Preparationofculturemedium

MEFmediumDMEMcontaining10%FBS,2mMglutamine,1x10^-4Mnonessentialaminoacids,and50Uand50mg/mlpenicillinandstreptomycin.
MouseESmediumKO-DMEMcontaining15%ES-FBS,2mMglutamine,1x10^-4Mnonessentialaminoacids,1x10^-4M2-mercaptoethanol,1x10³U/mlLIF,and50Uand50mg/mlpenicillinandstreptomycin.
2XFreezingmedium20%DMSOplus80%FBS

Crowthconditionformousefibroblasts

Gelatintreatmentofplates

1.Addenoughsterile/autoclaved0.1%gelatintocoverthebottomofthewells.

Approximateamounts:

Plate/dish	Amount/Well
96well	100μl
48well	300μl
24well	0.5ml
12well	1.0ml
6well	1.5-2.0ml
30mm	1.5-2.0ml
60mm	3.0ml
100mm	4.0-5.0ml

2.Incubatethegelatin-coateddishesforatleast15minat37°C.

3.Aspirateexcessgelatinsolutionbeforeusing.

ThawingMEFcells

Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingitonthecells.Cellsshouldbeplatedataminimumcelldensityof1x10⁴cells/cm².

1.Removethevialfromliquidnitrogenandthawquicklyin37°Cwaterbath.

2.Removethevialfromthewaterbathassoonasthecellsarehalfwaythawed,andsterilizebysprayingwith70%ethanol.

3.Transferthecellswith10mlofMEFmediumtoa15-cmconicaltubeandpelletthecellsbycentrifugationat200xgfor5min.

4.DiscardthesupernatantandresUSPendthecellswith10mlfreshMEFmediumandplatethecellsatseeddensityof1x10⁴cells/cm².

5.Incubateat37°Cwith5%CO₂inairatmosphere,untilthecellsreach80-90%confluency.

6.ChangemediatwiceaweekorwhenpHdecreases.

PassageofMEFcells

Cellsshouldbesplitwhentheyreachconfluency.Asplitbasedonseeddensityof0.5x10⁴cells/cm²isrecommended.

1.DiscardthemediumandwashthecellstwicewithPBS.

2.AspiratePBS,andadd1mlperT75flaskof0.05%trypsin-EDTA,andincubatefor2min.

3.Add5mlofMEFmedium,andbreakupthecellclumpsbygentlypipettingupanddownseveraltimes.

4.Transfercellsintoaconicaltubeandcentrifugeat200gfor5min.

5.Discardthesupernatant,andresuspendthecellpelletin10mlMEFmedium.

6.Countthenumberofcells,platecellsat0.5x10⁴cells/cm²,andincubateat37°Cwith5%CO₂.

FreezingMEFcells

1.Followsteps1-4fromthePassageofCellsabove.

2.Discardthesupernatant,andresuspendthepelletinMEFmedium.Addapproximately1mlforeachT75flask.

3.Countthenumberofcellsanddilutethecellsuspensionto1x10⁷cells/ml.

4.Addanequalvolumeofcold2XFreezingMedia(containing20%DMSOand80%FBS)tothecellsuspension.

5.Aliquot1mlofsuspensionintoeachcryovial(5x10⁶cells/vial).

6.Placevialsintoanisopropanolfreezingcontainerandplacethecontainerat-80°Covernight.

7.Transferthevialstoaliquidnitrogentankforlong-termstorage.

MitomycinCtreatmentofMEF

Atconfluence,MEFcellsaretreatedwithmitomycinCtohaltthedivisionofthecellswhentheyarestillabletoconditionthemediumasthefeederlayersforiPSorEScells.

1.Add6mLoffreshMEFmediumcontaining50μlofmitomycinCsolution(1mg/ml)tooneT75flaskofconfluentMEFcells,andswirlitbriefly.

2.Incubateat37°Cforatleast3h.

3.Afterincubation,aspiratethemitomycinC-containingmediumoffthecells,andwashthecellstwicewith10mlofPBS.

4.AspirateoffPBS,add1mlof0.05%trypsin-EDTA,swirltocovertheentiresurface,andincubatefor2minatroomtemperature.

5.Add5mlofMEFmedium,andbreakupthecellstoasinglecellsuspensionbypipettingupanddown.Countthenumberofcells.Seedthecellsongelatin-coateddishes(1x10⁶cellsper100-mmdish,or1.5x10⁵cellsperwellof6-wellplate).

6.Cellsshouldbereadytousebythenextday.

CultureconditionformouseiPScells

ThawingmouseiPScells

Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingitonthecells.

1.Removethevialfromliquidnitrogenandthawquicklyin37°Cwaterbath.

2.Removethevialfromthewaterbathassoonasthecellsarehalfwaythawed,andsterilizebysprayingwith70%ethanol.

3.Transferthecellswith10mlofmouseESmediumtoa15-cmconicaltubeandpelletthecellsbycentrifugationat200Xgfor5min.

4.Discardthesupernatant,resuspendthecellswithfreshmouseESmedium,andplatethecellsinthewellsof6-wellplatewithMEFfeedercells.

5.Incubateat37°Cwith5%CO_{2inairatmosphere,untilthecellsreach80%confluency.}

6.ChangethemediumeverydayorwhenpHdecreases.

MaintenanceofmouseiPScells

ItisimportanttonotethatdoNOTkeepmouseiPScellsincultureforlongperiodsinordertomaintainthepluripotency.

1.Aspiratethemedium,andwashthecellstwicewith1mlofPBS.

2.RemovePBScompletely,add0.5mlof0.25%trypsin-EDTAsolution,andincubateat37°Cfor2min.

3.Whileincubating,removea6-wellplatewithmitomycinC-treatedMEFsfromtheincubator.AspirateMEFmediumandadd2mlofmouseESmediumtoeachwell.

4.RemovetheplatecontainingmouseiPScellsfromtheincubatorandswirltodislodgethecellsfromthebottomoftheplate.

5.Add1mlofESmediumtotheplateandsuspendthecellsbypipettingupanddowntosinglecellsuspension.

6.Distribute0.2mlofthemouseiPScellsuspensiontoeachwellofthe6-wellplate.RightafterplatingiPScells,gentlyswirltheplateback-and-forthandside-to-sideandincubateat37°C.TheESmediamustbechangedeverydayandmouseiPScellssub-culturedevery2-3d.TrackpassagenumberofiPScells.

FreezingmouseiPScells

1.Growcellstotheexponentialphaseina6-wellplate.

2.Aspiratethemedium,andwashthecellstwicewith2mlofPBS.

3.Add0.5mlof0.25%trypsin-EDTAandincubate2minat37°C.

4.Add2mlofmouseESmedium,andsuspendthecellsbypipettingupanddowntosinglecellsuspension.

5.Transferthecellsuspensiontoa15-mlconicaltube,countthenumberofcellsandspinthecellsat200xgfor5min.

6.Discardthesupernatant,andresuspendthecellswithmouseESmediumtotheconcentrationat1x10⁶cellsperml.

7.Addequalvolumeof2Xfreezingmedium(20%DMSOand80%FBS),andaliquotitat0.5mlpervial.

8.Putthevialsinacell-freezingcontainer,andstorethevialsat–80°Covernight.

9.Transferthevialstoliquidnitrogenforlong-termstorage.