Y. Pu et al., GROWING A STRATIFIED, CORNIFIED PRIMARY CULTURE OF RAT KERATINOCYTES WITH EPIDERMIS-LIKE WATER PERMEATION BARRIER FUNCTION, In vitro cellular & developmental biology. Animal, 31(4), 1995, pp. 283-287
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.