Immortalizing mouse melanocytes

Suggested reference: Sviderskaya EV et al, J. Inv. Dermatol. 108, 30-34 (1997).

Primary culture is described on a separate page. The cells can be subcultured when approaching confluency or - if they are growing in isolated colonies - when the colonies get large (say 1000 cells or more each). This may be 3 weeks or more after primary culture. It is advisable to subculture cells at least every 2-3 weeks in any case, as mould spores can accumulate on outsides of dishes and eventually get inside [1].

Subculture by method described for immortal melanocytes, except plate on to fresh feeders, either at about 3-5x104 melanocytes/ml or at 1:1 [2], until immortal cells take over. This can be recognised because once the cells have senesced (around 4-6 weeks of culture - they get large, flat and well-pigmented), many cells fail to reattach at each subculture and only a small fraction even of surviving melanocytes grow and form colonies. The overall growth rate can fall to zero or below for some weeks or several passages, but do not discard. We think around 1 in 106 normal mouse melanocytes spontaneously becomes immortal. When immortal cells appear, they form one or more healthy, growing colonies of small, less-pigmented cells, overgrow the static diploid cells, and virtually all survive and proliferate after subculture. If any melanocytes are proliferating (as opposed to surviving) by 3 months after primary culture, they are almost certainly immortal.

In general, after immortalization, feeder cells are no longer needed, and the cholera toxin can also be omitted. However feeder cells are needed again if cloning the immortal cells. Cloning is often useful to ensure homogeneity.

Hints:

[1] Watch for mould colonies on walls or shelves of incubator, and wipe away with 70% ethanol; in any case swab outsides of all long-term cultures and their incubator trays once a week with 70% ethanol.)

[2] Some early subcultures may have to be at 1:1 or thereabouts, when melanocytes are sparse. We suspect trypsin may then not be sufficiently diluted, as cell growth seems better on adding soybean trypsin inhibitor to resuspension medium. We use full amount needed to neutralise the trypsin. No need to remove it before plating cells. 1 mg/ml inhibitor stock in PBS is stable at 4°C for at least 2 months.

[3] FCS can be used at 5%, as higher concentrations often lead to appearance and overgrowth of fibroblast-like cells; but 10% gives faster growth of melanocytes and can be used once they are immortal and cloned.

[4] If fibroblasts do appear, they can be killed selectively by treatment with geneticin (G418) at 100-150 µM for 3-4 days (no more). Melanocytes are very resistant to G418. Can be repeated after an interval. Physical scraping is sometimes useful to remove contaminating cells.

[5] Diploid melanocytes are even more sensitive to high pH than immortal ones. To help ensure cells are not exposed to alkaline medium during handling, we add extra phenol red indicator dye to the RPMI-1640 medium. RPMI-1640 contains 5 µg/ml phenol red (hard to see); MEM has 10 µg/ml and DMEM has 15 µg/ml. We add an extra 7.5 µg/ml to RPMI-1640. (We use water-soluble phenol red (sodium salt); can make stock at 3 mg/ml (400x); filter-sterilise; stable at room temperature).