Growth and differentiation of human keratinocytes on extracellular matrix

Extracellular matrices (ECM) have been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro. Since ECM components are physiological constituents of the dermoepidermal basement membrane, we studied the growth and differentiation of human keratinocytes on ECM in order to determine the benefits of culturing epidermal epithelial cells (keratinocytes) on reconstituted basement membranes. Disaggregated epidermal cells were grown in primary and subcultures in liquid medium; the attachment of the cells was greatly enhanced by ECM and noted within the first few hours after seeding; cells formed small islets that reached confluence within 2-12 days depending upon the plating density and the type of culture (primary or passages). Histological and ultrastructural cross-sections of the cultures clearly indicated that a multilayered epithelium can be obtained including a basal cell layer, several intermediate cell layers with cytoplasmic organelles, intermediate size filaments, desmosomes, and keratohyaline granules, and an upper layer of anucleated cells. Using immunofluorescence, both pemphigus and pemphigoid (basal membrane zone) antigens were expressed. The keratin pattern noted indicated that these epithelia differentiate and keratinize but do not express a complete program of keratinization, a finding usually noted when cells are grown submersed. These data show that ECM favor epidermal cell proliferation and differentiation and suggest that they may be used to obtain large amounts of epidermal equivalent suitable for grafting and/or in vitro studies.