生物反應(yīng)器 Different cell properties Select cell culture methodppt課件

1、Different cell propertiesSelect cell culture methodContent Introduction Cell culture specification Selection of bioreactorMicro organismsBacteria (0.5 2 m) Escherichia coli (lower intestine)Yeast (2 5 m) Saccharomyces cerevisiae (bakers yeast)Fungi (2 15 m) Penicillium spec. (Cheese and antibiotic)M

2、ammalian (7 20 m) Chinese Hamster Ovary Cells (Insuline)Animal cells vs. microbial cells PROTEINS - Post translational modifications Glycosylation, phosphorylation etc. Proper folding environment Efficient secretion Extra-cellular matrix medium for cells to interact and migrate Anchorage dependant o

3、r independantAnimal cell culture or the ability to continuously culture cells Platform to investigate normal physiology and biochemistry Test the effects of drugs and compounds in vitro Tissue engineering Synthesize valuable products Models for studying diseasesCell Structure Wide variety of cell li

4、nes cultured (epithelial, muscle etc.) Larger than microbial cells (10-30m) Many internal organelles No cell wall susceptible to shear forces Cytoskeleton division and migrationAnchorage dependantCommon cell lines and their applicationsCell Growth conditions Growth temperatureMammalian 37CInsect 27

5、C Dissolved oxygen Media requirements (insect, mammalian, serum etc.)Basic Media Differ For Mammalian and Insect CultureComponentMammalianInsectGlucose4 g/l2.5 g/lAmino acids0.01-0.05 g/l0.1-0.5 g/lGlutamine1 g/l1 g/lHCO33.5 g/l0.35 g/lH2PO40.1 g/l1 g/lSalts4.5 g/l NaCl1 g/l Mg2SO4, KClVitaminsMoreL

6、esspH7.26.4Osmolarity300 mOsm350 mOsmSerum AdditionUsed at 0-20% SerumIt contains: Growth factorsTransferrin (Fe)LipidsInsulinAlso important for:Shear protectionDetoxificationProblems:Infectious agents (viruses, mycoplasm, prions)Serum composition is poorly defined and the batches vary.ExpensiveGrow

7、th curves in yeasts and mammalian cells are different0.000010.00010.0010.010.11.010.0050100 YeastMammalian MinimumdensityLag phaseExponentialStationaryDeclineCell Metabolism Toxic metabolites Lactate from glucose metabolism (lowers pH) Ammonia from catabolism of glutamate and amino acidsType of cult

8、ivation BATCH FERMENTATION. Duration: 0.5-1 weeks Cell density 4*106 c/ml Finish: no more nutrientsType of cultivation FED-BATCH Addition of concentrated nutrients = higher product concentration Duration: 1-1.5 weeks Cell density 5*106 c/ml Finish: Viability higher cell density Duration: 1-6 months

9、Cell density 20*106 c/mlPerfusion Used to achieve high cell densities Fresh medium added as spent medium is removed Cell separation required (spin filters, hollow-fiber filters, centrifugal separation, BioSep) Remove toxicsAdditional considerations with a bioreactor? No anchorage Carriers adapt to s

10、uspension Shear Reactor design, Pluorinic F68 Waste accumulation Perfusion cultureMicrocarrier cultures Microcarriers provide a good surface are for attachment per volume. Frequently used with suspension cultures of anchorage dependant cells Solid microcarriers suffer from fluid mechanical damage Ma

11、croporous microcarriers have increased surface area (gelatin, collagen, cellulose, polystyrene, polyethylene etc.)Microcarrier culturesPacked bedHollow fiberSuspension cellsEasier scale upAeration and AgitationStirring provides: Suspension of cells and solid nutrients Dispersion of immiscible liquid

12、s Equalization of temp. and nutrient conc. Dispersion of airAeration and Agitation OTR = KLa (C* - CL) CL = concentration of dissolved oxygen in the fermentation broth C* = is the saturated dissolved oxygen concentration a = is the gas / liquid interface area per liquid volume KL = is the mass trans

13、fer coefficient Bioreactor designAgitation Seals Mechanical Face Seals Lip Seals Magnetic Drives Impellers Turbine Marine Pitched Blade OthersAgitationAgitation - turbine impeller For unaerated water 20 C in a fully baffled tank the following formula applies: P= 4.5 x 10-13 * Di5 * N3 * FDi = Impell

14、er Diameter in InchN = RPM of ShaftF = One (1) for one impeller (1.8) for two impellers (2.4) for three impellersAgitation - turbine impellerTip Speed should also be considered because of tendency to shear cells. Do not exceed 1 m/s for cell cultureAnd 5 - 6 m/s for microbial cultures.Bioreactor des

15、ign considerations Shape ASME Bottoms Hemispherical Bottoms Jacket Design Conventional Dimpled Half Pipe Ports Tri-Clamp, DN, NA-connect, Ingold Type, Flange typeBioreactor design considerationsASME Head(s)Hemispherical Head(s)Temperature controlTemperature controlCooling System: remove 50 to 100 Wa

16、tts/Liter of Volume.Heating Systems: One (1) degree C per minute between 25 and 45 CSpargersRing TypeSingle OrificeSinteredSterility Considerations Killing Rates Dead Legs Fitting Types Piping ConsiderationsSterility ConsiderationsCritical Elements of Steam Sterilization Time Temperature MoistureAir

17、 RemovalSterility ConsiderationsSterility Considerations Killing rate F0 = ti * 10(Ti - 121) / Z F0 = Integrated amount of lethality delivered during a cyclei.e. A cycle with F0 = 17 minutes has a lethality equivalent to 17 minutes at121 C.CIP considerations Sterile Piping should be pitched for drainability. CIP FluidsDetergentAlkaline AcidWFI rinse Clean Steam Circuit for Distribution Sprayballs should be designed to deliver 30 LPM per meter of vessel circumfer