I do, but I use DIspase instead. This is how we do it:
1. Rince specimens 3X in PBS supplemented with 1% antibiotics (pen-strep, GIBCO)
2. Incubate in 1mL DMEM with 2.5mg/mL Dispase (filtered) overnight at 4-8 .C
3. Remove Dispase solution, rince with PBS. Separate dermis from epidermis using sterile forceps
4. Mince the dermis using a sterile scalpel, cutting it into small fragments (around 0.5 - 1 mm2 in diameter)
5. Place fragments into a 25cm2 culture flask, or 6-well plates (or anything equivalent), around 5-7 pieces per flask/well
6.Briefly transfer to incubator at 37.C (~2mins), so that the fragments attach to the plastic (do not let them dry out)
7. Add culture medium (DMEM-High Glucose, 15% Foetal Bovine Serum, 1% antibiotics)
8. Replace medium every 3-4 days. Fibroblasts should start irradiating within 2 weeks.
9. Subculture when 70% confluent
Is it possibile that human dermal fibroblasts that were isolated from a skin sample differentiate and stop their proliferation?
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