Nakajima Ryota, Takeda Shizu
Central Research Laboratory, Hitachi Ltd., 2520 Hatoyama, Saitama 350-0395, Japan.
J Dermatol Sci. 2014 Dec;76(3):246-54. doi: 10.1016/j.jdermsci.2014.09.006. Epub 2014 Oct 2.
Epidermal cell sheets have been utilized for regeneration of skin when skin defects occur and prevention of esophageal stricture after endoscopic submucosal dissection. To reduce the cost of cultivation, a novel culture method to shorten a culture process needs to be developed.
To shorten a culture process of epidermal cell sheets, we developed a novel culture method to accelerate the fabrication of epidermal cell sheets using γ-secretase inhibitor.
Normal human epidermal keratinocytes (NHEKs) were cultured using γ-secretase inhibitor, DAPT, during expansion of the cells to confluence and culture without DAPT during stratification. The cell growth, quantitative gene expression of stem/progenitor or differentiation markers, and protein expression of these markers were analyzed to verify the effectiveness of the novel method.
The proliferation of NHEKs on cell-culture inserts was promoted using DAPT. However, NHEKs were not stratified completely in the presence of DAPT. In contrast, NHEKs cultured using DAPT were stratified and differentiated by eliminating the inhibitor after the cells reached confluence. Real-time PCR analyses showed that the gene expressions of putative epithelial stem/progenitor cell markers and epidermis differentiation markers in the cell sheets fabricated using this novel method were significantly higher than those in the cell sheets fabricated without DAPT. Histological and immunofluorescence analyses revealed that it was possible to fabricate well-differentiated epidermal cell sheets efficiently by the novel culture method. The culture period was shortened to 67% of the time required for the control group. In feeder-free conditions, stratified epidermal cell sheets were also fabricated using DAPT.
The novel culture method using γ-secretase inhibitor, DAPT, was found to be effective for fabricating epidermal cell sheets.
当出现皮肤缺损时，表皮细胞片已被用于皮肤再生，以及在内镜黏膜下剥离术后预防食管狭窄。为降低培养成本，需要开发一种新的培养方法来缩短培养过程。
为缩短表皮细胞片的培养过程，我们开发了一种新的培养方法，使用γ-分泌酶抑制剂来加速表皮细胞片的制备。
在细胞扩增至汇合期时，使用γ-分泌酶抑制剂DAPT培养正常人表皮角质形成细胞（NHEKs），在分层培养期间不使用DAPT。分析细胞生长、干细胞/祖细胞或分化标志物的定量基因表达以及这些标志物的蛋白质表达，以验证该新方法的有效性。
使用DAPT可促进NHEKs在细胞培养插入物上的增殖。然而，在存在DAPT的情况下，NHEKs未完全分层。相反，使用DAPT培养的NHEKs在细胞达到汇合后通过去除抑制剂进行分层和分化。实时PCR分析表明，使用这种新方法制备的细胞片中假定的上皮干细胞/祖细胞标志物和表皮分化标志物的基因表达显著高于未使用DAPT制备的细胞片。组织学和免疫荧光分析表明，通过这种新的培养方法可以有效地制备分化良好的表皮细胞片。培养时间缩短至对照组所需时间的67%。在无饲养层条件下，也使用DAPT制备了分层的表皮细胞片。
发现使用γ-分泌酶抑制剂DAPT的新培养方法对制备表皮细胞片有效。
