Cultivation, serial transfer, and differentiation of epidermal keratinocytes in serum-free medium.
We describe serum-free culture conditions for human epidermal keratinocytes using lethally treated 3T3 cells as feeder layers and normal Ca++ concentrations (1.2 mM), in a DMEM/F12-Ham nutrient mixture supplemented with several additives, and 10 mg/ml bovine serum albumin instead of animal serum. Keratinocytes were serially grown to 15-18 cell generations (4 subcultivations) and formed a stratified squamous epithelium that could be detached as a graftable epithelial sheet. EGF and TGF alpha significantly increased keratinocyte proliferation under these conditions; EGF reduced the expression of keratin K1, which is specific for stratified and terminally differentiated epidermal keratinocytes. In contrast with previous reports, the serum-free medium we describe here supports serial growth and normal differentiation of human epidermal keratinocytes, and the formation of graftable stratified epithelia; it also supports the assay of a variety of cytokines or compounds that modulate epidermal keratinocyte proliferation and differentiation.
Castro-Muñozledo
F
F
Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Mexico City, Mexico.
Hernández-Quintero
M
M
Marsch-Moreno
M
M
Kuri-Harcuch
W
W
D016475:3T3 Cells; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D003470:Culture Media; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice
D003470:Culture Media