I.Purpose:

A:Skintissuemaybeusedforchromosomeanalysisinspecialcaseswhentheresultsfromperipheralbloodareinconclusive,e.g.sUSPectedmosaicism,confirmationofanewchromosomedisorder,orspecialdermatologicaldisorders.Tissuemayalsobeusedwhenbloodculturesarenotavailable,suchasonstillbirths.B:Theskinbiopsyshouldbeperformedbyaphysician,withscrupulousaseptictechnique.Thebiopsysiteisthoroughlywashedwithanantisepticsoapandthenwipedwith70%ethanol,whichisallowedtodrybeforethebiopsyistaken.IodineandMercurochrome-likeantisepticsshouldnotbeused.Mostphysiciansprefertousealocalanesthetic;thisdoesnotaffectthesuccessoftheculture.A4mmpunchbiopsyshouldbetakenandplacedinavialcontaining8mloftransportmedia(F-10,Eagle"s,MEM,etc.).Forcasesofstillbirth,thepreferredtissuesareskin,fascia,kidney,andlung.Thetubeshouldbelabeledwiththepatient"snameanddeliveredtothecytogeneticslabwithoutdelay.

II.CultureProcedure:

A.Aseptictechniquemustbeusedwhensettingupthecultures,preferablyunderalaminarflowhood.Foreachtypeoftissuelabel1100mmpetridishand2-3T-25tissueflaskswiththepatientnumber,patientname,typeoftissue,anddate.Flasksshouldalsobelabeled(usinggreekletters)toidentifyprimarycultureswhenflasksaresub-cultured;thisallowsforidentificationofculturalartifactsandpsuedovsrealmosaicism.B.CulturesaregrownincompleteNutrientMixF-10.Mediashouldbefresh(lessthan4daysold)andbeprewarmedandatpH7.0-7.5.Allculturesareincubatedinawet,5%CO2incubatorat37C.Allnewculturesaretobeplacedinthe"alien"incubatorandcheckedthenextdayforcontamination.Oncecellsaregrowinganddeterminedtobeaseptic,theyaretransferredtothe"tissueculture"incubator.C.Usingforcepstransfertissuetothepetridish,asample2mmx2mmx2mmissufficient.Ifthesampleislarge,cutoffasection.Add0.5mlofmediatothedish.Usingscissors,cuttissueintosmallbits,usecleanstrokesandavoidmashingortearingthetissue.Addmediasothereisenoughtotransfer2mlintoeachflaskandleave2mlinthedish.Gentlyrocktheflaskstodistributethemedia.Screwthecapsonloosely,andplacetheflasksintheincubatorfor2-3daysforthetissuetoadheretothesurface.Carefullyaddanadditional3mlofmediatoeachflaskand5mltoeachdishandreturntheculturestotheincubator.D.Culturesshouldbeexaminedforgrowthafter4days.When5to10coloniesof50-100cellshaveformed,changethemediabyaspiratingofftheoldmediaandgentlyadding5mloffreshmedia.Culturesshouldbeharvestedassoonastherearesufficientcells(10-15coloniesofseveralhundredcells).FlasksfrommorethanoneprimarycultureandwhenpossIBLe,morethanonetypeoftissue,shouldbeharvestedtomaximizedetectionofmosaics.

III.HarvestProcedure:

A.Onetotwodayspriortotheharvestthecellsaredispersedintheflasktomaximizethemitoticindex.B.Aspiratethemediaoutoftheflask.Add0.5mlofprewarmed1XTrypsinEDTA,gentlyrocktheflasktodistributethetrypsinoverthesurface.Incubatetheflaskfor20-30secondsatroomtemperature(thisisdonetoremoveproteinsfromthemediaandcomplexanti-trypsinproducedbythecells).Aspirateoffthetrypsin.Add0.5mloffresh1XTrypsinEDTA,incubatetheflaskfor1-5minutesat37C.Examinetheflaskonthemicroscopetodetermineifthecellshavebecomedetachedfromthedish,tappingtheflaskmaybenecessarytodetachallthecells.Add4.5mloffreshmediatotheflask.Theculturemayneedtobesplitinto2or3flasks,ormayneedtobetransferedtoasmallerpetridishdependingonthenumberofcells.Theflasksshouldbeat50-75%confluency.Ifcultureissplit,addmediatobringeachflaskto5ml.Returntheculturestotheincubator.C.Thenextmorningchangethemediaandchecktheculturestoseeiftheyarereadyforharvest.Thecellsshouldbenearconfluentandshouldshowactivelydividingcells.Ifthecultureisnotconfluentenoughwaituntilthenextdaytoharvest.D.Add0.3mlofworkingcolcemidsolutiontoeachflask.Incubatetheculturesat37Cfor2-8hours.Checktheculturesafter2hourstoseeiftherearesufficientcellsinmitoticarrest(cellswillberoundedoffandmaybefloating),ifnot,continuetoincubateandcheckeveryhalfhour.E.Whenculturesareready,removemediawithapipetandtransfertoalabeled15mlconicalcentrifugetube.Add0.5mltrypsin,incubatefor20-30seconds.Transfertrypsintothetubeusingapipet.Add0.5mloffreahtrypsin,incubateat37Cfor4-5minutes.Checkonmicroscopetoseeifcellsaredetached.Add2mlofmediatoeachflask,pipetupanddowngently3-4timestoremoveanyremainingcellsfromthedishandtoformasinglecellsuspension.Transferthemediatothetube.Repeatthisstepwithanadditional2-3mlofmedia.F.Centrifugethetubesfor6-8minutesat150xg(900rpm).G.Aspirateanddiscardallbut0.3mlofsupernatant.Resuspendthecellpelletbymixingbyhand,donotuseamechanicalvortexerasthismaybreakthecells,thisisespeciallyimportantinstepI.Add3mlofprewarmedhypotonicsolutionandmix.Incubatethetubesat37Cfor8-10minutes.H.Centrifugethetubesfor6-8minutesat150xg.I.Aspirateanddiscardallbut0.3mlofsupernatant.Mixbyhandtobreakupthecellpellet.Itisimportanttogetthecellpelletcompletelyresuspended,butthecellsarefragileandeasilybroken.J.Slowlyadd2mloffixative(1pasteurpipetful)lettingitrundownthesideofthetubesothatitlayersontopofthecellsuspension.Capthetube.Mixrapidlybyhandsothatallthecellsuspensionisfixedevenly;ifitismixedtooslowly,clumpsmayform.Letsitatroomtemperaturefor15-20minutes.K.Prefixbycarefullyadding1pasteurpipetfuloffixandmixing.Centrifugeimmediatelyfor6minutesat150xg.L.Aspirateanddiscardallbut0.3mlofsupernatant.Resuspendthecellpellet.Add2mloffixativeandmix.Letsitatroomtemperature15-20minutes.M.RepeatstepsK,L1or2timesuntilthepelletiscleanandwhite.Thecultureisnowreadytomakeslides.Forbestresults,slidesshouldbemadethesamedayastheharvest.

IV.SlidePreparation:

SeeRegularProcedureforCulturingLymphocytes.

V.Solutions:

Colcemidworkingsolution:10mcg/mlColcemidinHank"sBalancedSaltSolution,storeat4C.CompleteNutrientMixF-10:100mlNutrientMixF-10,20mlFetalcalfserum,1mlPenicillin/Streptomycinsolution,1mlL-Glutaminesolution,storeat4C.Fixative:30mlMethanoland10mlGlacialAceticAcid,preparedfresh.Hypotonicsolution0.075MKCl:2.8gPotassiumchloridedissolvedin500mldH2O.1XTrypsin-EDTASolution:10mlstocktrypsinsolution(10Xtrypsininsaline),0.1gramEDTA(disodiumsalt),dissolvedin490mlHank"sBalancedSaltSolution.Sterilefiltered,50mlaliqots.Storefrozen

VI.Reagents:

Colcemid:Gibcocat#120-5210.10mcg/mlinHank"sBalancedSaltSolution,10mlbottle,storeat4C.EDTA:SIGMAcat#ED2SS.Ethylenediaminetetraaceticacid,disodiumsalt,dihydrate,100grambottle.Fetalcalfserum:HYCLONE/STERILESYSTEMSINC.cat#A-1111-D.definedfetalbovineserum100mlbottle,storefrozen.Hank"sBalancedSaltSolution:GIBCOcat#310-4170.Hak"sBalancedSaltSolution,withoutCaCl2,MgCl2,MgSo4,500mlbottle.Storeat4C.GlacialAceticacid:AMERICANSCIENTIFICPRODUCTScat#9508-2.Aceticacid,GlacialACS(Aldehydefree),500mlbottle.L-Glutamine:GIBCOcat#320-5030.L-Glutaminesolution100X(200mM),20mlbottle,storefrozen.Methanol:AMERICANSCIENTIFICPRODUCTScat#3016-1.MethylalcoholanhydrousAR(ACS)(Absolute)Acetonefree,500mlbottle.NutrientMixF-10:GIBCOcat#430-1200.NutrientMixtureF-10(HAM)powderedform,preparedtoinstructions,10x1literpackage.Penicillin/Streptomycin:GIBCOcat#600-5140.Penicillin/Streptomycinsolution,10,000units/ml/10,000mcg/ml,20mlbottle,storefrozen.PotassiumChloride:COLUMBUSCHEMICALINDUSTRIES,INC.ACSgranular,500glots.TrypsinStockSolution:GIBCOcat#610-5090.Trypsin2.5%(10X)insaline.Storefrozen.