flexcell FX5K Tension FlexLink and the FX 5000 FlexSoft Software see Part B pg 7 or the Flexcell FX 5000 Tension System This tension system can be used to apply a regimen of controlled elongation strain frequency or duration with added rest periods The regimen parameters must be tested beforehand to determine if the cell constructs can withstand the applied strain regimen U
25. cuum is being applied to the membrane to stretch it downwards The scale on the x and y axes can be changed by clicking on one of the two small double arrow buttons at either end of the axis The button with arrows pointing away from each other increases the axis E FLEXCELL INTERNATIONAL CORPORATION scale while the button with arrows pointing toward each other decreases the scale Below the plot is a timeline that displays the percentage of the regimen that has been completed To the right of the plot is a set of command buttons which allow the user to 1 Start a regimen 2 Stop a regimen 3 Pause a regimen 4 Resume a paused regimen 5 Simulate a regimen 6 Reset the controller 7 Control the vacuum pump 8 Access Help 9 Cancel the program 3 4 CONFIGURING USERS Prior to creating and downloading a regimen a user needs to be registered Once this task is completed regimens can be personalized under the registered user name In the Configure menu select User Editor A new window will appear entitled Users Fig 14 To add a user type in a user name must be at least 4 characters in the provided space and click on Add User The new name should appear in the Existing Users box 16 Existing Users Flexcell Shutdown User Name Flexcell Delete User Figure 14 FX 5000 user editor To delete a user click on the appropriate user name in the Existing Users box so
26. eaning and disinfecting seal baseplate and gasket in a plastic bag Store baseplate flat and be sure that the entire bottom of baseplate is supported to prevent warping Store gasket away from UV light exposure RUNNING LOW FREQUENCY REGIMENS WITH BIOFLEX CULTURE PLATES If running a low frequency or constant stretch regimen with the BioFlex culture plate without Loading Stations keep in mind that the 2 2 5 mL of medium in the well may not cover the entire surface area of the BioFlex well under stretch When the membranes are stretched and held for long periods of time the cells around the outer edge of the well will be exposed to open air and will be in danger of drying out It is therefore necessary to use additional medium 3 5 mL when running low frequency and high strain regimens on the BioFlex plate without the Loading Stations Running Low Frequency Regimens With BioFlex Culture Plates Flexcell International Corporation July 1997 1p zz FLEXCELL INTERNATIONAL CORPORATION WAVEFORM SHAPE DEFINITIONS 14 Sine Wave Sine Wave This waveform provides a smooth This waveform provides a smooth physiological variation in pressure at the physiological variation in pressure from max with a quick transition at the min min to max Fe RN TT Er k Loading Staton 25mm r F aT Jiwa Line Ere Ronse uae m i jll SN 2 MSS ELI i i
27. eb ee div a aede nba dit 14 3 4 Config rin 9 Sef Ss EET E I ET 16 2 Creatine Re SERIE TIS or e nee Ree he ve yeu m de e aeons sage GA Ke ved cess mra ete Rd 16 3 6 Creating a Regimen with a Custom Waveform Shape sse 18 Su Assigning Regimens oos bani ok ena den neqne etude m sue reti eet vn qal ud 19 3 8 Starting Rees elisa au iic d DH qd wir HA Nab N En ki kek dte bod p ba qd diui 19 3 9 5 mula ng Regens 2 5 os eden ca haee n tb edad aote ea e RUE a nena ond n D 20 3 10 Pausing Regimens uoce dr na dre trece A en Delo HHHH HHHH 20 3 1 1 Re suming ResHmetls oet erit eb et e nt tae cert E EU Ite 20 asl 2 Stopping Regimens eisene en eben ds sei cerca beeen au iou doe eL eer 20 3 13 Determine the Progress ot the Regimen iuo ucc en a cte ut et eas 20 3 14 Resetting the FlexLink uS ERN e e NON levees RR ed 21 3 15 Controlling the Vacuum Pump u ekere kek keke kek ee No ee k ea tees 21 Sd D ta Anab SIS ou aba re Solace edendi NAE bia NL RE EUN Du este M UD N 21 3 17 Shutting Down the System etc decere ce ertet dt vod etie de kek eq Lotta due 22 3 18 Exiting the FX 5000 Program tette tette teet 23 4 Addendums to the Tissue Train SAN DD nnn 24 For Optimal Performance of the Tension Un t kk k k k k 24 5 Systemi TrouDleslhOOtill co oso oos cenda n w d l W k s k erk Wad b e ed 25 6 APD CAtION NO eS s dear eb ted Q n Nro eie i r y
28. ened To turn OFF this feature uncheck the box marked Show Tutorial at Startup 3 3 MAIN DISPLAY WINDOW The main display window Fig 13 for each FXSK Tension FlexLink has several features that allow more precise monitoring of the regimen To access this window select the appropriate FlexLink FlexLink n from the Controllers menu where n is the FlexLink number to which the regimen will be downloaded The FlexLink number can be found on the LCD screen on the front of the FlexLink controller box FX 5000 Controllers Configure Regimen Data Analysis Window Help Welcome to FX 5000 Tutorial Configure Users Create Regimen Start Regimen Figure 13 Main display window All of the remaining information and command button states in the main display window reflect the current state of the system for the currently selected FXSK Tension FlexLink It is possible to simultaneously assign and run a different regimen for each of the attached FlexLinks In the upper left hand corner of the window is a section labeled Plot Units This function allows users to change the units of the y axis elongation kPa strain 14 E FLEXCELL INTERNATIONAL CORPORATION millistrain mstrain or microstrain ustrain To select a particular unit click on the appropriate button beside the unit label The next block to the right is a section labeled Smoothing One expects data
29. eplate should be dried and stored on a flat surface Do not hang the red gasket on pegs to dry as the gasket can be stretched Protect both pieces from UV light exposure To disinfect use a mild bleach commercial bleach solution such as Clorox or other laundry bleach solution is normally 5 25 sodium hypochlorite solution 1 500 bleach to water or a 1 benzalkonium chloride solution which can be sprayed poured or wiped on both pieces and allowed to stand on surfaces for at least 5 min Rinse with distilled or deionized water The above solutions are effective against yeast and fungi A 70 ethanol solution also can be applied and pieces allowed to air dry do not wipe dry Tubing on the baseplate may be flushed with 70 ethanol but be sure tubing is completely dry before connecting to the FX5K Tension FlexLink Contact time time surface remains wet of the above solutions is critical to the effectiveness of disinfecting Times given are minimal and longer contact times are recommended in cases where contamination is severe The above decontamination procedure should be performed before and after each use of the 28 baseplate and gasket or whenever media is spilled onto these surfaces Be sure that the openings and the channels in the baseplate are dry and clear of any obstructions before using Do not store baseplate or gasket in incubator as the warm humid conditions will promote growth of microorganisms After cl
30. f media containing 10 fetal calf serum 70 matrix protein gel fluid and 20 5X MEM The objective is to achieve an overall gel MEM concentration of 1X The suggested formula for the cell matrix protein gel combination is as follows e 70 by volume matrix protein gel e 20 by volume of 5X MEM to yield an overall 1X concentration by total volume e 10 fetal calf serum e Cells For circular shaped constructs a Trough Loader and vacuum source are not required to create the 3D gels The foam annulus in the Tissue Train Circular Foam culture plate serves as both the anchor and forms a well into which the cells and gel are cast Pipette the cell and matrix protein gel solution into the space in the center of the foam anchor and around the inner perimeter of the annulus anchor approximately 1 5 mm wide Use the pipette tip to compress the edge of the foam so that the cell and matrix protein gel solution is drawn into it This step ensures that the matrix and cells will integrate with the structure of the foam and form a secure bond to support the artificial tissue disk during mechanical loading Place the baseplate with culture plates in a 37 C incubator and allow the solution to gel After the gel has set release the vacuum and add 3 mL of serum E FLEXCELL INTERNATIONAL CORPORATION containing media to each well Remove culture plates from the BioFlex baseplate if needed 10 Culture constructs according to the la
31. fore data can be viewed or before a regimen can be downloaded again It is recommended that the monitor be put in sleep mode when not in use and that the computer be allowed to run at full power during an experiment 2 8 LCD DISPLAY ON THE FX5K TENSION FLEXLINK The front of the FX5K Tension FlexLink box has an LCD graphic display When the FXSK Tension FlexLink is turned ON the LCD will display the FlexLink number i e 1 2 3 or 4 and the regimen status i e Idle Ready or Left the time remaining in minutes of a running regimen When a regimen has been downloaded and or is running the LCD will also display the user name platform and regimen name See Table 1 below for ig FLEXCELL INTERNATIONAL CORPORATION the display messages with various FlexLink statuses Table 1 Messages displayed on the FX5K Tension FlexLink LCD FlexLink Status LCD message Power ON 1 Regimen Idle Flexcell International Corp www flexcellint com Regimen downloaded 1 Regimen Ready User name Platform Regimen Name Flexcell International Corp www flexcellint com Regimen is 5 min running or being simulated 1 Regimen Left User name Platform Regimen Name Flexcell International Corp www flexcellint com Regimen has stopped 1 Regimen Idle User name Platform Regimen Name Flexcell International Corp www flexcellint com 2 0 USING ADDITIONAL FLEXLINKS
32. ft large blue tubing 9 5 mm O D for vacuum source connection Flexcell FX 5000 Tension System User s manual Culture Plate amp Loading Stations manual Vacuum Source Connection Manual Water Traps and Drying Filter Tech Report and other ancillary manuals for computer and monitor Surge protected power outlet strip TISSUE TRAIN ACCESSORY KIT OPTIONAL Note If purchased as a system the Tissue Train Accessory Kit is substituted for the Flexcell vacuum pressure baseplate gaskets and acrylic window for BioFlex culture plates and BioFlex Loading Stations Flexcell vacuum pressure baseplate gaskets and acrylic window for Tissue Train culture plates Arctangle Loading Stations and Lubricant Trough Loader Loading Stations FX 5000 TENSION COMPUTER SPECIFICATIONS Dell OptiPlex Small Form Factor Intel Core i5 processor 3 2 GHz 250 GB SATA 6 0 Gb s hard drive DVD RW Drive 32 E FLEXCELL INTERNATIONAL CORPORATION e 2 GB of DDR3 RAM e Integrated video Intel HD2500 Operating System e Windows 7 Professional FX5K TENSION FLEXLINK COMPONENT SPECIFICATIONS e Custom design vacuum controller board o Microprocessor sampling rate for display data transducer 200 Hz o Microprocessor sampling rate for feedback error valve adjustment transducer 1 KHz e 2 proportional solenoid valves e Ethernet port RJ45 FX5K TENSION FLEXLINK CALIBRATION SPECIFICATIONS Unconstrained Static Performance lo
33. fter a one day run or if the regimen runs for a longer period of time the Drying Filter can be used at the conclusion of the usage period The device and shutdown program will help to remove any moisture entrained into the valves during normal operation This will greatly prolong the life of the valves and reduce expensive repairs To use the Drying Filter first remove the orange plug in the bottom of the filter Attach the fittings of the Drying Filter to the proper ports on the rear of the FlexLink by replacing the lines running to the baseplate FLEX IN and FLEX OUT Assign the Drying regimen under the Shutdown user using the BioFlex Plate no Loading Stations platform E FLEXCELL INTERNATIONAL CORPORATION If the Drying regimen is not in the system or is erased create a regimen with the following specifications sine wave 0 28 0 5 Hz 1000 cycles all other parameters are standard The shutdown program will run for about 30 minutes Dry room air will help to reduce moisture entrained into the lines and valves from the incubator atmosphere Once the beads in the Drying Filter become pink the filter is saturated and needs to be replaced The baseplate should be removed from the incubator and stored flat to prevent warping Do not hang the red gaskets on pegs to dry They will stretch over time Figure 18 System Drying Filter 3 18 EXITING THE FX 5000 PROGRAM To exit the FX 5000 software in
34. g the gasket out of the well they should seal correctly 5 Place the Plexiglas sheet with weights on top 5 10 lbs over the culture plates to help seal the baseplate NOTE After a regimen is run for an hour or so there should be little or no moisture visible in the lines or Water Traps Condensation early on in the experiment indicates an air leak If condensation is present recheck the seals around each of the culture plates 11 2 6 CONNECTING THE SYSTEM VACUUM LINES Connect the blue 9 5 mm vacuum inlet tubing to a vacuum source A vacuum pump capable of producing a maximum vacuum of 100 kPa and a flow rate of at least 5 7 ft min 161 L min is necessary for optimal performance Keep the tubing length between the vacuum source and FlexLink as short as possible Also keep in mind that some house vacuum systems will vary in flow rate and or pressure level To eliminate or reduce this effect use of a pressure reservoir between the vacuum source and the FX5K Tension FlexLink is recommended Flexcell International Corp recommends the Leybold Trivac D8B Pump vacuum pump includes the D8B pump system power cable gas ballast valve and AR 4 8 return filter as a vacuum source as well as using a pressure reservoir The vacuum pump and pressure reservoir can be purchased directly from Flexcell International Corp NOTE Even with an optimal vacuum source there will still be some system limitatio
35. gaskets and plates from baseplate Check to see that each gasket is firmly seated in each plate without any folds in the thin bottom lip of the gasket and with each inside corner butted up against each outside comer of the Tissue Train plate Place each plate and gasket carefully into each baseplate well Check to be sure that the gaskets are flatly seated in the wells i e none of the sides touching the baseplate to the point that the gasket is not able to fully enter the well Move each gasket forward and back and side to side within the well to be sure that they are somewhat loose in the plane of the baseplate This will allow the vacuum pressure to pull the gaskets and plates firmly down to seal flat against the baseplate surface Run a regimen The plates and gaskets may require some initial hand pressure to start the seal Place the Plexiglas cover and a couple of lead weights approx 10 Ibs total variable on top of the plates If there is still a noticeable leak check all sides of each Tissue Train plate to see if the membrane is protruding from any side If there is any noticeable membrane protrusion remove this with a razor blade Problem 2 Regimen wave is flatlining Solution 2 Remove the SYSTEM vacuum line at the back of the FlexLink and check to be sure that there is vacuum coming through the line Check the VENT port at the back of the FlexLink when the system is running to see if air is moving in or out If
36. gned regimen click on Start in the main display window Fig 13 after the regimen has finished downloading Be sure that the vacuum pump is ON and all E FLEXCELL INTERNATIONAL CORPORATION other connections are made The membranes should begin flexing and the assigned regimen will be in progress The progress of the regimen for the currently selected FlexLink will be monitored continuously The displays for Run Time Step Cycle Jump and Min and Max will update as will the actual waveform data returned by the FlexLink Additionally a progress bar below the graph will display the percentage of the regimen that has been completed 3 9 SIMULATING REGIMENS The Simulate button is used to simulate a regimen graphically without actually running it Simulating a regimen will give the user an idea of the waveform output before actually running the regimen To start a simulation click on Simulate in the main display window A waveform will begin moving across the graphic display This waveform corresponds to the ideal waveform as generated by the FX 5000 software and is the actual waveform pattern sent to the FlexLink during the download process For certain waveforms e g square waves the idealized pattern may appear to have some unusual elements e g rounded corners The purpose of these unusual elements is to accommodate for the lag time during sudden changes in pressure e g rising falling edge of a square
37. il 2 GOVERNING LAW The performance of the duties and liabilities of the parties under the terms and conditions of this Limited Warranty shall be governed in all respects by the laws of the State of North Carolina the United States of America APPLICATION OF STATE LAWS Some states do not allow the exclusion or limitation of consequential damages nor do some states allow limitations on how long an implied warranty lasts so the above limitations may not apply to you This warranty gives you specific legal rights and you may also have other rights that vary from state to state 3 INTERNATIONAL CUSTOMERS The full text of the foregoing limited warranty and all disclaimers is applicable to international customers purchasers except when the purchase was made from an international distributor or reseller the warranty will be covered through your distributor or reseller If technical advisory support service is not available through your distributor or reseller for service contact warranty headquarters by phone or fax Within the United States only 1 800 728 3714 toll free Fax 1 919 732 5196 International Customer s telephone 01 919 732 1591 Fax 01 919 732 5196 Email info flexcellint com FLEXCELL INTERNATIONAL CORPORATION 2730 Tucker Street Suite 200 Burlington NC 27215 1 800 728 3714 within the U S only 1 919 732 1591 fax 1 919 732 5196 http www flexcellint com Customers should contact Dell Inc for all warranty
38. ir regimens In the Existing Users box click on the user name under which the regimen is to be created A list of current regimens created under this user will appear in the Regimens for Selected User box unless no regimens have been saved under this user in which case this box will be empty To create a new regimen click on Run Wizard in the bottom left hand corner This option will bring up a wizard to create a new regimen Or follow the sequence of steps below 1 Choose Custom from the list of waveform shapes under Shape by clicking on the downward arrow and selecting this entry from the drop down list E FLEXCELL INTERNATIONAL CORPORATION 2 Choose the appropriate file name under File by clicking on the downward arrow and selecting an entry from the drop down list 3 Type a regimen name in the box labeled Regimen Name 4 Enter the time interval in milliseconds between two elongation values in the box under Time 5 Enter the number of times for the regimen to repeat 0 65 535 in the box under Cycles 6 When the regimen step is complete click on Add Step at the bottom Every time a new edited step is saved the overall duration of the regimen will be calculated and displayed in the box labeled Duration next to the Step number box If the total duration exceeds the maximum allowable duration this value will be MAXIMUM and the regimen cannot be saved Adjust the time and or cycle number to decrease
39. l not create any breaks in the regimen Simply divide the total time in half or as many times as is necessary and make additional steps with the same parameters See section 3 5 of the manual for 26 instructions on programming additional steps Problem 5 Plate wells are drying up during the experiment Solution 5 When vacuum is pulled to expand the silicone membranes at the bottom of the Tissue Train wells the volume of the well increases pulling air into the well from the atmosphere When the membranes contract the air is pushed back out Because of this cyclic airflow during any experiment the fluid evaporation rate in the Tissue Train wells will increase The best solution to this problem is to use more culture medium Problem 6 An invalid argument was encountered error is shown when attempting to assign a regimen Solution 6 This problem is caused when a user name is entered into the software without any regimens listed under his her name If the assign function sees that a user has nothing to assign an error is displayed Simply delete the user s without a regimen or make and save a regimen under the user s name s Problem 7 Water is accumulating quickly in the Water Traps Solution 7 This problem is likely due to a poor gasket seal on the BioFlex baseplate See the solution to Problem 1 to correct this Be sure to empty the Water Traps frequently if this problem occurs Water accum
40. let on a different circuit from the receiver Ie mes lt Consult with the dealer or an experienced radio TV technician for advice on ways to reduce interference E FLEXCELL INTERNATIONAL CORPORATION TISSUE TRAIN QUICK START INSTRUCTIONS 1 Choose a location for the Tissue Train system that is near the incubator 2 Place the computer monitor keyboard mouse and FX5K Tension FlexLink on a sturdy flat surface 3 Connect the keyboard mouse and monitor to the computer according to the manufacturer s instructions 4 Connect the FlexLink to the Ethernet port on the back of the computer using the Ethernet cable supplied see Figs 6 and 7 pg 9 5 Connect the computer monitor and FlexLink to a power source using a surge protector 6 Connect the system vacuum to the SYSTEM port on the back of the FlexLink and check the system vacuum supply see manual section 2 6 for pump specifications recommendations 7 Cut two lengths of tubing from those supplied cut a 10 3 m length of the 4 6 4 mm and cut a 10 length of the 9 5 mm tubing Connect each length of tubing to the rear connection of the FlexLink in the FLEX IN and FLEX OUT ports accordingly Attach provided quick disconnects to the other end of each piece of tubing 8 Place the Water Traps see Fig 5 pg 9 inline with the FLEX IN and FLEX OUT tubing a Make cuts in the tubing for the Water Traps to be positioned as near as pos
41. more than 5 1 5 m longer or shorter Cut the tubing after the system is fully set up to ensure of the proper length The Tissue Train system will require a space 28 75 73 03 cm wide by 22 44 57 00 cm deep if the computer FX5K Tension FlexLink and monitor are placed side by side with the keyboard and mouse in front The total weight of the system is 48 37 lbs 21 96 kg Power Surge Protector SOS Vacuum Pump Pressure Reservoir Vacuum Line To k SYSTEM Port Blue Tubing Computer FLEX OUT Clear Tubing FLEX IN Blue Tubing g Water to Trap Incubator Access Port CO Baseplate Flexcell Intamational Corp 437 Dimmocks Ml Road Hisborough NC 27278 Inus oak 1 800 728 3714 12197321891 www flexcellint com PUMP CONTROL Figure 6 FX5K Tension FlexLink rear view Power Connector Voltage Selector Switch l Ethernet Port 6 USB Ports 4 Available 1x Keyboard 1 x Mouse Monitor Connection Ethemet Port Figure 7 Dell OptiPlex rear view 2 3 SETUP OF THE COMPUTER AND FX5K TENSION FLEXLINK Setup the Dell OptiPlex desktop computer and peripherals according to the manufacturer s instructions Refer to Figures 5 9 for proper setup of the Tissue Train system instruments Place the FXSK Tension FlexLink next to the computer and connect the FlexLink to the co
42. mputer via the Ethernet cable provided If additional FlexLinks have been purchased they are to be placed side by side For multiple FlexLinks to be controlled by one computer connect the Ethernet cable from each FlexLink to an Ethernet Hub Then connect the hub to the Ethernet port on the back of the computer Connect the system tubing as follows E FLEXCELL INTERNATIONAL CORPORATION 1 Connect the 9 5 mm O D blue tubing from the vacuum source to the SYSTEM port on the back of the FlexLink Fig 5 amp 6 2 Attach the free end of the clear 34 9 5 mm tubing to the FLEX OUT port on the back of the FlexLink Fig 6 3 Attach the free end of the blue 6 4 mm tubing to the FLEX IN port on the back of the FlexLink Fig 6 The VENT port should be left open Fig 6 4 Connect a Water Traps inline with the clear tubing and the blue 4 tubing See the Addendum pg 24 B for further details concerning the placement of the Water Traps NOTE When connecting the tubing to the back of the FlexLink slip the brass nut with the nylon ferrule over the tubing then push the tubing onto the fitting as far as possible and thread the nut onto the fitting Tighten each nut by hand until the tubing is crimped to minimize vacuum leakage Connect the power cables 115 VAC or 230 VAC to the rear power inlet FlexLink Power of the FlexLink Fig 6 If the available power supply is 115 VAC or 230 VAC
43. n process and minimizes potential sources of contamination Tissue Train Culture System with Arctangle Loading Post and Trough Loader Figure 1 Tissue Train culture plate for 3D culture of cell matrix constructs The top left well has a Trough Loader beneath the flexible membrane The four adjacent wells show the anchors for attachment of cells and gel for a uniaxial construct The bottom left well shows an Arctangle loading post to deliver uniaxial tension 10 Pipette the cell and matrix protein gel solution into the trough in each Tissue Train well Fig 2 First pipette a small drop of gel at each end of the trough under the anchor stems Then press the anchor stems into the trough and pipette over top of them Finally fill the middle of the trough with gel moving the pipette back and forth to create a uniform strip of gel in the well see video of Tissue Train FLEXCELL INTERNATIONAL CORPORATION Bioartificial Tissue Fabrication with Uniaxial Strain on Flexcell s web site www flexcellint com TISSUE TRAIN CULTURE SYSTEM Bioartifical Linear Tissue Development Rubber Membrane Side view Anchor Stem UL Potted Anchor Rubber Membrane Trough Bay id Trough Loader n Vacuum Hole e Add cells in a gel matrix to trough bay e Rubber membrane deflected into trough Vacuum e diee Cells anc C02 incubator Figure 2 The top view shows a cell gel construct in a Tissue Tr
44. ns due to airflow Combinations of high frequencies and 96 elongations may not be achievable due to the high vacuum level in the short period of time requested An example would be a regimen programmed to run a sine wave from 0 20 elongation at 1 0 Hz using the Arctangle Loading Stations The system is not able to achieve the vacuum required to reach 20 in 0 5 seconds because of airflow limitations The maximum elongation may only reach 15 The exact output is dependent on many system variables See the Addendum on page 24 for suggestions on maximizing system performance To use the auto control pump feature plug the vacuum pump power cable into the E FLEXCELL INTERNATIONAL CORPORATION PUMP CONTROL CONNECTION outlet on the rear of the FX5K Tension FlexLink Fig 6 and 10 NOTE When using the auto pump control feature the power cable from the FX5K Tension FlexLink must be connected directly into the mains power Failure to do so will cause damage to the FX5K Tension FlexLink Power Surge Protector Source Vacuum Pump Pressure Reservoir Vacuum Line To SYSTEM Port 34 Blue Tubing Computer FLEX OUT Clear Tubing FLEX IN 4 Blue Tubing Incubator Access Port CO Incubator Baseplate Figure 10 Flexcell Tissue Train System power set up with auto control pump feature See page 21 for additional comments on setting up the vacuum po
45. nt to the top surface of the Trough Loader prior to placement of the Tissue Train culture plate on top The lubricant will facilitate uniform and unrestricted conformation of the membrane in the trough Place the Tissue Train culture plate on a BioFlex baseplate with gaskets and connect to the Flexcell FX5K Tension FlexLink or other regulated vacuum source Vacuum should be applied to the baseplate in a steady hold mode so that the flexible membrane is deformed and held in the space in the Trough Loader To supply the proper vacuum level with the FX 5000 system it is recommended that a maximum of 20 elongation be used with the Tissue Train Loading Station 24 mm platform setting This is the equivalent of 90 kPa Be sure that you allow enough vacuum tubing for your baseplate to reach from your incubator to your tissue culture hood NOTE When using the Flexcell Tissue Train System use the minimal amount of tubing required as longer tubing decreases cyclic strain performance It may be more practical to use two sets of tubing to supply vacuum to the Tissue Train baseplate One set should be used for 3D gel formation and should be long enough to span the distance from the vacuum source and both the cell culture hood and the incubator The second vacuum line set should be dedicated to the cyclic strain process and should be routed through the incubator portal Such an arrangement optimizes the cyclic strai
46. or the currently selected FlexLink will be monitored continuously The displays for Run Time Step number Cycle number Jump to number and Min and Max elongations will be updated in the upper right hand corner of the main display window The actual waveform data returned by the controller will also be continuously updated on the graph A timeline progress bar below the plot E FLEXCELL INTERNATIONAL CORPORATION illustrates the percent of the regimen that has been completed 3 14 RESETTING THE FLEXLINK Resetting a simulated or running regimen causes the current activity to stop at the next cycle of the current regimen step and disassociate the regimen from the FlexLink controller To reset click on Reset in the main display window Click on Yes when the program asks for confirmation The system will now be in state similar to that which exists when the FX5K Tension FlexLink is first turned ON 3 15 CONTROLLING THE VACUUM PUMP On the right side of the FlexLink window the box labeled Pump allows the user to control power to the vacuum pump NOTE The vacuum pump must be plugged into the back of the FX5K Tension FlexLink in the appropriate 110 220V plug prior to system power up for this option to work and the ON OFF switch on the vacuum pump must be in the ON position For manual control of the pump through the FlexLink use the On and Off options If On is selected the FlexLink will turn the pump
47. patterns to represent normal distributions of a function describing a straight line sinusoidal curve etc Therefore the plotted points of a mathematical function should be a predictable data set that is smooth i e a curve comprised of points whose coordinates are predicted by the function For a straight line the equation would be y mx b for a power function the equation may be y x or some derivative thereof In general data patterns may be confounded with noise For the case of the pressure plot as a function of voltage from the Tissue Train System pressure transducer the rough data yield a plot with a coefficient of variation of only 0 01 Therefore the signal to noise ratio for the combined Flexcell system of circuits hardware and transducers is high lending confidence in the displayed plot Nevertheless the user can select a data smoothing option that is a simple weighted average of the points The Smoothing section contains the option of either applying no smoothing None to the data points returned from the FlexLink or of applying a three point moving average filter Moving Average to the incoming data Choose the desired affect by clicking on the appropriate button Below the box for Smoothing is a section used to show the curent X and Y coordinates for the waveform These are displayed when the left mouse button is clicked and held down in the graph area When this is done a pair of crosshair
48. pend on what order it appears in the connection chain but according to the configuration of each FlexLink as FlexLink 1 FlexLink 2 etc during production When powering up the FlexLink s and computer turn the FlexLink s ON before the computer To use a particular FlexLink select the appropriate FlexLink number 1 2 3 or 4 from the Controllers menu Assign download and run regimens just as would be done when using one FlexLink E FLEXCELL INTERNATIONAL CORPORATION 3 OPERATION INSTRUCTIONS 3 1 STARTING FX 5000 PROGRAM THE The Flexcell system should now be set up and ready for operation Be sure that the system vacuum supply is adequate to operate the instrument s see section 2 6 Turn ON the power switch on the computer monitor and FX5K Tension FlexLink The system should then boot and load Windows Select and double click on the FX 5000 icon Fig 11 on the desktop to start the FlexSoft FX 5000 software A main display window will appear see Fig 13 Ez B DU Figure 11 FX 5000 icon 3 2 USING THE FX 5000 TUTORIAL In the Help menu click on View Tutorial to view the FX 5000 tutorial at any time Fig 12 The tutorial will appear on the left side of the screen The tutorial explains the main steps involved in configuring users creating regimens and starting regimens The tutorial will start automatically as a default setting when the software is op
49. questions or concerns with regards to the Dell OptiPlex computer and monitor iii E FLEXCELL INTERNATIONAL CORPORATION Dell COMPUTER WARRANTY INFORMATION Dear Customer Congratulations on your purchase of the Flexcell Tissue Train system This system comes with a one 1 year warranty The Dell computer that is included with this equipment will be transferred into the Principal Investigator and University s name at the time of purchase This computer comes with an excellent warranty through Dell If you have any problems with this computer you will contact Dell directly The details of the warranty are listed below You will need to have the service tag number and Express Service Code number which are located on the side of the computer e Dell Hardware Limited Warranty Plus Onsite Service Extended Year e Dell Hardware Limited Warranty Plus Onsite Service Initial Year e Thank you choosing Dell ProSupport For tech support visit http support dell com ProSupport or call 1 866 516 3115 e ProSupport for IT Next Business Day Parts and Labor Onsite Response 2 Year Extended e ProSupport for IT Next Business Day Parts and Labor Onsite Response Initial Year e ProSupport for IT 7x24 Technical Support for certified IT Staff 2 Year Extended e ProSupport for IT 7x24 Technical Support for certified IT Staff Initial year E FLEXCELL INTERNATIONAL CORPORATION FEDERAL COMMUNICATIONS COMMISSION RADIO FREQUENCY INTERFERENCE STATEME
50. r work Problem 1 Gaskets on baseplate are not sealing Symptoms a System is not performing to specified parameters i e not reaching maximum elongation value entered in regimen b Water Traps are filling with water quickly during use with incubator Test to determine if this is the problem If experiencing symptom a there is likely a leak or crimped tubing somewhere in the system First check all tubing to be sure that it is straight and not bent to restrict air flow To find the location of any vacuum leaks do the following First check all vacuum connections at the back of the FlexLink If these are tight and without noticeable leaks then run a regimen with the baseplate and take note of the max and min elongations Disconnect the quick disconnects from the baseplate and connect them together Take note again of the max and min elongations These values should change slightly because of the decrease in system volume however not by a significant amount 296 elongation or greater The wave may also be somewhat choppy because of the low system volume If the outputs were originally only slightly lower or higher than the inputs the problem is likely not a leak but calibration Call Flexcell for further instructions If the output max elongation increases significantly when the quick 25 disconnects are connected together the leak is most likely in the baseplate or gaskets Solution 1 Remove
51. r and the shutdown program whenever the unit has 24 been operating continually for more than one day and will not be operated again for more than one day See section 3 17 for instructions Place the enclosed Plexiglas sheet over the culture plates and distribute a weight of about 5 10 Ibs 2 25 4 5 kg evenly across the sheet as necessary to prevent vacuum leaks between the Tissue Train plate gasket and baseplate Lead donuts are recommended As the gaskets are worn in this will no longer be necessary Make sure that the brass fittings on the back of the unit are tight and that the tubing is secure Since the FX5K Tension FlexLink is calibrated with 4 Tissue Train plates on the baseplate Flexcell recommends having 4 Tissue Train plates on the baseplate during strain even if only one plate has been seeded with cells Achieving the highest possible combinations of elongation and frequency a Shorten the FLEX IN FLEX OUT and SYSTEM tubing to their minimum possible lengths See section 2 2 for minimum lengths b Use a more powerful vacuum source if recommended specs are not being reached with the current vacuum source see section 2 6 E FLEXCELL INTERNATIONAL CORPORATION 5 SYSTEM TROUBLESHOOTING NOTE If the Tissue Train system is not functioning properly and the solution is not clearly outlined or defined below please contact Flexcell before proceeding with any repai
52. r plating By day one the cells will begin to attach E FLEXCELL INTERNATIONAL CORPORATION to and spread into the matrix Cells will 4 then form attachments to each other and intercommunicate By days 3 5 cells will 5 reorganize and contract the matrix Fig 4 Bioartificial Tendon BAT Development Day 0 and Day 5 Figure 4 Contraction of a bioartificial tissue construct over time 2 2 PREPARATION OF CELLS IN 3D CIRCULAR GELS IN A TISSUE TRAIN CULTURE PLATE s 1 Prepare cells according to the laboratory s established protocol for primary cultures or continuous cell lines in the medium of choice 2 Release cells from their substrates with 0 05 trypsin trypsin EDTA 0 05 g bacterial collagenase or other means Add serum containing media to the cells to neutralize the trypsin or collagenase 3 Count cells and determine the number of cells needed e Circular gel approximately 1 2 million cells in 1 mL for each well of a 6 well Tissue Train Circular Foam culture plate NOTE Cell seeding density will vary depending on cell type We recommend testing cell seeding 9 densities to determine the best cell number for your application and cell type Wash cells 2 times with medium to remove trypsin or collagenase Before adding cells the matrix protein gel solution should be neutralized to pH 7 0 using 1 M sodium hydroxide Cells may be reconstituted in one volume o
53. rogram will calculate the number of cycles 8 To make a regimen step except for the first step return Jump back to a previous regimen step enter the return step number in the box under Back To Then type the number of times to repeat this process in the box under Repeat For instance if the user was entering Step 4 and wanted to return to Step 2 and repeat Steps 2 through 4 three times enter 2 under Back To and 3 under Repeat when inputting Step 4 9 When the regimen step is complete click on Add Step at the bottom Every time a new edited step is saved the overall duration of the regimen will be calculated and displayed in the box labeled Duration next to the Step number box If the total duration exceeds the maximum allowable duration this value wil be MAXIMUM and the regimen cannot be saved Adjust the time and or cycle number to decrease the total duration time 10 To add other steps to a regimen click in the box labeled Step and type in the next step number For example the first step will automatically be Step 1 To add another step delete the 1 and replace it with a 2 E FLEXCELL INTERNATIONAL CORPORATION After this is completed change the parameters as needed for the new step according to steps 2 to 9 above 11 To delete a step that has already been added click on the step in the window labeled Regimen Steps which contains the list of steps This will cause the step to be highlighted
54. s appears and the coordinates at the intersection of the crosshairs are shown in the X and Y spaces The crosshairs and values will track the mouse as long as the left mouse button is held down The next block to the right is a section used to display the current User Regimen Platform and Run time Run time has two blocks The one on the left displays the elapsed regimen time and the one on the right displays the remaining regimen time using the following format dd hh mm ss days hours minutes seconds The block at the top right contains the current regimen Step Cycle Jump when a regimen step is using the Jump Back to option minimum level Min and maximum level Max The minimum and maximum levels will be listed according to the units specified under the Plot Units section as the minimum and maximum points at which the membrane stretches with each cycle The major portion of the main display window is taken up with a plot designed to show the current state of a simulated or running regimen This plot is generated from the data returned by the FlexLink during regimen activity When using a platform configuration that uses vacuum to generate a downward stretching of the membrane elongation the vertical scale represents increasing elongation vacuum or strain When the selected unit of display is kPa the vertical scale will start at 0 0 and move upwards as an increasing negative pressure level indicating that a va
55. s a stand alone device to create three dimensional geometries ie linear trapezoidal or circular for cell culture in a matrix gel or allow the cells to build a self assembled matrix that connects to the anchors in the Tissue Train culture plate The investigator can also apply regulated strain to the growing tissues A user can define a frequency elongation and duration of strain in a regimen that simulates the strain environment of a tissue in the body The strain regimen is controlled by the Flexcell Tension System see Part B pg 7 that can apply static or cyclic equibiaxial or uniaxial strain The following is a method by which to culture cells in a 3D matrix 2 METHOD TO CREATE 3D CELL SEEDED HYDROGEL 24 PREPARATION OF CELLS IN 3D LINEAR OR TRAPEZOIDAL GELS IN A TISSUE TRAIN CULTURE PLATE 1 Prepare cells according to the laboratory s established protocol for primary cultures or continuous cell lines in the medium of choice 2 Release cells from their substrates with 0 0596 trypsin trypsin EDTA 0 05 bacterial collagenase or other means Add serum containing media to the cells to neutralize the trypsin or collagenase 3 Count cells and determine the number of cells needed e Linear gel approximately 50 000 200 000 cells in 150 200 uL for each well of a 6 well Tissue Train culture plate e Trapezoidal gel approximately 700 000 cells in 700 ul for each well of a 6 well Trapezoidal Tissue Train c
56. sible to the baseplate outside of the incubator so that the water will be trapped away from the FlexLink b Place the Water Traps at a position below the FlexLink unit 9 Place the baseplate s in the incubator a Insert the 4 6 4 mm and 9 5 mm tubing into the access opening of the incubator b Connect the quick disconnects for the 6 4 mm and 9 5 mm tubing to the baseplate s c Place four Trough Loaders into the four baseplate wells optional d Place four gaskets on four Tissue Train plates and place a Tissue Train plate and gasket in each baseplate well 10 Turn the FlexLink and computer system ON The Windows desktop should appear on the monitor Double click on the FX 5000 icon on the left hand side of the screen to start the software vi 11 12 13 14 15 16 17 18 E FLEXCELL INTERNATIONAL CORPORATION In the Configure menu select User Editor and register as a user In the Regimen menu select Create new and create a regimen by entering values into the appropriate spaces or click on the Run Wizard button in the bottom left hand corner for a step by step procedure When completed click on Save Regimen Exit the regimen editor In Controllers menu select the appropriate FlexLink number to which the regimen will be downloaded In the Regimen menu select Assign Choose the proper platform user and regimen see section 3 7 for assignment instruc
57. sually the cell matrix constructs can be stretched at 1 3 elongation for several minutes to several hours per day without matrix failure In the case of cardiac myocytes the myocyte matrix construct can maintain its beat frequency for a week or more in the matrix Uniaxial strain can be applied to linear and trapezoidal shaped constructs For application of uniaxial strain use Arctangle Loading Stations with the Tissue Train plates Equibiaxial strain can be applied to circular shaped constructs For application of equibiaxial strain use 25 mm cylindrical Loading Stations with the Tissue Train plates Regimens can be programmed with strains ranging from 0 20 and frequencies from 0 01 5 0 Hz When downloading regimens choose the Tissue Train Loading Station 24mm platform setting for applying uniaxial strain to linear and trapezoidal shaped constructs and choose the BioFlex Loading Stations 25mm platform setting for applying equibiaxial strain to circular shaped constructs NOTE Higher frequencies will limit the system s ability to maintain higher strain ranges Il FLEXCELL INTERNATIONAL CORPORATION PART B SET UP OF THE TISSUE TRAINS SYSTEM TENSION EQUIPMENT E FLEXCELL INTERNATIONAL CORPORATION 1 FLEXCELL TISSUE TRAIN SYSTEM COMPONENTS 1 Dell OptiPlex desktop computer Do not delete any software or change any settings on this computer It has been pre configured to operate the FlexSoft FX
58. t Flexcell International Corp immediately Copyright 2009 Flexcell International 2 2 LOCATION OF THE TISSUE TRAIN SYSTEM Choose a location for the Tissue Train system Fig 5 that is adjacent and as close as possible to the CO incubator and vacuum source A shorter distance in vacuum lines decreases the air flow resistance for elongation and release of the flexible bottomed culture plates allowing for a greater range of flexing at higher frequencies The length of the vacuum line should be a minimum of 4 1 2 m All tubing and the Ethernet communications cable connect to the rear of the FX5K Tension FlexLink Fig 6 All cables mouse monitor keyboard and Ethernet communications cables will connect to the rear of the computer Fig 7 The length of FLEXCELL BioFlex FlexLink and FlexSoft are trademarks of Flexcell International Corporation MS Windows and MS Office are trademarks of the Microsoft Corporation Adobe Acrobat is a trademark of Adobe Systems Inc Dell OptiPlex is a trademark of Dell General Notice Other product names mentioned in this manual are for identification purposes only and may be trademarks of their respective owners Flexcell International disclaims any and all rights in those marks E FLEXCELL INTERNATIONAL CORPORATION the clear and blue tubing from the FlexLink to the baseplate should be 10 3 m long to assure proper system performance This length can vary if needed but no
59. the Controllers menu select Exit E FLEXCELL INTERNATIONAL CORPORATION 4 ADDENDUMS TO THE TISSUE TRAIN SYSTEM FOR OPTIMAL PERFORMANCE OF THE FX5K TENSION FLEXLINE A Place the FlexLink higher than the baseplate If the FlexLink can safely sit on top of the incubator put it there This will reduce the amount of moisture reaching the valves in the system B It is necessary to place the included Water Traps inline between the FlexLink and the baseplate Be sure to place the Water Traps below the FX5K Tension FlexLink Humid air from the incubator tends to condense within the tubing lines This moisture may in turn lead to problems with long term regimens This fluid collection can be prevented from entering the FlexLink by the insertion of the Water Traps The Water Traps need to be placed so that they are maintained in a vertical position They should be checked frequently twice a day initially while a regimen is running and should be emptied when the Water Traps become half full On the first trial monitor the Water Traps every 15 minutes for the first two hours to be sure that a significant amount of water is not accumulating If so there may be a leak in the platform and or gasket Tissue Train plate assembly See System Troubleshooting Problem 1 pg 25 to correct this problem The valves and transducers may be damaged if moisture is allowed to accumulate C Use the system Drying Filte
60. the unit case or violating unit components will void the warranty This warranty is extended only to the original purchaser customer unless a transfer of ownership is approved by Flexcell in writing e LIMITED LIABILITY Flexcell or its distributor s only liability shall be to remedy any defect to comply with its warranty and return the repaired equipment to function as designed Under no circumstances shall Flexcell or its distributors be liable for any special incidental or consequential damages based upon breach of warranty or contract or negligence Such damages include but are not limited to loss of profits revenue loss of data down time customer s material or time f DISCLAIMER OF WARRANTIES The Limited Warranty expressed in the foregoing language is the only warranty applicable to this product Any other warranty expressed or implied warranty or of merchantability or fitness for a particular purpose are hereby disclaimed No oral or written information or advice provided by Flexcell through its agents or employees in the use and functioning of the equipment shall in any way create a warranty or in any way increase the scope of this limited warranty g DISCLAIMER LANGUAGE This warranty document accompanying instruction manual and supplemental applicable laws appear in the English language In the event of any inconsistency in the meaning of the words and terminology and any foreign language translation the English language shall preva
61. tions from the drop down menus Confirm with a Yes to download the regimen Once the program is downloaded click on Simulate in the main window display Once satisfied that the regimen is correct click on Stop Turn ON the vacuum system Click on Start to run the regimen The plates should begin flexing Make a visual check to verify membrane has been pulled downward into the troughs To pause or stop the regimen click on Pause or Stop at any time Otherwise the program will stop when the regimen is completed Check the Water Traps prior to each experiment to see if water is accumulating Empty the Water Traps before they are more than half full vii Il FLEXCELL INTERNATIONAL CORPORATION PART A CREATING 3D CELL SEEDED HYDROGELS g FLEXCELL INTERNATIONAL CORPORATION 1 INTRODUCTION Flexcell s Tissue Train culture system utilizes a novel method to culture and mechanically load cells in a three dimensional 3D hydrogel or cell assembled matrix Mechanical load may be applied using the Flexcell Tension System with a user defined program Cell gel constructs or cell assembled matrices may be mechanically loaded uniaxially or equibiaxially using special Arctangle or cylindrical Loading Stations respectively A program defining the elongation frequency and duration of mechanical load can be regulated by the user in the FX 5000 FlexSoft software Flexcell s Tissue Train Culture System can be used a
62. ulation in the FlexLink can damage the valves Problem 8 One or more of the FX5K Tension FlexLinks connected to the system are not being recognized by the software This is usually represented by E FLEXCELL INTERNATIONAL CORPORATION the error message Cannot Connect to Controller when attempting to connect a FlexLink to assign a regimen Solution 8 This problem is usually due to a lack of communication between the computer and one or more FX5K Tension FlexLinks Check the FX5K Tension FlexLink Ethernet port connection on the computer 27 and the communications connection on the back of the FlexLink to be sure that these are secure Check the FlexLink to see if the power is ON the FlexLink screen should power on If both of these conditions are true close and restart the software Try downloading the regimen again If the problem is not resolved call Flexcell for further instructions E FLEXCELL INTERNATIONAL CORPORATION 6 APPLICATION NOTES BASEPLATE AND GASKET CLEANING AND DISINFECTING The black urethane baseplate should not be autoclaved or washed in a dishwasher because the high temperature will cause warping The red rubber gasket should also not be autoclaved or washed in a dishwasher Both baseplate and gasket should be cleaned with regular laboratory detergent rinsed with warm tap water followed by distilled or deionized water and allowed to dry The bas
63. ulture plate NOTE Cell seeding density will vary depending on cell type We recommend testing cell seeding densities to determine the best cell number for your application and cell type 4 Wash cells 2 times with medium to remove trypsin or collagenase 5 Before adding cells the matrix protein gel solution should be neutralized to pH 7 0 using 1 M sodium hydroxide 6 Cells may be reconstituted in one volume of media containing 10 fetal calf serum 70 matrix protein gel fluid and 20 5X MEM The objective is to achieve an overall gel MEM concentration of 1X The suggested formula for the cell matrix protein gel combination is as follows ig FLEXCELL INTERNATIONAL CORPORATION e 70 by volume matrix protein gel e 20 by volume of 5X MEM to yield an overall 1X concentration by total volume e 10 fetal calf serum e Cells The Tissue Train culture plate in either the linear or trapezoidal anchor configuration can be used for creating the 3D constructs For linear shaped constructs use a linear Trough Loader with a standard Tissue Train culture plate For trapezoidal shaped constructs use a Trapezoidal Trough Loader with a Trapezoidal Tissue Train culture plate Place the appropriate shaped Trough Loader in a Loading Station beneath the flexible membrane of the Tissue Train culture plate so that the anchor stems are aligned with the long axis of the Trough Loader Fig 1 Apply a thin layer of lubrica
64. w side transducers Maximum allowable waveform variance from static line 0 2596 elongation Maximum elongation error actual vs programmed 0 6 elongation Maximum allowable transducer inaccuracy 1 0 kPa Unconstrained Dynamic Performance low side transducers Maximum elongation error actual vs programmed 0 5 elongation at the following frequencies lt 5 elongation 0 5 and 1 0 Hz 10 elongation 0 5 and 0 75Hz gt 10 elongation 0 5 Hz Maximum allowable transducer inaccuracy 1 0 kPa BioFlex 25mm Loading Station Static Performance high side transducers Maximum allowable waveform variance from static line 0 25 elongation Maximum elongation error actual vs programmed 0 5 elongation Maximum allowable transducer inaccuracy 2 0 kPa BioFlex 25mm Loading Station Dynamic Performance high side transducers Maximum elongation error actual vs programmed 0 75 elongation at the following frequencies lt 15 elongation 0 5 and 1 0 Hz gt 17 5 elongation 0 5Hz Maximum allowable transducer inaccuracy 2 0 kPa LOADING STATIONS SPECIFICATIONS 25mm Diameter Loading Stations equibiaxial strain Minimum achievable elongation with the FX 5000 0 8 Maximum achievable elongation with the FX 5000 21 8 28mm Diameter Loading Stations equibiaxial strain Minimum achievable elongation with the FX 5000 1 0 Maximum achievable elongation with the FX 5
65. wave Without these unusual elements the valve controllers may oscillate for a period of time e g dampened sine wave during beginning of plateau in a square waveform 3 10 PAUSING REGIMENS To pause a simulated or running regimen click on Pause in the main display window Click on Yes when the program asks for confirmation Pausing will cause the current 20 simulation or actual running of the regimen to stop at the next cycle of the current regimen step Pausing is different from stopping in that pausing leaves the regimen at its current position whereas stopping causes the regimen position to be set to the beginning of the first regimen step 3 11 RESUMING REGIMENS To resume a paused regimen click on Resume in the main display window This function is available only when a simulated or running regimen has been paused The regimen will restart its prior activity simulation or actual run from its last point 3 12 STOPPING REGIMENS To stop a simulated or running regimen click on Stop in the main display window Click on Yes when the program asks for confirmation Stopping causes the current activity to cease immediately and sets the regimen position back to the beginning of the first regimen step Stopping is different from pausing in that pausing leaves the regimen at its current position so that it can be resumed from where it stopped 3 13 DETERMINE THE PROGRESS OF THE REGIMEN The progress of the regimen f
66. wer to use the auto control feature 2 FLEXSOFI FX 5000 SOFTWARE AND SYSTEM POWER UP The FlexSoft FX 5000 software Flexcell Copyright Protected Software Property of Flexcell International Corporation 2730 Tucker Street Suite 200 Burlington NC 27215 has been pre installed on the hard drive Keep the CD package included with your system and store it in a safe place Be sure to shutdown Windows properly by using 12 the Shut Down command activated by the Start button Failure to follow the above directions can cause software and hardware malfunctions The computer has Windows 7 installed on the hard drive All backup software is provided with the system Check all connections and connect the surge protector into an appropriate power outlet 115 VAC or 230 VAC Turn ON the surge protector strip and check that the indicator light on the strip is lit If the power light does not come on the surge protector has failed Replace the surge protector and attempt to determine what caused the surge protector to fail Turn ON the power switches at the computer monitor and FX5K Tension FlexLink The system should then boot and load Windows Select and double click on the FX 5000 icon NOTE Flexcell recommends that the power saving or screen saver features in the Windows software are disabled Allowing the hard drive to power down while an experiment is running may create the need to reboot the computer be

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