The overall goal of this procedure is to generate primary human keratinocytes that can be used as a model to study cutaneous biology in vitro. This method can help answer key questions in the epidermal cell biology field, such as how human keratinocyte differentiation is regulated or as a model for studying inflammatory skin diseases. The main advantages of this technique are that primary human keratinocytes can be conventionally isolated from adult skin of both men and women and can include skin from people of any age older than 18.

Demonstrating the procedure will be Annette Blak Rasmussen, a technician from my laboratory. To begin, prepare a 50 milliliter solution of 0.25%trypsin and 0.1%glucose in DPBS. Then, mix and filter sterilize the solution.

Prepare 10 milliliters of RPMI 1640 plus 2%FBS, 50 milliliters of DPBS and keratinocyte serum free medium, or KSFM. Add KSFM supplements and 250 microliters of gentamicin to 500 milliliters of KSFM. Then, pre-warm the solutions to 37 degrees Celsius before use.

Collect 10 centimeter by 15 centimeter skin samples from healthy adult volunteers undergoing plastic surgery. Keep the skin cool by transporting the skin samples in a styrofoam box containing a cooling element. If necessary, store the skin sample at four degrees Celsius overnight.

Using sterilized scissors, scalpels and forceps, remove fat from underneath the skin section. Then, using needles, buckle out the skin section on a sterile cover on top of a plate. With a dry, sterilized gauze pad, clean the skin.

Then, follow with 70%ethanol on a sterilized gauze pad. Next, using a foot planer, cut off the upper epidermal layer of the skin section and transfer it into a nine centimeter Petri dish. Then, immediately add 25 milliliters of the previously prepared DPBS trypsin glucose solution to the Petri dish.

Incubate the samples at 37 degrees Celsius for 30 minutes. Using a pipette, remove the DPBS trypsin glucose solution from the Petri dish and add 10 milliliters of RPMI 1640 plus 2%FBS to inactivate the trypsin. Now, with two forceps, release the epidermal cells into the medium by gently scraping and agitating both the epidermal and the dermal compartment of the skin sections.

Filter the epidermal cell suspension through a metal filter and collect the filtrate into a 50 milliliter tube. Add the remaining skin sections to a 50 milliliter tube containing 10 milliliters of RPMI 1640 without FBS and vortex for 10 seconds. Filter this suspension through the metal filter into a 50 milliliter tube containing the epidermal cell suspension.

Then, add DPBS to a total volume of 50 milliliters. Centrifuge the cell suspension at 450 times G and room temperature, for 10 minutes. Then, remove the supernatant and depending on the size of the cell pellet, re-suspend the epidermal cell pellet in approximately 10 milliliters of 37 degrees Celsius KSFM.

Using the trypan blue staining method, count the cells under a microscope. Then, to each 75 square centimeter culture flask, transfer eight times 10 to the sixth cells, together with 12 milliliters of 37 degrees Celsius KSFM. Gently shake the culture flasks to ensure uniform distribution of the cells.

Incubate the keratinocytes in a 37 degree Celsius incubator with 100%humidity and 5%CO2. Change the medium after two days and then three times weekly. When the cells reach 70-80%confluency, keep the appropriate amount of 0.05%trypsin EDTA solution to 37 degrees Celsius in an incubator.

Remove the medium from the cells and add 4.5 milliliters per 75 square centimeter culture flask of pre-warmed 0.05%trypsin EDTA solution to the cells. After placing the culture flask in the 37 degree Celsius incubator for approximately five minutes, check under the microscope if the cells have started to loosen. When approximately 50%of the cells have loosened, gently hit the culture flask against the hand to loosen the remaining cells.

Then, to inactivate the trypsin, add six milliliters of 37 degree Celsius RPMI 1640 plus 2%FBS to the 75 square centimeter culture flask and transfer the cell suspension to 50 milliliter tubes. Rinse the culture flasks with three milliliters of RPMI 1640 plus 2%FBS and add the medium to the 50 milliliter tubes. Then, centrifuge the cells for 10 minutes at 450 times G and room temperature.

Use 10 milliliters of 37 degree Celsius KSFM to re-suspend the pelleted cells. Then, count and transfer three times 10 to the sixth cells to a 150 square centimeter culture flask, together with 20 milliliters of 37 degree Celsius KSFM. Gently shake the culture flask to ensure uniform distribution of the cells, before returning the cells to the incubator.

When the culture is 80-90%confluent, after trypsinizing and transferring the cells to 50 milliliter tubes, as demonstrated earlier in this video, rinse the culture flasks with six milliliters of RPMI 1640 plus 2%FBS and add it to the 50 milliliter tubes. Centrifuge the tubes for 10 minutes at 450 times G and four degrees Celsius. Then, prepare ice cold cell freezing medium.

Re-suspend the cell pellet in KSFM plus 10%DMSO. Then, after counting the cells, freeze them at a density of six times 10 to the sixth cells per milliliter, using standard, slow-freezing cryopreservation methods. Store the keratinocytes in liquid nitrogen until ready to use.

As seen here, human keratinocytes were isolated and cultured, as demonstrated in this video and then stimulated with Calcium. These images show the morphological changes due to the addition of Calcium on days one and two, compared to controlled treated cells. In addition to morphological changes, the addition of Calcium also resulted in an increase in transcription of mRNA for the differentiation marker Involucrin, 5.5-fold after 24 hours and 3.5-fold after 48 hours.

In this experiment, cultured keratinocytes were stimulated with IL-1 beta for various amounts of time. As seen by Western blotting, within five minutes, IL-1 beta stimulation led to a rapid activation phosphorylation of p38 MAP kinase as IL-1 beta stimulation had no effect on p38 MAP kinase total protein levels. After one hour, IL-1 beta induced p38 MAP kinase phosphorylation returned to basal levels.

After it's development, this technique paved the way for researchers in the field of human epidermal cell biology to explore the role of keratinocytes in innate immune responses, as well as in skin development. After watching this video, you should have a good understanding of how to isolate and culture primary keratinocytes from adult human skin, which can be used as a model to study cutaneous biology in vitro.