Isoprenoids: Biosynthesis and Biomedical Applications ...
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Fromearlypost-implantationstagestoapproximately18weeks"gestation,cytotrophoblastatthetipsofanchoringvilliattheplacentalperipheryproliferatetoformcolumns.Fromthedistaledgesofthese,singlecellsbreakawayandinfiltratetheadjacentmaternaldecidualstroma,progressingasfarastheinneronethirdofthemyometrium.Thesecellsinvadematernalspiralarteriesandtransformtheirwallswithlossofsmoothmuscleandtheassociatedelasticandcollagenousextracellularmatrixwhichisreplacedbyafibrinoidsubstance.ThismethodenablestheresearchertoexplantterminalplacentalvilliontoathreedimensionalmatrixofeithercollagenorMatrigelandmonitortheformationofanchoringcytotrophoblastcolumns.Theresultingcellscanbestimulatedtoassumeamigratoryphenotypebygrowthfactors.TGFb1inhibitsoutgrowth.ExplantcultureoffirsttrimesterplacentaltissuefordenovogenerationofextravilloustrophoblastTheprotocolsdescribedincludeexplantoneithercollagenIorMatrigel.Theresultsaresimilar,buttrophoblastoutgrowthoncollagenoccursrADIallyatthesurfacewhileonMatrigelitoccurspredominantlyinadownwarddirectionintothegel.Theformerexperimentisthereforemoreconvenientlymonitoredbywholemountmicroscopy(Figure6).Undercontrolconditionsof20%oxygenandserumfreemedium,cellproliferationoccursduringthefirst24hafterwhichitceasesandfurtheroutgrowthoncollagen,whichcontinuesforuptoabout5days,representsthereorganisationofanexistingpoolofcellsfromathickcolumnatthevilloustiptoasheetonlyafewcellsthickatthecollagengelsurface.ExplantculturemethodEquipmentandreagents- 6,12or24wellcultureplates(Costar)
- Pastettes
- 9cmpetridishes
- 5cmpetridishes
- RattailcollagentypeI(BectonDickenson,distributedbyStratech)
- Matrigel(BectonDickenson,distributedbyStratech)
- 10xDMEM(Sigma)7.5%sodiumbicarbonate(Gibco/BRL)
- Culturemedium:1:1mixtureofDMEMandHam"sF12
- 25gaugeneedles
- 1mlsyringes
- 10mlstripettes
- Antibiotic,antimycoticmixAAM(Sigma)
- Fetalcalfserum(Gibco/BRL)
- Growthfactors
- Humidbox
- 70%ethanol
- 50mgCollagenase(SigmatypeIA)
MethodCollagengelCollagengelsshouldbepreparedthedaybeforetheexperiment.Storinggelsforalongertimeperiodthanmayresultindeteriorationasindicatedbyastringyappearance.Foreach12wellplate,1mlofcollagentypeIisrequired.Ideallythecollagenconcentrationshouldbemorethan3mg/ml.Gelsbecomelessfirmwithstoragetimeofthecollagen,andmoredilutestocksolutionstendtohaveashortershelflife.Ifmorethan3-4mlofcollagenaretobeusedthenitisrecommendedthatitbepreparedinseparate1-2mlbatchestoensureevenmixing.Forbestresultsusecollagen,10xmediumandsodiumbicarbonatedirectlyfromthefridge.For1plate:- Withdraw1mlofcollagenfromthecontainerusinga1mlsyringeandneedle.Discardtheneedlebeforetransferringtoa1.5mlEppendorftube(thispreventstheintroductionofairbubbles).
- Add100mlof10xDMEMandmixcarefullywithadisposablepastette,takingcarenottointroducebubbles(solutionisayellowcolour).
- Addapproximately200mlof7.5%sodiumbicarbonatetoneutralisethecollagenandcauseittoset(thecolourchangesfromyellowtopink).Again,mixthiscarefullywithapastetteasthesolutionwillsetfromthebottom.
- Usethepastettetotransfer1drop(approximately80ml)tothecentreeachwelloftheplate.Avoidincludingairbubblesbycarefulpipetting.Thecultureplatecaneitherbeleftinthehoodorcarefullytransferredtoa5%CO2incubator.Thecollagenshouldtakearound10minutestogel.
- Thenaddculturemediumtoeachwelltocoverthegelandincubatetheplateinhumidboxat37°CinaCO2incubatoruntilrequired.
Gelscanalternativelybesetusingammonia.Thisisaccomplishedbymakingupthesolutionasaboveandspottingintotheplateswhicharethencarefullyplacedinsideabox.Afewdropsofammoniaareplacedonasmallpetridishinsidetheboxwhichisthensealedandincubatedfor15-30minutesat37°C.Caremustbetakennottodisturbthecollagendropletsandcausethemtospread-theywillbemorefluidthaninthenormalmethod--andnottoletthegelsdryout.Aftersettingthegelsareincubatedasbeforeinaboxfreefromammoniavapour.MatrigelMatrigelshouldbepreparedatleast24hpriortosettingupexplantcultures.Matrigelsetsatroomtemperature,thereforeallpreparationshouldbeperformedoniceorat4°C.- Frozen(-20°C)aliquotsofMatrigelshouldbedefrostedovernightat4°C.Pipettetipsandcultureplatesshouldalsobepre-cooledto4°CtopreventprematuregellingoftheMatrigel.
- Mix1mlofMatrigelwith100mlof10xDMEMina1.5mlEppendorfandstartthegellingprocessbyadding200mlof7.5%sodiumbicarbonateensuringcompletemixingbeforeplacingadropofapproximately80mlofMatrigelmixinthecentreofeachculturewell.
- Incubatethecultureplatesat37°Cfor10-15minandthencoverthegelin1mlofSFM,andstoreinasealedhumidboxat4°Cuntilrequired.
SelectionoftissueUsingscissors,cutnumerousterminalportions(~2-3mm)ofthevilloustreeforexplantculture.Foroptimalresults,villishouldbeeithertrimmedfromtheplacentalperipheryunderadissectingmicroscopeorthepiecescheckedforsuitABIlityusingthemicroscope.Optimalattachmentisobtainedwhenvillousclustersincludeseveralbranches.Observethefrondlikestructureofthevilliaftergentleteasingoutwithfineforceps.Ifthemesenchymal(terminal)villiappearelongatedandstringytothenakedeye,thensuccessofattachmentandoutgrowthtendstobelow.Useaportionthatincludesanintermediatevilluswithseveralmesenchymalbranchesemanatingfromit.Selectedportionscanbeplacedinadropofculturemediuminapetridishuntilenoughmaterialhasbeencollected.ExplantAttachment- Oncetheplacentaltissuehasbeendissectedintoappropriatesizedpieces,drainthegelscompletelyofmedium;duringincubationtheywillhavecontractedslightlyandbecomefirm,aidingtissueattachment.
- Transferonepieceofvilloustissuetoeachwellusingfineforcepsandcarefullyteaseoutthebranchesoverthegelsurface,takingcarenottoscorethegel.Alternatively,leavethemediuminthewellandaddtheselectedvilloustissuetothemedium.
- Carefullydrainthemediumfromthewellgentlyandlowerthetissueontothecollagendrop.Ifcorrectplacementdoesnotoccurthenuseapastetteandaminimalvolumeofmediumtogentlyliftandplacethevilloustissueontothedrop.Thismethodavoidspotentialscoringofthecollagengel.Ideallythevillousterminishouldbearrangedradiallyoverthesurfaceofthegeldrop.
- Followingplacement,covereachpieceoftissuewith20mlofpre-warmedserum-freeculturemediumandincubateat37°Cin5%CO2inahumidboxfor3-12hours.Afterthisperiod,initialoutgrowthissometimesseenusingahighmagnificationphasecontrastobjective.Formaximumattachment,thevilliareincubatedovernightat37°Cwith50mlofserum-freemedium.Attachmentmaybeindicatedbytheappearanceofradialstresslinesofbundledcollageninthegel.Followingthisinitialperiod,gentlyimmerseculturesin1mlofserum-freemedium,directingapipettetothesideoftheculturewelltoavoiddetachmentofthetissuefromthegel.Atthisstagegrowthfactors,bromodeoxyuridineoradhesion-modulatingantibodiesmaybeaddedtotheexplantintheserumfreemediumatsuitableconcentrations(32).
- Transferthecultureplatecarefullybacktothehumidboxintheincubator.Afterafurther12h,initialoutgrowthfromviableattachedterminalvillistructuresshouldbeapparent.Iftissuebecomesdetachedwhen1mlmediumisaddedorduringtheovernightincubationthatfollows,thenreattachmentcanbeattemptedbydrainingthewellandrepositioningtheexplant.However,repositionedtissueoftenhaspoorviabilityandislikelytodetachagainaswellasexhibitingdelayedgrowthcharacteristics.
CytotrophoblastColumnGrowthCytotrophoblastcolumnoutgrowthandsubsequentcellmigrationfromgel-attachedvilliismonitoreddailyusinglowandhighpowerwholemountmicroscopy.Fordailycomparisonsofcolumngrowth,lowpowerimagecaptureisrecommended(Figure6).Theareaofoutgrowingcellsisveryvariable,beinghighlydependentonthesizeoftheinitialvillus-gelcontact(32).Outgrowthislimitedtothetipsofthevillibutcellsfromadjacenttipsmergetoformashell-likestructuresurroundingtheexplant(35).Recoveryofcytotrophoblasts- Removaloftissuefromthesurfaceofacollagengelafterupto7days"cultureleavesapurepopulationofextravillouscytotrophoblastatthesurface.Toreleasecytotrophoblasts,removetheexplantconditionedmedium,filterandstoreitat-20°C,andwashtheexplant3xwithPBS.
- Removethevilloustissuefromthecollagenwithfineforcepsandincubatetheremainingcellswith100mg/mlcollagenase(Sigmatype1A)at37°C.CollagenaseshouldbereconstitutedinDEPC-treatedsterilePBSiftheisolatedcytotrophoblastsaretobeusedforRNAextraction.Cytotrophoblastcellsarereleasedfromthecollagenbya10minincubationat37°C.Gentleagitationbypipettingcanaidcellrelease.
- Pelletthecytotrophoblastsbycentrifugationat1500rpmfor10minat4°C,andwashwithPBStwicetoremovealltracesofcollagenase.
- Trypsinisationisalsoaneffectivemethodforrecoveryofcytos.
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