ISSN:
0021-9541
Keywords:
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12*) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 μg/ml hydrocortisone, 5 μg/ml transferrin, 10 μg/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10-4 M ethanolamine or 10-4 M phosphoethanolamine, and 5 × 10-8 M selenium. HUC grown in F12* on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serumfree F12* on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12* did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12* was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 μg/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12* decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 μg/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12*. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serumfree medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12* (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12* medium did not confer a long-term growth advantage. On the contrary, optimal long-term growth of HUC serially passaged in F12* was obtained using a moderate calcium concentration (0.3 mM) which supported morphological differentiation and stratification of HUC in postconfluent cultures, showing that these two parameters are not mutually exclusive in this system and that there exists a delicate calcium-regulated balance between growth of HUC in nonconfluent cultures and stratification and differentiation of HUC in postconfluent cultures.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcp.1041310302
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