The derivation and characterization of pig keratinocyte cell lines that retain the ability to differentiate

Pig skin may be a very useful model for studying human skin biology, since its morphology closely resembles that of human skin. To manipulate pig keratinocytes in vitro, we have analyzed different culture conditions for optimal pig keratinocyte growth and describe here a simple method for culture and extended passage of primary pig keratinocytes on collagen substrates. The colony-forming efficiency and proliferative capacity of primary pig keratinocytes were readily supported by Type I collagen and a final calcium concentration of 0.075 microM. These culture conditions permitted efficient gene transfer into keratinocytes using various cationic lipids at a 4:1 ratio (lipid: DNA). In addition, immortalized pig keratinocyte cell lines, which maintained a normal phenotype, were derived using these optimized culture conditions. By karyotype analysis, two independently derived cell lines had the same chromosomal abnormalities, suggesting a causal role in their immortalization. The keratinocyte cell lines exhibited a differentiated phenotype in response to elevated calcium concentration and were nontumorigenic in in vivo tumor assays. Immortalized pig keratinocyte cell lines that maintain the ability to differentiation may become a valuable tool in the study of epidermal differentiation both in vitro and in vivo. In addition, methods using keratinocytes to deliver genes to pigs in vivo could also be enhanced with these pig keratinocyte cell lines.