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- Inventor Info
|Antigen/Gene or Protein Targets||Melanocyte|
|Parental Line||Mouse melanoblasts|
|Disease Keywords||Cancer; Melanoma|
Melan-a cell line with use to investigate melanoma skin cancer.
Background and Research Application
Skin cancers are skin growths with differing causes and varying degrees of malignancy. The three most common malignant skin cancers are basal cell carcinoma, squamous cell carcinoma, and melanoma. To understand what is abnormal in melanomas, it is clearly desirable to make cellular and molecular comparisons between melanoma cells and the equivalent normal cells, melanocytes. The melanocyte line, melan-a, is derived from the embryonic skin of C57BL mice. Cells retain all tested characteristics of normal melanocytes except senescence and a proliferative response to cholera toxin in the presence of tetradecanoyl phorbol acetate (TPA). They have the diploid chromosome number and are non-tumorigenic in syngeneic and nude mice. They are therefore suitable for a range of comparative studies, in vitro and in vivo, with sublines of the B16 melanoma with which they are also syngeneic.
|Production Details||Primary culture: Trunk skin from a litter of 18-day-old embryos was split with trypsin (5mg/mL) in PBSA at 4°C. Epidermal sheets were washed in medium (SMEM and 10% FCS), minced finely in a drop of medium with 2 scalpels, and triturated vigorously in medium with a siliconized Pasteur pipette. The suspension was supplemented with 10 nM cholera toxin, divided between 3 50-mm dishes of XB2 feeder cells in only 2 ml medium per dish, and incubated. TPA (200nM) was added only after 3 days’ incubation, as preliminary work suggested that TPA inhibits the attachment of epidermal fragments from which many of the melanoblasts emerge. The medium was changed weekly. For subculture, cell suspensions were prepared as described above for melan-a cells, but were replated usually on to XB2 feeder cells and in medium with both TPA and cholera toxin.|
|Research Area||Cancer, Cell Cycle, Cell Type or Organelle Marker, Developmental Biology, Drug Discovery & Development|
Points of Interest
The primary culture (“Methods”) was typical; many growing, unpigmented cell colonies, with the characteristic appearance of melanoblasts were seen after a few days. These matured to pigmented melanocytes over about 3 weeks. A few colonies of fibroblastoid cells were also observed, but these soon senesced and were lost. After 3 weeks one dish was subcultured (“Methods”) at a ratio of 1 :4, the other cultures being frozen. About 95% of the harvested cells were visibly pigmented at this stage. Most of the remainder appeared to be keratinocyte feeder cells. After another week the cells were again subcultured at 1:2, but virtually all cells had senesced (stopped growing). Phenylthiourea (PTU) was added to 2 dishes. PTU is an inhibitor of melanin synthesis by the enzyme tyrosinase, and can increase the proliferation of highly melanogenic melanoma cells. Three weeks later, one black colony of 2-3mm diameter was observed on a PTU-containing plate. This was taken to be an immortalized clone, since the cultures had clearly reached the “crisis” phase. As no other proliferating cells were observed, the colony was subcultured, by local application of trypsin-EDTA solution, into 2 6-mm wells (counted as passage 4). The resulting cultures consisted homogeneously of pigmented cells. These cells continued to proliferate through more than 30 subcultures and many months, and are clearly established. Feeder cells were omitted from passage 6 onwards. Later, cholera toxin was found to be no longer mitogenic in the presence of TPA, and PTU was found to be slightly inhibitory so these were omitted. However, PTU is added for one passage before preparation of frozen stocks, as this modestly increases the yield of viable cells after thawing, from about 30% to about 40%.
Vial has between 1-5 million cells as standard, however this may vary.
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