A serum-free clonal growth assay for limbal, peripheral, and central corneal epithelium.
A serum-free clonal growth assay for limbal, peripheral, and central corneal epithelium.
复制标题
DOI：
--
复制
发表时间：
1991-06
影响因子：
4.4
通讯作者：
F. E. Kruse;Scheffer C. G. Tseng
中科院分区：
文献类型：
--
作者：
F. E. Kruse;Scheffer C. G. Tseng
角膜上皮的干细胞和瞬时扩增细胞被认为分别位于角膜缘和角膜基底上皮中。为了研究这些祖细胞增殖的差异调节，开发了一种用于中央（CC）和外周（PC）角膜和角膜缘（L）上皮细胞的定义的无血清克隆生长测定法。在Dispase II中孵育（1.2 U/ml; PC和CC为1小时，L为3小时）并随后进行短暂的胰蛋白酶-乙二胺四乙酸消化后，将18或180个单细胞/cm 2接种到补充有胰岛素、转铁蛋白、硒、氢化可的松、表皮生长因子（EGF）、磷酸乙醇胺、乙醇胺和钙的MCDB 151培养基中。在培养的第一周，细胞逐渐形成越来越多的集落，第6天L的平均集落形成效率为4.2 +/-2.4%，显著低于PC的11.4 +/- 5.9%和CC的12.8 +/- 7.6%（P <0.003）。L、PC和CC中的集落形态相同，其中小的细长细胞在中心更具粘性，但在外围更具迁移性。用单克隆抗体AE-5进行的免疫荧光染色没有差异，表明所有集落的角膜来源。培养物可以在第14天传代，并生长超过3周，伴随着越来越多的脱落。添加微量金属的混合物以产生MCDB 153并没有增强生长;增加硒浓度是抑制性的。从补充剂中消除EGF消除了大部分克隆生长。L增殖率较低可能是由于缺乏血清和基质促分裂原。该培养系统似乎优先支持瞬时扩增细胞，并允许研究分离的角膜干细胞向瞬时扩增细胞的分化或瞬时扩增细胞通过各种因素的增殖和分化，而不需要不确定的血清组分的相互作用或来自其他细胞的旁分泌影响。
The stem cells and transient amplifying cells of the corneal epithelium are thought to be localized in the limbal and corneal basal epithelium, respectively. To study the differential regulation of proliferation of these progenitor cells, a defined, serum-free, clonal growth assay was developed for central (CC) and peripheral (PC) corneal and limbal (L) epithelial cells. After incubation in Dispase II (1.2 U/ml; 1 hr for PC and CC and 3 hr for L) and subsequent brief trypsin-ethylenediaminetetraacetic acid digestion, 18 or 180 single cells/cm2 were seeded in MCDB 151 medium supplemented with insulin, transferrin, selenium, hydrocortisone, epidermal growth factor (EGF), phosphoethanolamine, ethanolamine, and calcium. During the first week of culture, the cells gradually developed an increasing number of colonies, and the mean colony-forming efficiency on day 6 for L was 4.2 +/- 2.4%, significantly lower than 11.4 +/- 5.9% for PC and 12.8 +/- 7.6% for CC (P less than 0.003). Colony morphology was identical in L, PC, and CC with small, elongated cells more cohesive in the center but more migratory in the periphery. There were no differences in immunofluorescent staining with monoclonal antibody AE-5, indicating the corneal derivation of all colonies. Cultures could be passaged on day 14 and grown for more than 3 weeks with increasing desquamation. Addition of a mixture of trace metals to yield MCDB 153 did not enhance growth; increased selenium concentrations were inhibitory. Elimination of EGF from the supplement abolished most of the clonal growth. The lower rate of L proliferation might be explained by the absence of serum and stromal mitogens. This culture system seems preferentially to support transient amplifying cells and allows investigation of the differentiation of isolated corneal stem cells to transient amplifying cells or the proliferation and differentiation of transient amplifying cells by various factors without the interaction of undefined serum components or paracrine influences from other cells.