3D Perfusion Bioreactor™ User Manual and 3D Cell
Contents
1. 10 Version 1 4 Updated 12 1 2014 2D Biotek 13 At this point the bioreactor is ready for media equilibration Place the bioreactor into a humidity free incubator and set the pump to 20 rpm While the media equilibrates the next is to seed cells into the bioreactor chamber following with 3 hours static seeding Media Out from Bioreactor Chamber Media Out from Media Reservoir Bioreactor CO Filter Chamber CO Filter Out CO In to Media Reservoir CO Out from Humidifier In Out In Out Media Reservoir Cap Co Humidifier Cap Figure 3 Tubing loop Assembly 4 Cell Culture Static Cell Seeding and Dynamic Cell Culture A successful 3D dynamic cell culture using 3D Perfusion Bioreactor requires the following steps 1 Media Equilibration prior to 3D Perfusion 2 3D Bioreactor Cell Seeding 3 Short Term Slow 3D Perfusion 4 Extreme aseptic conditions Figure 4 is a suggested time line for conducting a 3D cell culture experiment using 3D Perfusion Bioreactor 11 Version 1 4 Updated 12 1 2014 SD Biotek Cell Seeding Ready for End User 3D Scaffolds Experiments 3 hr Buttered Perfusion 21pm 24 hr 3D Perfusion Starts Figure 4 Time line for a successful perfusion bioreactor cell culture While the media equilibrates in the bioreactor there are general steps to successfully set up a 3D dynamic culture cell system 4 1 Direct Static Cell Seeding into Bioreactor Chamber 1 Depending on the seed2. ES 3DB 10 04 Bioreactor Polycarbonate Chambers 4 Eg 3DB 10 05 Silicon Rubber O rings 11 chamber x 4 chambers to separate individual scaffolds within the chamber 3DB 10 06 Pre packagedsets of tubing 4 to close loop the chambers with the media reservoir and Co humidifier glass bottle through the pump Each packaged sethas 2 pre assembled tubing for media and CO2 perfusion and 3 pieces of clear tubing 1 mid size with white stoppers on one end and 2 short tubing pieces for the air filters There are additional barbed tube fittings 5 for connecting bioreactor tubing circuitry during bioreactor equilibration See Lil inom anise 10 3DB 10 10 Co Humidifier glass bottle 4 Humidified CO is pumped into the medium reservoir item 7 Version 1 4 Updated 12 1 2014 2B Biotek 3 2 Perfusion Bioreactor Assembly Note All 3D Perfusion Bioreactor parts except peristaltic pump need to be autoclaved before any experiment The autoclavable parts can be autoclaved using autoclaving bags at 121 C for 30 minutes and 30 minutes drying The bioreactor tubing and syringe filters are autoclavable 5 cycles maximum For further technical questions on autoclavable syringe filters visit Millipore s Technical Library at http www millipore com search do q SLFG02550 0 0 The position of reservoir bottles need to be organized as 1 2 3 and 4 starting from left to right when assembling the 3D Perfusion Bioreactor Please ref
3. The 3D scaffold is engineered using 3D Biotek s Proprietary Precision Microfabrication Technology Unlike other sponge like porous scaffolds the porous 3D Insert scaffolds have 100 open and interconnected pores therefore cell culture medium can easily perfuse through the porous structure to eliminate the poor nutrient waste exchange phenomena often encountered with sponge like scaffolds The scaffolds are available in both biodegradable and non biodegradable polymers The entire scaffold is composed of polymer struts of uniform size Struts on different layers of the scaffold are 90 relative to each other creating a 3 dimensional and 100 interconnected porous structure Figure 1 Other important features of the 3D scaffolds e Pre sterilized and Ready to Use e Well Defined Pore and Porous Structure e Improved Cell Culture Efficiency e Easy Separation of Cytokines and Secreted Growth Factors e Produced from an Organic solvent Free Fabrication Process Figure 1 CAD images of 3D Insert PS scaffold Different color struts represent different layering in the scaffold This 4 layered model shows a top view a and b and side view C shows from bottom to top layer 1 Green layer 2 Yellow layer 3 Aquamarine and layer 4 Red Each layer is 90 and offset in reference to each other Version 1 4 Updated 12 1 2014 2B Biotek 3 3D Perfusion Bioreactor Parts and Assembly Note The 3D Perfusion Bioreactor i
4. of the cassette with 70 Ethanol Insert the aluminum stand holder into the aluminum base frame Refer to Figure 2 for assembly Media reservoir bottles item 7 should be placed in the front of the aluminum stand holder item 3 3 Place the Co humidifier chambers item 11 on the back holders in the aluminum stand Fill each humidifier bottle with 25 ml of sterile water 4 3D Perfusion Tubing Bioreactor Description From this point on refer to figure 3 for assembly of media and CO O tubing The tubing has been labeled with autoclavable color tape to match with the imprinted letter on the media reservoir Red R Orange O Yellow Y Green G and Co humidifier metal caps Green G and B Blue Start tubing assembly with reservoir bottle 1 from left to right Soray the autoclave bags and bottles with 70 ethanol and wipe Below is an itemized description of the tubing followed by detailed description on tubing assembling o Pre assembled Bioreactor Chamber Medium Loop This tubing is composed of small clear tubing orange labeled on one end Orange arrow Fig 3 This piece is attached to beige bioreactor tubing Dotted black lines Fig 3 which connects to large clear tubing Black arrow Fig 3 with red media stoppers on one end Media In towards bioreactor chamber As pre assembled tubing the Media In towards bioreactor chamber Red solid line Fig 3 is connected to medium size clear tubing using a large plastic tube fit
5. table 8 give the values of velocities as a function of chamber format and rpm aa IN Media Media Equilibration Bioreactor Perfusion Figure 5 Position of tube fitting and Bioreactor Chamber 14 Version 1 4 Updated 12 1 2014 2D Biotek Table 3 Total surface growth area on 3D PS scaffold Scaffold Format 2D Growth Area 3D Growth Area 3D Growth Area 3D 2D Ratio 3D 2D Ratio ene eee _ aa B E 24 Well 10 20 cm 12 Well 21 08 cm 19 65 cm a 6 Well 54 02 cm 52 10 cm Table 4 Total surface growth area on 3D PCL scaffolds Scaffold Format 2D Growth Area 3D Growth Area 3D Growth Area 3D 2D Ratio 3D 2D Ratio oe eae a aa ac an 24 Well 18 28 cm 13 74 cm 12 Well 39 27 cm 27 90 cm DEE MER E 6 Well 99 21 cm 75 62 cm Table 5 Seeding volume on scaffold as a function of scaffold format Seeding Volume ul PS 3040 PCL 3030 Oo 24w y 80 OO OS 20 6wel 30 6o 69 15 Version 1 4 Updated 12 1 2014 SD Biotek 5 Perfusion Velocity and Revolution per Minute RPM Graph and Table 55 24 Well Format E 12 Well Format 45 q 6 Well Format 40 35 RPM 25 20 15 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 Velocity cm min Figure 6 Range of rmp value for a range of biologically relevant flow velocities In figure 6 the rom values were calculated from flow velocity of interest and the scaffold cross sectional area by using following eq
6. then pressing down at the oval end until there is a clicking sound Bring the remaining length of the tubing from the back to the front towards the media bottle reservoir First secure the tubing on the right hook on the metal stand Second pass the tubing through the bioreactor holder hoop Then connect the red taped end with the letter R Red on the media bottle reservoir This completes the medium loop without the bioreactor chamber Later the bioreactor chamber will replace the large plastic tube fitting connecting the Media into Bioreactor and Media out to Reservoir Bottle 7 Bioreactor CO O Gas Loop connect the blue labeled end of the tubing to the letter B Blue on the humidifier bottle Unhook cassette number 2 following cassette next to media perfusion tubing pressing the oval mound always on the opposite side Each cassette has an internal channel where the beige tubing will pass through The beige pump tubing has two plastic stoppers The first stopper secures the tubing on one end Following place the tubing through the internal channel Then the second stopper will secure the beige tubing at the distal end At this point the beige tubing should be under tension Place the cassette back into the pump hooking the proximal side opposite to the oval mound first and then pressing down at the oval end until there is a clicking sound Bring the remaining of the tubing from the back to the front towards the media bottle reservoir Pa
7. BD Biotek 3D Perfusion Bioreactor User Manual and 3D Cell Perfusion Handbook Version 1 4 Updated 12 1 2014 2B Biotek Note This manual will be revised and updated without prior notice To check if the latest version comes with your 3D Bioreactor System please visit our website at http www 3dbiotek com web prod 3dbioreactor aspx Registration Registration of your purchase is recommended in order to receive product update and promotional discount code for PS PCL scaffolds specifically made for use in our 3D Bioreactor Please follow the link below to register http www 3dbiotek com web register4bioreactor aspx gt A Pate Sarr eee IF eee TE Fp 7 f Y 7 f ps 3 t A gA jm E Ace V Spel Magn Dot WD i 500 um QOORV AD 4 GSE 341 T2Terreorer Version 1 4 Updated 12 1 2014 2D Biotek Contents 1 3D Pertusion Bioreactor OverviCW oicsecccisteccaccnaseswstpanscortundvececsasincsumessectuanvexecavenimsceastinetsheesneniacs 4 2 3Dinsen TECHNOIOLY sivacovecoosnesecavcntesvedactaesencensewenesd calectivndensohettouncssegevensetenss syncevesteutewswavecateesues 5 3 3D Perfusion Bioreactor Parts and Assembly ccccsccccscsccccccscsccccccscscccsccccecscececcecscscecescecscesess 6 3 1 3D Perfusion Bioreactor Parts List esssseoseseosessosessossosssossosossosessosessosessosessosessosessosossos 7 3 2 Perfusion Bioreactor Assembly cccsccccscscccsccscsccccccscscscsccccc
8. cscnceccecscscsceccecsceseccccscscececescecs 8 4 Cell Culture Static Cell Seeding and Dynamic Cell Culture sccscscscsccscscsccccccscnccceccecscsceseeeees 11 4 1 Direct Static Cell Seeding into Bioreactor Chamber sesessesessesessssessssesssoesesoesseossoessssesseseo 12 5 Perfusion Velocity and Revolution per Minute RPM Graph and Table cscsccscsssscsssscsceccsces 16 6 Pump Tubing Sterilization sessesesessessssessssecosscesseossseossseossseossssossssossssossssoesossssossssossssoesssossssseoe 16 7 Tubing Connection Circuit Diagram csceccsceccccccscsceccccscscsceccccecsceccececscececcecscecescecscececesescecscesess 16 B APPEN a A T E 19 8 1 Pr Installation Check LiStrisesccioscccegesnssenuenscuccacatcncavesnouansancencaaonescaucaneadedeansnmsyescauonmeneccessanes 19 8 2 Pump s Power Cord Through Incubator s Double Doors scscscsscscsceccccscscecsccscsceseccececs 20 For Latest Updates on the Bioreactor Manual Please Visit our Website at http www 3dbiotek com web prod_3dbioreactor aspx Version 1 4 Updated 12 1 2014 2D Biotek 1 3D Perfusion Bioreactor Overview Congratulations on the purchase of this 3D perfusion bioreactor This bioreactor is specifically designed for conducting physiology relevant cell culture in a 3D in vivo like microenvironment under dynamic perfusion conditions This bioreactor has integrated our proven 3D Insert technology with a uniqu
9. e dynamic perfusion system to allow mammalian cells to grow in a 3D in vivo like micro environment Users will obtain the following distinct advantages by using this 3D perfusion bioreactor 1 Wide range of 3D surface areas for initial cell seeding 3D Perfusion Bioreactors are available in different configurations that provide cell growth area from 9 5 cm to 3960 cm wide enough to meet various lab research needs 2 Compatible with wide range of 3D Insert scaffolds A very unique feature about the 3D Perfusion Bioreactor is that a wide range of 3D Insert scaffolds from 24 well to 6 well configurations can be used in the bioreactor all with a simple change of a chamber 3 Autoclavable All parts and tubing except pump are autoclavable 4 Forced CO and O exchange to maintain better cell growth environment CO and O are forced to bubble through the cell culture medium to better maintain an ideal cell culture environment 5 Adjustable medium flow rate to better meet individual needs Medium perfusion flow rate can be adjusted to meet individual needs This also makes it possible to study the flow rate or shear force effects on cell growth The pump s digital display and control make it easier to adjust the flow rate Version 1 4 Updated 12 1 2014 2B Biotek 2 3D Insert Technology One of the novelties of the 3D Bioreactor is the 3D Insert porous polymer scaffolds that sit inside the bioreactor chambers
10. er to Figure 2A The perfusion bioreactor assembly has been optimized to achieve media equilibration and bioreactor chamber static cells seeding at the same time These two steps are required for final 3D perfusion Each non sterile pre assembled tubing set comes with different lengths to account for the positions of the media reservoir and humidifier bottles on the aluminum metal stand Make sure you don t mix all the different pre assembled pump tubing before or after an experiment Figure 2A Designation of Bottle Location Autoclaving of all the parts except the pump and 3D Insert scaffolds is needed before assembling the bioreactor system because the polycarbonate chambers 4 tubing fittings Co humidifier bottles and Version 1 4 Updated 12 1 2014 2D Biotek media reservoir bottles are non sterile Before autoclaving mark the autoclaving bags containing the tubing with the number associated to the position of the media reservoir bottle to avoid tubing mismatch Assembly of the Perfusion Bioreactor should be done in a sterile hood Refer to figure 2 for visual description of bioreactor tubing assembly 1 Wipe the sterile hood surface with 70 ethanol prior to assembly and let the hood surface air dry 2 Place all the sterile parts inside the hood Wipe the aluminum stand holder pump and pump s cassettes Unlock the cassette channels from the pump by clicking downward on the oval mound on each cassette distal end
11. ing density of the experiment cells need to be expanded under regular tissue culture conditions Tables 3 and 4 show the different surface areas of the 3D Insert as a function of scaffold format fiber diameter and fiber to fiber configurations These tables help determine the seeding density on 3D scaffolds from already established 2D seeding densities into seeding densities 2 Follow standard protocols for cell detachment and cell counting Make sure the total cell suspension volume is scaled up for your specific cell number needs and 3D Biotek s suggested seeding volume Refer to Table 5 for seeding volume values 3 Once cells are in suspension take the bioreactor chamber and seal the bottom with the provided short clear tubing with a stainless steel ball Then place the bioreactor chamber on the chamber stand 13 On the opening of the bioreactor chamber cap place another small clear tubing following with a 0 20 um air syringe filter These will prevent any potential contamination in transit to the incubator and during the three hour incubation period 4 Using the forceps pick an O ring and place it on the base of the bioreactor chamber Using the O ring pusher slide the O ring evenly using rotating motion until it reaches the bottom of the bioreactor chamber 5 Next grab a 3D Insert scaffold using the appropriate forceps along the scaffold s diameter and from the easy handle sides small cut outs for stable scaffold grip Slide
12. onnection Circuit Diagram The following diagram Figure 8 shows the tubing connection circuit for the system which should be used in conjunction with the schematic diagram Figure 3 To connect the tubing circuit for each bottle chamber location also refer to Figure 2A l a Culture a eean h i Chambel N A a qj H wm IVICU IUI 1 I gt le D atetl on gt pee DULLIC Figure 8 Tubing Connection Circuit for the system 18 Version 1 4 Updated 12 1 2014 2B Biotek 8 Appendix 8 1 Pre Installation Check List Before the bioreactor system is installed it is recommended to check if the following items are available e Bioreactor Parts all the items listed in Section 3 1 on Page 7 e Scaffolds scaffolds ordered from 3D Biotek or your own scaffolds e Cells amp Media from your laboratory e Incubators either option 1 or 2 below o 1 Tall incubation with 2 independent compartments each with standard internal space 18 W x 18 D x 20 H see Figure 9 One compartment for Media Equilibration in non humidified condition and one for Static Cell Seeding in humidified condition o 2 1 standard size incubator with single compartment required minimum internal space 16 W x 15 D x 14 H for Media Equilibration in non humidified condition and 1 desktop size incubator no minimum internal space requirement for Static Cell Seeding in humidified condition o For the overall dimensions of the bi
13. oreactor assembly refer to Figure 2 on Page 6 Figure 9 Incubator with 2 independent compartments 19 Version 1 4 Updated 12 1 2014 82B Biotek 8 2 Pump s Power Cord Through Incubator s Double Doors The following diagram Figure 10 shows the setup of the pump s power cord through the double doors of the incubator Figure 10 Pump s Power Cord Through Incubator s double doors 20 Version 1 4 Updated 12 1 2014
14. s designed to use within a cell culture incubator at 37 C Please do not use the pan humidifier of the incubator because the pump is not designed for use in a high humidity environment The bioreactor comes with its own humidifier to reduce the evaporation of the cell culture medium The 3D Perfusion Bioreactor consists of 4 independent autoclavable polycarbonate chambers The chambers are available in different sizes to accommodate 3D Insert of 24 12 and 6 well configurations An 8 channel peristaltic pump which has an adjustable speed range from O to 60 RPM to recirculate cell culture medium through the 4 cell culture chambers at the same time The system is equipped with a build in CO humidifier system that constantly pumps CO O directly into the cell culture media Figure 2 and Table 1 give a detailed list of part number and description of all individual components of the 3D Perfusion Bioreactor Figure 2 Illustration of bioreactor Parts Bioreactor s max dimensions Width X 14 Depth Y 13 and Height 12 Z Version 1 4 Updated 12 1 2014 2D Biotek 3 1 3D Perfusion Bioreactor Parts List Table 1 Catalog number and description of 3D Perfusion Bioreactor Parts Fal call e 3DB 10 01 Peristaltic Pump with 8 channels ES 3DB 10 02 Aluminum Base Frame 3DB 10 03 Aluminum stand for holding 4 bioreactor chambers item 4 4 glass reservoirs item 7 amp 4 small humidifier glass bottles item 10
15. s is to maintain constant pressure across the bioreactor 13 Grab one bioreactor chamber remove the bottom clear tubing with the stainless steel ball Secure the bioreactor chamber on the metal stand hoop and tighten using the metal slider 14 Unhook the tubing end with the red stoppers Then connect it to the bottom of the bioreactor chamber 15 Next remove the clear tubing with the syringe filter from the top of the bioreactor chamber Remove the large tube fitting from the other end of the remaining tubing with the white stoppers Connect this tubing end to the top of the bioreactor chamber At this point the bioreactor chamber is ready for 3D perfusion See Figure 5 to see the large tube fitting position and bioreactor chamber for 3D perfusion white and red asterisks 16 Repeat steps 12 15 for each bioreactor chamber 3 h incubation 47 C 5 CO Cell Seeding O Ring 3D Insert 7 93 rpm 24h Slow Perfusion Ed Media In 3D Seeding 3D Perfusion Figure 4 3D Seeding and 3D Perfusion Diagram 13 Version 1 4 Updated 12 1 2014 2B Biotek 17 After connecting all 4 bioreactor chambers unhook the red and white plastic stoppers Warning Failure to release the stoppers will cause pressure buildup during perfusion and experimental failure Bring the bioreactor to a humidity free incubator and set rom to 1 9 to 2 1 Figure 4 3D Perfusion Press start and allow slow perfusion for 24 hours Figure 6 and
16. ss the tubing through the right metal hook connecting the green labeled end with the letter G Green on the media reservoir This completes the Bioreactor CO 0O Loop humidified CO O will be actively pumped from the humidifier bottle to the media reservoir bottle during perfusion 8 CQO gt O Filters the remaining short clear tubing i e yellow and green labeled place a 0 20 um pore syringe filter on each end and connect the color labeled end to the media reservoir and the humidifier bottles yellow Y on media reservoir bottle and blue B on humidifier bottle 9 Repeat steps 6 through 8 for the remaining bioreactor bottles from left to right or 1 to 4 10 After connecting all tubing fill the media reservoir bottles with cell culture media Media bottle max volume 90 ml Table 2 gives approximate values of bioreactor chamber and media perfusion tubing pre loading volumes This take into account the extra volume to add in addition to media for extended cell culture conditions 11 Remove the silicon stoppers and place on sterile surface Using a pipette draw desired amount of media and dispense into the media reservoir bottle After completion wipe the rim of the media orifice and the silicon stopper with 70 ethanol 12 Place the silicon stopper tightly into the media reservoir orifice Table 2 Approximate volume required to fill each bioreactor chamber format and tubing Chamber Tubing Total Volume 6 Well 45 ml 12 Well 15 ml
17. ting The medium size clear tubing has white media stoppers and red labeled at the end This will connect to the media reservoir cap Red arrow Fig 3 o Pre assembled Reservoir CO O In This tubing is composed of small blue labeled end clear tubing connecting the humidifier cap Blue arrow Fig 3 This piece is attached to beige bioreactor tubing Dotted black lines Fig 3 which connects to large green labeled clear tubing connecting to the media reservoir cap Green arrow Fig 3 o CO O Filters Short Clear Tubing to attach Syringe Filters Yellow and Green solid lines with circle ends Fig 3 5 The following is the methodology to assemble and connect all bioreactor tubing 6 Bioreactor Cell Culture Media Loop connect the orange labeled end of the tubing to the letter O Orange on the media reservoir cap Secure the tubing using the left metal hook Unhook cassette number 1 the oval mound always on the opposite side Each cassette has an internal channel where the beige tubing will pass through The beige pump tubing has two plastic stoppers The first stopper secures the tubing on one end Following place the tubing through Version 1 4 Updated 12 1 2014 2D Biotek the internal channel Then the second stopper will secure the beige tubing at the distal end At this point the beige tubing should be under tension Place the cassette back into the pump hooking the proximal side opposite to the oval mound first and
18. uation EQ 1 RPM K x Velocity x cross section area of scaffold FQ 1 Where K 8 547 this is an experimentally determined constant from the linear fit between flow rate ml min and rpm for the 3D bioreactor system R 0 999 Table 7 Cross section Area of Scaffold as a function of format 16 Version 1 4 Updated 12 1 2014 2B Biotek Table 8 Relationship between pump s rpm and biologically relevant flow velocities O F ee a Velocity m min em m m E a a ee a ee D Y S YC po PB 86 28 PBB 9888 OAB O 6 Pump Tubing Sterilization The tubing for the pump refers to the one with 2 stops that are used with the cartridge of the pump head The 2 stops are attached to the tubing by adhesive Figure 7 Figure 7 Pump Tubing with Stops During successive autoclave procedures under high temperature these stops may drop off the tubing due to failure of the adhesive Given below are some recommended methods to sterilize this particular tubing e Gas sterilization with ethylene oxide e Gamma Irradiation e Autoclave at 1 BAR and 120 C for 30 minutes e Flush ethanol and then rinse with cell media through the tubing using the pump Consider that the tubing will mechanically degrade over time as it is repeatedly squeezed by the rollers There comes a time when it is necessary to replace the tubing independent of cleaning considerations 17 Version 1 4 Updated 12 1 2014 82B Biotek 7 Tubing C
19. without releasing the scaffold until it evenly touches the first O ring at the bottom Release the forceps and retract them from the chamber 6 Take another O ring and repeat step 4 12 Version 1 4 Updated 12 1 2014 2D Biotek 7 Using the pipette aliquot the correct volume Table 5 containing the specified number of cells Center the pipette tip and lower it carefully and gently until touching the scaffold Do not push polystyrene scaffolds will break Release the cell suspension volume Bring the pipette tip up 8 The previous O ring scaffold O ring cell seeding is a sequence that ensures minimal disturbance to the seeded cells below for piece of mind Figure 4 3D Seeding 9 Repeat steps 5 through 7 until loading a total of 10 scaffolds and 11 O rings for each chamber Figure 4 3D Seeding 10 Close tight the bioreactor chamber using the bioreactor cap make sure it has the syringe filter Bring the bioreactor chamber in its holder to a humidified incubator at normal culture conditions for 3 hours static seeding 11 After 3 hours turn off the bioreactor and bring it out from the non humidified incubator into the sterile hood Then bring all the 3D seeded and loaded bioreactor chambers to the sterile hood as well 12 Before removing the large plastic fitting Refer to Section 3 2 step 6 press the red and white plastic stoppers on each side of the large plastic fitting before unplugging each clear tubing end Thi
Download Pdf Manuals
Related Search