School of Biological and Biomedical Sciences, Durham University, South Road, Durham, United Kingdom.
PLoS One. 2013;8(1):e52494. doi: 10.1371/journal.pone.0052494. Epub 2013 Jan 11.
Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.
从皮肤组织中分离出来的原代人表皮干细胞，并在组织培养中进行扩增，用于人类治疗，以重建患者的皮肤，并在培养中生成用于学术和商业研究的人工皮肤。经典上，表皮细胞，称为角质形成细胞，需要成纤维细胞饲养支持和含血清的培养基进行连续繁殖。为了与全球去除潜在动物污染物的努力保持一致，已经开发了许多无血清、无饲养的培养方法，这些方法支持这些细胞在二维培养中的衍生和生长。在这里，我们表明，在无血清和无饲养条件下连续生长的角质形成细胞无法在皮肤等效模型中形成分层、成熟的表皮。这并不是由于细胞潜能的丧失，因为在无血清、无饲养条件下增殖的角质形成细胞在重新引入经典含血清培养基时保留了形成分层表皮的能力。细胞外钙补充未能改善表皮发育。相比之下，向商业、无血清培养生长培养基中添加血清，促进了我们的皮肤等效模型中 3 维分层。此外，添加热灭活血清可改善表皮结构和厚度，表明血清中含有促进和抑制分层的因子。
