微载体细胞培养放大手册解读Word格式.docx

微载体细胞培养放大手册解读Word格式.docx

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ProteinAmplificationandSimplePurification18-1142-75

ProteinPurification

Handbook18-1132-29

IonExchangeChromatography

PrinciplesandMethods18-1114-21

AffinityChromatography

PrinciplesandMethods18-1022-29

HydrophobicInteractionChromatography

PrinciplesandMethods18-1020-90

GelFiltration

PrinciplesandMethods18-1022-18

Handbooks

fromAmershamBiosciences

ReversedPhaseChromatography

PrinciplesandMethods18-1134-16

ExpandedBedAdsorption

PrinciplesandMethods18-1124-26

Chromatofocusing

withPolybufferandPBE50-01-022PB

Microcarriercellculture

PrinciplesandMethods18-1140-62

1

MicrocarrierCellCulture

Scale-UpMethods

Contents

Chapter1

Microcarriercellculture...................................................3Introduction.....................................................................................3Applications.....................................................................................3Cellattachment................................................................................4Requirementsforanoptimummicrocarrier..........................................6Propertiesandcharacteristics............................................................6Instructionsforuse...........................................................................8Preparation...........................................................................................................8Inoculum..............................................................................................................8CultureProcedure..................................................................................................8Monitoringcellgrowth............................................................................................8Harvestingcells.....................................................................................................9Scalingupbyserialsubcultivation..........................................................................9Equipmentrecommendations................................................................................10Industrialapplications..........................................................................................11TroubleShooting:

............................................................................12

Chapter2

AnAnalysisofAlternativesandProcessDevelopment........15Introduction...................................................................................15Processoverview.............................................................................16Provisions...........................................................................................................16Processthemestobeevaluated............................................................................17Principalprocessflow..........................................................................................17Equipmentused..................................................................................................17Processoutlineandsuggestedexperiments.......................................18Process1a..........................................................................................................18Process1b..........................................................................................................22Process2a..........................................................................................................24Process2b..........................................................................................................27

References....................................................................28

2

Introduction

Animalcellculture,vitaltothestudyofstructure,function,anddifferentiation,alsoprovidesimportantbiologicalsforthepharmaceuticalindustry,includingviralvaccines,enzymes,hormonesandantibodies.Microcarriercellculturetechnologymeansthatanchoragedependentanimalcellsaregrownonthesurfaceofsmall（approximately150microndiametersphereswhicharemaintainedinstirredsuspensioncultures.Thistechnologyisreplacingconventionalmonolayercellculturemethodsastheextremelyhighsurfaceareatovolumeratioaffordedbymicrocarriersisalsousedfor:

•Routinecellculture

•Newresearchapplications

•Highdensityculture

•Productionculturevolumesgreaterthan1000litres

Someoftheadvantagesofthemicrocarriersystemare:

•Efficientmonitoringandculturecontrol

•Reducedcostsandcontamination

Applications

Cytodex™enablesallanchoragedependentanimalcellstogrowinsuspensioncultures,andisusedinbothbatchandperfusionculturesystems,aswellastoincreasethesurfaceareaoftraditionalmonolayercultures.Instirredsuspensioncultures,cellsgrowinahomogeneousenvironmentwherethecultureparametersareeasilymonitoredandcontrolled.Culturesmaybesampled,atwill,toexaminecellmorphologyandtodeterminecellviability.Microcarriertechniquesarethereforethenaturalchoicewherecellsareusedfortheproductionofbiologicals.Attheresearchlevel,applicationswithCytodexinclude:

•Studiesofcellstructure,functionanddifferentiation

•Enzymefreesubcultivation

•Implantationstudies

•Harvestingofmitoticcells

•Lightandelectronmicroscopy

•Transportationandstorage

IndustrialscaleapplicationsutilizingCytodexincludetheproductionofcells,cellproductsandviralvaccines（seeIndustrialapplications,Page11.

3

4

Cellattachment

Inordertocultureanchoragedependentcellsinvitroitisessentialtoknowhowthecellsinteractwiththeirenvironment.Thecellmembraneissurroundedbya"

coat"

calledtheglycocalyx

（Figure1.Theglycocalyxconsistofglycolipids,glycoproteinsandtransmembraneproteoglycans.Alargenumberofthesemoleculescontainsugarswithanegativecharge.Sothenetchargeontheglycocalyxisnegative.Thatmeansthatifaculturesurfaceispositivelychargedtherewillbeanelectrostaticattractionbetweenthecellandtheculturesurface.Ithasbeenshownthattheattachmenttogelatinmicrocarrierscomparedtochargedcarriers,isslowerbecausethesurfacechargeisnotoptimal（1.

Fig.2.Thesubunitstructureofacell-surface

fibronectinreceptor.Bybindingtofibronectinoutsidethecellandtothecytoskeleton（viatheattachmentproteintalininsidethecell,theproteinservesasatransmembranelinker.Thefibronectinreceptorbelongstoalargesuperfamilyofhomologousmatrixreceptorscalledintegrins,mostofwhichrecognizeRGDsequencesintheextracellularproteinstheybind.©

GarlandPublishingInc.

Fig.1.Diagramofthecellcoat（glycocalyxwhichismadeupoftheoligosaccharidesidechainsofglycolipidsandintegralmembraneglycoproteins,andpolysaccharidechainsonintegral

proteoglycans.Inaddition,adsorbedglycoproteinsandabsorbedproteoglycans（notshowncontributetotheglycocalyxinsomecells.©

Amongtheglycoproteinsinthemembrane,thereisafamilyofheterodimericglycoproteinreceptorscalledintegrins（Figure2.TheseintegrinshelpbindcellstomainlyRDGsequencesinextracellularmatrix（ECMproteins.TheintegrinreceptorsrequiredivalentionssuchasCa2+,Mg2+,Mn2+forbindingtotheirligands（2.ThisisthereasonwhyEDTAandCa2+,Mg2+freebuffersareusedwhenharvestingcellsinculture.Theinternalpartoftheintegrinreceptor,interactswiththecytoskeleton,whichmakesitpossibleforthecelltospreadoutafterattachment.Themembraneintegral

proteoglycanmoleculesalsobindtoextracellularmatrixcomponents.（Recentlyithasbeenshownthatmembraneboundproteoglycansbindto

growthfactorsandtherebymodulatetheiractivity.Syndecan,aheparansulfateproteoglycanbindstobasicFGFandDecorinandBetaglycanbindsTGF-b（3.Therearealsounrelatedtransmembraneglycoproteinsthatbindtocollagen.

TheECMconsistmainlyoffibrousproteinsembeddedinapolysaccharidegel,whicharesecretedlocally.ThemainECMcomponentsarecollagen,elastin,fibronectin,laminin,andtheglycosaminoglycans.Glycosaminoglycansconsistsoflongunbranchedpolysaccharidechainscomposedofrepeatingdisaccharideunits.Alargenumberofthesugarsaresulfatedorcontaincarboxylgroups.ThisgivestheGAG'

shighnegativecharges.Thefactthatthecellsinvivo,aresurroundedbyhydratedsugargelsandfibrousproteinsisinterestingwhenchoosingmaterialsformicrocarriers.ThefactthatcellsalsointeractwithcollagenhavebeenutilizedwhendesigningCytodex3.

TheattachmentprocessinvitroissummarizedinFigure3.

Fig.3.Simplifiedoutlineofstepsinvolvedinadhesionofanimalcellstoculturesurfaces.Thewholeinvolvesdivalentcationsandglycoproteins（mainlyfibronectin（Fnandvitronectinadsorbedtotheculturesurface.Undercultureconditionstheattachmentglycoproteinoriginatesfromtheserumsupplementinthemedium.MHSissynthesizedbythecells.Withanchorage-dependentcellsproliferationoccursonlyafterthespreadingstep.MHS–multivalentheparinsulphate.

5

Requirementsforanoptimummicrocarrier

Propertiesandcharacteristics

TwotypesofCytodexareavailabletosupportthegrowthofallanchoragedependentanimalcellsforamultitudeofapplications,andthemicrocarriersaredesignedtomeetthespecialrequirementsforthistechnology.Theoptimizedbeadsizeanddensitysupportmaximumcellgrowthrateandcellyield.Abiologicallyinertmatrixprovidesastable-butnonridgid-substrateforstirredcell

6

7

Sizeis