Clonal growth of normal XXXX uroepithelial cells.

@entity1 @entity8398 @entity19 @entity332 @entity291
We report the development of culture conditions which routinely support clonal growth of normal @entity1 uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3 X 10(5) lethally irradiated @entity19 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1 +/- 2.5%. A cloning efficiency of 4.9 +/- 1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM @entity8398 . The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for @entity1 epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of @entity19 feeder cells resistant to 5 micrograms/ml @entity332 (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9 +/- 2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 micrograms/ml @entity291 pituitary extract. The average cloning efficiency using these conditions was 5.7 +/- 1.7%.
