Comparative Study of Xenobiotic-Free Media for the Cultivation of Human Limbal Epithelial Stem/Progenitor Cells.
Comparative Study of Xenobiotic-Free Media for the Cultivation of Human Limbal Epithelial Stem/Progenitor Cells.
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DOI：
10.1089/ten.tec.2016.0388
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发表时间：
2017-04
期刊：
影响因子：
--
通讯作者：
Deng SX
中科院分区：
文献类型：
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作者：
González S;Chen L;Deng SX
在含有动物成分的情况下培养人角膜缘上皮干/祖细胞(LSCs)在临床应用中存在跨物种污染的风险。我们定量比较了不同的无外源培养基人LSCs的培养效果。取2 × 2 mm角膜缘组织块，接种于裸露的人羊膜上，分别加入含0%、1%、5%、10%人血清的CNT-PRIME(CNT-PR)、单独或与标准激素上皮(SHEM，对照)1：1稀释液混合的胚胎干细胞(ESCM)和去除霍乱毒素和二甲基亚砜(DMSO)的改良SHEM(改良SHEM)，加入异丙肾上腺素，降低表皮生长因子浓度。定量评价LSC的表型：细胞形态、细胞生长、细胞大小、突起大小、未分化LSC标志物细胞角蛋白(K)14和p63α高表达细胞(p63αBright)、成熟角质形成细胞标志物K12、上皮标志物泛角蛋白(PANK)和基质细胞标志物波形蛋白(Vim)的表达。与标准SHEM对照组相比，CNT-PR基础培养液中，无论HS添加量多少，细胞生长和干细胞比例均降低(p <； 0.05)；ESCM导致Pank−/Vim+基质细胞比例增加(p <； 0.05)，p63α亮细胞比例降低(p <； 0.05)；mSHEM支持相似的细胞生长(p >； 0.05)，增加小细胞数量(Diameter≤12 μm；p <； 0.05)，并提供类似比例的p63α明亮细胞(p >； 0.05)。在所测试的所有条件中，mSHEM在支持LSC的表型和生长方面是最有效和一致的。
The culture of human limbal epithelial stem/progenitor cells (LSCs) in the presence of animal components poses the risk of cross-species contamination in clinical applications. We quantitatively compared different xenobiotic-free culture media for the cultivation of human LSCs. LSCs were cultured from 2 × 2 mm limbal tissue explants on denuded human amniotic membrane with different xenobiotic-free culture media: CnT-Prime (CnT-PR) supplemented with 0%, 1%, 5%, and 10% human serum (HS), embryonic stem cell medium (ESCM) alone or in combination with the standard supplemented hormonal epithelium medium (SHEM, control) at a 1:1 dilution ratio, and modified SHEM (mSHEM), in which cholera toxin and dimethyl sulfoxide (DMSO) were removed, isoproterenol was added, and the epidermal growth factor concentration was reduced. Several parameters were quantified to assess the LSC phenotype: cell morphology, cell growth, cell size, outgrowth size, and expression of the undifferentiated LSC markers cytokeratin (K) 14, and p63α high-expressing (p63αbright) cells, a mature keratinocyte marker K12, epithelial marker pancytokeratin (PanK), and stromal cell marker vimentin (Vim). Compared with the standard SHEM control, CnT-PR base medium was associated with a lower cell growth and reduction in the proportion of stem cells generated regardless of the amount of HS supplemented (p < 0.05); ESCM resulted in an increased proportion of PanK−/Vim+ stromal cells (p < 0.05) and a decreased proportion of p63αbright cells (p < 0.05); mSHEM supported a similar cell growth (p > 0.05), increased the number of small cells (diameter ≤12 μm; p < 0.05), and provided a similar proportion of p63αbright cells (p > 0.05). Among all the conditions tested, mSHEM was the most efficient and consistent in supporting the LSC phenotype and growth.