GROWING A STRATIFIED, CORNIFIED PRIMARY CULTURE OF RAT KERATINOCYTES WITH EPIDERMIS-LIKE WATER PERMEATION BARRIER FUNCTION

The culture of cutaneous keratinocytes grown on a Puropore nylon micro porous membrane at the air-liquid interface has been shown to be simil ar to the epidermis in a number of molecular and morphologic character istics but to exhibit a significantly greater degree of tritiated wate r permeation. Various culture conditions have been altered in an effor t to improve the water barrier properties. A Kp value in the range of 5.5 +/- 1.6 X 10(-3) has been obtained for 79% of the cultures a) by p lating 0.9 X 10(6) viable basal cells on a piece (13-mm diameter) of m embrane for 7 days of submerged growth, b) by placing two membranes on two stacked glass fiber filters (47-mm extra-thick) in a culture dish (60 mm) for 14 days of growth at the air-liquid interface, c) by repl acing the growth medium, i.e., 1 mi of complete minimum essential medi um (CMEM) every 24 h after lifting, d) by using 10% fetal bovine serum (FBS) in the CMEM during the submerged culture period and 15% FBS in tile CMEM during the lifted culture period, and e) by adding a dialysi s membrane on top and a Puropore nylon membrane below tile culture whe n the cultures were inserted in the permeation cell for testing. The p ercentage of cultures with this value for kp can be increased to 90% i f only cultures with yellow, smooth, and shiny surfaces are tested. Th is system should be useful as a replacement for skin in testing the cu taneous permeation of some chemicals.