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.

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