Primary culture of melanoblasts/melanocytes from mouse skin
Suggested reference: Sviderskaya EV et al, J. Inv. Dermatol. 108, 30-34 (1997).
Late embryos (14 days to term) can be used, but newborn mice up to 3 days old (no hair) give better yields. The younger, the better - the most melanocytes are obtained from newborn animals. One newborn mouse gives about 12ml of culture.
Melanocyte growth medium [for primary and early-passage cells] is RPMI-1640 medium with FCS (10%) (NOT heated - important!), TPA (200 nM) and cholera toxin (200 pM). Note that both TPA and cholera toxin are hazardous. Incubation is at 37°C with 10% CO2, pH 7.0-7.1. This low pH is important for pigmented normal melanocytes, especially in early passages when they get very pigmented, as melanin synthesis becomes too rapid (toxic) at high pH. If you cannot use a 10% CO2 incubator, you can adjust the bicarbonate level with acid (separate protocol available), but growth is less good that way. If you have repeated trouble with fibroblasts, try 5% FCS in place of 10%.
1 Prepare cultures of feeder cells (mitomycin C-treated XB2 keratinocytes) 1-3 days (preferably 1 day) earlier, in RPMI-1640 medium with 5% FCS.
2 Kill the animal(s), e.g. by decapitation.
3 Sterilise the skin of postnatal animals (not embryos) by immersing in 70% ethanol for 5-10 seconds (depending on age - shorter time for younger skin which is thinner; too long will damage the skin). All subsequent procedures are sterile.
4 Transfer to PBSA (Dulbecco's phosphate-buffered saline lacking Ca++ and Mg++), to wash off the ethanol.
5 Dissect off trunk skin in one piece, avoiding muscle (pinkish). Keep wet.
6 Collect skin(s) in PBSA and remove any bits of muscle using fine forceps and a dissecting microscope.
7 Transfer to concentrated trypsin solution, 5 mg/ml in PBSA (about 5 ml per skin).
8 Incubate at 37°C for about 1 hr for newborn skin. Embryonic skin is best trypsinized on ice until it will split: 15 min to 3 hr for 14-18 day embryos.
9 Transfer to PBSA, say 20 ml, in a large culture dish.
10 [The critical bit!] Split off epidermis (thin, silvery-white layer) under dissection microscope using 2 pairs of fine ("watchmaker's") forceps. It should come off very easily, otherwise trypsinize for longer. Use only epidermal tissue that splits off without any force - or you will get fibroblast contamination. Remove any obvious pieces of dermis (whitish), but don't scrape; melanocytes are loosely attached at this stage. (So don't over-trypsinize either.) This step generally takes some trial and error to learn.
11 Wash epidermis in clean PBSA.
12 Transfer to trypsin-EDTA solution as used for subculture (PBSA with trypsin, 250 µg/ml, and EDTA, 200 µg/ml), about100 µl/skin, on a culture dish.
13 Chop quite finely with a curved scalpel.
14 Add 5 ml growth medium (lacking TPA and cholera toxin), with 5 µg/ml soybean trypsin inhibitor (yes, despite the serum), and pipette vigorously, 3-5 times, with a 5-ml pipette or a 2.5-ml Combitip.
15 Make up to about 12 ml with more growth medium and pipette 1-2 more times. Note: if feeder cells were plated only 1 day ago, it is beneficial to use the conditioned medium off the feeders for steps 14-15, instead of fresh medium (and work quickly to avoid drying-out of feeders).
16 Add TPA (200 nM) and cholera toxin (200 pM) and plate on to the feeder cells.
17 Change medium initially twice a week, but reduce frequency if cells become very sparse through loss of feeders. Unknown conditioning /autocrine factors seem important at this stage. Add new TPA twice a week when not changing medium, as it is unstable. Unpigmented melanoblasts are present initially, but groups of pigmented melanocytes should appear (readily identified by bright-field microscopy) from about 1-2 weeks. Skin keratinocytes also grow in the first week, but then differentiate and die.
Further procedures for early subcultures and establishing a line are here.