Note on frozen storage of pigmented melanocytes [Updated 09/07 /DCB]

This information is provided because it is sometimes difficult to freeze these cells. We have no evidence whether our basic method is any better for melanocytes than any other; if you have a different method, it may be fine. We only know that the "special precautions" (below) do help.

(1) Our basic method for cryopreservation (from M. Vogt)

Harvest cells as usual with trypsin and EDTA, resuspending in culture medium and serum and counting cell number.

Centrifuge (at about 1000 rpm for 7 min). Meanwhile prepare culture medium with serum (5% or 10%, as usual for that line) and DMSO, 7.5% v/v. Don't add neat DMSO to medium with cells in it. (Use glass pipette or micropipette tip for 100% DMSO - it dissolves something toxic out of other plastics such as plastic pipettes. So don't put neat DMSO in polystyrene plastic bottles or tubes either. Glass is ok.)

Remove supernatant and resuspend cells in the DMSO medium, at required density. We typically freeze 5-15 aliquots of 1-3x106 cells each. Aliquot to freezing vials at 1 ml per vial.

Immediately place vials in insulated container at -70° C. Leave overnight (or up to a few days is ok without loss of viability), then transfer to liquid nitrogen freezer, ensuring their temperature does not rise above -40° C, which can kill cells. Use dry ice if necessary.

To recover stocks: vial is thawed quickly, e.g. in warm waterbath. Cells are resuspended from bottom of vial by gentle pipetting, transferred to a 20-ml centrifuge tube and diluted by gradually adding 20 ml medium with serum. Important: dilute very slowly, dropwise. E.g. add first 1 ml with mixing over about 30 sec, second 1 ml over 10 sec, then remaining 18 ml over 30 sec. (Note that freezing medium is highly hypertonic: over 4 x isotonic.) The DMSO needs time to diffuse out of the cells, or they burst. Centrifuge, resuspend in more medium and plate out at desired density (generally higher than normal).

(2) Special precautions for pigmented melanocytes, especially mouse :

We expect around 90-98% viability for most cell types frozen as above, but for very pigmented melanocytes (melan-a, melan-rs, melan-si-1, etc.) it can drop to 10% or even 1%. This can be improved by:

(1) Use of the tyrosinase inhibitor phenylthiourea (PTU), sold by Sigma as phenylthiocarbamide.
Cells are grown with PTU (100-300 µM depending on level of pigmentation) for a few days, ideally a whole passage, before freezing; also after thawing for a few hours or days.
We also add PTU at 200 µM to the solutions used for freezing and thawing the cells.
This combination significantly increases recovery rates. The cells become depigmented. Pigmentation returns a few days after removing PTU. We dissolve PTU in ethanol at 10-100 mM and store aliquots at -20° C, or briefly at 4° C (The 100 mM solution precipitates when chilled, and should be warmed before use).

If cells are not completely depigmented, the following are also important:

(2) Ensure pH of culture medium is not too high (even for a few minutes) - do not use partially de-gassed medium, especially for the actual freezing medium.

(3) Work quickly, while cells are out of freezer but in the presence of DMSO, which is highly toxic to pigmented cells. (So are other cryopreservatives. We presume they permeabilize the melanosome membrane, with release of toxic melanin intermediates into the cytosol.)