M. Schorkhuber et al., SURVIVAL OF NORMAL COLONIC EPITHELIAL-CELLS FROM BOTH RATS AND HUMANSIS PROLONGED BY COCULTURE WITH RAT EMBRYO COLONIC FIBROBLASTS, Cell biology and toxicology, 14(3), 1998, pp. 211-223
Primary cultures of normal colonic epithelial cells from both humans (
HCEC) and rats (RCEC) have been established using coculture with colon
fibroblasts isolated from rat term embryos. While no other factors we
have analyzed had any effect on the survival of epithelial cells, whi
ch is normally 3-4 days, coculture with viable fibroblasts extended th
is period to at least 2 weeks. The effects depended on early passages
and low seeding densities of the fibroblasts and on direct cell-cell c
ontact. We have obtained cultures of epithelial cells expressing kerat
in, laminin, and uvomorulin, displaying a polygonal, epithelial morpho
logy and forming microvilli. DNA synthesis as measured by BrdU uptake
into DNA varied widely between colonies of the same culture depending
on cell morphology: flat colonies of RCECs contained 5.7% +/- 0.56% Br
dU-positive cells, while the proportion in dense three-dimensional col
onies reached 50.3% +/- 2.6%. In HCECs the growth fraction was lower,
but showed the same distribution between classes of colonies. In the p
resence of rat embryonic colon fibroblasts, growth factors exerted sur
vival activity on colonic epithelial cells. Consecutive addition of in
sulin and epidermal growth factor/fibroblast growth factor (EGF/ FGF)
increased colony number (15.0 +/- 1.0 and 23.0 +/- 2.0 colonies/well r
espectively; p less than or equal to 0.05 increased above control) and
size (1022 +/- 155 and 1207 +/- 158 cells/colony respectively; p less
than or equal to 0.05 increased above control) compared to serum-free
control medium and basic MEM without growth factors. BrdU labeling in
dex was not increased, however: EGF/FGF actually decreased BrdU labeli
ng from 33.2% +/- 3.9% in controls to 21.3% +/- 3.8% in the EGF/FGF gr
oup (p less than or equal to 0.05) owing to the high proportion of fla
t colonies consisting of resting cells. The newly established culture
model can now be used to investigate growth control mechanisms in colo
nic mucosa and the effects of toxic and/or tumor-promoting substances
on these mechanisms.