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InVitro Adipocytes Differentiation
Introduction
Obesityisasignificantclinicalproblemthatcontributestolife-threateningdiseasessuchasdiabetesandatherosclerosis.Withanincreasingincidenceofobesityworldwide,rationalstrategiesareneededtocontrolADIpogenesis.Cellsthatundergodeterminationtotheadiposelineagecalledadipoblastsandthisprocesscalledadipogenesis.Adipogenesishasbeenextensivelystudiedusinginvitromodelsystemsconsistingeitherofestablishedadipogeniccelllines(clonallines3T3-L1,3T3-F442A,Ob17,BFC-1,ST13,A31T,...)andprimarycultureofadipocyteprecursorsandpre-adipocytes.GrowtharrestofadipoblastsattheG1/SphaseofthecellcycleappearstoberequiredforthecommitmentofadipoblaststopreadiposecellswhichisaccompaniedbytheemergenceofearlyMarkersofadipgenesis.Usingcellculturesystemscultivatedeitherinthepresenceofserumorindefinedmedium,ithasbeenpossIBLetoidentifyfactorsregulatingeitherpositivelyornegativelyadiposedifferentiation.Thepositiveregulatorsincludeseveralhormonessuchasinsulin,growthhormone,andtriiodothronine.Inaddition,serum,partiallypurifiedserumfactors,andconditionedmediumfromseveraladipogeniccelllineshavebeenshowntostimulateadiposedifferentiationinvitroandassumedtocontainadipogenicregulatorsdistinctfromthehormones.Mammalian3T3-L1cellsdifferentiateintoadipocytesaftercontinuousexposuretopharmacologicaldosesofinsulinorphysiologicaldosesofinsulin-likegrowthfactorI.
Principle
Whenadipoblaststreatedwithacombinationofdexamethasone,isobutylmethylxanthine(IBMXorMIX)andinsulin,cellsstarttoadoptaroundedphenotypeandwithin5-8daysbegintoaccumulatelipidsintracellularlyintheformoflipiddroplets.Theinductioncondtionsandmediavaryaccordingtothecelllines.TreatmentofcellswithdexamethasoneactivatesthetranscriptionfactorCCAAT/enhancer-bindingproteinb(C/EBPb).IBMXinhibitssolublecyclicnucleotidephosphodiesterasesandresultsinincreasedintracellularcAMPlevels.Atthenuclearlevel,treatmentwithIBMXresultsinactivationoftherelatedtranscriptionfactorC/EBPd.C/EBPbanddinturninducetranscriptionofC/EBPaandPPAR.Within3daysofexposuretoinducers,thecellsundergotworoundsofmitosis,termedmitoticclonalexpansion,whicharerequiredfordifferentiation.Insulinorinsulin-likegrowthfactor-1promoteadipocytedifferentiationbyactivatingPI3-kinaseandAktactivity.ModulationoftheactivityoftheforkheadtranscriptionfactorFoxo1appearstobenecessaryforinsulintopromoteadipocytedifferentiation.C/EBPaandPPARdirectthefinalphaseofadipogenesisbyactivatingexpressionofadipocyte-specificgenes,suchasfattyacidsynthetase,fattyacidbindingprotein,leptinandadiponectin.
Reagentsandrequirements
Materialandsources:
- DMEM(GibcoBRL-Cat#11965-084)
- CalfSerum(GibcoBRL-Cat#16170-078)
- FetalBovineSerum(GibcoBRL-Cat#10437-028)
- Isobutylmethylxanthine(IBMX;SigmaI-7018)
- Dexamethasone(SigmaD-4902)
- Insulin(Bovine;SigmaI-5500)
- MEMSodiumPyruvate(100mM;GibcoBRLCat#11360-070)
- Pen/Strep/Glutamine(100xP/S/G;GibcoBRLCat#10378-016)
- Sterilizedpetriplates
- Leminarairhood
- CO2incubator
- SterilizedPipettes
Preparationofsolutions
- 10%CalfSerum/DMEM60mL:CalfSerum6mL:100mMMEMSodiumPyruvate6mL:100xP/S/G500mL:DMEM
- 10%FBS/DMEM60mL:FetalBovineSerum(FilterSterilized)6mL:100mMMEMSodiumPyruvate6mL:100xP/S/G500mL:DMEM
- IBMXSolution(makefresh)DissolveIBMXinasolutionmadeof0.5NKOHtoafinalconcentrationof0.0115g/mL.Filtersterilizethrougha0.22mmsyringefilter.
- InsulinStockSolution167mM(1mg/mL)in0.02MHClFiltersterilizedthrough0.22mmfilterCanstoreat-20°Cforlongterm,4°Cshortterm.
- DexamethasoneStockSolutionsFreezerStock:10mMofDexin100%ethanol(storeat-20°C)WorkingStock:DiluteFreezerstockto1mMinPBSFiltersterilizeandstoreat4°C.
- MDIInductionMedia(10mL/10cmplate;5mL/6cmplate)Torequiredvolumeof10%FBS/DMEMadd:1:100IBMX1:1000Insulin1:1000Dexamethasoneworkingstock
- InsulinMedia(10mL/10cmplate;5mL/6cmplate)Torequiredvolumeof10%FBS/DMEMadd:1:1000Insulin
- OilredOstocksolution(0.5g/100mlisopropanol)Justbeforestaining:mix60mlofstockwith40mlofH20,letitsitfor1hratRTandfilterthroughwhatmanpaper3MM.
Procedures
Preadipocytemaintenanceandpassage:
Platethecellsin10%CS/FBS-DMEMonculturedishesandincubatethemat37°Cin10%CO2.Itisimportanttofeedthepreadipocyteseverycoupleofdaysandavoidlettingthemgettooconfluent(>70%),ifyouwanttocontinuetopassagethemanddifferentiatethematalaterdate.So,takecaretosplitthemappropriately.Theycanbesplitasfaras1:15,thoughweusuallydo1:10orlessdependingonneed.
AdipocyteDifferentiationProtocol
- Growpreadipocytes/fibrobalsttoconfluencyin10%FBS-DMEM.
- Aftertwodaysofpostconfluency(whichiscountedasday0),stimulatethecellswithMDIinductionmedia.Youwillnoticeadistinctchangeinthemorphologyofthecells(becomemorespindly)inthenext2days.
- AftertwodaysofMDIaninductionmedium(whichiscalledasday2)replacetheMDIinductionmediawithInsulinMedia.Themediawillbegintogetmoreviscousasfreefattyacidsareproducedbythecellsandsecretedintothemedia.
- Twodayslater(whichiscalledasday4)changemediato10%FBS-DMEM.Feedcellswith10%FBS-DMEMeverytwodays.Fulldifferentiationisusuallyachievedbyday8.
Stainingprocedure:
- Aspiratemedia,addformaldehydeslowlyandletsitfor30min.
- AspirateformaldehydeandaddOilredOsolutiontocoverthewell,leave1hratRT.
- Removethestainandwashwithdistilledwatertwice.
- Letitdryforpictures.
Precautions:
- DexamethasoneSolution,IBMXSolutionandInsulinSolutionshouldbestoredat-20ºC.
- OilRedOSolution,WashSolution,andDyeExtractionSolutionshouldbestoredatroomtemperature.StorageofOilRedOSolutionandDyeExtractionSolutionat-20ºCmayresultinformationofinsolubleprecipitatesandisnotrecommended.IfOilRedOsolutionformsaprecipitate,removeparticulatesbypassagethrougha0.22or0.45micronfilter.OilRedOstainsskinandclothing.
- IBMXanddexamethasoneareirritantsandpotentiallytoxic.DMSOisreadilyabsorbedthroughtheskin.Wearalabcoatandgloveswhenhandlingthesesolutions.
- Isopropanolisflammable.Keepsolutionscontainingisopropanol(OilRedOSolution,WashSolutionandDyeExtractionSolution)awayfromopenflames.
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