生物反应器课件-Different-cell-properties-Select-cell-culture-method.ppt

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)Mammalian(

2、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 or independantAnim

3、al 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 lines cultured(epit

4、helial,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 C Dissolved oxygen Med

5、ia 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,KClVitaminsMoreLesspH7.26.4Osmolarity300 m

6、Osm350 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.ExpensiveGrowth curves in yeasts and mammalia

7、n 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 cultivation BATCH FERMENTATION.Duratio

8、n: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 Cell density 20*106 c/mlPerfusion Used to

9、 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 suspension Shear Reactor design,Pluorinic F68 Wa

10、ste 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 Macroporous microcarriers have increased surface ar

11、ea(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 liquids Equalization of temp.and nutrient conc.Dispersion of

12、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 transfer coefficient Bioreactor designAgitation Seals Mechanical Face Seals Li

13、p 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=Impeller Diameter in InchN =RPM of ShaftF =One(1)for one impeller (1.8)for two impellers (2

14、.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 design considerations Shape ASME Bottoms Hemispherical Bottoms Jacket Design Conventional Dimpled H

15、alf 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 Watts/Liter of Volume.Heating Systems:One(1)degree C per minute between 25 and 45 CSpargersRing TypeSin

16、gle OrificeSinteredSterility Considerations Killing Rates Dead Legs Fitting Types Piping ConsiderationsSterility ConsiderationsCritical Elements of Steam Sterilization Time Temperature MoistureAir RemovalSterility ConsiderationsSterility Considerations Killing rate F0=ti*10(Ti-121)/Z F0 =Integrated

17、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 designe

18、d to deliver 30 LPM per meter of vessel circumference.Minimum flow rate of 1.5 meter per second for pipe or tubing for effective cleaning.EP surface finishingCIP Considerations ElectropolishingConclusions Many factors involved in bioreactor design Supplier has experience Cell culture aspect determined in R&DQuestions?