1. -embryosaredissectedfromtimed-pregnantmicefrom11.5d.p.c.to13.5d.p.c.
2. -metanephroiandassociateduretericbudsaremicrodissectedandplacedinholdingmedium(L15mediumsupplementedwith1xMITO+serumextender)(allmediaissupplementedwith100Upenicillin/mland0.1mgstreptomycin/ml)
3. -metanephroiareallowedtositinholdingmediumanywherefrom20minto2hours
4. -0.2mlofserum-freeexplantmedia(DMEM-F12mediumsupplementedwith5xMITO+withorwithout10%fetalbovineserum)isaddedtoeachwellofasix-welltissuecultureplatecontainingCyclopore(polyethyleneterphthalate;FalconLabware)inserts
5. -metanephroiaretransferredfromtheholdingmediaontothesurfaceoftheCycloporemembranes(fortransfer,theendofa200祃Pipettetipisremovedwithasterilerazorblade(toenlargethehole),andmetanephroiaretakenupin5–10祃ofholdingmedia)
6. -metanephroiaregrownonthetissuecultureinsertsina37oCincubatorunderanatmosphereof5%CO2
7. -explantmediaisreplacedevery48hours

Metanephroicanbesuccessfullygrownfor7–10days

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