This protocol presents a novel method for the cultivation and maintenance of epidermal sphere cultures, and the strategy to closely investigate them using the spheroid replating assay. Begin by preparing wash medium as described in the text manuscript. In a laminar flow hood, wash neonatal foreskin twice with five milliliters of wash media in a five milliliter conical tube, then transfer the washed foreskin to a sterile Petri dish.
Using a scalpel and forceps, scrape off adipose tissue and loose connective tissues from the dermal layer, then rewash the foreskin with wash media. Place the foreskin epidermis side up in a six-well plate containing two milliliters of Dispase enzyme diluted in wash media. Transfer the plate to an incubator for four hours.
After incubation, transfer the foreskin to a Petri dish and use fine-tip forceps to separate the epidermis from the dermis layer. Place the epidermis into a 15 milliliter conical tube containing two milliliters of 0.25%Trypsin-EDTA. Crush the floating epidermis using a five milliliter serological pipette and incubate it for 15 minutes at 37 degrees Celsius with periodic vortexing for five seconds every five minutes.
After incubation, add two milliliters of soybean trypsin inhibitor and mix it by pipetting to neutralize the trypsin, then centrifuge the cell suspension. Resuspend the pellet in 12 milliliters of complete KSFM-SCM medium and plate the cells in a 10 centimeter dish to incubate overnight at 37 degrees Celsius and 5%carbon dioxide. On the next day, remove the medium using an aspirating pipette and replace it with 12 milliliters of complete KSFM-SCM.
Repeat this on days four and seven. Prepare 5%agarose mixture by adding 2.5 grams of agarose to 50 milliliters of PBS in a 100 milliliter glass bottle. Autoclave the bottle onto the liquid cycle and allow it to cool to room temperature.
To prepare plates, place the cooled glass containing agarose solution in a one liter beaker filled with 200 milliliters of deionized water. Melt the agarose mixture in a research-grade microwave for up to two minutes, mixing the agarose every 60 seconds by gently tilting the bottle side to side. Add three milliliters of melted 5%agarose to 12 milliliters of prewarmed KSFM-SCM at 42 degrees Celsius for a final concentration of 1%agarose.
Add 200 microliters of the 1%agar mix to each well of a 96-well plate using a multi-channel pipetter. Then leave the plate in a sterile environment at 25 degrees Celsius for four hours. Passage spheroid-forming normal human keratinocytes by aspirating media and washing cells in two milliliters of PBS.
Aspirate PBS and add two milliliters of 0.25%Trypsin-EDTA to the washed cells. Then incubate for five minutes at 37 degrees Celsius. After the incubation, add two milliliters of soybean trypsin inhibitor to the plate and wash the cells from the plate into a 15 milliliter tube.
Centrifuge at 250 times G for five minutes. Resuspend the cell pellet in one milliliter of PBS and quantify the cells using Trypan Blue staining. Aliquot 20, 000 normal human keratinocytes in 100 microliters of KSFM-SCM and seed the cells in each well of the previously prepared 96-well plate.
Then incubate the plate overnight. Using an inverted phase contrast microscope, analyze the seeded wells for epidermis sphere formation. 24 to 48 hours after 3D epidermis sphere formation, add four milliliters of prewarmed KSFM-SCM at 37 degrees Celsius to a six centimeter dish.
Using a wide bore one milliliter pipette tip, transfer a single spheroid to the plate and incubate the plate overnight. Analyze the seeded spheroid for attaching or propagating cells using an inverted phase contrast microscope. Feed cells every 96 hours by removing the media and adding two milliliters of fresh KSFM-SCM media to the plate.
Passage 70 to 80%confluent spheroid-derived normal human keratinocytes and 2D monolayer cultures as previously demonstrated. Then quantify cell viability using Trypan Blue and an automated cell counter. Aliquot 100 microliters containing 100, 000 to 4 million normal human keratinocytes into 1.5 milliliter microcentrifuge tubes.
Place the tubes on ice in a dark environment by turning off the bright lights within the laminar hood. Add two microliters of FITC-conjugated anti-integrin alpha six and two microliters of PE-conjugated anti-EGFR to the tubes to achieve a one to 50 dilution. Prepare a tube with no antibodies added to serve as the unstained control.
Incubate the tubes on ice in the dark for 30 minutes. Perform flow cytometry analysis with appropriate lasers. Use the negative and positive controls to establish gates.
Sort the subpopulation of epidermal stem cell fraction, the proliferative progenitor cell fraction, and committed progenitor cell fraction. Test for proliferative capacity of sorted cell subpopulations by transferring the content of each respective sorting tube into a 15 milliliter conical tube containing 10 times the volume of sorted cells in PBS. Centrifuge the tubes at 250 times G for five minutes.
Remove the supernatant and resuspend the pellet in four milliliters of KSFM-SCM. Transfer the resuspended cells to a six centimeter plate and incubate overnight at 37 degrees Celsius in a 5%carbon dioxide incubator with 95%humidity. On the next day, remove media from the plates and add four milliliters of prewarmed KSFM-SCM at 37 degrees Celsius every three days until 70 to 80%confluency is reached.
Autonomous epidermal spheroid-forming ability of normal human keratinocytes in 3D culture was assessed using the skin epidermis sphere assay. Non-spherical cell aggregation was not considered as adequate epidermis sphere formation. Dense sphere-shaped aggregation was considered a hallmark of spontaneous spheroid formation.
It was necessary to use more than two times 10 to the four cells to ensure proper spontaneous aggregation. Planting of epidermis spheres in 2D culture resulted in the proliferation of a small-sized viable normal human keratinocytes. It's important to maintain these cultures below 100%confluency as this can considerably impair their growth and stem cell state in culture.
The process of epidermal spheroid formation was functionally tracked at the single cell level by transfected cells with a fluorescent reporter. Spheroid-derived normal human keratinocytes subpopulation are integrin alpha six high and EGF are low cells. The cells generally make up about 25%of cultures.
Characterization of this stem-like keratinocyte subpopulation was achieved by immunostaining analysis of basal cytokeratin 14 and tumor protein 63. The spheroid replating assay is an effective strategy for epidermal stem cell enrichment from neonatal human skin. This system can be employed as a high-throughput cell-based model to investigate cutaneous diseases such as wound healing and cancer.
