Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Our method for harvesting epidermal cells from adult mice is useful for downstream applications such as keratinocyte stem cells. Our technique is highly reproducible and can be used in conjunction with in vivo procedures in mice in the context of the skin carcinogenesis model. After harvesting dorsal skin samples from euthanized adult mice, use autoclaved forceps and a scalpel to place one dorsal skin at a time hairy side down into a thin Petri dish and scrap off the subcutaneous tissue including the fat from the ventral skin tissue until the tissue is semi translucent.

Place the scraped skin in PBS until all other remaining skins are processed. Then use a scalpel to slice the skin samples into 0.5 by one to 1.5 centimeter strips. Place the strips hairy side up in a sterile Petri dish before floating the samples hairy side up on the surface of a 20 milliliter PBS plus two x gentamicin solution, supplemented with 0.25%trypsin in a plastic 100 by 20 millimeter Petri dish for two hours at 32 degrees Celsius.

At the end of the incubation, place a sterile, plastic, square Petri dish containing 15 milliliter of harvesting medium at a 30 degree incline and use curved forceps to carefully transfer a floating skin strip into the dish. Holding a new scalpel blade at a perpendicular angle to the skin and using sufficient but not excessive force, scrap off the epidermis and the hairs from the sample into the medium. When all the strips have been scraped, carefully decant the epidermal cell containing supernatant into a sterile 60 milliliter jar containing a 1.5 inch magnetic stir bar and rinse the Petri dish with additional harvesting medium to collect any remaining epidermal cells.

Bring the final volume in the jar to 30 milliliters with fresh medium, and stir the epidermal cell solution at 100 rotations per minute for 20 minutes at room temperature. At the end of the stirring incubation in a biosafety cabinet remove the stir bar and filter the cell solution through a 70 micrometer strainer into a 50 milliliter conical tube. Use forceps and then a pipette to press the hairs and the stratum corneum materials through the strainer to manipulate the tissue to release the trapped hair cells and use an additional five milliliters of harvesting medium to release the remained trapped hair cells into the tube.

It is critical that the epidermal cells and the hairs are manipulated so as to release the cells from the hair follicles. Bring the total volume in the tube up to 50 milliliters with fresh medium and collect the cell filtrate by centrifugation. Resuspend the pellet in 5 milliliters of fresh harvesting medium in about 20 triturations with a 5 milliliter pipette.

Take 0.5 milliliters of the one to 20 dilution and transfer it to a 2 milliliter microfuge tube. After mixing the sample carefully, take 200 microliters from the microfuge tube and mix gently with an equal volume of 0.4%trypan blue solution. Gently mix this solution three times and transfer the cells to a hemocytometer for counting nucleated keratinocytes.

Score all dark blue cells as nonviable cells and score small gold and pinkish cells as viable cells. The viability averages about 80%and the final keratinocyte yield per mouse should be about 30 million viable cells. After counting, collect the cells with another centrifugation, and for mass culture resuspend two to four times 10 to the sixth viable keratinocytes per 35 millimeter Petri dish in 2 milliliters of cell culture medium.

For a clonogenic colony formation assay, resuspend the cells at a one times 10 to the third keratinocytes per four milliliters of modified William's E Medium with supplements in serum concentration per collagen coated 60 millimeter Petri dish on X-ray irradiated Swiss mouse 3T3 feeder layers. For mass culture, grow the cultures at 32 degrees Celsius in 5%carbon dioxide for the appropriate cell culture period. Changing the medium 24 hours after the initial seeding for mass culture and three times a week thereafter.

At the end of the clonal culture aspirate the medium and fix the colonies in 10%buffered formalin over night at room temperature. The next morning, stain the colonies with 0.5%rhodamine B in autoclaved water for one hour, before rinsing the dishes in cold autoclaved water until the water runs clear. Then incline the dishes on their lids to dry before counting the colonies.

Here, typical results from keratinocyte colony formation assays after various topical treatments are shown. Hair follicle stem cells typically make up approximately 9%of the cells isolated from adult C57BL/6 mouse dorsal skin as assessed by flow cytometric staining for mouse hair follicle stem cell markers. In this figure the growth characteristics of keratinocyte stem cell colonies after culture under four different medium conditions can be observed.

Following this procedure, the cells can be used for flow cytometry, fluorescence-activated cell sorting, cell culture, and molecular biological analyses. This technique has enabled us to determine that the number of epidermal stem cells is a quantitative and complex trait leading to the identification of a new stem cell regulatory gene.