Organotypic raft culture of PHKs¶

Contributed by Matt Lewis <hop2kin@yahoo.co.uk>

Organotypic raft culture of primary human keratinocytes.

Trypsinize the PHKs off the flask and resuspend in (say) 2ml raft medium.
Count cells via haemocytometry and isolate 106 keratinocytes per raft from cell source to fresh medium.
Remove the medium from the collagen plugs and overlay with the new medium containing PHKs.
Incubate for 1-2 days to allow the PHKs to settle and become confluent.
Separate the edge of the collagen plug from the wall of the well by going round it with a sterile scalpel.
Allow the collagen plug to contract away from the walls for a few hours.

*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 optionally rinse in PBS. Deposit onto the mesh in the petri-dish, keeping everything sterile.

This transfer is the trickiest part of the whole procedure. If you get it intact, right side up (ie. PHKs up), and roundish on the mesh, without having scraped off all the PHKs, then you’ve done well. Usually it ends up on the floor. Sometimes the PHKs all come away like a membrane; try to spread it back onto the collagen.*

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. *

Re-feed with raft media; wait 2 days
Re-feed with raft media; wait 2 days
Re-feed with raft media; wait 2 days
After 8-14 days of growth/differentiation add in BrdU 8-12 hours before harvesting; see [Brdu protocol](http://lab.methodmint.com/tasks/38/)

This method is based, with permission, on an original protocol available here.