Exploring the potentials of nurture: 2(nd) and 3(rd) generation explant human skin equivalents

Explant human skin equivalents (Ex-HSEs) can be generated by placing a 4mm skin biopsy onto a dermal equivalent. The keratinocytes migrate from the biopsy onto the dermal equivalent, differentiate and form the epidermis of 1(st) generation Ex-HSEs. This is especially suitable for the expansion of skin material from which only small fragments of skin can be harvested e.g. diseased skin.

We evaluated whether 2(nd) and 3(rd) generation Ex-HSEs can also be generated from a single skin biopsy whilst maintaining the epidermal properties of 1(st) generation Ex-HSEs and native human skin.

2(nd) generation Ex-HSEs were produced by placing a biopsy from the 1(st) generation Ex-HSE onto a new dermal equivalent. Likewise, the 3(rd) generation Ex-HSEs were generated from a 2(nd) generation Ex-HSE biopsy.

We show for the first time that Ex-HSEs can be passaged to the 2(nd) and 3(rd) generation and display similar epidermal morphology and expression of differentiation markers as in native human skin and 1(st) generation Ex-HSEs except for involucrin. The 2(nd) and 3(rd) generation Ex-HSEs also show many similarities with 1(st) generation Ex-HSEs in lipid properties e.g. presence of all lipid classes, similar fatty acid chain length distribution and lamellar lipid organization. However, some differences arise in increased level of hexagonal lateral packing and a change in ceramide profiling. The changes in specific lipid classes were also accompanied by changes in the expression of the enzymes responsible for their synthesis.

The expansion of skin biopsies to the 2(nd) and 3(rd) generation Ex-HSEs could be a promising method to expand valuable epidermal tissue to analyze morphological and differentiation parameters in the native epidermis.