SURVIVAL OF NORMAL COLONIC EPITHELIAL-CELLS FROM BOTH RATS AND HUMANSIS PROLONGED BY COCULTURE WITH RAT EMBRYO COLONIC FIBROBLASTS

Citation
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
Citations number
36
Categorie Soggetti
Cell Biology",Toxicology
Journal title
ISSN journal
07422091
Volume
14
Issue
3
Year of publication
1998
Pages
211 - 223
Database
ISI
SICI code
0742-2091(1998)14:3<211:SONCEF>2.0.ZU;2-1
Abstract
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.