Hengge U R, Chan E F, Hampshire V, Foster R A, Vogel J C
Dermatology Branch, National Cancer Institute, National Institutes of Health (NIH), Bethesda, Md 20892-1908, USA.
J Invest Dermatol. 1996 Feb;106(2):287-93. doi: 10.1111/1523-1747.ep12340722.
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.
猪皮可能是研究人类皮肤生物学的一种非常有用的模型，因为其形态与人类皮肤极为相似。为了在体外操作猪角质形成细胞，我们分析了不同的培养条件以实现猪角质形成细胞的最佳生长，并在此描述一种在胶原蛋白底物上培养原代猪角质形成细胞并进行长期传代的简单方法。原代猪角质形成细胞的集落形成效率和增殖能力很容易在I型胶原蛋白和最终钙浓度为0.075微摩尔的条件下得到支持。这些培养条件允许使用各种阳离子脂质以4:1的比例（脂质:DNA）将基因有效地导入角质形成细胞。此外，利用这些优化的培养条件获得了维持正常表型的永生化猪角质形成细胞系。通过核型分析，两个独立获得的细胞系具有相同的染色体异常，这表明这些异常在它们的永生化过程中起到了因果作用。角质形成细胞系在钙浓度升高时表现出分化表型，并且在体内肿瘤试验中不具有致瘤性。能够维持分化能力的永生化猪角质形成细胞系可能会成为体外和体内表皮分化研究中的一种有价值的工具。此外，利用这些猪角质形成细胞系，也可以改进在体内将基因传递给猪的角质形成细胞的方法。
