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首页> 外文期刊>Journal of dermatological science >Isolation of small-sized human epidermal progenitor/stem cells by Gravity Assisted Cell Sorting (GACS)
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Isolation of small-sized human epidermal progenitor/stem cells by Gravity Assisted Cell Sorting (GACS)

机译：通过重力辅助细胞分选（GACS）分离小型人表皮祖细胞/干细胞

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Background: Small diameter characterizes epidermal progenitor/stem cells. We have developed Gravity Assisted Cell Sorting (GACS) to simply enrich small-sized epidermal progenitor/stem cells. Objective: The cells sorted by GACS were characterized by fluorescence-activated cell sorting analysis, and cultured for up to 7 weeks. The cultured cells were then used for reconstruction of skin equivalent. Methods: GACS was performed on primary cultures (primary cell) and passage 6-7 cultures (cultured cell) of keratinocytes. A keratinocyte suspension was sized into two groups: cells trapped by a 20 μm filter (trapped cells), and cells flowing through both a 20 and 11 μm filter (non-trapped cells). Results: In the primary cell groups, viability of the trapped cells was 62.5 ± 7.2% compared to 77.0 ± 3.7% for the non-trapped cells. In the cultured cell groups, viability of the trapped cells was 64.3 ± 14.9%, compared to the non-trapped cells (93.1 ± 2.0%). Flow cytometric analysis showed better discrimination by cell size between trapped and non-trapped cells in culture than in the primary cell suspension. Non-trapped cells contained a larger number of cells with high levels of α6 integrin and low levels of CD71 (α6 integrinbriCD71dim), indicating an enriched progenitor/stem cell population. The difference in these markers between the non-trapped and trapped cells was seen in both the primary and cultured cell groups although this difference was more distinct in cultured cells. Culture of both groups showed that cultures originating from the trapped cells senesced after approximately 15 days while the non-trapped keratinocytes grew for up to 40 days. Manufacture of an epidermis/dermal device (artificial skin) showed that non-trapped cells formed a significantly thicker epithelial layer than the trapped cells, demonstrating the enhanced regenerative capability of the smaller diameter, α6 integrinbriCD71dim cells separated by GACS. Conclusion: These results indicate that GACS is simple and useful technique to enrich for epidermal progenitor/stem cell populations, and is more efficient when used on cells in culture.
机译：背景：小直径表征表皮祖细胞/干细胞。我们已经开发了重力辅助细胞分选（GACS），以简单地富集小型表皮祖细胞/干细胞。目的：通过荧光激活细胞分选分析表征通过GACS分选的细胞，并培养长达7周。然后将培养的细胞用于重建皮肤等效物。方法：对角质形成细胞的原代培养物（原代细胞）和传代6-7培养物（培养细胞）进行GACS。将角质形成细胞悬浮液分为两组：被20μm过滤器捕获的细胞（捕获的细胞）和流过20和11μm过滤器的细胞（未捕获的细胞）。结果：在原代细胞组中，被捕获细胞的活力为62.5±7.2％，而未捕获细胞的活力为77.0±3.7％。在培养的细胞组中，与未捕获的细胞（93.1±2.0％）相比，被捕获的细胞的生存力为64.3±14.9％。流式细胞仪分析显示，与原代细胞悬浮液相比，培养物中捕获和未捕获细胞之间的细胞大小更好的区分。未捕获的细胞包含大量具有高水平α6整联蛋白和低水平CD71的细胞（α6整联蛋白briCD71dim），表明祖细胞/干细胞富集。在原代和培养细胞组中都观察到了非标记细胞和标记细胞之间这些标记的差异，尽管这种差异在培养细胞中更为明显。两组的培养结果均显示，源自捕获细胞的培养物在约15天后会衰老，而未捕获的角质形成细胞最多生长40天。表皮/真皮装置（人造皮肤）的制造显示，未捕获的细胞比捕获的细胞形成了明显更厚的上皮层，这证明了通过GACS分离的较小直径的α6整合蛋白CD71dim细胞的再生能力增强。结论：这些结果表明，GACS是富集表皮祖细胞/干细胞群体的简单而有用的技术，并且在用于培养的细胞时更有效。

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