Keratinocyte feeder cells for melanocytes (Updated 11/2007 - DCB)

XB2 is an immortal line of mouse keratinocytes that differentiated in culture from teratoma cells (J G Rheinwald & H Green, Cell 6, 317-330 (1975)). Provided to us by JGR.
XB2 cells were originally grown on feeder cells (3T3 fibroblasts) themselves, but we have selected a variant line which grows ok without fibroblasts (see Bennett et al, Int. J Cancer 39, 414-8 (1987)).

(A) Growth of XB2 stock cells

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

Medium:
DMEM with 10% foetal calf serum, in 10% CO2.
Note: Standard DMEM medium is buffered for use with 10% CO2, giving pH 7.4. With 5% CO2 it is too alkaline (about pH 7.7). If you cannot get a 10% incubator, instead you can buy bicarbonate-free DMEM and make up the medium with about 25 mM bicarbonate instead of 45 mM, then gas with 5% CO2 before use.
On XB2 cultures, change medium 3- 4 days after plating. Subculture when cells virtually confluent but not yet at saturation density. Usually about 5-6 days.

Subculture procedure for a 9-cm dish:
XB2 keratinocytes attach very firmly and need initial rinses in EDTA and a long time in trypsin to harvest them. (Solutions take a while to get underneath cells.) Also ensure that the cells are still growing, not at saturation density, when they get even harder to detach.

1) 2 washes (5 ml each) in Dulbecco's PBSA (PBS without Ca or Mg) containing EDTA (200  µg/ml).

2) 1 wash in the same EDTA solution with trypsin (250 µg/ml) (less trypsin than for fibroblasts, to allow for long exposure). Remove all but 0.75 ml of trypsin-EDTA.

3) Incubate at 37°C (preferably in air, not CO2) until cells are completely detached, as judged by tilting dish. If a very small number of cells are still attached after a long time (say 15-20 min), stop incubation anyway. However the cells that take longest to detach seem to be the stem cells (small, flat, round, epithelioid, healthy-looking), so ensure nearly all cells are harvested. It may improve the culture to discard the quickest-detaching fraction every few subcultures, once familiar with the cells' appearance and the time taken to detach. The more-differentiated cells have a scrappy appearance and tend to fragment.

4) Resuspend in growth medium, count, replate at 3x104/ml (10ml / 9cm dish). With a healthy culture we get about 4-5x105/ml after 6 days, i.e. doubling time about 1.2 days. It is worth counting the cells, as they don't do well at densities that are either too low or too high.

(B) Preparation of feeders

Plate about four to six 9-cm dishes of cells as above. When they are ready to subculture, but still growing, remove medium and add 5 ml fresh medium containing 8 µg/ml mitomycin C*. Incubate for 3 to 3.5 hr (timing is important). Wash in DMEM and incubate in fresh DMEM+FCS without mitomycin, for 10 min, to elute any remaining drug. Then subculture as usual. Either replate at about 3x104/ml for immediate use, or freeze aliquots of about 106 cells in liquid nitrogen, and plate at about 5x104/ml on thawing. Check viability of each batch on thawing (i.e. floating versus attached cells - none of them should divide); and adjust plating density for that batch if necessary.

Plate melanocytes on top within next 3 days, ideally after 1 day. Feeders gradually die off as melanocytes grow up. For preparative studies of immortal melanocytes that are feeder-dependent, like melan-si, these can be plated for 1 passage without feeders.

* Mitomycin C:- we get it from Sigma. Dissolved at 500 µg/ml in water. Protect from light - wrap vials in foil during use. Can store in fridge for up to about 1 month only or indefinitely at -70C or less. However if freezing it, freeze rapidly to avoid precipitation, e.g. drop vials into liquid nitrogen. (We freeze it.)