engleski

Human keratinocyte culture in vitro

Human epidermal cells, keratinocytes, can be cultured in vitro using a selective defined keratinocyte serum-free medium (DKSFM) or a feeder-layer of irradiated murine fibroblasts. We describe our experience with keratinocyte cultures in DKSFM. Keratinocytes were isolated from human foreskins obtained at routine pediatric circumcisions. Epidermis was separated from dermis by enzymatic digestion in dispase and dissolved into single cell suspensions by trypsin. Keratinocyte suspensions in DKSFM were seeded into 25 cm2 tissue culture flasks or into 35 mm, 60 mm or 100 mm Petri dishes and maintained at 37 °C, 5% CO2 and maximal humidity. The medium was renewed every 2 days until the cells reached confluence. In successful cultures that occurred 2-3 weeks after the initiation. A satisfactory growth was obtained with one-third (35%) of the isolates. The failures were due to contamination (32%), inappropriate plastics (13%) or for reasons not sufficiently clear (19%). Primary cultures that reached confluence were trypsinized and the cells frozen in liquid nitrogen or re-seeded into secondary cultures for expansion. Samples of primary or secondary keratinocytes were cytospinned for immunocytochemical examination. The keratinocyte proliferation rate was measured in a microplate assay using a colorimetric method. DNA extracted from cultured keratinocytes showed by PCR the presence of the gene coding the membrane metallopeptidase CD10 (EC 3.4.24.11). Thus, primary cultures of human keratinocytes in DKSFM can be a convenient experimental model provided a satisfactory growth is obtained.

keratinocytes; cell culture; cell proliferation; defined keratinocyte serum-free medium (DK-SFM); CD10/NEP

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