Rapid Culturing of Primary Murine Melanocytes and Fibroblasts: Isolating Melanocytes and Fibroblasts from Whole Murine Skin Sample

- The skin consists of various cells populating its different layers. Melanocytes, the melanin-producing cells, localize at the epidermal-dermal junction, while fibroblasts, the connective tissue generating cells, reside in the dermis.

To rapidly isolate melanocytes and fibroblasts, begin by taking the trunk of a euthanized mouse pup. Cut the skin ventrally through the length of the trunk. Peel out the skin containing the epidermal and dermal layer and mince the tissue into small pieces.

Now, treat with a digestion buffer containing trypsin and collagenase enzymes that degrade the extracellular matrix, releasing the cells into suspension. Centrifuge to pelletize the cells and discard the supernatant. Resuspend the cells in melanocyte media and transfer the suspension into a multiwell plate.

During culture, most of the fibroblasts adhere to the well bottom, while the melanocytes and other epidermal cells remain in suspension. Aspirate the suspended cells and add fibroblast-specific media to the fibroblast culture to promote their growth. Transfer the aspirated suspension to a fresh collagen-coated well to aid the formation of an adherent culture.

Supplement with geneticin, an antibiotic, and add fresh melanocyte growth media. Geneticin selectively eliminates any remaining fibroblasts, while the media promotes melanocytes' growth over other epidermal cell types. In the following protocol, we will isolate melanocytes and fibroblasts from murine pup skin.

- In a laminar flow cabinet, briefly roll the trunk of each euthanized mouse in a sterile Petri dish containing 70% ethanol. Remove the mouse from the ethanol and place it into an empty sterile Petri dish. Next, sterilize surgical scissors in 70% ethanol. Use these scissors to make an incision in the skin on the ventral side of the trunk, starting from the neck and continuing to the tail. Using sterile forceps, peel the skin away from the trunk of the mouse and remove excess adipose tissue from the dermal side of the skin.

Place the skin, dermis side down, into a 6-well dish containing 3 milliliters of 1x antibiotic/antimycotic solution. Incubate at room temperature for 2 to 3 minutes. Then, turn on the tissue chopper and adjust the settings to those shown here.

- Be sure to exercise caution when installing the tissue chopper blade and when operating the machine.

- Transfer the skin, dermis side down, to a sterile tissue chopper dish and pass the skin completely through the tissue chopper three times. After this, transfer the homogenized skin to a sterile 15 milliliter conical tube containing 3 milliliters of skin digestion buffer.

Using a P1000 micropipette, mix the resulting suspension by pipetting up and down vigorously ten to fifteen times. Cap the conical tube and incubate the sample in a water bath at 37 degrees Celsius for 15 minutes, making sure to invert the tube every 3 to 5 minutes.

Next, centrifuge the tube in a swinging bucket rotor at 750 x g at room temperature for 5 minutes to pellet the cells in the skin homogenate. Use a P1000 micropipette to slowly and completely remove the skin digestion buffer, being careful not to disturb the pellet.

- Be sure to aspirate off all the skin digest buffer. If you're having trouble, wash the cell pellet with 2 to 3 milliliters of melanocyte media and repeat centrifugation and remove excess skin digest buffer.

- Then, thoroughly resuspend the cell pellet in 1 milliliter of melanocyte media by pipetting up and down fifteen to twenty times with a P1000 pipette. Transfer the resulting cell solution drop-wise to an uncoated well in a 6-well dish that contains 1 milliliter of melanocyte media. Incubate the plated skin homogenate in a tissue culture incubator at 37 degrees Celsius with 5% carbon dioxide for 40 minutes.

After this, transfer the culture supernatant from the uncoated dish to one well of a prewashed collagen coated 6-well dish. Add G418 to the media such that the final concentration is 100 nanograms per milliliter. Add 2 milliliters of fibroblast media to one well of the uncoated dish, now containing adherent fibroblasts. Incubate both cultures overnight in a tissue culture incubator at 37 degrees Celsius with 5% carbon dioxide.

After 16 to 24 hours of incubation, separately aspirate the media and any debris from each culture. Wash each dish twice, using 1 milliliter of PBS for each wash. Then, add 2 milliliters of fresh melanocyte media to the melanocyte culture, and add 2 milliliters of fresh fibroblast media to the fibroblast culture.