Heat Inactivation— Are You Wasting Your Time? - Thermo Scientific

theproblemsinceprolongedincubationonlyresultsintheformationofadditionalprecipitate.Thisprecipitateresemblesamicrobialcontaminanttotheextentthatevenexperiencedcellculturistsandmicrobiologistsoftencannotdistinguishbetweenthetwo.Extensivetesting(electronmicroscopy,platingonbacterialgrowthmedia,Gramstaining,etc.)isrequiredtoconfirmthepresenceofaprecipitate.Thesetestswasteconsiderabletimeandresourcessimplytoconvincecellculturiststhattheserumisnotcontaminatedwithbacteria.Precipitatesarealsomorelikelytoformifserumisheatinactivatedattemperaturesabove56°Corformorethan30minutesorifthecontentsofthebottlearenotproperlymixed.Although56°Cisthemostcommontemperatureforheatinactivation,someprotocolslisthighertemperatures,whichfurthercompromisethegrowth-promotingabilityofserum,inadditiontoresultingintheformationofadditionalprecipitates.ThegrowthofMRC-5andMDBKcellsdecreasesdramaticallyinFBSthathasbeenheatinactivatedat65°Cfor30minutes(Figure3).FBSheatinactivatedat65°CfailstosupportthegrowthofHBAEcells,eventhoughthegrowthofSp2/0Ag14hybridcellsisnotaffected.Technical Information171

Serumproductsareoftenexposedtohightemperaturesformorethan30minutes,eitherintentionallyorunintentionally,e.g.,serumismistakenlyleftinthewaterbathovernight.Extendedheatinactivationalsoincreasestheformationofprecipitatesanddecreasesthegrowthpromotioncapacityoftheserum.Figure4showsthegrowthofHBAE,MRC-5,MDBK,andSp2/0Ag14hybridcellsinserumthathasbeenheatedat56°Cfor30,75,120,and1,080minutes.ThegrowthofHBAE,MRC-5,andMDBKcellsisaffectedadverselywitheachincreaseinexposuretime.TheHBAEcelllineisverysensitivetoheatinactivatedserumandfailedtogrowinFBSheatinactivatedfor1,080minutes.ThegrowthofMRC-5andMDBKcellswasalsoimpairedsignificantly,althoughtoalesserdegreethantheHBAEcells.Again,asintheexposuretohighertemperatures,thegrowthofSp2/0Ag14hybridsdidnotseemtobeaffectedbyincreasedexposuretimes.Severalfactorscaninfluencethetotaltimethatserumisexposedtohightemperatures.Glassandplastic(PETG)differintheirheatcapacities,whichdirectlyaffectsheatingrates.Toreducebreakageandfacilitatestorage,plasticbottleshavelargelyreplacedborosilicateglassbottles.Theseplasticbottlesincreasethetimerequiredforthecontentstoreach56°Cbyapproximately30percent.Thisinsulatingeffectalsoincreasestheamountoftimerequiredforthecontentsofplasticbottlestocoolfollowingheatinactivation.Thecoolingratealsodependsonwhether172

Insummary,mostcellcultureapplicationsdonotrequireheatinactivationofserum.Inmostcases,heatinactivationdoesnotimprovethegrowthpromotioncapabilityoftheserumandmayactuallyhaveadverseeffects.Inthefewinstancesinwhichheatinactivationimprovedperformance,theimprovementwasminimal.Moreover,heatinactivationcausesanincreaseintheformationofprecipitateswhicharefrequentlymistakenforamicrobialcontaminant.Thiscreatesunnecessaryconcernsandalsoconsumesvaluableresourcesonbehalfoftheuseraswellasthesupplier.theserumisplacedintoarefrigerator,freezer,oricebathfollowingheatinactivation(Figure5).Heatinactivatedserumrequired30minutestocooltoatemperatureof10°Cinanicebathversus330minuteswhensimplyplacedintoarefrigerator.Dividingtheserumintoaliquotsfacilitatesheatingandcooling.Thewaterlevelintheheatinactivationbathwillalsoaffectheatingratesoftheserum.Itisofteninconvenienttofillthewaterbathsuchthatitequalsthelevelofseruminthebottlesincethebottleswillhaveatendencytofloat.Floatingcanbepreventedbyplacingcommerciallyavailableleadweightsaroundthenecksofthebottles.Figure6illustratestheheatingratesinwaterbathsinwhichlevelswereequaltoeitherthe300mLorthe500mLgraduationofastandardplasticbottle.Atthe500mLgraduation,theserumrequired40minutestoreach56°C.Atthe300mLgraduation,theserumrequired60minutestoreach56°Cthusincreasingthetotalexposuretimetoelevatedtemperatures.Customerswhocurrentlyheatinactivatetheirserumshoulddetermineifthisprocedureisreallynecessarywiththeirparticularcelllinesorculturesystems.Serumisofteninadvertentlyheatedforlongerperiodsoftimeandathighertemperaturesthanrecommendedintraditionalprotocols.Ifheatinactivationisnecessary,itshouldbestrictlymonitoredandperformedusingaprocedurethatisrepeatable.References:1. Triglia,R.P.,Linscott,W.D.1980.TitersofNineComplementComponents,ConglutininandC3b-InactivatorinAdultandFetalBovineSera.Mol.Immunol.17:741-748.2. Pinyopummintr,T.,andBavister,B.D.1994.DevelopmentofBovineEmbryosinaCell-FreeMedium—EffectsofTypeofSerum,TimingofitsInclusionandHeatInactivation.Theriogenology.41:1241-1249.3. Giard,D.J.1987.RoutineHeatInactivationofSerumReducesitsCapacitytoPromoteCellAttachment.InVitroCellular&DevelopmentalBiology.23:691-697.4. Invitrogen.1995.EXPRESSIONS.Vol.2Issue2.p11.Technical Information173