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ContentIntroductionCell culture specificationSelection 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(7 20 m)Chinese Hamster Ovary Cells(Insuline)Animal cells vs.microbial cellsPROTEINS-Post translational modificationsGlycosylation,phosphorylation etc.Proper folding environmentEfficient secretionExtra-cellular matrix medium for cells to interact and migrateAnchorage dependant or independantAnimal cell culture or the ability to continuously culture cellsPlatform to investigate normal physiology and biochemistryTest the effects of drugs and compounds in vitroTissue engineeringSynthesize valuable productsModels for studying diseasesCell StructureWide variety of cell lines cultured(epithelial,muscle etc.)Larger than microbial cells(10-30m)Many internal organellesNo cell wall susceptible to shear forcesCytoskeleton division and migrationAnchorage dependantCommon cell lines and their applicationsCell Growth conditionsGrowth temperatureMammalian 37CInsect 27 CDissolved oxygenMedia 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 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.ExpensiveGrowth curves in yeasts and mammalian cells are different0.000010.00010.0010.010.11.010.0050100 YeastMammalian MinimumdensityLag phaseExponentialStationaryDeclineCell MetabolismToxic metabolitesLactate from glucose metabolism(lowers pH)Ammonia from catabolism of glutamate and amino acidsType of cultivationBATCH FERMENTATION.Duration:0.5-1 weeksCell density 4*106 c/mlFinish:no more nutrientsType of cultivationFED-BATCHAddition of concentrated nutrients=higher product concentration Duration:1-1.5 weeksCell density 5*106 c/mlFinish:Viability higher cell densityDuration:1-6 monthsCell density 20*106 c/mlPerfusionUsed to achieve high cell densitiesFresh medium added as spent medium is removedCell separation required(spin filters,hollow-fiber filters,centrifugal separation,BioSep)Remove toxicsAdditional considerations with a bioreactor?No anchorage Carriers adapt to suspensionShear Reactor design,Pluorinic F68Waste accumulation Perfusion cultureMicrocarrier culturesMicrocarriers provide a good surface are for attachment per volume.Frequently used with suspension cultures of anchorage dependant cellsSolid microcarriers suffer from fluid mechanical damageMacroporous 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 nutrientsDispersion of immiscible liquidsEqualization of temp.and nutrient conc.Dispersion of airAeration and AgitationOTR=KLa(C*-CL)CL=concentration of dissolved oxygen in the fermentation brothC*=is the saturated dissolved oxygen concentration a=is the gas/liquid interface area per liquid volume KL=is the mass transfer coefficient Bioreactor designAgitationSealsMechanical Face SealsLip SealsMagnetic DrivesImpellersTurbineMarinePitched BladeOthersAgitationAgitation-turbine impellerFor 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 (2.4)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 considerationsShapeASME BottomsHemispherical BottomsJacket DesignConventionalDimpledHalf PipePortsTri-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 TypeSingle OrificeSinteredSterility ConsiderationsKilling RatesDead LegsFitting TypesPiping ConsiderationsSterility ConsiderationsCritical Elements of Steam SterilizationTimeTemperatureMoistureAir RemovalSterility ConsiderationsSterility Considerations Killing rateF0=ti*10(Ti-121)/ZF0 =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 considerationsSterile Piping should be pitched for drainability.CIP FluidsDetergentAlkaline AcidWFI rinseClean Steam Circuit for DistributionSprayballs should be designed 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 ConsiderationsElectropolishingConclusionsMany factors involved in bioreactor designSupplier has experienceCell culture aspect determined in R&DQuestions?Thank you拯畏怖汾关炉烹霉躲渠早膘岸缅兰辆坐蔬光膊列板哮瞥疹傻俘源拯割宜跟三叉神经痛-治疗三叉神经痛-治疗 拯畏怖汾关炉烹霉躲渠早膘岸缅兰辆坐蔬光膊列板哮瞥疹傻俘源拯割宜跟三叉神经痛-治疗三叉神经痛-治疗
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