TYPESOFCELLSGROWNINCULTURE

Tissuecultureisoftenagenerictermthatreferstobothorgancultureandcellcultureandthetermsareoftenusedinterchangeably.CellculturesarederivedfromeitherprimarytissueexplantsorcellsUSPensions.Primarycellculturestypicallywillhaveafinitelifespaninculturewhereascontinuouscelllinesare,bydefinition,abnormalandareoftentransformedcelllines.

II.WORKAREAANDEQUIPMENT

A.Laminarflowhoods.Therearetwotypesoflaminarflowhoods,verticalandhorizontal,andbothtypesofhoodsareavailableintheAppliedMolecularBIOLOGylaboratory.Theverticalhood,alsoknownasabiologysafetycABInet,isbestforworkingwithhazardousorganismssincetheaerosolsthataregeneratedinthehoodarefilteredoutbeforetheyarereleasedintothesurroundingenvironment.Horizontalhoodsaredesignedsuchthattheairflowsdirectlyattheoperatorhencetheyarenotusefulforworkingwithhazardousorganismsbutarethebestprotectionforyourcultures.BothtypesofhoodshavecontinuousdisplacementofairthatpassesthroughaHEPA(highefficiencyparticle)filterthatremovesparticulatesfromtheair.Inaverticalhood,thefilteredairblowsdownfromthetopofthecabinet;inahorizontalhood,thefilteredairblowsoutattheoperatorinahorizontalfashion.NOTE:thesearenotfumehoodsandshouldnotbeusedforvolatileorexplosivechemicals.Theyshouldalsoneverbeusedforbacterialorfungalwork.Thehoodsareequippedwithashort-waveUVlightthatcanbeturnedonforafewminutestosterilizethesurfacesofthehood,butbeawarethatonlyexposedsurfaceswillbeaccessIBLetotheUVlight.DonotputyourhandsorfacenearthehoodwhentheUVlightisonastheshortwavelightcancauseskinandeyedamage.Thehoodsshouldbeturnedonabout10-20minutesbeforebeingused.Wipedownallsurfaceswithethanolbeforeandaftereachuse.Keepthehoodasfreeofclutteraspossiblebecausethiswillinterferewiththelaminarflowairpattern.

B.CO₂Incubators.Thecellsaregrowninanatmosphereof5-10%CO₂becausethemediumusedisbufferedwithsodiumbicarbonate/carbonicacidandthepHmustbestrictlymaintained.Cultureflasksshouldhaveloosenedcapstoallowforsufficientgasexchange.Cellsshouldbeleftoutoftheincubatorforaslittletimeaspossibleandtheincubatordoorsshouldnotbeopenedforverylong.Thehumiditymustalsobemaintainedforthosecellsgrowingintissueculturedishessoapanofwateriskeptfilledatalltimes.

C.Microscopes.Invertedphasecontrastmicroscopesareusedforvisualizingthecells.Microscopesshouldbekeptcoveredandthelightsturneddownwhennotinuse.Beforeusingthemicroscopeorwheneveranobjectiveischanged,checkthatthephaseringsarealigned.

D.Preservation.Cellsarestoredinliquidnitrogen(seeSectionIII-Preservationandstorage).

E.Vessels.Anchoragedependentcellsrequireanontoxic,biologicallyinert,andopticallytransparentsurfacethatwillallowcellstoattachandallowmovementforgrowth.Themostconvenientvesselsarespecially-treatedpolystyreneplasticthataresuppliedsterileandaredisposable.Theseincludepetridishes,multi-wellplates,microtiterplates,rollerbottles,andscrewcapflasks-T-25,T-75,T-150(cm²ofsurfacearea).Suspensioncellsareeithershaken,stirred,orgrowninvesselsidenticaltothoseusedforanchorage-dependentcells.

III.PRESERVATIONANDSTORAGE.LiquidN₂isusedtopreservetissueculturecells,eitherintheliquidphase(-196EC)orinthevaporphase(-156EC).Freezingcanbelethaltocellsduetotheeffectsofdamagebyicecrystals,alterationsintheconcentrationofelectrolytes,dehydration,andchangesinpH.Tominimizetheeffectsoffreezing,severalprecautionsaretaken.First,acryoprotectiveagentwhichlowersthefreezingpoint,suchasglycerolorDMSO,isadded.Thefreezingmediumwetypicallyuseis90%serum,10%DMSO.Inaddition,itisbesttousehealthycellsthataregrowinginlogphaseandtoreplacethemedium24hoursbeforefreezing.Also,thecellsareslowlycooledfromroomtemperatureto-80ECtoallowthewatertomoveoutofthecellsbeforeitfreezes.Theoptimalrateofcoolingis1E-3ECperminute.Somelabshavefancyfreezingchamberstoregulatethefreezingattheoptimalratebyperiodicallypulsinginliquidnitrogen.WeusealowtechdevicecalledaMr.Frosty.TheMr.Frostyisfilledwith200mlofisopropanolatroomtemperatureandthefreezingvialscontainingthecellsareplacedinthecontainerandthecontainerisplacedinthe-80ECfreezer.Theeffectoftheisopropanolistoallowthetubestocometothetemperatureofthefreezerslowly,atabout1ECperminute.Oncethecontainerhasreached-80EC(about4hoursor,moreconveniently,overnight)thevialsareremovedfromtheMr.Frostyandimmediatelyplacedintheliquidnitrogenstoragetank.Cellsarestoredatliquidnitrogentemperaturesbecausethegrowthoficecrystalsisretardedbelow-130EC.Tomaximizerecoveryofthecellswhenthawing,thecellsarewarmedveryquicklybyplacingthetubedirectlyfromtheliquidnitrogencontainerintoa37ECwaterbathwithmoderateshaking.Assoonasthelasticecrystalismelted,thecellsareimmediatelydilutedintoprewarmedmedium.

IV.MAINTENANCE

Culturesshouldbeexamineddaily,observingthemorphology,thecolorofthemediumandthedensityofthecells.Atissueculturelogshouldbemaintainedthatisseparatefromyourregularlaboratorynotebook.Thelogshouldcontain:thenameofthecellline,themediumcomponentsandanyalterationstothestandardmedium,thedatesonwhichthecellsweresplitand/orfed,acalculationofthedoublingtimeoftheculture(thisshouldbedoneatleastonceduringthesemester),andanyobservationsrelativetothemorphology,etc.

A.Growthpattern.Cellswillinitiallygothroughaquiescentorlagphasethatdependsonthecelltype,theseedingdensity,themediacomponents,andprevioushandling.Thecellswillthengointoexponentialgrowthwheretheyhavethehighestmetabolicactivity.Thecellswillthenenterintostationaryphasewherethenumberofcellsisconstant,thisischaracteristicofaconfluentpopulation(whereallgrowthsurfacesarecovered).

B.Harvesting.Cellsareharvestedwhenthecellshavereachedapopulationdensitywhichsuppressesgrowth.Ideally,cellsareharvestedwhentheyareinasemi-confluentstateandarestillinlogphase.Cellsthatarenotpassagedandareallowedtogrowtoaconfluentstatecansometimelagforalongperiodoftimeandsomemayneverrecover.Itisalsoessentialtokeepyourcellsashappyaspossibletomaximizetheefficiencyoftransformation.Mostcellsarepassaged(oratleastfed)threetimesaweek.

1.Suspensionculture.Suspensionculturesarefedbydilutionintofreshmedium.

2.Adherentcultures.Adherentculturesthatdonotneedtobedividedcansimplybefedbyremovingtheoldmediumandreplacingitwithfreshmedium.Whenthecellsbecomesemi-confluent,severalmethodsareusedtoremovethecellsfromthegrowingsurfacesothattheycanbediluted:

Mechanical-Arubberspatulacanbeusedtophysicallyremovethecellsfromthegrowthsurface.Thismethodisquickandeasybutisalsodisruptivetothecellsandmayresultinsignificantcelldeath.Thismethodisbestwhenharvestingmanydifferentsamplesofcellsforpreparingextracts,i.e.,whenviabilityisnotimportant.

Proteolyticenzymes-Trypsin,Collagenase,orpronase,usuallyincombinationwithEDTA,causescellstodetachfromthegrowthsurface.Thismethodisfastandreliablebutcandamagethecellsurfacebydigestingexposedcellsurfaceproteins.Theproteolysisreactioncanbequicklyterminatedbytheadditionofcompletemediumcontainingserum

EDTA-EDTAalonecanalsobeusedtodetachcellsandseemstobegentleronthecellsthantrypsin.

Thestandardprocedurefordetachingadherentcellsisasfollows:

1.Visuallyinspectdaily

2.Releasecellsfrommonolayersurface

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

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

	a.RemovemediumfromculturedishandwashcellsinabalancedsaltsolutionwithoutCa++orMg++.Removethewashsolution.
	b..Addenoughtrypsin-EDTAsolutiontocoverthebottomoftheculturevesselandthenpourofftheexcess.
	c.Placecultureinthe37ECincubatorfor2minutes.
	d.Monitorcellsundermicroscope.Cellsarebeginningtodetachwhentheyappearrounded.
	e.Assoonascellsareinsuspension,immediatelyaddculturemediumcontainingserum.Washcellsoncewithserumcontainingmediumanddiluteasappropriate(generally4-20fold).

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

	a.Removecellsfromfrozenstorageandquicklythawina37ECwaterbathbygentlyagitatingvial.
	b.Assoonastheicecrystalsmelt,pipetgentlyintoacultureflaskcontainingprewarmedgrowthmedium.
	c.Logoutcellsinthe"LiquidNitrogenFreezerLog"Book.

	a.Harvestcellsasusualandwashoncewithcompletemedium.
	b.Resuspendcellsincompletemediumanddeterminecellcount/viability.
	c.Centrifugeandresuspendinice-coldfreezingmedium:90%calfserum/10%DMSO,at106-107cells/ml.Keepcellsonice.
	dTransfer1mlaliquotstofreezervialsonice.
	e.PlaceinaMr.Frostycontainerthatisatroomtemperatureandthathassufficientisopropanol.
	f.PlacetheMr.Frostyinthe-70ECfreezerovernight.Note:Cellsshouldbeexposedtofreezingmediumforaslittletimeaspossiblepriortofreezing
	gNextday,transfertoliquidnitrogen(DON"TFORGET)andloginthe"LiquidNitrogenFreezerLog"Book.

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