MaterialsTrypsin(Gibco25200-023)3T3Medium:500mLDME(Invitrogen)+50mLFBS(Hyclone)+5mL100xPen/Strep2xFreezingMedium:3T3Medium+20%DMSO:Filtersterilized.SterilePBSP100andP60tissueculturedishesDissectingtools:Scissors,forceps,scalpel95%ethanolinasqueezebottleandinabeakerMousebreeders

PROCEDURE

Note:ThisprotocoldescribestheisolationofprimaryMEFsandpassageaccordingtothe3T3protocol.Passagingprimarycellsonthe3T3protocolwillmaximizethegrowthpriortothedevelopmentofcellularsenescence.The3T3protocolwasoriginallydescribedasthepassageof3x10⁵cellsevery3dayson50mm.disheswhichisequivalentto1.2x10⁶cellsonaP100dish(NilausenK,GreenH.ExpCellRes.1965).Ifcellsarepassagedcontinuouslyeverythreedaysundernormoxicconditionstheywillexperienceaperiodofrapidgrowth(passage1-5),progressivelyslowergrowth(passage5-10)andsenescence(littleornogrowth,passage10-25).Afterthistimetheasubsetofcellswillemergefromcrisis,throughtheselectionforamorerapidlygrowing,immortalizedsubclone.Thisistypicallyaccompaniedbythedevelopmentofananeuploidkaryotype.EarlypassageprimaryMEFsandimmortalizedMEFsareusuallyspindleshapedandformdenselypackedmonolayers,whereassenescentcellstendtoflattenandspreadout.Somelinesmaybecometransformedanddisplayneuron-likeorrefractilemorphology,growthdespiteserumstarvation,growthinsUSPensionorinsoftagar,andtheABIlitytoformfibrosarcomatumorsinmice.Senesenceinmurinefibroblastsisaresultofoxidativestressasgrowthinphysiologic(3%)O2allowscontinualgrowthofmurinecellssimilartohumanfibroblasts(ParrinelloS.,etal.NatureCellBiol,2003).

A.IsolatingPrimaryMEFs:

1. Setupmousebreederpairsintheafternoon.Ideallyonemaleshouldbesetupwithoneortwovirgin7-8weekoldfemales.
2. Checkfemalesforcopulatoryplugsthefollowingmorning.Ifpresentthisshouldbemarkedasday+0.5(orroundofftoday+1).Removepluggedfemalesfromthemalesbreedercages.Checktheremainingfemalesonadailybasis.
3. Embryosshouldbeharvestedonday11.5to13.5asfollows:
   1. Placecleanforcepsandscissorsintoabeakerwithethanol.FillasecondbeakerwithsterilewaterorPBStowashinstrumentsbetweenembryos.AliquotsterilePBSintoP60dishes(2-3perembryo).
   2. Euthanizethepregnantfemaleaccordingtostandardprotocols.CO₂inhalationshouldbeavoidedbecausethisinducesacidosis.
   3. Saturatefurwithethanol.Incisetheskinverticallyandpintotheside.
   4. Usingafreshpairofforcepsandscissorsopenthebodywallandpinthisbackaswell.
   5. Extracttheintactuterusbygraspingonehornwithforceps.WithdrawtheuterinehornfromtheaBDomenwithoutlettingittouchingskinornon-sterileareas.Cutthecervixandbloodvesselsandandtransfertheuterustoasterilepetridish.
   6. Theembryoswillappearlikespearlsonastring.Cuttheuterusintosectionsbetweeneachembyo.PlaceauterinesectionwithasingleembryointoafreshpetridishwithsterilePBS.Squeezetheuteruswithforcepstoexpelltheembryothroughthecutportionoftheuterus.
   7. WashtheembryoinafreshdishwithsterilePBStowashawayanyremainingmaternalbloodcells.
   8. TransfertheembryotoafreshPetridishwithsterilePBS.Removethemembranesandumbilicalcord.Withascalpelverticallyincisetheabdominalwallandremovethepinkhematopoietictissue(liverandspleen)aswellasthetubularintestine.
   9. TrimoffthebulkoftheCNStissuebydissecingawaytheheadabovetheleveloftheoralcavity.
   10. SaveCNSorlivertissueina1.5mLtubeforDNAextractionandgenotyping.EmbryosshouldbesignedamouseIDandgenotypedaccordingtostandardprotocols.ItmaybenecessarytodilutetheDNAorlimitthePCRcyclenumberbecauseofthelargeamountofDNAandthepossibilityofmaternalbloodcontamination.
4. TransfertheembryobodytothetissueculturehoodinaP100dishwith5mLof1xtrypsin.
5. Minceeachembryofor2-3min.withasterileforcepsandscissorsorascalpeltocreatebitssmallerthan2mm.Placethedishina37ºCincubator.Rinsetheinstrumentsinsterilewaterandthen95%ethanoltocleanandairdrythembetweeneachembryo.
6. Afteratotalof15minutestransferthecellsandtissuebitswitha10mLpipettoa15mLconicaltube.Pipetthecellsseveraltimestofurtherdissociatetissuebits.Add5mLofmedia(DME+10%FBS).
7. Spinat1000RPMinatabletopcentrifugefor5min.
8. Aspiratethesupernatant.Resuspendtheembryoniccellpelletin10mLfreshmediaandtransfertoacleansterileP100dish.SmallbitsoftissueinthecellsuspensionareOK.MEFswillmigrateoutoftissuebitsontothedish.
9. Marktheplatesas"passage0",withthedate,andtheembryoID.Evenlydistributethecellsandtissuebitsbyhorizontalagitationandthenincubateat37ºCin5%CO2.(Optional:Tominimizesenescencegrowcellsin3%O₂,seeabove).

B.PassagingprimaryMEFS:

1. Checktheplatesdailyuntiltheyarenearlyconfluent(usually1-3days).
2. Totrypsinizethecells:
   1. Aspirateawaythemedia.Add5mLofPBS.SwirltorinseandthenaspirateoffthePBS.
   2. Add3-4mLof1xtrypsin(orenoughtocovertheplate).Incubateat37ºCfor2min.
   3. Taptheplatetodislodgecells.Continueincubatinguntilstarttocomeoffinlargesheets.(Forthe1stpassageitissufficienttoloosenupthecellsinthemonolayer.Donotdislodgelargerchunksoftissue,ifpresent.)
   4. Add5mLofmedia(DME+10%FBS)andtransfertoa15mLconicaltube.Pipettocreateasinglecellsuspension.(Iflargerchunksoftissuearepresentinthefirstpassage-allowthemtosettleoutofsuspensionfor1minute.Transferthesuspendedcellstoafreshconicaltube.)
3. Determinetheviablecellcount:
   1. Add10µLofcellssuspensiontoamultiwelldish.Add10µLof2xTrypanBluestain.
   2. Mixbypipettingandtransfer10µLtoeachsideofahemocytometer.
   3. Countwhite(live)andblue(dead)cellsoneachsideofthehemocytometer.Countatotalof100-200cellstobereasonablyaccurate.
   4. Multiplytheaveragenumberoflivecellsineachlargesquarex10⁴tocalculatethenumberofcellspermL.
   5. Keeparecordthenumberofcellspresentandamountthattheywillbedilutedtobeabletocalculatedoublingtimes.
4. Meanwhilespintheremainingcellsfor5min.at1000RPM.
5. Aspirateoffthesupernatantleaving~0.2mLofmediabehind.Resuspendpelletbyflickingbottomoftube.Addfreshmediatobringthecellstoadensityof10⁷cells/mL.
6. Add9mLofmediatoeach100mmdish+1mLofcellsuspensiontoplate1.2x10⁶cellsperdish.
7. Mixthecellsonthedishbyswirlingtheplates.Horizontallyagitatetheplatesintheincubatortoensureanevendistributionofcells.Incubate37ºC+5%CO2.(Optional:Tominimizesenescencegrowcellsin3%O₂.)
8. Passagethecellsevery3daysasdescribedabove(B.2-B.7)evenifthereislittleornogrowthsincethelastpassage.

1. PrimaryMEFsdonotdofreezerandthawwellandarelikelysenesceatalowerpassagethanarecellskeptincontinuousculture.Iftheyaretobefrozenitshouldprobablybeatpassage1-2.
2. TofreezeMEFs:
   1. TrypsinizecellsasaboveB.2-B.4.Resuspendthecellsatadensityof4x10⁶livecells/mL.Addanequalvolumeof2xFreezingMedia(media+20%DMSO).
   2. Aliquot1mLofcellsuspensioninfreezingmediaintocryovials.Capsecurely,beingcarefulnottocontaminatetheedgesofthevialorcap.LabeltubesasMEFs,passage#,date,andyourinitials.
   3. Placevialsinastyrofoamcontaineroracellfreezingjarwithisopropanol.Placeat-80ºCovernight.
   4. Transfervialstoacryofreezer.RecordthelocationintheFreezerdatabase.
3. TothawMEFs:
   1. Pre-warmmediumat37ºC.
   2. Removeavialofcellsfromthecryofreezerandplacein37ºCbath.
   3. DeletethevialentryfromtheFreezerdatabase.
   4. Wipethevialwithethanolandplaceinthetissueculturehood.
   5. Transferthecellstoasterileconicaltubewith10mLofmedium.
   6. Counttheviablecellswithtrypanblueasabove(B.3).
   7. Platethecellsatdensityof1.2x10⁶cellsperP100(or1.5x10⁴cells/cm²inasmallerdish).
   8. Incubate37ºC+5%CO2.(Optional:Tominimizesenescencegrowcellsin3%O₂.)