a.washoncewithabuffersolutionb.treatwithdissociatingagentc.observecellsunderthemicroscope.Incubateuntilcellsbecomeroundedandloosenwhenflaskisgentlytappedwiththesideofthehand.d.Transfercellstoaculturetubeanddilutewithmediumcontainingserum.e.Spindowncells,removesupernatantandreplacewithfreshmedium.f.Countthecellsinahemacytometer,anddiluteasappropriateintofreshmedium.

C.Mediaandgrowthrequirements

1.Physiologicalparameters

A.temperature-37Cforcellsfromhomeother

B.pH-7.2-7.5andosmolalityofmediummustbemaintained

C.humidityisrequired

D.gasphase-bicarbonateconc.andCO₂tensioninequilibrium

E.visiblelight-canhaveanadverseeffectoncells;lightinducedproductionoftoxiccompoundscanoccurinsomemedia;cellsshouldbeculturedinthedarkandexposedtoroomlightaslittleaspossible;

2.Mediumrequirements:(oftenempirical)

A.Bulkions-Na,K,Ca,Mg,Cl,P,BicarborCO₂B.Traceelements-iron,zinc,seleniumC.sugars-glucoseisthemostcommonD.aminoacids-13essentialE.vitamins-B,etc.F.choline,inositolG.serum-containsalargenumberofgrowthpromotingactivitiessuchasbufferingtoxicnutrientsbybindingthem,neutralizestrypsinandotherproteases,hasundefinedeffectsontheinteractionbetweencellsandsubstrate,andcontainspeptidehormonesorhormone-likegrowthfactorsthatpromotehealthygrowth.H.antibiotics-althoughnotrequiredforcellgrowth,antibioticsareoftenusedtocontrolthegrowthofbacterialandfungalcontaminants.

3.Feeding-2-3times/week.

4.Measurementofgrowthandviability.Theviabilityofcellscanbeobservedvisuallyusinganinvertedphasecontrastmicroscope.Livecellsarephasebright;suspensioncellsaretypicallyroundedandsomewhatsymmetrical;adherentcellswillformprojectionswhentheyattachtothegrowthsurface.Viabilitycanalsobeassessedusingthevitaldye,trypanblue,whichisexcludedbylivecellsbutaccumulatesindeadcells.Cellnumbersaredeterminedusingahemocytometer.

V.SAFETYCONSIDERATIONS

	Assumeallculturesarehazardoussincetheymayharborlatentvirusesorotherorganismsthatareuncharacterized.Thefollowingsafetyprecautionsshouldalsobeobserved:
	pipetting:usePipetteaidstopreventingestionandkeepaerosolsdowntoaminimum
	noeating,drinking,orsmoking
	washhandsafterhandlingculturesandbeforeleavingthelab
	decontaminateworksurfaceswithdisinfectant(beforeandafter)
	autoclaveallwaste
	usebiologicalsafetycabinet(laminarflowhood)whenworkingwithhazardousorganisms.Thecabinetprotectsworkerbypreventingairbornecellsandvirusesreleasedduringexperimentalactivityfromescapingthecabinet;thereisanairbarrieratthefrontopeningandexhaustairisfilteredwithaHEPAfiltermakesurecabinetisnotoverloadedandleaveexhaustgrillsinthefrontandthebackclear(helpstomaintainauniformairflow)
	useaseptictechnique
	disposeofallliquidwasteaftereachexperimentandtreatwithbleach

REFERENCES:

R.IanFreshney,CultureofAnimalcells:Amanualofbasictechniques,Wiley-Liss,1987.

VI.TISSUECULTUREPROCEDURES

Eachstudentshouldmaintainhisowncellsthroughoutthecourseoftheexperiment.Thesecellsshouldbemonitoreddailyformorphologyandgrowthcharacteristics,fedevery2to3days,andsubculturedwhennecessary.Aminimumoftwo25cm²flasksshouldbecarriedforeachcellline;thesecellsshouldbeexpandedasnecessaryforthetransfectionexperiments.Eachtimethecellsaresubcultured,aviablecellcountshouldbedone,thesubculturedilutionsshouldbenoted,and,afterseveralpassages,adoublingtimedetermined.Assoonasyouhaveenoughcells,severalvialsshouldbefrozenawayandstoredinliquidN₂.Onevialfromeachfreezedownshouldbethawed1-2weeksafterfreezingtocheckforviability.Thesefrozenstockswillprovetobevitalifanyofyourculturesbecomecontaminated.

Procedures:1.Mediapreparation.Eachstudentwillberesponsibleformaintaininghisownstockofcellculturemedia;theparticulartypeofmedia,theseratypeandconcentration,andothersupplementswilldependonthecellline.Donotsharemediawithyoupartner(oranyoneelse)becauseifacultureorabottleofmediagetscontaminated,youhavenoback-up.Mostofthemediacomponentswillbepurchasedpreparedandsterile.Ingeneral,allyouneedtodoissterilycombineseveralsterilesolutions.Totestforsterilityafteraddingallcomponents,pipetseveralmlsfromeachmediabottleintoasmallsterilepetridishorculturetubeandincubateat37ECforseveraldays.Useonlymediathathasbeensterilitytested.Forthisreason,youmustanticipateyourcultureneedsinadvancesoyoucanpreparethereagentsnecessary.But,pleasetrynottowastemedia.Anticipateyourneedsbutdon"tmakemorethanyouneed.Tissueculturereagentsareveryexpensive;forexample,bovinefetalcalfserumcost~$200/500ml.Somecellcultureadditiveswillbeprovidedinapowderedform.Theseshouldbereconstitutedtotheappropriateconcentrationwithdouble-distilledwater(ormedium,asappropriate)andfiltered(inasterilehood)througha0-22μmfilter.

Allmediapreparationandothercellcultureworkmustbeperformedinalaminarflowhood.Beforebeginningyourwork,turnonblowerforseveralminutes,wipedownallsurfaceswith70%ethanol,andethanolwashyourcleanhands.Useonlysterilepipets,disposabletesttubesandautoclavedpipettipsforcellculture.Allculturevessels,testtubes,pipettipboxes,stocksofsterileEppendorfs,etc.shouldbeopenedonlyinthelaminarflowhood.Ifsomethingisopenedelsewhereinthelabbyaccident,youcanprobablyassumeitscontaminated.Ifsomethingdoesbecomecontaminated,immediatelydiscardthecontaminatedmaterialsintothebiohazardcontainerandnotifytheinstructor.

2.Growthandmorphology.Visuallyinspectcellsfrequently.Cellcultureissometimesmoreanartthanascience.Gettoknowwhatmakesyourcellshappy.FrequentfeedingisimportantformaintainingthepHbalanceofthemediumandforeliminatingwasteproducts.Cellsdonottypicallyliketobetooconfluentsotheyshouldbesubculturedwhentheyareinasemi-confluentstate.Ingeneral,mammaliancellsshouldbehandledgently.Theyshouldnotbevortexed,vigorouslypipettedorcentrifugedatgreaterthan1500g.

3.Cellfeeding.Useprewarmedmediaandhavecellsoutoftheincubatorforaslittletimeaspossible.Use10-15mlforT-25"s,25-35mlforT-75"sand50-60mlforT-150"s.a.Suspensioncultures.Feedingandsubculturingsuspensionculturesaredonesimultaneously.Aboutevery2-3days,dilutethecellsintofreshmedia.Thedilutionyouusewilldependonthedensityofthecellsandhowquicklytheydivide,whichonlyyoucandetermine.Typically1:4to1:20dilutionsareappropriateformostcelllines.b.Adherentcells.Aboutevery2-3days,pouroffoldmediafromcultureflasksandreplacewithfreshmedia.Subculturecellsasdescribedbelowbeforeconfluencyisreached.

4.Subculturingadherentcells.Whenadherentcellsbecomesemi-confluent,subcultureusing2mMEDTAortrypsin/EDTA.

Trypsin-EDTA:

	a.RemovemediumfromculturedishandwashcellsinabalancedsaltsolutionwithoutCa++orMg++.Removethewashsolution.
	b.Addenoughtrypsin-EDTAsolutiontocoverthebottomoftheculturevesselandthenpourofftheexcess.
	c.Placecultureinthe37°Cincubatorfor2minutes.
	d.Monitorcellsundermicroscope.Cellsarebeginningtodetachwhentheyappearrounded.
	e.Assoonascellsareinsuspension,immediatelyaddculturemediumcontainingserum.Washcellsoncewithserumcontainingmediumanddiluteasappropriate(generally4-20fold).

EDTAalone:

	a.Preparea2mMEDTAsolutioninabalancedsaltsolution(i.e.,PBSwithoutCa++orMg++).
	b.Removemediumfromculturevesselbyaspirationandwashthemonolayertoremovealltracesofserum.Removesaltsolutionbyaspiration.
	c.DispenseenoughEDTAsolutionintoculturevesselstocompletelycoverthemonolayerofcells.
	d.Thecoatedcellsareallowedtoincubateuntilcellsdetachfromthesurface.Progresscanbecheckedbyexaminationwithaninvertedmicroscope.Cellscanbegentlynudgedbybangingthesideoftheflaskagainstthepalmofthehand.
	e.Dilutecellswithfreshmediumandtransfertoasterilecentrifugetube.
	f.Spincellsdown,removesupernatant,andresuspendinculturemedium(orfreezingmediumifcellsaretobefrozen).Diluteasappropriateintocultureflasks.

5.Thawingfrozencells.

	a.Removecellsfromfrozenstorageandquicklythawina37°Cwaterbathbygentlyagitatingvial.
	b.Assoonastheicecrystalsmelt,pipetgentlyintoacultureflaskcontainingprewarmedgrowthmedium.
	c.Logoutcellsinthe"LiquidNitrogenFreezerLog"Book.

6.Freezingcells.

	a.Harvestcellsasusualandwashoncewithcompletemedium.
	b.Resuspendcellsincompletemediumanddeterminecellcount/viability.
	c.Centrifugeandresuspendinice-coldfreezingmedium:90%calfserum/10%DMSO,at106-107cells/ml.Keepcellsonice.
	d.Transfer1mlaliquotstofreezervialsonice.
	e.PlaceinaMr.Frostycontainerthatisatroomtemperatureandthathassufficientisopropanol.
	f.PlacetheMr.Frostyinthe-70°Cfreezerovernight.Note:Cellsshouldbeexposedtofreezingmediumforaslittletimeaspossiblepriortofreezing
	gNextday,transfertoliquidnitrogen(DON"TFORGET)andloginthe"LiquidNitrogenFreezerLog"Book.

7.Viablecellcounts.USINGAHEMOCYTOMETERTODETERMINETOTALCELLCOUNTSANDVIABLECELLNUMBERS(Reference:Sigmacatalogue)Trypanblueisoneofseveralstainsrecommendedforuseindyeexclusionproceduresforviablecellcounting.Thismethodisbasedontheprinciplethatlivecellsdonottakeupcertaindyes,whereasdeadcellsdo.

1.Prepareacellsuspension,eitherdirectlyfromacellcultureorfromaconcentratedordilutedsuspension(dependingonthecelldensity)andcombine20μlofcellswith20μloftrypanbluesuspension(0.4%).Mixthoroughlyandallowtostandfor5-15minutes.

2.Withthecoverslipinplace,transferasmallamountoftrypanblue-cellsuspensiontobothchambersofthehemocytometerbycarefullytouchingtheedgeofthecoverslipwiththepipettetipandallowingeachchambertofillbycapillaryaction.Donotoverfillorunderfillthechambers.3.Startingwith1chamberofthehemocytometer,countallthecellsinthe1mmcentersquareandfour1mmcornersquare.Keepaseparatecountofviableandnon-viablecells.4.Iftherearetoomanyortoofewcellstocount,repeattheprocedureeitherconcentratingordilutingtheoriginalsuspensionasappropriate.5.Thecircleindicatestheapproximateareacoveredat100Xmicroscopemagnification(10Xocularand10Xobjective).Includecellsontopandlefttouchingmiddleline.Donotcountcellstouchingmiddlelineatbottomandright.Count4cornersquaresandmiddlesquareinbothchambersandcalculatetheaverage.6.Eachlargesquareofthehemocytometer,withcover-slipinplace,representsatotalvolumeof0.1mm³or10^-4cm³.Since1cm³isequivalenttoapproximately1ml,thetotalnumberofcellspermlwillbedeterminedusingthefollowingcalculations:Cells/ml=averagecellcountpersquarexdilutionfactorx10⁴;

Totalcells=cells/mlxtheoriginalvolumeoffluidfromwhichthecellsamplewasremoved;%Cellviability=totalviablecells(unstained)/totalcellsx100.