Culture of rat keratinocytes with acellular pig dermis

This study evaluated the growth rate and the cell activity of cultured keratinocytes on acellular pig dermis in order to develop a composite skin in vitro for burn injuries or other skin defects. Full thickness skin was cultivated from neonatal SD rats, and separated into epidermal layer and dermal layer with enzyme digestion. The keratinocytes were then seeded on the prepared acellular pig dermis soaked in the culture medium. The cultures were incubated and the growth status of keratinocytes on acellular pig dermis evaluated by phase contrast microscope, histological examination with hematoxylin-eosin staining and acridine orange staining, immunohistochemistry, observation of growth curve plotted by MTT colorimetry and analysis of changes in keratinocytes proliferation cycle with flow cytometer. Almost all keratinocytes anchored in 48-72 h, and most inosculated at days 6 and 7. The growth curve showed that the keratinocytes grew in logarithmic phase at days 3-6 after seeding. More than four layers of keratinocyte structure and the basement membrane between keratinocytes and porcine dermis were observed. Pancytokeratin was strongly positive in the cultured keratinocytes. Laminin and collagen IV were positive in the basement membrane. It is concluded that the cultured keratinocytes on acellular pig dermis grow well and the structure of composite skin which has been established is satisfactory.