TISSUE CULTURE ON COVERSLIPS

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.Foreachtypeoftissuelabel1100mmpetridishwiththepatientnumber,patientname,typeoftissue,anddate.Labelasterile15mlconicalcentrifugetubewithpatient"snumber,name,"Collagenase",timeofday,anddate.

B.CulturesaregrownincompleteChang/F-10media.Mediashouldbefresh(lessthan4daysold)andbeprewarmedandatpH7.0-7.5.Allculturesareincubatedinawet,5%CO2incubatorat37°C.Allnewculturesaretobeplacedinthe"alien"incubatorandcheckedthenextdayforcontamination.Oncecellsaregrowinganddeterminedtobeaseptic,theyaretransferredtothe"tissueculture"incubator.

C.Usingsterileforceps,transfertissuetothepetridish,a2mmcubedsampleissufficient.Ifthesampleislarge,cutoffasection.Add2.0mlofmediatothedish.Usingscissors,cuttissueintosmallbits,usecleanstrokesandavoidmashingortearingthetissue.Add4mlmediaandtransfer2mlintothecentrifugetubeandleave4mlinthedish.Gentlyrockthep-dishtodistributethemedia.Placethep-dishintheincubatorfor2-3daysforthetissuetoadheretothesurface;thenadd1mlfreshmedia.Thep-dishwillbeyourback-upifthecoverslipsdon"tgroworgetcontaminated.

D.Allowthetissueinthecentrifugetubetosettlewhileyouprepareandsterilefilterthecollagenasesolution.(Theconcentrationofyourcollagenasesolutioncanvarydependinguponthetypeandamountoftissueyoureceive.Placentaltissuesbreakupeasierandrequirelessconcentratedcollagenase.).RemovethesupernatantfromthecentrifugetubewithasterilePipetteanddiscard.Addthefilteredworkingcollagenasesolution.Incubatefor1to3hours,mixingoccasionally,untilthetissueisbrokenup.

E.Whenthetissueincollagenaseissufficientlybrokenup;thesolutionshouldbecloudy,labelculturedishes.For1typeoftissuereceived,setup8dishes.For2typesoftissuereceived,set6oneach;for3ormoretypesoftissue,set4oneach.Centrifugethetissueincollagenasefor6minutesat150Xg(900rpminSorvalGLC-2B).Removethesupernatant,resuspendthepelletandadd2.5mlofcompleteChang/F-10media.NOTE:Ifyoursamplesizeislargeorthetissuedissolvesnicelyinthecollagenase,youmaywanttoaddmoremediatomakeyourtissuesuspensionmoredilute.Plateoutwith0.3mloneachof2coverslips.Add0.5mlofmediatothecentrifugetubeandplateout2morecoverslips.Continuetodothisuntilallcoverslipshavebeenplatedout.Carefullyplacethecoverslipsintheincubator.Checkthemonaninvertedmicroscopeafter2hours,ifpossIBLe.Ifthereisadherence,add1.5mlofmediatoeachdishandrecheckthecoverslips.(Sometimes,itisdifficulttoseethroughthe0.3mlofsampletogetagoodestimateofadherencesothedishesneedtorechecked).Ifthereissignificantadherenceorgrowth,removethismediaandadd2mloffreshmedia.Youmaywanttosavethesupernatantandcentrifugeittoreplateiftheoriginalcoverslipsaretoodense.Withtheremainingtissuesuspensioninthecentrifugetube,youcaneitherdiscardortransfertoalabeledflaskandsaveasanadditionalback-upculture.

F.Ifthereisnoadherenceorthesamplewassetlateintheday,incubateovernight.Thenextday,floodthecoverslipswith1.0mloffreshmediaandcheckforgrowth.Ifthereissufficientgrowth,immediatelychangethemedia.Ifthereisnogrowth,returntotheincubator.Thefollowingday,add0.5mlofmediaandcheckforgrowth.Checkthecoverslipseachdayforgrowth.Sometissueswillgrowasindividualcells,otherswillgrowascolonies.Ifcellsaretoodense,metaphasesmaynotspreadwell.Iftissuesgrowasdensecolonies,itmaybebesttosubcultureontoadditionalcoverslips.YourmitoticindexwillbegreaterandspreADIngwillimprove.

III.HarvestProcedure,In-Situ:

A.Add20µlEthidiumBromideworkingsolutiontoeachdish,incubatefor40minutes.Add40µlofcolcemidworkingsolution,incubateforanother20minutes.

B.Tenminutesbeforeendofincubation,startupTECANharvestersothatitwillbeready.

C.HarvestusingTECAN.

Ifyouneedtoharvestbyhand:

1.Removemedia;add2mlhypotonicfor20minutes.

2.Removehypo;add2mlfreshhypofor20minutes.

3.Add2mlfixativetohypotonic.

4.Remove2mloffixative/hypo;add2mlfixative.

5.Removeall;add2mlfixativefor15minutes.

6.Removeall;add2mlfixativefor15minutes.

7.Removeall;add4mlfixativefor15minutes.

8.Removeall;dryindryingchamberorinahumidenvironment.

D.Dryingconditionsareveryimportantfortheproperspreadingofmetaphases.UsingtheTECANdryprogram,removefixativefromthedish.Usingtheaspirator,removealmostallthefix,goingaroundtheedgeofthecoverslip.Allowtodryinahumidenvironment,55-60%humidity.Ifthecoverslipdriestoorapidly,allthecellswillbetrappedinthemembranes.Ifitdriestooslowly,thechromosomeswillfloatawayfromthemetaphase.

E.Removethecoverslipfromthepetridish,keepingitright-sideup.Ithelpstoholdthedishwithyourthumbandmiddlefingerandbendupthebottomofthedishwithyourindexfingersothatthecoverslipisliftedup.Gently,wipeoffanyfixativefromthebottomofthecoverslipwithatissue.Mountthecoversliponalabeledmicroscopeslideusingadropofmountingmedia.(IfFISHistobeperformedontheseslides,usesupergluetomountthecoverslips.)

F.Allowtheslidestodryatroomtemperatureforatleast30minutes.Slidescannowbebakedandbanded.

IV.Solutions:

Colcemidworkingsolution:10µg/mlColcemidinHank"sBalancedSaltSolution,storeat4°C.

CompleteCHANG/F10media:45mlChangbasalmediaB,42mlNutrientMixF-10,5mlChangsupplementA,8mlFetalcalfserum,1mlPenicillin/Streptomycin,solution,1.1mlL-Glutaminesolution,storeat4°C.

EthidiumBromideworkingsolution:2mg/mlEthidiumBromideinRPMI-1640,storedindark.

Fixative:30mlMethanoland10mlGlacialAceticAcid,preparedfresh.

Hypotonicsolution:0.8%NaCitrate/0.075MKCl:1.6gNaCitrate,1.68gPotassiumChloridedissolvedin500mldH₂O.

1XTrypsinEDTAsolution:10mlstocktrypsinsolution(10Xtrypsininsaline),0.1gEDTA(disodiumsalt),dissolvedin490mlHank"sBalancedSaltSolution.Sterilefiltered,50mlaliquots.Storefrozen.

Collagenase

Stocksolution:add16.5mlcompleteChang/F-10toa100mgcollagenasebottle,dissolve(6mg/ml).Aliquotintomicrocentrifugetubestostoreat-20degreesCforupto6months.

Workingsolution:add0.5mlto1.0mlthawedcollagenasestocksolutionto2.5mlcompleteChang/F-10media.Filter,usinga3mlsyringeandanAcrodiscfilter,intoasterile15mlconicalcentrifugetube.

V.Reagents:

Changmedia:Irvinecat#T100-018,liquidandfrozensupplement,100mlbottle,storesupplementfrozen,basalmediaat4°C.

Colcemid:Gibcocat#15210-040.10µg/mlinHank"sBalancedSaltSolution,10mlbottle,storeat4°C.

Collagenase:SIGMAcat#C-9263,100mgbottle.

EDTA:SIGMAcat#ED2SS.Ethylenediaminetetraaceticacid,disodiumsalt,dihydrate,100gbottle.

EthidiumBromide:SIGMAcat#E-8751.2,7-diamino-10-ethyl-9-phenyl-phenanthridiniumbromide,250mgbottle.

Fetalcalfserum:HYCLONE,INC.cat#A-1111-D.Definedfetalbovineserum,100mlbottle,storefrozen.

Hank"sBalancedSaltSolution:GIBCOcat#14170-112.Hank"sbalancedsaltsolution,withoutCaCl₂,MgCl₂,MgSO₄,500mlbottle,Storeat4°C.

GlacialAceticAcid:FISHERSCIENTIFICcat#A38-500.Aceticacid,GlacialACS(Aldehydefree),500mlbottle.

L-Glutamine:GIBCOcat#25030-032.L-Glutaminesolution100X(200mM),20mlbottle,storefrozen.

Methanol:FISHERSCIENTIFICcat#A412-500.Methylalcoholanhydrous(ACS)(Absolute)Acetonefree,500mlbottle.

NutrientMixF-10:GIBCOcat#81200-040.NutrientMixtureF-10(HAM)powderedform,preparedtoinstructions,10x1literpackage.

Penicillin/Streptomycin:GIBCOcat#15140-031.Penicillin/Streptomycinsolution,10,000units/ml;10,000µg/ml,20mlbottle,storefrozen.

PotassiumChloride:UW-STORES.cat#2485ACSgranular,500gbottle.

RPMI-1640:GIBCOcat#31800-022RPMI-1640powderedform,preparedtoinstructions,10x1literpackage.

TrypsinStockSolution(1X):GIBCOcat#25300-013.Trypsin2.5%(10X)insaline.Storefrozen.

SodiumCitrate:UW-Stores,cat#2588.Crystal,ACS,Na₃C₆H₅0₇^.2H₂0,500gbottle.