A method for culturing chick melanocytes: the effect of BRL-3A cell conditioning and related additives.
A method for growing chick embryo melanocytes is described that utilizes medium conditioned by Buffalo Rat liver (BRL-3A) cells. The dissected trunk region of each 72 h (Stages 14 to 19) embryo produces approximately 200,000 melanocytes (purity, 80%) when processed and cultured for 8 d. Thus, a typical experiment involving 20 embryos would produce a total of 4 x 10(6) melanocytes. Choice of serum, serum concentration, and cell density were determined experimentally. Partially purified multiplication stimulating activity (MSA) from BRL-3A cells and insulin were also tested as medium additives. MSA was not stimulatory, whereas insulin gave a positive response in 2% but not 10 or 0% serum. The final protocol used a modified F12 medium with 10% bovine calf serum conditioned by BRL-3A cells. Cultures were fed every other day. Small colonies of cells became evident by culture Day 3 and increased rapidly to Day 5 when pigmentation became obvious. Colony size continued to increase but more slowly from Days 5 to 8, whereas pigmentation increased rapidly and maximized on Day 8. There is a factor, or factors, present in BRL-3A conditioned medium that stimulates embryonic chick melanocytes to divide preferentially over contaminating cell types. This results in cultures that can provide adequate numbers and purity for biochemical studies.
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D003470:Culture Media; D007328:Insulin / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II; D008099:Liver / Q000166:cytology; D008544:Melanocytes / Q000166:cytology*; D008722:Methods; D010455:Peptides / Q000494:pharmacology; D051381:Rats; D011915:Rats, Inbred BUF
D003470:Culture Media; D007328:Insulin; D010455:Peptides; D007335:Insulin-Like Growth Factor II