Peh Gary S L, Toh Kah-Peng, Ang Heng-Pei, Seah Xin-Yi, George Benjamin L, Mehta Jodhbir S
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, 11 Third Hospital Ave, #06-00, Singapore 168751, Singapore.
BMC Res Notes. 2013 May 3;6:176. doi: 10.1186/1756-0500-6-176.
Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.
Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.
Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.
供体角膜的全球短缺极大地限制了每年进行的角膜移植数量。原代人角膜内皮细胞的体外扩增有限，因此利用培养的角膜内皮细胞开发组织工程构建体具有相当大的临床意义。本研究的目的是研究从配对供体角膜分离的人角膜内皮细胞的密度依赖性生长，并阐明其在体外扩展培养时保持独特细胞形态的最佳接种密度。
已建立的原代人角膜内皮细胞在用于本研究之前传代至第二代（P2）。将汇合的P2细胞解离并以四种接种密度接种：每平方厘米2500个细胞（“低”）；每平方厘米5000个细胞（“中”）；每平方厘米10000个细胞（“高”）；以及每平方厘米20000个细胞（“高（×2）”），随后分析它们的增殖倾向。当每种培养物汇合时，还对它们进行形态计量分析，比较细胞大小、变异系数以及细胞圆形度。在两个较低密度下，增殖率高于以较高密度接种的细胞，尽管无统计学意义。然而，以较低密度接种的角膜内皮细胞尺寸明显更大，形状异质且圆形度较低（成纤维细胞样），并且在培养一个月后仍呈肥大状态。相比之下，以较高密度接种的细胞在汇合时明显均匀、紧密且呈圆形。潜在地，在每平方厘米10000个细胞的最佳接种密度下，第三代时有可能获得1000万至2500万个细胞。更重要的是，这些扩增的人角膜内皮细胞保留了其独特的细胞形态。
我们的结果证明了原代人角膜内皮细胞培养中的密度依赖性。接种密度次优会导致细胞饱和密度降低以及增殖潜能丧失。因此，我们建议原代人角膜内皮细胞常规传代的接种密度不低于每平方厘米10000个细胞。
