(updated 3/09 - DCB). This method is for normal human skin.
For sources and more information about materials, see Materials page.
Suggested basic growth medium:-
RPMI 1640, bicarbonate-buffered for pH 7.0-7.1 in your incubator, and supplemented witrh FCS and several growth factors - details of supplements in Culture and Passaging protocol.
1 Provide nearly-full 20-ml tube of culture medium (with glutamine/Glutamax and antibiotics but serum and growth factors not needed), on ice, for collection of skin. (Neonatal or infant skin gives much better-proliferating cultures than adult: circumcisions a good source. Neonatal melanocytes are often unpigmented for first few passages.)
2 Soak tissue in 10 ml of same medium with 10x normal [penicillin & streptomycin] for 10 min. All procedures sterile from here. Remove blood & blood clots (serum inhibits trypsin). Can trim to remove hypodermis (fatty layer).
3 Leave overnight ("16 hr") in 10 ml of ice-cold concentrated trypsin solution, 1.5 mg/ml in PBSA (calcium- & magnesium-free Dulbecco's phosphate-buffered saline, pH 7.2).
4 Transfer to 9-cm dish containing PBSA. Peel epidermis (thin, whitish) off dermis with sterile forceps. Transfer epidermis to universal.
5 Pipette well in medium + 10% FCS to disrupt tissue.
6 Make approximate cell count with haemocytometer. Make cell suspension to 5-10x 104 cells/ml in complete growth medium. (around 1 ml per 5cm2 growth area). Plate on dishes or flasks. If flasks, it is helpful to gas flask with right % CO2 from cylinder, before adding cells (since diffusion through neck of flask is slow), to avoid high-pH shock before equilibration. Incubate.
Note: for a small sample it can increase yield and growth rate to plate on to mitomycin-treated feeder cells from an immortal line, XB2 mouse keratinocytes. Not essential for human melanocytes where SCF is used. See other protocol.
7 Change medium 2x weekly. Melanocytes should be apparent after 48 hrs, increasing to become predominant or only population.
8 If colonies of contaminating fibroblasts appear, treat with G418 (geneticin) at 75-150 µM for 3-4 days. Repeat if necessary, but allow recovery for a few days between treatments.
9 Subculture (passage) when approaching confluence (cell yield =around 1.5 -2 x105/ml). See Culture and Passaging. Avoid allowing normal cells to get confluent and quiescent - makes them harder to detach and reduces overall lifespan.
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.