Culture and passaging of human melanocytes

(updated 3/2009-DCB)

Separate protocol available for primary culture.

Suggested growth medium:-
RPMI 1640, bicarbonate-buffered for pH 7.0-7.1 in your incubator.

It is easiest to use standard RPMI 1640 + 10% CO₂. If you cannot use a 10% CO₂ incubator, you can adjust the pH using acid or by using bicarbonate-free medium and adding less bicarbonate than normal; and pre-equilibrate with 5% CO₂ (protocol available for that). However 10% gives best results.
If pH of medium rises during storage, readjust pH by gassing from cylinder of 10% (or 5% etc if using that) or 100% CO₂ before adding to cells - alkaline medium is quite toxic to pigmented melanocytes. If gassing with 100% CO₂, it is important not to over-gas (stop at orange color of phenol red, not yellow).

Supplements:-
Penicillin, 10⁵ U/L
Streptomycin, 100 mg/L
Glutamax or glutamine, 2 mM
Extra phenol red, 7.5 µg/ml^a
10% foetal calf serum (not heated - this impairs its stimulatory activity)
TPA, 200 nM^b,c
Cholera toxin, 200 pM^c
endothelin 1, 10 nM^c,d
human stem cell factor (SCF), 10 ng/ml^c

(higher concentrations better still, but it's expensive!).

Change medium 2 x per week.

Subculture procedure

• Subculture (passage) when approaching confluence (cell yield =around 1.5 -2x10⁵/ml). Wash gently 1x in PBSA, then 1x in PBSA containing EDTA (200 µg/ml) and trypsin (125 µg/ml: less than for other cell types).
  
• Remove trypsin immediately; add a small fresh amount of same solution to plate (e.g. 0.75 ml for a 10-ml plate).
  
• Incubate, preferably in air, until cells completely detached, as seen by gently tilting dish (4-10 min); do not attempt to detach them by pipetting or tapping - tends to kill them by breaking their processes.
  
• Resuspend in growth medium, count and replate at 5x10⁴ cells/ml or more. If diluting less than 1:4, add some soybean trypsin inhibitor (use full amount to neutralise trypsin remaining on dish), or else centrifuge to remove trypsin. See also Materials.

Note: If you use flasks, it is helpful to gas flask with right %CO₂ from cylinder, before adding cell suspension for plating out (since diffusion through neck of flask is slow), to avoid high-pH shock before equilibration in incubator.

Time between passages varies between strains, from a few days to several weeks. After a number of passages, growth rate starts falling as culture senesces, eventually to zero. Neonatal/infant melanocytes grow more quickly and for longer than adult melanocytes; they can reach 20-30 passages, 50-70 doublings in the recommended medium.

Notes:

^aPhenol red: we add this to help with monitoring pH of medium; RPMI has only 5 µg/ml, which is hard to see, especially in the presence of serum. We use water-soluble phenol red; see Materials.

^bHandling of TPA: TPA (tetradecanoyl phorbol acetate) is a potent tumour promoter - care! It is also light-sensitive and quite unstable. A concentrated stock can be made at 2 mM in pure ethanol, stored at -70°C. (Do not use DMSO, which is toxic to melanocytes.) A working stock at 40 µM (200x) is prepared in phosphate-buffered saline containing BSA (say 1 mg/ml) 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 dark for up to 2 weeks. TPA is added to medium on the day of use. TPA solutions and media are inactivated with bleach before disposal.

^cSee Materials for sources of supplements etc. We use PBS + BSA carrier (as in note b) to dilute growth factor stocks. Aliquotted peptide solutions are kept at -70°C, or in fridge for up to 2 weeks

^dWe have not tested endothelin 1 on primary explant cultures. It is a growth factor for some fibroblasts too, so it may stimulate contaminating cell growth.