Formation of keratinocyte multilayers on filters under airlifted or submerged culture conditions in medium containing calcium, ascorbic acid, and keratinocyte growth factor
Formation of keratinocyte multilayers on filters under airlifted or submerged culture conditions in medium containing calcium, ascorbic acid, and keratinocyte growth factor
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DOI：
10.1007/s00418-016-1472-1
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发表时间：
2016-11-01
影响因子：
2.3
通讯作者：
Inai, Tetsuichiro
中科院分区：
文献类型：
--
作者：
Seo, Akira;Kitagawa, Norio;Inai, Tetsuichiro
三维细胞培养是研究角质形成细胞分层和分化的有效体外技术。然而，培养条件，包括培养基、补充剂和支架（例如，具有或不具有成纤维细胞的胶原凝胶）可以有相当大的变化。在这里，我们评估了钙，l-抗坏血酸磷酸镁盐n-水合物（APM），和角质形成细胞生长因子（KGF）在化学成分确定的培养基，EpiLife，在3D培养的原代人表皮角质形成细胞直接接种在聚碳酸酯过滤器插入在空气提升或淹没条件下的作用。检查了含有这三种补充剂的各种组合的八种培养基。根据角蛋白和外皮蛋白的定位，钙对于角质形成细胞的分层和分化是必需的。然而，空运后3周，角蛋白和整合素β 4的定位模式被部分破坏，Ki 67阳性基底细胞几乎消失。添加KGF而不是APM阻止了这些变化。进一步添加APM显著改善了组织结构，包括基底细胞形态和透明角质颗粒的出现以及上部基底上细胞中的局部外皮蛋白，即使在1周后也是如此。虽然浸没式培养也形成了皮质上皮样多层膜，外皮蛋白定位于皮质层，在那里经常发现的核。基于这些结果，它是最有效的培养角质形成细胞在空气-液体界面的EpiLife培养基中补充钙，APM，和KGF形成良好的组织和orthokeratinized多层膜的皮肤模拟物。
Three-dimensional (3D) cell culture is a powerful in vitro technique to study the stratification and differentiation of keratinocytes. However, culture conditions, including culture media, supplements, and scaffolds (e.g., collagen gels with or without fibroblasts), can vary considerably. Here, we evaluated the roles of calcium, l-ascorbic acid phosphate magnesium salt n-hydrate (APM), and keratinocyte growth factor (KGF) in a chemically defined medium, EpiLife, in 3D cultures of primary human epidermal keratinocytes directly plated on polycarbonate filter inserts under airlifted or submerged conditions. Eight culture media containing various combinations of these three supplements were examined. Calcium was necessary for the stratification and differentiation of keratinocytes based on the localization of keratins and involucrin. However, the localization patterns of keratins and integrin beta 4 were partially disrupted and Ki67-positive basal cells almost disappeared 3 weeks after airlift. The addition of KGF, but not APM, prevented these changes. Further addition of APM markedly improved the tissue architecture, including basal cell morphology and the appearance of keratohyalin granules and localized involucrin in the upper suprabasal cells, even after 1 week. Although the submerged culture also formed cornified epithelium-like multilayers, involucrin was localized in the cornified layer, where nuclei were often found. Based on these results, it is most effective to culture keratinocytes at the air-liquid interface in EpiLife medium supplemented with calcium, APM, and KGF to form well-organized and orthokeratinized multilayers as skin analogues.