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Noelia J Kunzevitzky, Alena Bartakova, Katarzyna Wilczek, Jonathan Sheu, Jonathan Van Dyke, Gerhard Bauer, Jeffrey L Goldberg; Culture, functional characterization and stability of human corneal endothelial cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1165. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelial dysfunction is a leading cause of blindness. Surgical treatment is limited by scarcity of donor tissue and cost. Cell therapy could overcome these limitations. Several methods have been developed for human corneal endotheial cell (HCEC) culture; however, with increased number of passages, HCECs undergo endothelial-to-mesenchymal transition (EnMT) and lose function. Here we present a method that stabilizes HCEC morphology and renders functional cells after several passage which can be shipped for use in cell therapy.
HCECs isolated from cadaveric donor corneas were expanded in vitro following the Joyce method (2004). At confluence, HCEC morphology was assessed by microscopy, identity by flow cytometry and RT-PCR, and function by the trans-epithelial electrical resistance (TEER) assay. HCECs were next subcultured using either proliferative or stabilization growth media, or a serial combination of them. HCEC morphology, identity and function were compared daily for 7 days. A fraction of the cells were re-suspended in BSS+ and shipped at 40C. Cell viability, morphology, identity and function were assessed for 7 days upon arrival.
HCECs underwent EnMT after 3 to 4 passages. Switching to stabilization medium before each passage helped HCECs maintain their morphology, identity and function over time.<br /> When HCECs cultured in stabilization medium were exposed to proliferative medium, HCECs became fibroblastic, and exhibited a decreased expression of canonical markers and function. Finally, HCECs transported in 1ml syringes with BSS+ were viable (>70%) for up to 96 hours when shipped and stored at 4°C, expressed canonical HCEC markers and maintained their barrier function.
In later passages, HCECs undergo endothelial-to-mesenchymal transition. Modifications to the traditional HCEC culture protocol to include a stabilization step prior to each passage helps maintain the HCECs’ canonical morphology, identity and function. Thus, these novel HCEC culture and shipping methods may be an important milestone towards successful HCEC manufacturing for clinical use.
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