Separation of Epidermis from Dermis Using Dispase Solution (Matrix Metalloprotease)

Protocol Overview

• Skin samples are cut into small section (3-4cm) in form of long strips. The sections are immediately placed into either Hank’s Balanced Salt Solution (HBSS) or appropriate media (Epilife media/ KGM media) with antibiotics/antifungal (pen-strep + amphotericin B) in standard concentration. All work starting from first step till the end should be done in sterile conditions (biosafety cabinet). • Tissue sections are placed in a 60mm petri plate containing 1ml of dispase solution (5000 caseinolytic unit) to 9 ml of appropriate media with antibiotics (already mentioned). The section should be placed such that the epidermis layer (darker side) is at the bottom and well covered with fluid. (Add more dispase solution if needed). The petri dish is placed overnight (about 14-16 hrs.) in 4 degree C. • Next day, place the petri plate in a 37 degree incubator (sterile) for 1-2 hrs. Take out the plate and inside a sterile hood, peel off the epidermis from dermis using a pair of fine forceps. Immediately place the peeled epidermis (extremely thin) layer in fresh media in a separate petri plate. Discard the dermis (white gooey part) or it can be utilized for fibroblast isolation. • The thin epidermis is then cut into very small pieces (1mm) and trypsinized to obtain keratinocytes.