Ingeneraltermsculturedcellsrequireasterileenvironmentandasupplyofnutrientsforgrowth.InadditionthecultureenvironmentshouldbestableintermsofpHandtemperature.Overthelastthirtyyearsvariousdefinedbasalmediatypeshavebeendevelopedandarenowavailablecommercially.OriginallybalancedsaltsolutionswereusedtomaintaincontractilityofmammalianhearttissueandTyrode’ssaltsolution(Prod.No.T2397)wasdesignedforuseinworkwithprimarymammaliancells.Thesehavesincebeenmodifiedandenrichedwithaminoacids,vitamins,fattyacidsandlipids.Consequentlymediasuitableforsupportingthegrowthofawiderangeofcelltypesarenowavailable.Theprecisemediaformulationshaveoftenbeenderivedbyoptimizingtheconcentrationsofeveryconstituent.Examplesofthedifferentmediaandtheirusesaregiveninthetablebelow(Table2).

Table2.Differenttypesofculturemediumandtheiruses

1BasicConstituentsofmedia

• Inorganicsalts
• Carbohydrates
• AminoAcids
• Vitamins
• Fattyacidsandlipids
• Proteinsandpeptides
• Serum

Eachtypeofconstituentperformsaspecificfunctionasoutlinedbelow:

2InorganicSalts

Theinclusionofinorganicsaltsinmediaperformsseveralfunctions.Primarilytheyhelptoretaintheosmoticbalanceofthecellsandhelpregulatemembranepotentialbyprovisionofsodium,potassiumandcalciumions.Allofthesearerequiredinthecellmatrixforcellattachmentandasenzymecofactors.

3BufferingSystems

MostcellsrequirepHconditionsintherange7.2-7.4andclosecontrolofpHisessentialforoptimumcultureconditions.Therearemajorvariationstothisoptimum.FibroblastspreferahigherpH(7.4-7.7)whereas,continuoustransformedcelllinesrequiremoreacidconditionspH(7.0-7.4).RegulationofpHisparticularlyimportantimmediatelyfollowingcellseedingwhenanewcultureisestablishingandisusuallyachievedbyoneoftwobufferingsystems;(i)a"natural"bufferingsystemwheregaseousCO₂balanceswiththeCO₃/HCO₃contentoftheculturemediumand(ii)chemicalbufferingusingazwitterioncalledHEPES(Prod.No.H4034).

Culturesusingnaturalbicarbonate/CO₂bufferingsystemsneedtobemaintainedinanatmosphereof5-10%CO₂inairusuallysuppliedinaCO₂incubator.bicarbonate/CO₂islowcost,non-toxicandalsoprovidesotherchemicalbenefitstothecells.

HEPES(Prod.No.H4034)hassuperiorbufferingcapacityinthepHrange7.2-7.4butisrelativelyexpensiveandcanbetoxictosomecelltypesathigherconcentrations.HEPES(Prod.No.H4034)bufferedculturesdonotrequireacontrolledgaseousatmosphere.

Mostcommercialculturemediaincludephenolred(Prod.No.P3532/P0290)asapHindicatorsothatthepHstatusofthemediumisconstantlyindicatedbythecolor.Usuallytheculturemediumshouldbechanged/replenishedifthecolorturnsyellow(acid)orpurple(alkali).

4Carbohydrates

Themainsourceofenergyisderivedfromcarbohydratesgenerallyintheformofsugars.Themajorsugarsusedareglucoseandgalactosehoweversomemediacontainmaltoseorfructose.Theconcentrationofsugarvariesfrombasalmediacontaining1g/lto4.5g/linsomemorecomplexmedia.Mediacontainingthehigherconcentrationofsugarsareabletosupportthegrowthofawiderrangeofcelltypes.

5Vitamins

Serumisanimportantsourceofvitaminsincellculture.However,manymediaarealsoenrichedwithvitaminsmakingthemconsistentlymoresuitableforawiderrangeofcelllines.Vitaminsareprecursorsfornumerousco-factors.ManyvitaminsespeciallyBgroupvitaminsarenecessaryforcellgrowthandproliferationandforsomelinesthepresenceofB12isessential.SomemediaalsohaveincreasedlevelsofvitaminsAandE.Thevitaminscommonlyusedinmediaincluderiboflavin,thiamineandbiotin.

6ProteinsandPeptides

Theseareparticularlyimportantinserumfreemedia.Themostcommonproteinsandpeptidesincludealbumin,transferrin,fibronectinandfetuinandareusedtoreplacethosenormallypresentthroughtheadditionofserumtothemedium.

7FattyAcidsandLipids

Likeproteinsandpeptidestheseareimportantinserumfreemediasincetheyarenormallypresentinserum.e.g.cholesterolandsteroidsessentialforspecializedcells.

8TraceElements

Theseincludetraceelementssuchaszinc,copper,seleniumandtricarboxylicacidintermediates.SeleniumisadetoxifierandhelpsremoveoxygenfreerADIcals.

Whilstallmediamaybemadefromthebasicingredientsthisistimeconsumingandmaypredisposetocontamination.Forconveniencemostmediaareavailableasreadymixedpowdersoras10xand1xliquidmedia.AllcommonlyusedmediaarelistedintheSigma-AldrichLifeScienceCatalogue.Ifpowderor10xmediaarepurchaseditisessentialthatthewaterusedtoreconstitutethepowderordilutetheconcentratedliquidisfreefrommineral,organicandmicrobialcontaminants.Itmustalsobepyrogenfree(Prod.No.W3500,water,tissueculturegrade).Inmostcaseswaterpreparedbyreverseosmosisandresincartridgepurificationwithafinalresistanceof16-18Mxissuitable.Oncepreparedthemediashouldbefiltersterilizedbeforeuse.Obviouslypurchasing1xliquidmediadirectfromSigmaeliminatestheneedforthis.

9Serum

Serumisacomplexmixofalbumins,growthfactorsandgrowthinhibitorsandisprobablyoneofthemostimportantcomponentsofcellculturemedium.Themostcommonlyusedserumisfetalbovineserum.Othertypesofserumareavailableincludingnewborncalfserumandhorseserum.Thequality,typeandconcentrationofserumcanallaffectthegrowthofcellsanditisthereforeimportanttoscreenbatchesofserumfortheirABIlitytosupportthegrowthofcells.Inadditionthereareotherteststhatmaybeusedtoaidtheselectionofabatchofserumincludingcloningefficiency,platingefficiencyandthepreservationofcellcharacteristics.

Serumisalsoabletoincreasethebufferingcapacityofculturesthatcanbeimportantforslowgrowingcellsorwheretheseedingdensityislow(e.g.cellcloningexperiments).Italsohelpstoprotectagainstmechanicaldamagewhichmayoccurinstirredculturesorwhilstusingacellscraper.Afurtheradvantageofserumisthewiderangecelltypeswithwhichitcanbeuseddespitethevaryingrequirementsofdifferentculturesintermsofgrowthfactors.Inadditionserumisabletobindandneutralizetoxins.However,serumissubjecttobatch-batchvariationthatmakesstandardizationofproductionprotocolsdifficult.Thereisalsoariskofcontaminationassociatedwiththeuseofserum.Theseriskscanbeminimizedbyobtainingserumfromareputablesourcesincesuppliersoflargequantitiesofserumperformabatteryofqualitycontroltestsandsupplyacertificateofanalysiswiththeserum.Inparticularserumisscreenedforthepresenceofbovineviraldiarrhoeavirus(BVDV)andmycoplasma.Heatinactivationofserum(incubationat56ºCfor30minutes)canhelptoreducetheriskofcontaminationsincesomevirusesareinactivatedbythisprocess.Howevertheroutineuseofheatinactivatedserumisnotanabsoluterequirementforcellculture.Theuseofserumalsohasacostimplicationnotonlyintermsofmediumformulationbutalsoindownstreamprocessing.A10%FBSsupplementcontributes4.8mgofproteinpermilliliterofculturefluid,whichcomplicatesdownstreamprocessingprocedures.

10Guidelinesforserumuse

Fetalbovineserum(FBS)hasbeenusedtoprepareanumberofBIOLOGicalandhasanexcellentrecordofsafety.TherecognitionofBovinespongiformencepalopathy(BSE)in1986andit’ssubsequentspreadintocontinentalEuropealongsidetheannouncementoftheprobablelinkbetweenBSEandanewvariantofCreutzfeldtJacobdiseaseinHumans,stimulatedanincreasedconcernaboutsafesourcingofallbovinematerials.In1993theFoodandDrugAdmiNISTration(FDA)"recommendedagainsttheuseofbovinederivedmaterialsfromcattlewhichhaveresidedin,ororiginatedfromcountrieswhereBSEhasbeendiagnosed.Thecurrent(EuropeanUnion)EUguidelinesonviralsafetyfocusonsourcing,testingandpayingparticularattentiontothepotentialriskofcrosscontaminationduringslaughteringorcollectionofthestartingtissue.AsfarasBSEisconcerned,theEUguidelinesonminimizingtheriskofBSEtransmissionviamedicinalproducts,CPMP/BWP/877/96,recommendsthemainmeasurestobeimplementedinordertoestablishthesafetyofbovinematerialversustheBSErisk.Again,similarlythefocusisongeographicalorigin,theageoftheanimals,thebreedingandslaughteringconditions,thetissuetobeusedandtheconditionsofit’sprocessing.

TheuseofFBSinproductionprocessesofmedicinalproductsisacceptableprovidedgooddocumentationonsourcing,ageoftheanimalsandtestingfortheabsenceofadventitiousagentsissubmitted.AllresponsIBLesuppliersofFBSforbio-pharmaceuticalapplicationswillprovidesuchdocumentation.

RecentregulatoryrequirementsinEuropestresstheimportanceofjustifyingtheuseofmaterialofbovine,caprineorovineoriginintheproductionofpharmaceuticalproducts.Thus,althoughFBShasbeenusedformanyyearsintheproductionprocessofmanymedicinalproductssuchasviralvaccinesandrecombinantDNAproducts,atpresentthereisajustifiedtrendtoremoveallmaterialofanimaloriginfrommanufacturingprocesses.Sigma-Aldrichhasrecognizedthisgrowingtrendandworkscloselywithcustomerstooptimizeanimalfreemediaformulationstomeeteachcustomer’scellculturerequirements.

SimilarlytheFDAhassimilarguidelineswhenacceptingregulatorysubmissions.TheFDAregulatesallmedicinalproductsforHumanuse,suchastherapeutics,vaccinesanddiagnostics,and,usually,theUnitedStatesDepartmentAgriculture(USDA)arenotinvolved.

TheUSDAregulatesallmedicinalproductsforveterinaryuseorforagriculturaluse.Similarly,theUSDAregulatesallproductsthatcontainaprimarycomponentofanimalorigin.Itisimportantthatyouareawareoftheuseandrestrictionswhenusingserum,forfurtherinformationcontactyoulocalSigma-Aldrichsalesofficeforspecificinformationontheserumbeingused(contactinformationisatthebackofthishandbook).WithspecificreferencetoserumtheUSDAhasdeclaredthatformaterialswhichfallundertheirjurisdiction,onlybiologicalproductsmanufacturedusingserumfromapprovedcountriesoforiginbeallowedintotheUSA.

11OriginofSerum

USA/Canada,NewZealand,FinlandandDenmark-Nosafetytestingrequired.Australia,Mexico,CentralAmerica-Safetytestingmayberequired,dependingonthegeographicalregionwheretheserumwascollected.

Sigma-AldrichcarriesoutallsafetytestrequirementsstipulatedbytheUSDAinUSAlaboratories.IfnecessarySigma-AldrichwillassistcustomersinobtainingapprovalfromtheUSDA,onanybatchofserumofAustralianoriginsuppliedbySigma-Aldrich.

Sigma-AldrichsourcesserumfromthestatesofVictoriaandTasmania.TasmaniawillshortlybeconsideredaseparategeographicalareawithinAustraliaeliminatingtherequirementforanysafetytesting.