A METHOD FOR THE ISOLATION AND SERIAL PROPAGATION OF KERATINOCYTES, ENDOTHELIAL-CELLS, AND FIBROBLASTS FROM A SINGLE PUNCH BIOPSY OF HUMAN SKIN

When multiple types of cells from normal and diseased human skin are r equired, techniques to isolate cells from small skin biopsies would fa cilitate experimental studies. The purpose of this investigation was t o develop a method for the isolation and propagation of three major ce ll types (keratinocytes, microvascular endothelial cells, and fibrobla sts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from th e dermis by treatment with dispase. Keratinocytes were dissociated fro m the epidermis by trypsin and plated on a collagen-coated tissue cult ure petri dish. A combination of two commercial media (Serum-Free Medi um and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated t issue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishm ent and growth of these cells. Epithelioid endothelial cells were sepa rated from spindle-shaped endothelial cells and from dendritic cells b y selective attachment to Ulex europeus agglutinin I-coated paramagnet ic beads. To establish fibroblasts, dermal explants depleted of kerati nocytes and endothelial cells were attached to plastic by centrifugati on, and fibroblasts were obtained by explant culture and grown in Dulb ecco's modified Eagle's medium (DMEM) containing fetal bovine serum (F BS). Using these isolation methods and growth conditions, two confluen t T-75 flasks of keratinocytes, one confluent T-25 flask of purified e ndothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This metho d should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical a nalysis.