The skin contains multiple cell types including, keratinocytes, fibroblasts, and nerve Schwann cells. This protocol allows isolation of each of these cell types for in vitro experiments. Isolation and establishment of individual primary cell cultures from a single donor allows for robust comparisons and co-culture experiments, while mitigating the effects of genetic variation.
With this protocol, the analysis of individual cells and their interactions allows the analysis of the role of Schwann cells in skin homeostasis and pathophysiology. These distinct cell populations can be used to study Schwann cells, fibroblasts, or keratinocytes either individually or in combination in normal skin physiology, wound healing or in skin diseased states. Jagadeeshaprasad M G, a post-doctoral scholar in my laboratory will be assisting me in demonstrating the procedure.
After acquiring the neonatal foreskin following standard of care procedures, place the excised foreskin in a micro centrifuge tube containing eight to 10 milliliters of ice cold DMEM basal medium, and immediately transport the foreskin to the cell culture laboratory for cells isolation. Rinse the foreskin twice with 20 to 25 milliliters of Dulbecco's Phosphate Buffered Saline or DPBS, containing antibiotics and antimycotic. Then soak the washed skin in 25 to 30 milliliters of ice cold DMEM basal medium in a 10 centimeter tissue culture dish.
After 15 minutes slice the foreskin open using a scalpel to expose the dermis and subcutaneous adipose tissue. Use forceps, a scalpel blade, and scissors to remove and discard the subcutaneous adipose tissue. Then cut the clean foreskin into three to five smaller pieces and incubate the skin pieces in 16 to 20 milliliters of Dispace one DMEM medium in a sterile conical tube.
During the first 30 minutes of incubation with Dispace one intermittently mix the skin pieces in the conical tube by inversion. Then place the conical tube at four degrees Celsius for 16 to 18 hours. The next day remove the skin pieces and place them on a dry sterile culture dish and unroll each piece of skin so that the outer epidermis layer is touching the culture dish.
Starting on the edge of the tissue peel the epidermis away from the the dermis using two sets of forceps. Add the separated epidermis fragments to five milliliters of HBS in a 10 centimeter culture dish to incubate at room temperature for 10 minutes. Then collect the HBS solution in a 50 milliliter conical tube and add five milliliters of trypsin neutralization buffer or T and B in the conical tube.
Set the tube aside. To the epidermal fragments in the culture dish add three milliliters of Trypsin solution to incubate at 37 degrees Celsius for 10 minutes. Following the incubation gently agitate the epidermal fragments with forceps until the trypsin keratinocytes solution becomes turbid.
Next add the trypsin keratinocyte solution to the HBS T and B tube. After dissolving undigested epidermal fragments with trypsin EDTA solution, add two milliliters of FBS to the HBS T and B tube containing trypsin EDTA solution, and centrifuge the two about 870 times G for five minutes at four degrees Celsius. Then aspirate the supernatant and resuspend the cell pellet in three milliliters of keratinocyte complete growth medium, followed by filtration of the cell suspension with a sterile 100 micrometer filter into a sterile 50 milliliter conical tube.
Once the cell number and cell viability are quantified, seed the cells in the culture plates to place in an incubator at 37 degrees Celsius in 5%carbon dioxide. After two days aspirate the non-adhered cells, and refresh the cell culture media every other day until keratinocytes reach 50 to 80%confluence for passaging or downstream experiments. Precoat T25 flasks with poly-l-lysine or PLL, using five milliliters of DPBS and place the precoated flasks at 37 degrees Celsius for three hours.
Later wash the PLL coating matrix twice with DPBS. After washing the isolated pieces of the dermis with five milliliters of DMEM basal medium, mince the dermis into small pieces with scissors. Digest the minced dermis with five milliliters of collagenase at 37 degrees Celsius for two and a half hours, with gentle titration using a pipette tip every 30 minutes until the dermis dissociates completely.
Once digested, filter the cell suspension with a 70 micrometer cell strainer, followed by dilution of the filtered cell suspension with five milliliters of complete DMEM medium to stop the enzymatic activity of collagenase. Next centrifuge the cell suspension at 870 times G for five minutes at room temperature and resuspend the cell ballot in two milliliters of DMEM complete medium to divide the cells. After repeating the centrification process as described, aspirate the supernatant to use the cell pellet to isolate the Schwann cells or fibroblasts.
To isolate the Schwan cells, resuspend the cell pellet in five milliliters of fresh DMEM complete medium and quantify the cell number and viability before seeding five milliliters of the resuspended cells in pre-coded T25 flasks at a density of 4.0 times 10 to the three cells per milliliter. Incubate the flask at 37 degrees Celsius and 5%carbon dioxide. After 16 hours confirm cell adhesion by microscopy at 10X magnification.
Then remove the non-adherence cells and wash the flask thrice with five milliliters of DPBS. Next incubate the cells with five milliliters of 10 micromolar cytosine arabinoside in a DMEM complete medium. After 24 hours aspirate the cytosine arabinoside containing medium before rinsing the flask as demonstrated before.
Refill the culture flask with five milliliters of Schwann cells complete culture medium to incubate 48 hours, while refreshing the medium every other day until Schwann cells reach 80%confluency. To isolate the fibroblasts, resuspend the collected pellet in five milliliters of fibroblasts complete medium. To culture the cells in non coded T25 culture flasks for 24 hours as demonstrated for the Schwann cells.
After aspirating the non-adherence cells and washing the flask, incubate the cells with five milliliters of fresh fibroblasts complete medium at 37 degrees Celsius and 5%carbon dioxide for 48 hours. As the isolated primary cells were cultured in respective cell culture media, the primary keratinocytes reached 85%confluency by day seven and exhibited characteristic cell morphology with a cobblestone shape. The Schwann cells reached 95%confluence by day five and appeared bipolar or tripolar in shape.
The fibroblasts cultures reached 95%confluence by day four and most cells exhibited a spindle shape morphology. The immunofluorescence staining confirmed cell type specific protein expression in the cell cultures. The keratinocytes were positive for K10 and K14 protein.
The merged images of keratin immunofluorescence and DAPI nuclear staining indicated 97.8%purity of the cell cultures. Similarly, the Schwann cells were positive for S100 and P75 NTR and had a purity of 95.2%in the culture. The fibroblasts positively expressed vimentin, but did not express alpha smooth muscle Actin, a myofibroblast marker.
The purity of the culture was around 97.2%To enhance Shanse's evidence, pre-coated poly-l-lysine dish can be used. And to further mitigate fibrous contamination, cytosine arabinoside and antimycotic agent was added after cells had added for a short time. This is a simple inexpensive experimental procedure to establish the three primary cells, such as shwann cells, keratinocytes, and fibroblasts, from a single human fore skin.
This protocol is ideal for in vitro experiments involving cell cell communication or crosstalk between cell types.
