1BasicTechniques-The?Do?sandDon"ts?ofCellCulture

Givenbelowareafewoftheessential"do?sanddon?ts"ofcellculture.Someofthesearemandatorye.g.useofpersonalprotectiveequipment(PPE).Manyofthemarecommonsenseandapplytoalllaboratoryareas.Howeversomeofthemarespecifictotissueculture.

TheDo?s

1. Usepersonalprotectiveequipment,(laboratorycoat/gown,glovesandeyeprotection)atalltimes.Inaddition,thermallyinsulatedgloves,full-facevisorandsplash-proofapronshouldbewornwhenhandlingliquidnitrogen.
2. Alwaysusedisposablecapstocoverhair.
3. WeardedicatedPPEfortissueculturefacilityandkeepseparatefromPPEworninthegenerallaboratoryenvironment.Theuseofdifferentcoloredgownsorlaboratorycoatsmakesthiseasiertoenforce.
4. Keepallworksurfacesfreeofclutter.
5. Correctlylabelreagentsincludingflasks,mediumandampuleswithcontentsanddateofpreparation.
6. Onlyhandleonecelllineatatime.Thiscommon-sensepointwillreducethepossibilityofcrosscontaminationbymislabelingetc.ItwillalsoreducethespreadofbacteriaandmycoplasmabythegenerationofaerosolsacrossnumerousopenedmediabottlesandflasksinthecABInet.
7. Cleantheworksurfaceswithasuitabledisinfectant(e.g.70%ethanol)betweenoperationsandallowaminimumof15minutesbetweenhandlingdifferentcelllines.
8. WhereverpossIBLemaintainseparatebottlesofmediaforeachcelllineincultivation.
9. Examineculturesandmediadailyforevidenceofgrossbacterialorfungalcontamination.Thisincludesmediumthathasbeenpurchasedcommercially.
10. QualityControlallmediaandreagentspriortouse.
11. Keepcardboardpackagingtoaminimuminallcellcultureareas.
12. Ensurethatincubators,cabinet,centrifugesandmicroscopesarecleanedandservicedatregularintervals.
13. Testcellsformycoplasmaonaregularbasis.

TheDon?ts

1. Donotcontinuouslyuseantibioticsinculturemediumasthiswillinevitablyleadtotheappearanceofantibioticresistantstrainsandmayrenderacelllineuselessforcommercialpurposes.
2. Don?tallowwastetoaccumulateparticularlywithinthemicroBIOLOGicalsafetycabinetorintheincubators.
3. Don"thavetoomanypeopleinthelabatanyonetime.
4. Don"thandlecellsfromunauthenticatedsourcesinthemaincellculturesuite.Theyshouldbehandledinquarantineuntilqualitycontrolchecksarecomplete.
5. Avoidkeepingcelllinescontinuallyinculturewithoutreturningtofrozenstock.
6. Avoidcellculturebecomingfullyconfluent.Alwayssub-cultureat70-80%confluencyorasadvisedonECACC"scellculturedatasheet.
7. Donotallowmediatogooutofdate.Shelflifeisonly6weeksat+4ºConceglutamineandserumisadded.
8. AvoidwaterbathsfrombecomingdirtybyusingSigmaClean(Prod.No.S5525).
9. Don?tallowessentialequipmenttobecomeoutofcalibration.Ensuremicrobiologicalsafetycabinetsaretestedregularly.

BacktoTop

2Protocol1-AsepticTechniqueandGoodCellCulturePractice

AimToensureallcellcultureproceduresareperformedtoastandardthatwillpreventcontaminationfrombacteria,fungiandmycoplasmaandcrosscontaminationwithothercelllines.

Materials

• Chloros/Preseptsolution(2.5g/l)
• 1%formaldehydebaseddisinfectante.g.Virkon,Tegador
• 70%ethanolinwater(Prod.No.R8382)

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Microbiologicalsafetycabinetatappropriatecontainmentlevel

Procedure

1. Sanitizethecabinetusing70%ethanolbeforecommencingwork.
2. Sanitizeglovesbywashingthemin70%ethanolandallowingtoairdryfor30secondsbeforecommencingwork.
3. Putallmaterialsandequipmentintothecabinetpriortostartingworkaftersanitizingtheexteriorsurfaceswith70%ethanol.
4. Whilstworkingdonotcontaminateglovesbytouchinganythingoutsidethecabinet(especiallyfaceandhair).Ifglovesbecomecontaminatedre-sanitizewith70%ethanolasabovebeforeproceeding.
5. Discardglovesafterhandlingcontaminatedculturesandattheendofallcellcultureprocedures.
6. Equipmentinthecabinetorthatwhichwillbetakenintothecabinetduringcellcultureprocedures(mediabottles,Pipettetipboxes,pipetteaids)shouldbewipedwithtissuesoakedwith70%ethanolpriortouse.
7. Movementwithinandimmediatelyoutsidethecabinetmustnotberapid.Slowmovementwillallowtheairwithinthecabinettocirculateproperly.
8. Speech,sneezingandcoughingmustbedirectedawayfromthecabinetsoasnottodisrupttheairflow.
9. Aftercompletingworkdisinfectallequipmentandmaterialbeforeremovingfromthecabinet.Spraytheworksurfacesinsidethecabinetwith70%ethanolandwipedrywithtissue.Disposeoftissuebyautoclaving.
10. Cellculturediscardinchloros(10,000)ppmmustbekeptinthecabinetforaminimumoftwohours(preferablyovernight)priortodiscardingdownthesinkwithcopiousamountsofwater.
11. PeriodicallycleanthecabinetsurfaceswithadisinfectantsuchasPresept,TegadororVirkonorfumigatethecabinetaccordingtothemanufacturersinstructions.Howeveryoumustensurethatitissafetofumigateyourownlaboratoryenvironmentduetothegenerationofgaseousformaldehyde,consultyouron-siteHealthandSafetyAdvisor.

3Protocol2-ResuscitationofFrozenCellLines

Clickhereforaschematicdiagramof"ResuscitationofFrozenCellLines"

AimManyculturesobtainedfromaculturecollection,suchasECACC,willarrivefrozenandinordertousethemthecellsmustbethawedandputintoculture.Itisvitaltothawcellscorrectlyinordertomaintaintheviabilityofthecultureandenabletheculturetorecovermorequickly.Somecryoprotectants,suchasDMSO(Prod.No.D2650),aretoxicabove4ºCthereforeitisessentialthatculturesarethawedquicklyanddilutedinculturemediumtominimizethetoxiceffects.

Materials

• Media?pre-warmedtotheappropriatetemperature(refertotheECACCCellLineDataSheetforthecorrectmediumandsizeofflasktoresuscitationinto.)
• 70%ethanolinwater(Prod.No.R8382)
• DMSO(Prod.No.D2650)

Equipment

• Personalprotectiveequipment(sterilegloves,Laboratorycoat,safetyvisor)
• Waterbathsettoappropriatetemperature
• Microbiologicalsafetycabinetatappropriatecontainmentlevel
• CO₂incubator
• Prelabeledflasks
• MarkerPen
• Pipettes
• AmpuleRack
• Tissue

Procedure

1. ReadTechnicaldatasheettoestablishspecificrequirementsforyourcellline.
2. Preparetheflasksasappropriate(informationontechnicaldatasheet).Labelwithcelllinename,passagenumberanddate.
3. Collectampuleofcellsfromliquidnitrogenstoragewearingappropriateprotectiveequipmentandtransfertolaboratoryinasealedcontainer.
4. Stillwearingprotectiveclothing,removeampulefromcontainerandplaceinawaterbathatanappropriatetemperatureforyourcelllinee.g.37ºCformammaliancells.Submergeonlythelowerhalfoftheampule.Allowtothawuntilasmallamountoficeremainsinthevial-usually1-2minutes.TransfertoclassIIsafetycabinet.
5. Wipetheoutsideoftheampulewithatissuemoistened(notexcessively)with70%alcoholholdtissueoverampuletoloosenlid.
6. Slowly,dropwise,pipettecellsintopre-warmedgrowthmediumtodiluteouttheDMSO(Prod.No.D2650)(flaskspreparedinStep2).
7. IncubateattheappropriatetemperatureforspeciesandappropriateconcentrationofCO₂inatmosphere.
8. Examinecellsmicroscopically(phasecontrast)after24hoursandsub-cultureasnecessary.

KeyPoints

1. Mosttextbooksrecommendwashingthethawedcellsinmediatoremovethecryoprotectant.Thisisonlynecessaryifthecryoprotectantisknowntohaveanadverseeffectonthecells.Insuchcasesthecellsshouldbewashedinmediabeforebeingaddedtotheirfinalcultureflasks.SeeProtocol7forfurtherdetails.
2. Donotuseanincubatortothawcellculturessincetherateofthawingachievedistooslowresultinginalossofviability.
3. IfaCO₂incubatorisnotavailablegastheflasksfor1-2minuteswith5%CO₂in95%airfilteredthrougha0.25mfilter.
4. Forsomeculturesitisnecessarytosubculturebeforeconfluenceisreachedinordertomaintaintheircharacteristicse.g.thecontactinhibitionofNIH3T3(Prod.No.93061524)cellsislostiftheyareallowedtoreachconfluencerepeatedly.

4Protocol3-SubcultureofAdherentCellLines

Clickhereforaschematicdiagramof"SubcultureofAdherentCellLines"

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

1. Viewculturesusinganinvertedmicroscopetoassessthedegreeofconfluencyandconfirmtheabsenceofbacterialandfungalcontaminants.
2. Removespentmedium.
3. WashthecellmonolayerwithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537)usingavolumeequivalenttohalfthevolumeofculturemedium.Repeatthiswashstepifthecellsareknowntoadherestrongly.
4. Pipettetrypsin/EDTA(Prod.No.T4049)ontothewashedcellmonolayerusing1mlper25cm₂ofsurfacearea.Rotateflasktocoverthemonolayerwithtrypsin.Decanttheexcesstrypsin.
5. Returnflasktotheincubatorandleavefor2-10minutes.
6. Examinethecellsusinganinvertedmicroscopetoensurethatallthecellsaredetachedandfloating.Thesideoftheflasksmaybegentlytappedtoreleaseanyremainingattachedcells.
7. Resuspendthecellsinasmallvolumeoffreshserum-containingmediumtoinactivatethetrypsin.Remove100-200uLandperformacellcount(Protocol6-CellQuantification).
8. Transfertherequirednumberofcellstoanewlabeledflaskcontainingpre-warmedmedium(refertoECACCCellLineDataSheetfortherequiredseedingdensity).
9. Incubateasappropriateforthecellline.
10. Repeatthisprocessasdemandedbythegrowthcharacteristicsofthecellline.

KeyPoints

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

5Protocol4-SubcultureofSemi-AdherentCellLines

Clickhereforaschematicdiagramof"SubcultureofSemi-AdherentCellLines"

AimSomeculturesgrowasamixedpopulation(e.g.B95-8-marmoset)whereaproportionofcellsdonotattachtothetissuecultureflaskandremaininsuspension.Thereforetomaintainthisheterogeneityboththeattachedcellsandthecellsinsuspensionmustbesubcultured.

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)
• Waterbathsetto37ºC
• Microbiologicalsafetycabinetattheappropriatecontainmentlevel
• Centrifuge
• Invertedphasecontrastmicroscope
• CO₂incubator
• Haemocytometer(Bright-line,Prod.No.Z359629,ImprovedNeubauerGrid,CamlabCCH.AC1)
• Pre-labeledflasks
• Tissues

Procedure

1. Viewculturesusinganinvertedphasecontrastmicroscopetoassessthedegreeofconfluencyandconfirmtheabsenceofbacterialandfungalcontaminants.Givetheflaskagentleknockfirst,thismaydislodgethecellsfromtheflaskandremovetheneedforatrypsinisationstepwiththesubsequentlossofsomecellsduetothewashings.
2. Decantspentmediumintoasterilecentrifugetubeandretain.
3. WashanyremainingattachedcellswithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537)using1-2mlforeach25cm₂ofsurfacearea.Retainthewashings.
4. Pipettetrypsin/EDTA(Prod.No.T4049)ontothewashedcellmonolayerusing1mlper25cm₂ofsurfacearea.Rotateflasktocoverthemonolayerwithtrypsin.Decanttheexcesstrypsin.
5. Returnflasktoincubatorandleavefor2-10minutes.
6. Examinethecellsusinganinvertedmicroscopetoensurethatallthecellsaredetachedandfloating.Thesideoftheflasksmaybegentlytappedtoreleaseanyremainingattachedcells.
7. Transferthecellsintothecentrifugetubecontainingtheretainedspentmediumandcells.
8. Centrifugetheremainingcellsuspensionat150gfor5minutes.AlsocentrifugethewashingsfromNumber3aboveiftheycontainsignificantnumbersofcells.
9. Decantthesupernatantsandresuspendthecellpelletsinasmallvolume(10-20mls)offreshculturemedium.Poolthecellsuspensions.Countthecells.
10. Pipettetherequirednumberofcellstoanewlabeledflaskanddilutetotherequiredvolumeusingfreshmedium(refertoECACCCellLineDataSheetfortherequiredseedingdensity).
11. Repeatthisprocessevery2-3daysasnecessary.

KeyPoints

1. AlthoughmostcellswilldetachinthepresenceoftrypsinalonetheinclusionofEDTAisusedtoenhancetheactivityoftheenzyme.
2. Trypsinisinactivatedinthepresenceofserum.Therefore,itisessentialtoremovealltracesofserumfromtheculturemediumbywashingthemonolayerofcellswithPBSwithoutCa₂₊/Mg₂₊(Prod.No.D8537).Repeatedwarmingto37ºCalsoinactivatestrypsin.
3. Cellsshouldonlybeexposedtotrypsin/EDTA(Prod.No.T4049)longenoughtodetachcells.Prolongedexposurecoulddamagesurfacereceptors.Ingeneralashortertimeofexposuretotrypsinisrequiredforsemiadherentcelllines.
4. Trypsinshouldbeneutralizedwithserumpriortoseedingcellsintonewflasksotherwisecellswillnotattach.
5. TrypsinmayalsobeneutralizedbytheadditionofSoybeantrypsinInhibitor(Prod.No.T6414),whereanequalvolumeofinhibitorataconcentrationof1mg/mlisaddedtothetrypsinisedcells.Thecellsarethencentrifuged,resuspendedinfreshculturemediumandcountedasabove.
6. IfaCO₂incubatorisnotavailablegastheflasksfor1-2minuteswith5%CO₂in95%airfilteredthrougha0.25mfilter.

[NextPage]

6Protocol5-SubcultureofSuspensionCellLines

Clickhereforaschematicdiagramof"SubcultureofSuspensionCellLines"

AimIngeneraltermsculturesderivedfromblood(e.g.lymphocytes)growinsuspension.Cellsmaygrowassinglecellsorinclumps(e.g.EBVtransformedlymphoblastoidcelllines).Forthesetypesoflinessubculturebydilutionisrelativelyeasy.Butforlinesthatgrowinclumpsitmaybenecessarytobringthecellsintoasinglecellsuspensionbycentrifugationandresuspensionbypipettinginasmallervolumebeforecounting.

Materials

• Media?pre-warmedto37ºC(refertotheECACCCellLineDataSheetforthecorrectmedium)
• 70%Ethanolinwater(Prod.No.R8382)

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Waterbathsetto37ºC
• Microbiologicalsafetycabinetatappropriatecontainmentlevel
• Centrifuge
• CO₂incubator
• Invertedphasecontrastmicroscope
• Haemocytometer(Bright-line,Prod.No.Z359629,ImprovedNeubauer,CamlabCCH.AC1)
• Pre-labeledflasks

Procedure

1. Viewculturesusinganinvertedphasecontrastmicroscope.Cellsgrowinginexponentialgrowthphaseshouldbebright,roundandrefractile.Hybridomasmaybeverystickyandrequireagentleknocktotheflasktodetachthecells.EBVtransformedcellscangrowinverylargeclumpsthatareverydifficulttocountandthecenterofthelargeclumpsmaybenon-viable.
2. DonotcentrifugetosubcultureunlessthepHofthemediumisacidic(phenolred=yellow)whichindicatesthecellshaveovergrownandmaynotrecover.Ifthisisso,centrifugeat150gfor5minutes,re-seedataslightlyhighercelldensityandadd10-20%ofconditionedmedium(supernatant)tothefreshmedia.
3. Takeasmallsampleofthecellsfromthecellsuspension(100-200uL-Protocol6-CellQuantification).Calculatecells/mlandre-seedthedesirednumberofcellsintofreshlypreparedflaskswithoutcentrifugationjustbydilutingthecells.Thedatasheetwillgivetherecommendedseedingdensities.
4. Repeatthisevery2-3days.

KeyPoints

1. Ifthecelllineisahybridomaorothercelllinethatproducesasubstance(e.g.recombinantproteinorgrowthfactor)ofinterestretainthespentmediaforanalysis.

7Protocol6-CellQuantification

Clickhereforaschematicdiagramof"CellQuantification"

AimForthemajorityofmanipulationsusingcellcultures,suchastransfections,cellfusiontechniques,cryopreservationandsubcultureroutinesitisnecessarytoquantifythenumberofcellspriortouse.Usingaconsistentnumberofcellswillmaintainoptimumgrowthandalsohelptostandardizeproceduresusingcellcultures.Thisinturngivesresultswithbetterreproducibility.

Materials

• Media?pre-warmedtoappropriatetemperature(refertotheECACCCellLineDataSheetforthecorrectmediumandtemperature)
• 70%ethanolinwater(Prod.No.R8382)
• 0.4%TrypanBlueSolution(Prod.No.T8154)
• Trypsin/EDTA(Prod.No.T4049)

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Waterbathsettoappropriatetemperature
• Microbiologicalsafetycabinetatappropriatecontainmentlevel
• Centrifuge
• CO₂incubator
• Haemocytometer(Bright-line,Prod.No.Z359629,ImprovedNeubauer,CamlabCCH.AC1)
• Invertedphasecontrastmicroscope
• Pre-labeledflasks

Procedure

1. Bringadherentandsemiadherentcellsintosuspensionusingtrypsin/EDTA(Prod.No.T4049)asabove(Protocol3and4)andresuspendinavolumeoffreshmediumatleastequivalenttothevolumeoftrypsin.Forcellsthatgrowinclumpscentrifugeandresuspendinasmallvolumeandgentlypipettetobreakupclumps.
2. Understerileconditionsremove100-200uLofcellsuspension.
3. AddanequalvolumeofTrypanBlue(Prod.No.T8154)(dilutionfactor=2)andmixbygentlepipetting.
4. Cleanthehaemocytometer.
5. Moistenthecoverslipwithwaterorexhaledbreath.Slidethecover-slipoverthechamberbackandforthusingslightpressureuntilNewton?srefractionringsappear(Newton?srefractionringsareseenasrainbow-likeringsunderthecover-slip).
6. Fillbothsidesofthechamber(approx.5-10uL)withcellsuspensionandviewunderalightmicroscopeusingx20magnification.
7. Countthenumberofviable(seenasbrightcells)andnon-viablecells(stainedblue)-(seebelow).Ideally>100cellsshouldbecountedinordertoincreasetheaccuracyofthecellcount(seenotesbelow).Notethenumberofsquarescountedtoobtainyourcountof>100.
8. Calculatetheconcentrationofviableandnon-viablecellsandthepercentageofviablecellsusingtheequationsbelow.

Where:

• Aisthemeannumberofviablecellscounted,i.e.TotalviablecellscounteddividedbyNumberofsquares
• Bisthemeannumberofnon-viablecellcounted,i.e.Totalnon-viablecellscounteddividedbyNumberofsquares
• Cisthedilutionfactorand
• Disthecorrectionfactor(thisisprovidedbythehaemocytometermanufacturer).

Concentrationofviablecells(cells/ml)=AxCxDConcentrationofnon-viablecells(cells/ml)=BxCxDTotalnumberofviablecells=concentrationofviablecellsxvolumeTotalnumberofcells=numberofviable+numberofdeadcellsPercentageViability=(Noofviablecellsx100)dividedbyTotalNoofcells

KeyPoints

1. TrypanBlue(Prod.No.T8154)istoxicandisapotentialcarcinogen.Protectiveclothing,glovesandface/eyeprotectionshouldbeworn.Donotbreathethevapor.
2. Thecentralareaofthecountingchamberis1mm2.ThisareaissuBDividedinto25smallersquares(1/25mm₂).Eachoftheseissurroundedbytriplelinesandisthenfurtherdividedinto16(1/400mm₂).Thedepthofthechamberis0.1mm.
3. Thecorrectionfactorof10₄converts0.1mm₃to1ml(0.1mm₃=1mm₂x0.1mm)
4. Thereareseveralsourcesofinaccuracy:
   • Thepresenceofairbubblesanddebrisinthechamber.
   • Overfillingthechambersuchthatsamplerunsintothechannelsortheotherchamber
   • Incompletefillingofthechamber.
   • Cellsnotevenlydistributedthroughoutthechamber.
   • Toofewcellstocount.Thiscanbeovercomebycentrifugingthecells,resuspendinginasmallervolumeandrecounting.
   • Toomanycellstocount.Thiscanbeovercomebyusingahigherdilutionfactorintrypanbluee.g.1:10

8Protocol7-CryopreservationofCellLines

Clickhereforaschematicdiagramof"CryopreservationofCellLines"

AimTheprotocolbelowdescribestheuseofpassivemethodsinvolvinganelectric-80ºCfreezerforthecryopreservationofcellcultures.ECACCroutinelyuseaprogrammableratecontrolledfreezer(PlanerSeriesTwo)fromPlanerProducts.Thisisthemostreliableandreproduciblewaytofreezecellsbutasthecostofsuchequipmentisbeyondthemajorityofresearchlaboratoriesthemethodsbelowaredescribedindetail.Iflargenumbersofcellculturesareregularlybeingfrozenthenaprogrammableratecontrolledfreezerisrecommended.

Materials

• Freezemedium(commonly70%basalmedium,20%FCS,10%DMSO(Prod.No.D2650)orglycerol,checkECACCdatasheetsfordetails).
• 70%ethanolinwater(Prod.No.R8382)
• PBSwithoutCa₂₊Mg₂₊(Prod.No.D8537)
• 0.25%trypsin/EDTAinHBSS,withoutCa₂₊/Mg₂₊(Prod.No.T4049)
• DMSO(Prod.No.D2650)
• Trypsin/EDTA(Prod.No.T4049)
• HL60(Prod.No.98070106-1v1)

Equipment

• Personalprotectiveequipment(sterilegloves,Laboratorycoat)
• Full-faceprotectivemask/visor
• Waterbathsetto37ºC
• Microbiologicalsafetycabinetatappropriatecontainmentlevel
• Centrifuge
• Haemocytometer(SigmaBright-lineProd.No.Z359629,ImprovedNeubauer?CamlabCCH.AC1)
• Prelabeledampules/cryotubes
• CellFreezingDevice(e.g.NalgeneMr.FrostyProd.No.C1562)

Procedure

1. Viewculturesusinganinvertedmicroscopetoassessthedegreeofcelldensityandconfirmtheabsenceofbacterialandfungalcontaminants.
2. Bringadherentandsemiadherentcellsintosuspensionusingtrypsin/EDTA(Prod.No.T4049)asabove(Protocol3and4?Subcultureofadherent/attachedandsemi-adherentcelllines)andre-suspendinavolumeoffreshmediumatleastequivalenttothevolumeoftrypsin.Suspensioncelllinescanbeuseddirectly.
3. Removeasmallaliquotofcells(100-200uL)andperformacellcount(Protocol6?CellQuantification).Ideallythecellviabilityshouldbeinexcessof90%inordertoachieveagoodrecoveryafterfreezing.
4. Centrifugetheremainingcultureat150gfor5minutes.
5. Re-suspendcellsataconcentrationof2-4x106cellspermlinfreezemedium.
6. Pipette1mlaliquotsofcellsintocyroprotectiveampulesthathavebeenlabeledwiththecelllinename,passagenumber,cellconcentrationanddate.
7. Placeampulesinsideapassivefreezere.g.NalgeneMr.Frosty(Prod.No.C1562).Fillfreezerwithisopropylalcoholandplaceat?80ºCovernight.
8. Frozenampulesshouldbetransferredtothevaporphaseofaliquidnitrogenstoragevesselandthelocationsrecorded.

KeyPoints

1. Themostcommonlyusedcryoprotectantisdimethylsulphoxide(DMSOProd.No.D2650),however,thisisnotappropriateforallcelllinese.g.HL60(Prod.No.98070106-1v1)whereDMSOisusedtoinducedifferentiation.Insuchcasesanalternativesuchasglycerolshouldbeused(refertoECACCdatasheetfordetailsofthecorrectcryoprotectant).
2. ECACCfreezemediumrecommendedabovehasbeenshowntobeagooduniversalmediumformostcelltypes.Anothercommonlyusedfreezemediumformulationis70%basalmedium,20%FCS,10%DMSObutthismaynotbesuitableforallcelltypes.Checkitworksforyourcellsbeforeusingonaregularbasis(Prod.No.C6164).
3. Itisessentialthatculturesarehealthyandinthelogphaseofgrowth.Thiscanbeachievedbyusingpre-confluentcultures(culturesthatarebelowtheirmaximumcelldensity)andbychangingtheculturemedium24hoursbeforefreezing.
4. Therateofcoolingmayvarybutasageneralguidearateofbetween?1ºCand?3ºCperminutewillprovesuitableforthemajorityofcellcultures.
5. AnalternativetotheMr.FrostysystemistheTaylorWhartonpassivefreezerwhereampulesareheldinliquidnitrogenvaporintheneckofDewar.Thesystemallowstheampulestobegraduallyloweredtherebyreducingthetemperature.Ratecontrolledfreezersarealsoavailableandareparticularlyusefuliflargenumbersofampulesarefrozenonaregularbasis.
6. Asalastresortifnootherdevicesareavailableampulesmaybeplacedinsideawellinsulatedbox(suchasapolystyreneboxwithsidesthatareatleast1cmthick)andplacedat?80ºCovernight.Itisimportanttoensurethattheboxremainsuprightthroughoutthefreezingprocess.Oncefrozen,ampulesshouldbetransferredtothevaporphaseofaliquidnitrogenstoragevesselandthelocationsrecorded.
7. Ifusingafreezingmethodinvolvinga-80ºCfreezeritisimportanttohaveanallocatedsectionforcelllinefreezingsothatsamplesarenotinadvertentlyremoved.Ifthishappensatacrucialpartofthefreezingprocessthenviabilityandrecoveryrateswillbeadverselyaffected.

[NextPage]

9Protocol8-TestingforBacteriaandFungi

AimIncasesofgrosscontaminationthenakedeyemayidentifythepresenceofbacteriaandfungi.However,itisnecessarytodetectlow-levelinfectionsbyincubationofcellculturesand/ortheirproductsinmicrobiologicalbroth.Equallythesesterilitytestscanbeusedtoconfirmtheabsenceofbacteriaandfungifromthepreparationwhichisimportantwhenpreparingcellbanksorcellcultureproducts.

Materials

• SoybeanCaseinDigest(TryptoneSoyaBroth,TSB)(15mlaliquots)(Prod.No.S1674)TSBPowder(Prod.No.T8907)
• FluidThioglycollateMedium(20mlaliquots)(TGM)(Prod.No.F4797)
• BacillussubtilisNCTC*
• CandidaalbicansNCTC*
• ClostridiumsporogenesNCTC*

Equipment

• Personalprotectiveequipment(latexmedicalgloves,laboratorycoat,safetyglasses)
• Waterbathsetto37ºC
• Microbiologicalsafetycabinetatappropriatecontainmentlevel
• Centrifuge
• Incubatorsetat32ºC
• Incubatorsetat22ºC

ClickhereforFigure12.FlowSchemeforBacteriaandFungiTesting

Procedure

1. Culturecelllineintheabsenceofantibioticsfor2passagespriortotesting.
2. Bringattachedcellsintosuspensionwiththeuseofacellscraper.Suspensioncelllinesmaybetesteddirectly.
3. Inoculate2xThioglycollateMedium(TGM)(Prod.No.F4797)and2xTryptoneSoyabroth(TSB)(Prod.No.T8907)with1.5mltestsample.
4. Inoculate2(TGM)and2(TSB)with0.1mlC.albicans(containing100colonyformingunits,cfu).
5. Inoculate2(TGM)and2(TSB)with0.1mlB.subtilis(containing100cfu).
6. Inoculate1TGMwith0.1mlC.sporogenes(containing100cfu).
7. Leave2(TGM)and2(TSB)un-inoculatedasnegativecontrols.
8. Incubatebrothsasfollows:
   • ForTSB,incubateonebrothofeachpairat32ºCtheotherat22ºCfor14days
   • ForTGM,incubateonebrothofeachpairat32ºCtheotherat22ºCfor14days
   • FortheTGMinoculatedwithC.sporogenesincubateat32ºCfor14days
9. ExamineTestandControlbrothsforturbidityafter14days.

CriteriaforaValidResultAllpositivecontrolbrothsshowevidenceofbacteriaandfungiwithin14daysofincubationandthenegativecontrolbrothsshownoevidenceofbacteriaandfungi.

CriteriaforaPositiveResultTestbrothscontainingbacteriaorfungishowturbidity.

CriteriaforaNegativeResultTestbrothsshouldbeclearandshownoevidenceofturbidity.

Notes

1. Thepositivecontrolsshouldbehandledinalaboratoryremotefromthemaintissueculturelaboratory.
2. Controlorganisms(Bacillussubtilis,ClostridiumsporogenesandCandidaalbicans)arealsoavailablefromtheNationalCollectionofTypeCultures(NCTC),UK*.
3. Thistestprocedureshouldbecarriedoutinamicrobiologylaboratoryawayfromthecellculturelaboratory.

10Protocol9-DetectionofMycoplasmabyCulture

AimDetectionofmycoplasmabycultureisthereferencemethodofdetectionandhasatheoreticallevelofdetectionof1colony-formingunit(cfu).Howevertherearesomestrainsofmycoplasmathatarenon-cultivable(certainstrainsofMycoplasmahyorhinis).Themethodissuitableforthedetectionofmycoplasmainbothcellculturesandcellculturereagentsandresultsareobtainedwithin4weeks.Mycoplasmacoloniesobservedonagarplateshavea`friedegg?appearance(seeMycoplasmapneumoniae","");">Figure14).

Materials

• 70%ethanolinwater(Prod.No.R8382)
• MycoplasmaPigAgarplates(in5cmpetridishes)
• MycoplasmaPigAgarbroths(in1.8mlaliquots)
• M.oraleNCTC*10112
• M.pneumoniaeNCTC*10119

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Waterbathsetto37ºC
• Microbiologicalsafetycabinetatappropriatecontainmentlevel
• CO₂Incubatorsetat32ºC
• GasJar(Gallenkamp)
• GasPakAnaerobicSystem(Gallenkamp)
• GasPakCatalyst(Gallenkamp)
• GasPakAnaerobicIndicator(Gallenkamp)

ClickhereforFigure13.FlowSchemeforDetectionofMycoplasmabyCulture

Procedure

1. Inoculate2agarplateswith0.1mloftestsample.
2. Inoculate1agarplatewith100cfuM.pneumoniae.
3. Inoculate1agarplatewith100cfuM.orale.
4. Leave1agarplateun-inoculatedasanegativecontrol.
5. Inoculate1brothwith0.2mloftestsample.
6. Inoculate1brothwith100cfuM.pneumoniae.
7. Inoculate1brothwith100cfuM.orale.
8. Leave1agarplateun-inoculatedasanegativecontrol.
9. Incubateagarplatesanaerobicallyfor14daysat37ºCusingagasjarwithanaerobicgaspakandcatalyst.
10. Incubatebrothsaerobicallyfor14daysat37ºC.
11. Between3and7daysand10and14daysincubation,subculture0.1mloftestbrothontoanagarplateandincubateplateanaerobicallyasabove.
12. Observeagarplatesafter14daysincubationatx300magnificationusinganinvertedmicroscopeforthepresenceofmycoplasmacolonies(seeMycoplasmapneumoniae","");">Figure14).

CriteriaforaValidResultAllpositivecontrolagarplatesandbrothsshowevidenceofmycoplasmabytypicalcolonyformationonagarplatesandusuallyacolorchangeinbroths.Allnegativecontrolagarplatesandbrothsshownoevidenceofmycoplasma.

CriteriaforaPositiveResultTestagarplatesinfectedwithmycoplasmashowtypicalcolonyformation.

CriteriaforaNegativeResultThetestagarplatesshownoevidenceofmycoplasma.

Notes

1. Mycoplasmacolonieshaveatypicalcolonyformationcommonlydescribedas?friedegg?(SeeFigure8)duetotheopaquegranularcentralzoneofgrowthpenetratingtheagarsurroundedbyaflattranslucentperipheralzoneonthesurface.Howeverinmanycasesonlythecontrolzonewillbevisible.
2. Positivecontrolsmaybeincludedataconcentrationtogive100colony-formingunits.Thesecontrolsshouldobviouslybehandledinalaboratoryremotefromthemaintissueculturelaboratory.
3. Controlorganisms(M.pneumoniae,andM.orale)areavailablefromNationalCollectionofTypeCultures(UK).
4. MycoplasmapneumoniaeisapotentialpathogenandmustbehandledinaclassIImicrobiologicalsafetycabinetoperatingtoACDPCategory2Conditions.
5. Thistestprocedureshouldbecarriedoutinamicrobiologylaboratoryawayfromthecellculturelaboratory.

Mycoplasmapneumoniae","");">ClickhereforFigure14-Typical?friedeggcolonies?Mycoplasmapneumoniae

[NextPage]

11Protocol10-TestingforMycoplasmabyIndirectDNAStain(Hoechst33258stain)

AimDNAstainingmethodssuchasHoechststainingtechniquesarequickwithresultsavailablewithin24hours,whichcomparesfavorablywith4weeksfordetectionbyculture.HoweverthestainingofculturesdirectlywithaDNAstain,resultsinamuch-reducedsensitivity(~10₆cfu/ml).Thismaybeimprovedbyco-culturingthetestcelllineinthepresenceofanindicatorcelllinesuchasVero(Prod.No.84113001-1v1).Thisenrichmentstepresultsinasensitivityof104cfu/mlofculture.Thisstepalsoimprovessensitivitybyincreasingthesurfaceareauponwhichmycoplasmacanadhere.Likedetectionbyculture,DNAstainingmethodsaresuitableforthedetectionofmycoplasmafromcellculturesorcellculturereagents.

Materials

• Media?pre-warmedto37ºC(refertotheECACCCellLineDataSheetforthecorrectmedium)
• 70%ethanolinwater(Prod.No.R8382)
• Methanol(Prod.No.175)
• AceticAcidGlacial(Prod.No.A6283)
• Hoechst33258stainsolution(Prod.No.H6024)
• Verocells(Prod.No.84113001-1v1)
• Mountant(Autoclave22.2ml0.2Mcitricacidwith27.8ml0.2Mdisodiumphosphate.Add50mlglycerol.Filtersterilizeandstoreat4ºC)(Prod.No.M1289)
• MycoplasmahyorhinisNCTC*10112

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Waterbathsetto37ºC
• Microbiologicalsafetycabinetofappropriatecontainmentlevel
• Centrifuge
• CO₂Incubatorsetat37ºC
• Microscope(uvEpi-Fluorescent.)
• 35mmplastictissueculturedishes(Prod.No.C6296)
• Multidish24well(Prod.No.M9655)
• Cellscraper
• Microscopeslidesand22mmcoverslips
• Aluminumfoil(Prod.No.Z185140)

ClickhereforFigure15.TestingforMycoplasmabyIndirectDNAStain

ProcedureEquipment

1. Foreachsampleandcontrolsterilize2coverslipsinahotovenat180ºCfor2hoursorbyimmersingin70%ethanol(Prod.No.R8382)andflaminginablueBunsenflameuntiltheethanolhasevaporated.Alsosterilize2coverslipstouseasanegativecontrol.
2. Placethecoverslipsin35mmculturedishes(Prod.No.C6296)(1perdish).
3. Storeuntilneeded.
4. TopreparetheVero(Prod.No.84113001-1v1)indicatorcellsadd2x104cellsin2mlofantibiotic-freegrowthmediumtoeachtissueculturedish.
5. Incubateat37ºCin5%CO₂for2?24hrstoallowthecellstoadheretothecoverslips.
6. Bringattachedtestcelllinesintosuspensionusingacellscraper.Suspensioncelllinesmaybetesteddirectly.
7. Remove1mlofculturesupernatantfromduplicatedishesandadd1mloftestsampletoeach.Inoculate2disheswith100cfuM.hyorhinisand2with100cfuM.orale..
org.uk
8. Leaveduplicatetissueculturedishesun-inoculatedasnegativecontrols.
9. Incubatedishesat37ºCin5%CO₂for1-3days.
10. After1dayobserveonedishfromeachpairforbacterialorfungalinfection.Ifcontaminateddiscardimmediately.Leavetheremainingdishofeachpairforafurther2days.
11. Fixcellstocover-slipbyaddingaminimumof2mloffreshlypreparedfixative(1:3glacialaceticacid:absolutemethanol)tothetissueculturedishandleavefor3to5minutes.
12. Decantusedfixativetotoxicwastebottle.Addanother2mlaliquotoffixativetocover-slipandleaveforafurther3to5min.Decantusedfixativetotoxicwaste.
13. Airdrycover-slipbyrestingitagainstthetissueculturedishfor30-120min.
14. Replacecover-slipindishandaddaminimumof2mlHoechststain(Prod.No.H6024).Leavefor5minutesshieldedfromdirectlightbyaluminumfoil(Prod.No.Z185140).
15. Decantusedandunusedstaintotoxicwaste.
16. Add1dropofmountanttoapre-labeledmicroscopeslideandplacecover-slip(cellsidedown)ontoslide.
17. Keepslidecoveredwithaluminumfoil(Prod.No.Z18514-0),allowingittosetforatleast15minat37ºCorfor30minatroomtemperature.
18. ObserveslideunderuvEpi-Fluorescenceatx1000.

CriteriaforaValidResultNegativecontrolsshownoevidenceofmycoplasmainfectionPositivecontrolsshowevidenceofmycoplasmainfectionVerocellsclearlyseenasfluorescingnuclei.

CriteriaforaPositiveResultSamplesinfectedwithmycoplasmaareseenasfluorescingnucleiplusextra-nuclearfluorescenceofmycoplasmaDNA(smallcocciorfilaments).

CriteriaforaNegativeResultUninfectedsamplesareseenasfluorescingnucleiagainstadarkbackground.Thereshouldbenoevidenceofmycoplasma.

Notes

1. DNAstainssuchasHoechststain(Prod.No.H6024)bindspecificallytoDNA.Inallculturescellnucleiwillfluoresce.Uncontaminatedcultureswillshowonlyfluorescentnucleiwhereasmycoplasmapositiveculturescontainsmallcocciorfilamentswhichmayormaynotbeadsorbedontothecells(seefigure16).
2. Hoechststainistoxicandshouldbehandledanddiscardedwithcare.
3. Culturedishesshouldbeplacedinasealedboxorculturedinlargepetridishestoreduceevaporation.
4. Positivesshouldobviouslybehandledinalaboratoryremotefromthemaintissueculturelaboratory.
5. Controlorganisms(M.hyorhinis)areavailablefromtheNationalCollectionofTypeCultures(UK).
6. Insomeinstancesresultsmaybedifficulttointerpretforthefollowingreasons:
   • Bacterial/yeast/fungalcontamination
   • Toomuchdebrisinthebackground
   • Brokennucleiascellsarealldead
   • Toofewornolivecells
7. Althoughthisprocedurerecommendsthesettingupofpositivecontrols,thismaynotnecessarilybefeasiblenordesirableinacellculturefacilitywithlimitedresources.Ifpositivecontrolsaretobesetuptheyshouldbedonesoinaseparatelaboratoryfromthemaintissueculturefacility.IfthisisnotpossiblethenpositiveslidescanbepurchasedfromECACC.Ifpositivecontrolsarenotbeingusedthenitisstronglyrecommendedthatyougetanindependenttestinglaboratorytoperiodicallytestyourcelllines.