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 mult