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Organotypicraftcultureofprimaryhumankeratinocytes(PHKs)

Notes

Use10⁶keratinocytesperraft.

TypicalProtocol

TrypsinizethePHKsofftheflaskandresUSPendin(say)2mlraftmedium.

Seeseparateprotocolforraftmedium

RemovethemediumfromthecollagenplugsandoverlaywiththenewmediumcontainingPHKs.Incubatefor1-2daystoallowthePHKstosettleandbecomeconfluent.Autoclavethefollowing;

SpatulasforscoopingoutthecollagenplugPBSStainlesssteelmeshgridinaglasspetri-dish.(Thoroughlyrinsetheglasspetri-dishtoremovedetergent.Makethefour"legs"forthemeshgridbyplacingitflatontoamicrofugetuberackandpressingatubeintoanedge,deformingitdownwards.Thisdownwarddeformedbitisaleg.)

WhenthePHKsareconfluent:-Separatetheedgeofthecollagenplugfromthewallofthewellbygoingrounditwithasterilescalpel.Allowthecollagenplugtocontractawayfromthewallsforafewhours.

Thisislikecuttingroundacakeinacaketin.ThePHK"membrane"maycontractfromtheedgealsoanddetachalittlefromthecollagen

Removemedia,scoopoutpluganddepositontothemeshinthepetri-dish,keepingeverythingsterile.

Optional-rinseinPBSbeforeplacingontomeshThistransferisthetrickiestpartofthewholeprocedure.ThereisnoeasywaytodoitalthoughIfoundthatthecollagengelisstrongerthanitlooks.Ifyougetitintact,rightsideup(ie.PHKsup),andlookingroundishonthemesh,withouthavingscrapedoffallthePHKs,thenyou"vedonewell.Usuallyitendsuponthefloor.SometimesthePHKsallcomeawaylikeamembrane;trytospreaditbackontothecollagen.Keepsterile

Feedtheraftbyadding12-15mlofraftmediumtothepetri-dish.

Theideaistowettheundersideofthemeshbutnottolettheliquidcomeontotheuppersurface.IfthetopoftheraftbecomeswetitwilldestroythegrADIentandspoiltheexperiment.Watchoutforairbubblesloiteringundertheraft.

RefeedeverytwodaysPeopleseemtoleavethemfor8-14daystogrow/differentiateAddinBrdU8-12hoursbeforeharvesting(seeseparateBrdUprotocol)

Suppliers

Stainlesssteelwiremesh3¹/₁₆inch40mesh.010(=wiresize)304SScircle,item#105493100forabout$25from;USFilter-Johnson/NiagaraScreen(acivilengineering,waterfiltertypecompany)NiagaraMeshSalesFax0017139391337Tel0017139391830

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...culture of primary human keratinocytes (PHKs) 细胞培养 生 ...

...culture of primary human keratinocytes (PHKs) 细胞培养生 ...