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Gary S. Peh, Kah Peng Toh, Fei Yi Wu, Donald T. Tan, Jodhbir S. Mehta; Comparison of Five Culture Media for the Isolation and Propagation of Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6595.
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This study evaluates five culture conditions for the isolation and propagation of human corneal endothelial cells (hCECs).
The Descemet’s membrane, together with the corneal endothelium, was carefully stripped off from paired research-grade human cadaver corneas deemed unsuitable for transplantation. Following an enzymatic dissociation, isolated hCECs were seeded equally into five culture dishes coated with FNC Mix®. Four of the culture media were previously published, coded here as: M1-DMEM (10% serum); M2-OptiMEM-I (8% serum); M3-DMEM/F12 (5% serum), and M4-Ham’s F12/M199 (5% serum). The fifth, coded as M5, is a supplemented basal medium (5% serum) that has not been described for the culture of hCECs. Corneal endothelial cells established in the five conditions were subsequently expanded for at least two passages, and assessed morphologically. The corneal endothelial cultures were also analyzed for their propensity to proliferate in the five conditions and analyzed for their expression of characteristic markers: ZO-1, and Na+K+ATPase.
Corneal endothelial cells established in the five conditions showed vast differences in their proliferation profiles with striking morphological differences. In the first two passages, hCECs expanded in M2 and M4 were generally small and extremely proliferative, whilst those cultured in M1, M3 and M5 were distinctively larger but retained a more polygonal shape. However, M1, and in most cultures of M3 was not able to support the continual propagation of hCECs beyond the second passage. By the third passage and beyond, corneal endothelial cells propagated in M2 and M4 became heterogeneous, whereby majority of the cells took up a spindly fibroblast-like morphology. Although not as proliferative compared to M2 or M4, corneal endothelial cells cultured in M5 maintained a homogeneous polygonal cellular morphology. We also showed that cultured hCECs express markers characteristics of the human corneal endothelium: ZO-1 and Na+K+ATPase.
The proliferative capacity and morphological appearances of hCECs are vastly affected by the various culture conditions. We propose the selective use of culture media for the propagation (M2 or M4) and maintenance (M5) of hCECs to preserve their morphological characteristic with multiple passages.
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