Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers
Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers
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DOI：
10.1007/s00417-008-0904-6
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发表时间：
2008-11-01
影响因子：
2.7
通讯作者：
Werner, Carsten
中科院分区：
文献类型：
--
作者：
Goetze, Thomas;Valtink, Monika;Werner, Carsten
最近，有可能表明人类角膜内皮细胞（HCEC）可以在热响应性聚合物基质上培养，并且可以作为完整的细胞片收获而不失去活力。我们试图在无血清条件下研究HCEC薄片在这种细胞培养载体上的培养，作为开发角膜内皮细胞移植方法的下一个重要步骤。方法将一个永生化异种HCEC群体和2个永生化克隆培养HCEC细胞系（HCEC- b4g12和HCEC- h9c1）在热反应性底物上进行补血清和无血清培养。细胞片通过相对比显微镜和ZO-1、Na+、K+- atp酶和vculin的免疫荧光染色进行表征。结果在血清补充条件下，所有测试的HCEC群体均能在热反应性底物上粘附、扩散和增殖。在无血清条件下，除了HCEC-B4G12细胞外，必须在聚合物底物上预先涂覆ECM蛋白，以促进细胞的附着和扩散。异质HCEC群体形成封闭的单层，将ZO-1适当定位到细胞外侧边界，在无血清培养条件下具有中等的病毒素水平，在血清补充培养条件下具有较高的病毒素水平。HCEC-B4G12细胞形成封闭的单层，ZO-1和Na+，K+- atp酶在细胞外侧边界有适当的定位，并且无论培养条件如何，都具有较高的血毒蛋白水平。相比之下，HCEC-H9C1细胞在补充血清条件下的球蛋白水平最低，而在无血清培养条件下的球蛋白水平较高。在两种培养条件下，细胞质中均检测到ZO-1。这些松散贴壁的细胞只能在血清补充条件下形成封闭的单层。结论无血清生产HCEC片是可行的。具有极强粘附性的克隆HCEC B4G12比其他两个测试的HCEC群体产生更高的病毒素水平，并且无论培养条件如何，都表现出对热反应性聚合物培养基质的强粘附性。该细胞系与体内终末分化的HCEC非常相似，特别适合于HCEC细胞片工程的进一步研究。
Background Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants.Methods An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na+,K+-ATPase, and vinculin.Results All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na+,K+-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions.Conclusions Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.