AimAdherentcelllineswillgrowinvitrountiltheyhavecoveredthesurfaceareaavailableorthemediumisdepletedofnutrients.Atthispointthecelllinesshouldbesub-culturedinordertopreventtheculturedying.TosubculturethecellstheyneedtobebroughtintosUSPension.Thedegreeofadhesionvariesfromcelllinetocelllinebutinthemajorityofcasesproteases,e.g.trypsin,areusedtoreleasethecellsfromtheflask.However,thismaynotbeappropriateforsomelineswhereexposuretoproteasesisharmfulorwheretheenzymesusedremovemembraneMarkers/receptorsofinterest.Inthesecasescellsshouldbebroughtintosuspensionintoasmallvolumeofmediummechanicallywiththeaidofcellscrapers.

Materials

• Media–pre-warmedto37ºC(refertotheECACCCellLineDataSheetforthecorrectmedium)
• 70%ethanolinwater(Prod.No.R8382)
• PBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537)
• 0.25%trypsin/EDTAinHBSS,withoutCa₂₊/Mg₂₊(Prod.No.T4049)
• Trypsin(Prod.No.T4424)
• SoybeantrypsinInhibitor(Prod.No.T6414)

Equipment

• Personalprotectiveequipment(sterilegloves,Laboratorycoat,safetyvisor)
• Waterbathsettoappropriatetemperature
• MicroBIOLOGicalsafetycABInetatappropriatecontainmentlevel
• CO₂incubator
• Pre-labeledflasks
• MarkerPen
• Pipettes
• AmpuleRack
• Tissue

Procedure

• Viewculturesusinganinvertedmicroscopetoassessthedegreeofconfluencyandconfirmtheabsenceofbacterialandfungalcontaminants.
• Removespentmedium.
• WashthecellmonolayerwithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537)usingavolumeequivalenttohalfthevolumeofculturemedium.Repeatthiswashstepifthecellsareknowntoadherestrongly.
• Pipettetrypsin/EDTA(Prod.No.T4049)ontothewashedcellmonolayerusing1mlper25cm₂ofsurfacearea.Rotateflasktocoverthemonolayerwithtrypsin.Decanttheexcesstrypsin.
• Returnflasktotheincubatorandleavefor2-10minutes.
• Examinethecellsusinganinvertedmicroscopetoensurethatallthecellsaredetachedandfloating.Thesideoftheflasksmaybegentlytappedtoreleaseanyremainingattachedcells.
• Resuspendthecellsinasmallvolumeoffreshserum-containingmediumtoinactivatethetrypsin.Remove100-200uLandperformacellcount(Protocol6-CellQuantification).
• Transfertherequirednumberofcellstoanewlabeledflaskcontainingpre-warmedmedium(refertoECACCCellLineDataSheetfortherequiredseedingdensity).
• Incubateasappropriateforthecellline.
• Repeatthisprocessasdemandedbythegrowthcharacteristicsofthecellline.

KeyPoints

• Somecultureswhilstgrowingasattachedlinesadhereonlylightlytotheflask,thusitisimportanttoensurethattheculturemediumisretainedandtheflasksarehandledwithcaretopreventthecellsdetachingprematurely.
• AlthoughmostcellswilldetachinthepresenceoftrypsinalonetheEDTAisaddedtoenhancetheactivityoftheenzyme.
• Trypsinisinactivatedinthepresenceofserum.Therefore,itisessentialtoremovealltracesofserumfromtheculturemediumbywashingthemonolayerofcellswithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537).
• Cellsshouldonlybeexposedtotrypsin/EDTA(Prod.No.T4049)longenoughtodetachcells.Prolongedexposurecoulddamagesurfacereceptors.
• Trypsinshouldbeneutralizedwithserumpriortoseedingcellsintonewflasksotherwisecellswillnotattach.
• Trypsinmayalsobeneutralizedbytheadditionofsoybeantrypsininhibitor(Prod.No.T6414),whereanequalvolumeofinhibitorataconcentrationof1mg/mlisaddedtothetrypsinisedcells.Thecellsarethencentrifuged,resuspendedinfreshculturemediumandcountedasabove.Thisisespeciallynecessaryforserum-freecellculture.
• IfaCO₂incubatorisnotavailablegastheflasksfor1-2minwith5%CO₂in95%airfilteredthrougha0.25mfilter.