The overall goal of this procedure is to isolate primary mouse keratinocytes from neonatal or adult mouse skin from their in-vitro differentiation or to study their innate immune responses to UVb exposure. This method can help answer key questions in the epidermal biology field about how keratinocyte differentiation is regulated or how epidermal keratinocytes respond to external stimuli. The main advantages of this technique are that the keratinocytes can be conventionally isolated from both mouse, neonatal and adult mouse skin and that they can be cultured be with our serum.

Demonstrating the procedure with me will be, Fengwu Li, a staff research associate from my laboratory. Begin by removing the limbs of a wild type C57 BL6 postnatal day zero to two mouse neonatal, just above the wrist entangled joints and cut off the tail completely leaving a small ball. Insert sharp scissors through the tail hole and cut the skin along the dorsal midline of the body to the opening on the neck.

Using one forceps, grasp the exposed body and use another forceps to grasp the skin. Then gently peel the whole skin off of the body and over the leg stumps in one continuous motion, taking care not to break the skin into pieces. Rinse the peeled skin with 50 millimeters of sterile PBS in a 10 centimeter petri dish and place the skin into a two milliliter tube containing ice cold, dispase digestion buffer, taking care that the skin is not folded.

When all of the neonatal skin sheets have been collected, place the tube on a rotator at four degree celsius overnight. The next morning, dispense the tube contents into a new petri dish. Then, transfer each piece of tissue into a new petri dish containing 50 milliliters of sterile PBS to remove any excess dispase.

Use two forceps, place the skin samples into individual petri dishes, epidermal side down and carefully stretch the skin folds so that the pieces are fully extended on the dish bottoms. Add 500 microliters of a trypsin based digestion solution to each dish and to use one forceps to slowly lift each dermis up and away from the epidermis, while holding the epidermis in place with a second forceps. Float the isolated epidermises in the trypsin solution with the basal levels facing down and cover the dishes.

Then incubate the skin pieces on a horizontal shaker at room temperature, with gentle agitation for 20 minutes. At the end of the incubation, add two milliliters of supplemented keratinocyte growth medium per epidermis to each petri dish and use forceps to vigorously rub the epidermal sheets releasing the single cells from the skin tissue. When the medium becomes turbid, tilt the dishes to allow the collection and transfer of the cell suspensions into a new tube.

Leaving the remaining epidermal tissue in the dish. After the third round of cell collection, gently triturate the pooled cell suspension a few times to break up any cell clumps, and filter the cells through a 100 micron strainer into a new 50 milliliter conical tube. Collect the cells by centrifugation and gently re-suspend the pellet in one milliliter of cold keratinocyte growth medium on ice, for counting.

Seed the cells at a 5x10 to the fourth centimeter squared density in keratinocyte growth medium in cultured dishes, pre-coated with an extra-cellular matrix product that promotes cell attachment. Then incubate the cells at 37 degrees celsius and 5%carbon dioxide, replacing the supernatant daily with fresh medium until the cells have reached the appropriate confluency. To isolate keratinocytes from an adult mouse, first remove the tail at its base.

Followed by removal of approximately two millimeters of the tail tip so there is visible hole. Use a sharp blade to cut along the tail skin from the base to the tip. Then, using one forceps to grasp the exposed tail bone and another forceps to grasp the skin, gently peel the skin tissue from the bone in one continuous motion.

Cut the peeled skin at the midline, so that each piece is less than two to three centimeters long, and rinse the pieces in 15 milliliters of sterile PBS in a 10 centimeter petri dish. Place the skins from up to five tails into a 15 milliliter tube containing up to 12 milliliters of ice cold, dispase digestion buffer and incubate the skins overnight at four degree celsius, with rotation. The next morning, isolate the epidermal cells from the partially digested skin sheets, as just demonstrated for the neonatal skin samples.

Then seed the isolated adult keratinocyte cells at 1x10 to the fifth power centimeter square density in kerationcyte growth medium in culture dishes pre-coated with collagen. Changing the supernatant daily, until the cells reach the appropriate confluency. To induce the terminal differentiation of either type of keratinocyte culture, add calcium chloride for the cells at 2 nanomolar concentrations.

For UVb irradiation induced death, replace the growth medium from a confluent keratinocyte culture, with 500 microliters of PBS and briefly expose the cells to 15 millijoules per square centimeter of UVb. Replace the PBS with fresh growth medium and return the cells to the cell culture incubator for six hours. After collecting the conditioned medium at each experimental time point, quantify the cell viability by the cell counting kit aid viability assay, according to the manufacturer's instructions.

Then measure the TNL for release by the irradiated keratinocytes into conditioned medium according to standard protocols. Primary mode's epidermal keratinocytes plated and maintained at 06 nanomolar calcium chloride, grew as a monolayer with the individual cells exhibiting a polygonal shape with distinct inter-cellular spaces and demonstrating an overall cobblestone appearance when confluent. Elevating the calcium chloride to 2 millimolar, induces a rapid morphology change in the cells.

With the cells becoming flattened and the distinct inter-cellular space becoming less apparent within eight hours. And the cell to cell adhesion between the tight junctions becoming more striking within 24 hours. By 48 to 72 hours, the formation of the cornified cell envelope and the vertical cell stratification, become apparent.

Phalloidin staining of high calcium treated keratinocytes, reveals the formation of actin fiber-rich filopodial projections between adjacent cells, as early as three hours, post calcium switch. Between six and 24 hours, further actin fiber remodeling becomes evident. With the prominent amount of cell stratification apparent at 48 hours post high calcium exposure.

UVb irradiation triggers the time dependent cell death of keratinocyte cultures as evidenced by both phase contrast imaging and cell viability assay. Indeed, by 24 hours, nearly all of the cells become rounded and detached, and are measured as unviable by cell viability assay. Most likely due to the high levels of TNF alpha, relieved by the keratinocytes, in response to UVb exposure.

While attempting this procedure, it's important to remember that to keep the dissociated keratinocyte on ice at all times and to plate the cells as soon as possible to avoid terminal differentiation of the isolated cells. After its development, this technique paved the way for researchers in the field of epidermal biology to explore the roles of keratinocytes in skin development and in an innate immune defense of the skin. After watching this video, you should have a good understanding of how to isolate and culture primary epidermal keratinocytes from neonatal or adult mouse skin.