Generation of Organotypic Full Skin Reconstructs: A Procedure to Establish 3D Skin Model Using Human Skin Samples

Skin primarily consists of an upper epidermal layer and a lower dermal layer. The epidermal cells are stratified into multiple layers ranging from the least differentiated cells present at the base to the highly differentiated cells at the periphery. To grow a skin tissue mimicking the morphological and biochemical aspects of normal skin, we can establish an organotypic skin model.

To begin, take a multi-well plate with a membranous insert. Add dermal fibroblasts mixed with collagen to the insert. Incubate to facilitate collagen matrix solidification to embed the fibroblasts and mimic the dermal compartment of the skin.

Next, add a keratinocyte cell suspension over the dermal layer. Incubate the plate to allow keratinocytes to adhere to the dermal compartment. Supplement the culture with an epidermalization medium to initiate keratinocyte proliferation and the formation of an epidermal layer.

Finally, transfer the inserts to a fresh multi-well plate. Add a stratification medium such that it covers only the base of the construct, thereby creating an air-liquid interface. Upon incubation, the air-liquid interface facilitates the differentiation of keratinocytes into a stratified multilayered epidermis.

In the following protocol, we will generate 3D organotypic skin reconstructs having dermal and epidermal counterparts from isolated human skin cells.

To generate the dermal counterpart of the skin reconstructs, place 8 micrometer pore size membrane inserts in a 24-well cell culture plate. For each insert, resuspend 100,000 fibroblasts in gel neutralization solution. Next, quickly mix the fibroblast suspension with collagen type 1 at a ratio of 1:3 in a final volume of 500 microliters without forming bubbles.

Then, use a pipette to add the suspension to each insert. Inside a sterile hood, place the inserts at room temperature without medium for 30 minutes to allow the dermal gels to settle. Next, cover the gels with DMEM and incubate overnight at 37 degrees Celsius.

After the overnight incubation, remove the DMEM from the dermal gels and equilibrate them with EGM for 2 hours at 37 degrees Celsius to generate the epidermal counterpart of the skin reconstructs. Next, use a pipette to remove the EGM from the gels.

Then, carefully seal 100,000 keratinocytes resuspended in 100 microliters of EGM on top of each gel. Incubate the reconstructs for 1.5 hours at 37 degrees Celsius to allow the keratinocytes to adhere to the dermal gel. Using a pipette tip, carefully remove the residual gel from the insert wall.

Next, cover each reconstruct with 800 microliters of EGM and culture for 7 days at 37 degrees Celsius in 5% carbon dioxide.

Carefully remove residual gel from the insert wall with a small white pipette tip.

On day 8, place the inserts into separate wells of a 6-well plate. Next, add 1.2 to 1.4 milliliters of metastatic melanoma medium to each well to cover only the base of the skin reconstruct. Then, incubate for 10 to 17 days at 37 degrees Celsius in 5% carbon dioxide.