EditedbyCherylD.HelgasonCindyL.MillerBasicCellCultureProtocolsTHIRDEDITIONVolume290METHODSINMOLECULARBIOLOGYTMMETHODSINMOLECULARBIOLOGYTMEditedbyCherylD.HelgasonCindyL.MillerBasicCellCultureProtocolsTHIRDEDITIONBasicCellCultureTechniques11From:MethodsinMolecularBiology,vol.290:BasicCellCultureProtocols,ThirdEditionEditedby:C.D.HelgasonandC.L.Miller©HumanaPressInc.,Totowa,NJ1CultureofPrimaryAdherentCellsandaContinuouslyGrowingNonadherentCellLineCherylD.HelgasonSummaryCellcultureisaninvaluabletoolforinvestigatorsinnumerousfields.Itfacilitatesanalysisofbiologicalpropertiesandprocessesthatarenotreadilyaccessibleattheleveloftheintactorganism.Successfulmaintenanceofcellsinculture,whetherprimaryorimmortalized,requiresknowledgeandpracticeofafewessentialtechniques.Thepur-poseofthischapteristoexplainthebasicprinciplesofcellcultureusingthemainte-nanceofanonadherentcellline,theP815mousemastocytomacellline,andtheisolationandcultureofadherentprimarymouseembryonicfibroblasts(MEFs)asexamples.Proceduresforthawing,culture,determinationofcellnumbersandviability,andcryopreservationaredescribed.KeyWords:Cellculture;nonadherentcellline;adherentcells;P815;primarymouseembryonicfibroblasts;MEF;hemocytometer;viability;subculturing;cryopreservation.1.IntroductionTherearefourbasicrequirementsforsuccessfulcellculture.Eachofthesewillbebrieflyreviewedinthisintroduction.However,amoredetaileddescriptionisbeyondthescopeofthischapter.Instead,thereaderisreferredtooneofanumberofvaluableresourcesthatprovidetheinformationnecessarytoestablishatissueculturelaboratory,aswellasdescribethebasicprinciplesofsteriletechnique(1–4).Thefirstnecessityisawell-establishedandproperlyequippedcellculturefacility.Thelevelofbiocontainmentrequired(Levels1–4)isdependentonthetypeofcellsculturedandtheriskthatthesecellsmightcontain,andtransmit,infectiousagents.Forexample,cultureofprimatecells,transformedhumancelllines,mycoplasma-contaminatedcelllines,andnontestedhumancellsrequireaminimumofaLevel2containmentfacility.Allfac...
