Maintenance of immortal mouse melanocytes such as melan-a (1-4).
Updated 3/2009 /DCB

See Sviderskaya et al (3) for most recent methods.

For culture of our existing melanocyte lines, see Cell Bank/Holdings web page for specific medium recommended for each different line.

For sources and more information about materials, see Materials page.

CULTURE MEDIUM: RPMI 1640, gassed with 10% CO2, pH 7.0-7.1. This low pH is essential only for pigmented (not albino) melanocytes. If pH of stored medium rises noticeably (red or pink colour), re-gas from cylinder of 10% CO2 before use.

We strongly recommend use of a 10% CO2 incubator for growing pigmented melanocytes. This should also be used for any cells grown in DMEM medium, as DMEM is buffered for 10% CO2. If this is impossible and you can only use 5% CO2 (or other concentrations of CO2), you can EITHER start with bicarbonate-free medium; the [bicarbonate] to be added can be determined by a simple pH titration (4); OR add HCl and equilibrate pH by gassing from cylinder - other protocol available. However results are less good than with 10% CO2.

Medium supplements:

penicillin, 100 000 U/L
streptomycin sulphate, 100 mg/L
glutamine 2 mM
Foetal calf serum, 10%. ( Important: do not heat the serum, nor buy heat-inactivated serum, as this destroys its stimulatory activity for melanocytes [ref. 2].)
TPA, 200 nM
For some lines: cholera toxin, 200 pM
We also add extra phenol red (water-soluble form, add 7.5
µg/ml from 3 mg/ml stock) to help monitor pH.

Hazard warning: TPA (tetradecanoyl phorbol acetate) is a potent tumour promoter - care with handling! It is also light-sensitive and quite unstable.
A concentrated stock can be made at 2 mM in pure ethanol, stored at -70°C. Not DMSO: do not use DMSO as solvent. It is highly toxic to melanocytes.
A working stock at 40
mM is prepared in phosphate-buffered saline containing 1 mg/ml BSA or 1% serum as carrier, otherwise TPA from dilute solutions can stick to glass or plastic. Aliquots are stored at ~70°C indefinitely, or at 4°C in the dark for up to 2 weeks.
TPA is added to medium on the day of use. TPA solutions and media are inactivated with chlorine bleach before disposal.

2-Mercaptoethanol (ME): This is required for melan-c cells, melan-si-1 cells, and some others - see other web pages or publications. Worth trying, for a new cell line of your own. We think it inactivates something toxic in serum. ME stock (1 M) in water is stable only for 2-3 weeks, but culture medium + serum once treated with ME (0.1 mM) is ok for at least 2 months. Avoid rubber-lined caps which can cause a precipitate with concentrated ME. Add ME to medium a day (or at least 3-4 hr) before use to allow reaction.

ME (1 M) = 4.65 ml sterile water + 0.35 ml pure ME.

Alternative medium for albino melanocytes: Dulbecco's (DMEM) with 10% CO2 (pH 7.4), with penicillin, streptomycin, pyruvate, glutamine (the 4 mM normal for DMEM is ok), 1% nonessential amino acids, mercaptoethanol (for melan-c and other cells that need it), FCS and TPA as above. Not suitable for pigmented melanocytes as pH 7.4 is high enough to be toxic for these.
(DMEM with 5% CO2 is even worse for melanocytes and at pH ~7.7 is not optimal even for fibroblasts etc, even though quite widely used).

 

PROCEDURES

Feeding: Medium is changed 2x weekly. If culture very sparse (under plating densities stated below) and fresh medium not needed after 3-4 days, add extra TPA as it is unstable.

Subculture:
- For a growing culture, rinse in Dulbecco's phosphate-buffered saline lacking CaCl2 and MgCl2 (PBSA), and then in PBSA containing EDTA (200
µg/ml) and trypsin (250 µg/ml: less than for other cell types). OR, for a slowly-growing or quiescent culture, rinse 2x in EDTA solution and 1x in trypsin-EDTA. (The second method is best for melan-b at all times).
- Leave a little trypsin-EDTA on dish (e.g. 0.75ml for a 9-cm dish); incubate in (preferably) air at 37°C.
- For a healthy single-cell suspension, do not pipette until the cells have not only detached from the dish but partly separated from each other. Check by microscope at intervals. Cells should dissociate sufficiently within about 4-6 min from a fast-growing culture, 8 min for more slowly-growing cells, 10-15 min for quiescent/confluent melanocytes. Best results are obtained with a growing culture - longer exposure to trypsin can cause some cell death.
- Melan-a cells can be replated at 1.5 x 104/ml every 5-7 days and should grow at least 20-fold; other lines generally at 3-5 x 104cells/ml whenever nearly confluent.

NOTE: Passage level: All these lines grow progressively faster with continued subculture. Melan-a cells, at least, become less pigmented. Especially if differentiated or "normal" cultures are important to you, it is recommended to freeze stocks immediately and use only passage numbers up to about 30.

References:

1 Bennett DC et al, Int. J. Cancer 39, 414-8 (1987)

2 Bennett DC et al, Development 105, 379-85 (1989)

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

4 Bennett DC et al, J. Cell Science 77, 167-83 (1985)