This protocol describes a rapid and simple method to generate primary melanocyte and fibroblast cultures from mice. Because this technique does not require that the epidermis and dermis are separated, it can be performed quickly and consistently with minimal training. This method can be used to study cutaneous melanocyte and fibroblast biology.
Experiments in these cultures can provide insights relevant to cancer, wound healing, and pigmentation defects. Before beginning, make sure to prepare all of the necessary reagents and check the temperature of the water bath. If you plan to process multiple samples, refer to the reagent scaling guide in Table One.
Demonstrating this procedure will be Brandon Murphy, a graduate student from my laboratory. 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 six well dish containing three milliliters of 1X antibiotic antimycotic solution. Incubate at room temperature for two to three 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 three milliliters of skin digestion buffer.
Using a P1000 micro-pipette, mix the resulting suspension by pipetting up and down vigorously 10 to 15 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 three to five minutes. Next, centrifuge the tube in a swinging bucket rotor at 750 times G at room temperature for five minutes to pellet the cells in the skin homogenate.
Use a P1000 micro-pipette 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 are having trouble, wash the cell pellet with two to three mils of melanocyte media and repeat centrifugation and remove excess skin digest buffer.
Then thoroughly resuspend the cell pellet in one milliliter of melanocyte media by pipetting up and down 15 to 20 times with a P1000 pipette. Transfer the resulting cell solution drop wise to an uncoated well in a six well dish that contains one 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 pre-washed collagen coated six well dish. Add G-418 to the media, such that the final concentration is 100 nanograms per milliliter. Add two 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 one milliliter of PBS for each wash.
Then, add two milliliters of fresh melanocyte media to the melanocyte culture and add two milliliters of fresh fibroblast media to the fibroblast culture. In this study, fibroblast and melanocyte cultures are simultaneously generated from the same skin sample. When the homogenized skin is first plated, the media appears cloudy.
However, after incubation larger cell conglomerates and adherent cell fibroblasts become visible in the dish. Non-adherent cells from the culture are transferred to a collagen coated dish and treated with G-418. Fibroblasts killed by G-418 are seen floating in the melanocyte culture medium for the next five days.
After four to five days in culture, the plated melanocytes begin to take on a dendritic phenotype with melanocytic granules. This phenotype persists upon passaging, while clusters of contaminating keratinocytes are lost. The purity of the resulting melanocyte and fibroblast cultures is confirmed using flow cytometry.
Expression of the melanocyte marker GP100 is specific to the melanocyte cultures, while the fibroblast marker, FSP1, is found solely in the primary fibroblasts. Control C59 keratinocyte cells are the only cultures that stain positive for the keratinocyte marker, K14. Additional analyses are performed to determine how long it takes to establish enriched melanocyte cultures.
GP100 positive cells begin to appear on day four and peak after 10 days in culture. This procedure can be used to rapidly generate melanocyte and fibroblast cultures for a variety of in vitro and high throughput omic assays.
