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Comparative Physiology @ Guelph

Keratinocyte Monolayers

From David Russell, who got the protocol from a dermatologist colleague. This is the protocol they use for preparing keratinocyte monolayers for grafting.

The keratinocytes to be grafted must be confluent otherwise they will disintegrate when the enzyme dispase is added. It has been reported that it is best to use graft within two days after reaching confluence for good results, though no clinical trial has been performed to prove this to date.

Cut the flask open (or use culture dish). Remove growth media and add the enzyme Dispase (2mg/ml in DMEM + 8% FCS) covering the whole area. This is approximately 10-15 ml for a T75 flask.

Incubate the flask in an incubator at 37C. Within an hour the graft will start detaching from the edges and curl inwards as the sheet shrinks towards the middle of the flask. When this starts happening place a piece of Tricotex (a non-adherent gauze dressing) on top of the graft. This will keep the graft evenly spread, provide support to the graft and preserve the orientation of the cell layers so tat the basal cells facing down at this stage will ultimately be applied to the wound surface.

Wash the graft 3 times with PBSA, remove graft with the dressing from the flask by holding the two edges of the graft with the dressing with a pair of sterile forceps, lifting it on to a sterile petri dish. The graft should then be stapled to the dressing with sterile ligaclips to the corners and sides. Add serum free media (enough to cover the graft) and transport it to theatres.

Grafting should be carried out as soon as possible but a total of 4-5 hours delay is not deleterious, in so far as can be determined from colony forming efficiency. Store it at 4C if possible during delay. It is important that all these procedures and handling of the graft is done in a sterile environment.