Survival of normal colonic epithelial cells from both rats and humans is prolonged by coculture with rat embryo colonic fibroblasts.
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, which is normally 3-4 days, coculture with viable fibroblasts extended this period to at least 2 weeks. The effects depended on early passages and low seeding densities of the fibroblasts and on direct cell-cell contact. We have obtained cultures of epithelial cells expressing keratin, laminin, and uvomorulin, displaying a polygonal, epithelial morphology 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% BrdU-positive cells, while the proportion in dense three-dimensional colonies reached 50.3% +/- 2.6%. In HCECs the growth fraction was lower, but showed the same distribution between classes of colonies. In the presence of rat embryonic colon fibroblasts, growth factors exerted survival activity on colonic epithelial cells. Consecutive addition of insulin and epidermal growth factor/fibroblast growth factor (EGF/FGF) increased colony number (15.0 +/- 1.0 and 23.0 +/- 2.0 colonies/well respectively; p < or = 0.05 increased above control) and size (1022 +/- 155 and 1207 +/- 158 cells/colony respectively; p < or = 0.05 increased above control) compared to serum-free control medium and basic MEM without growth factors. BrdU labeling index was not increased, however: EGF/FGF actually decreased BrdU labeling from 33.2% +/- 3.9% in controls to 21.3% +/- 3.8% in the EGF/FGF group (p < or = 0.05) owing to the high proportion of flat colonies consisting of resting cells. The newly established culture model can now be used to investigate growth control mechanisms in colonic mucosa and the effects of toxic and/or tumor-promoting substances on these mechanisms.
Schörkhuber
M
M
Institute of Tumor Biology-Cancer Research, University of Vienna, Austria.
Karner-Hanusch
J
J
Sedivy
R
R
Ellinger
A
A
Armbruster
C
C
Schulte-Hermann
R
R
Marian
B
B
D000818:Animals; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D003106:Colon / Q000166:cytology*; D004847:Epithelial Cells / Q000502:physiology; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology*; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D008648:Mesoderm; D051381:Rats; D013997:Time Factors
D006133:Growth Substances