Long term growth and maintenance of stratified rat urothelium in vitro.
Culture conditions that allow long term growth and maintenance of rat urothelium have been determined using short (3 to 8 days) and long (14 to 60 days) term measurements of cell density and tritiated thymidine incorporation as indices. The basal nutrient medium utilized was a mixture of 199 plus Ham's F 12 (1:1) supplemented with insulin (1 microgram/ml) and hydrocortisone (1 microgram/ml). Long term culture of urothelium seems to require porous collagen. Porous albumin, or plastic dishes thinly coated with albumin, collagen, fibronectin or mixtures thereof, did not support long term maintenance. Serum was required at a concentration of 5%, independent of other additives. Decreasing Ca++ levels below that normally found the basal medium (approximately 1 X 10(-3] to as low as 1 X 10(-4), resulted in increased short term proliferation, but decreased long term maintenance by causing a loss of stratification of the urothelium. Even a slight increase in Ca++ concentration from 1.0 to 1.5 X 10(-3) resulted in an inhibition of proliferation and an increase in the number of large flat cells which subsequently sloughed off in sheets. The deletion of either insulin, hydrocortisone or both, inhibited growth. The addition of epidermal growth factor (EGF) or its homologue, transforming growth factor (TGF-alpha), increased cell proliferation markedly and caused a variable increase in stratification. However, epithelium induced to rapid growth and proliferation with EGF, eventually exhausted its growth potential and died. TGF-beta 1, alone or in combination with either EGF or alpha-TGF, had no additional effect upon urothelial growth. Repeated transfers of urothelium by enzymatic dissociation led to decreased growth and maintenance potential. The data indicates that long term maintenance of stratified urothelium in culture requires a porous collagen substrate and fetal bovine serum together with hormonal requirements and concentrations of Ca++ that neither greatly stimulate nor inhibit growth.
Chlapowski
F J
FJ
Department of Biochemistry, University of Massachusetts, Medical School, Worcester 01655.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D046508:Culture Techniques; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006854:Hydrocortisone / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D008858:Microscopy, Phase-Contrast; D008940:Mitotic Index; D051381:Rats; D011916:Rats, Inbred F344; D013936:Thymidine / Q000378:metabolism; D015291:Transforming Growth Factors / Q000494:pharmacology; D001743:Urinary Bladder / Q000166:cytology* / Q000378:metabolism
D003470:Culture Media; D007328:Insulin; D004815:Epidermal Growth Factor; D015291:Transforming Growth Factors; D002118:Calcium; D013936:Thymidine; D006854:Hydrocortisone