|Matt Lewis||Department of Pathology |
University of Liverpool
Organotypic raft culture of primary human keratinocytes (PHKs)
Use 106 keratinocytes per raft.
Trypsinize the PHKs off the flask and resuspend in (say) 2ml raft medium.
Remove the medium from the collagen plugs and overlay with the new medium containing PHKs.
Spatulas for scooping out the collagen plug
When the PHKs are confluent:-
This is like cutting round a cake in a cake tin. The PHK 'membrane' may contract from the edge also and detach a little from the collagen
Remove media, scoop out plug and deposit onto the mesh in the petri-dish, keeping everything sterile.
Optional - rinse in PBS before placing onto mesh
Feed the raft by adding 12-15ml of raft medium to the petri-dish.
The idea is to wet the underside of the mesh but not to let the liquid come onto the upper surface. If the top of the raft becomes wet it will destroy the gradient and spoil the experiment. Watch out for air bubbles loitering under the raft.
Refeed every two days
Stainless steel wire mesh 3 1/16 inch 40 mesh .010 (=wire size) 304 SS circle, item #105493