Generation of Differentiating Human Epidermal Cells on Basement Membrane Matrigel: Potential for use as Autografts in Recurrent Ulcerative Lesions

Abstract

AbstractThe successful use of in vitro-generated skin autografts in the treatment of burns in humans (Green and O'Connor, 1984) has advanced the potential for application in the treatment of recurrent ulcerative lesions that occur in some patients with sickle cell disease and other disorders. Conventional grafts frequently break down in these patients, requiring repeated grafting that may eventually result in the lack of suitable donor sites. Preliminary studies show that Type I collagen (Collistat) promotes healing of sickle cell-lesions but with extensive scarring and loss of pigmentation (Adekile et al., 1987). Therefore, the primary aim of this study was the development of a culture procedure for the rapid generation of differentiating human epidermal cells on a biomatrix for use in conjunction with the Collistat, and later, the establishment of a frozen culture bank. Dispasefollowed by trypsin-dissociated inner epidermal cells from dermatome sections of skin of adult patients undergoing elective plastic surgery were cultured on basement membrane-derived Matrigel (Collaborative Research, Inc.) coated onto plastic coverslips. Growth was established in RPMI1640 or IMEM supplemented with insulin, transferrin, hydrocortisone and bovine pituitary extract or Keratinocyte Growth Medium (KGM)a nd from 0.5–2.0% fetal bovine serum. The epidermal cells exhibited active migration and the formation of bi- and tri-layered aggregates by day 3. Large multilayered aggregates were produced in KGM with 1.16 mM CaCl2 added. Electron microscopic observations revealed increased numbers of desmosomal junctions, interdigitations and apical-basal polarity in these aggregates. A distinguishing feature ofthese and other cells grown on Matrigel is the absence of the degree of spreading that is characteristic of cells grown on plastic. It is noteworthy that the epidermal cells did not invade through the Matrigel.