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Karen Alvarez-Delfin, Noelia Kunzevitzky, Alejandra Weisman, Richard Merkhofer, Jeffrey Goldberg; A cell therapy approach to address corneal endothelial dysfunction. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1648.
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© ARVO (1962-2015); The Authors (2016-present)
The endothelium is the innermost layer of the cornea and is responsible for maintaining transparency. Human corneal endothelial cell (HCEC) density gradually decreases with age, and their proper function is affected by genetic diseases, such as Fuchs’ dystrophy, or surgical trauma, ultimately leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a global shortage of tissue donors. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a first step toward cell therapy.
HCECs were isolated from cadaveric donor corneas and cultured following the method described by Zhu and Joyce (2004). In order to improve cell morphology and proliferation rate, different media additives and culture dish coatings were assayed. Cellular identity was assessed by morphology, RT-PCR, and immunohistochemistry. Viability was measured by Trypan blue staining after cells were stored at room temperature, 4oC or cryopreserved in liquid N2.
Cultured HCECs often demonstrated characteristic hexagonal-like morphology for 2-3 passages, reaching passage 8 in one culture. Time to reach confluence was highly influenced by donor age, with youngest donors exhibiting higher proliferative rates. Donor disease also affected culture quality. Overnight storage of HCECs at 4oC dramatically reduced viability, while cryopreservation maintained higher viability. Immunohistochemistry showed that cultured HCECs were positive for zonula occludens-1(ZO-1) and Na+K+ ATPase, common markers for HCEC. Fibroblastic-like morphologic conversion positive for the marker α-smooth muscle actin (α-SMA) was a common feature at late passages, appearing earlier in poor quality cultures.
The in vitro expansion of HCEC from donor corneas yields a number of suitable cells that could help treat patients otherwise in need of cornea transplantation. Ongoing experiments are addressing the ability of these cells to integrate into a host cornea and restore endothelial function.
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