{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T04:55:40Z","timestamp":1789707340861,"version":"4.0.1"},"reference-count":64,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2020,6,9]],"date-time":"2020-06-09T00:00:00Z","timestamp":1591660800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Health Research Endowment Fund (HREF), Singapore National Eye Centre","award":["R1488\/71\/2017"],"award-info":[{"award-number":["R1488\/71\/2017"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet\u2019s membrane\/corneal endothelium (DM\/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM\/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM\/CE incubated in M4-F99 medium (p &lt; 0.05). M5-Endo medium was hence selected as the DM\/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 \u00b1 147.8 \u03bcm on day 1, gradually thinned, and remained clear with a CCT of 385.5 \u00b1 38.6 \u03bcm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (p = 0.40, p = 0.17, and p = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (p &lt; 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation.<\/jats:p>","DOI":"10.3390\/cells9061428","type":"journal-article","created":{"date-parts":[[2020,6,9]],"date-time":"2020-06-09T06:34:16Z","timestamp":1591684456000},"page":"1428","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5475-8712","authenticated-orcid":false,"given":"Hon Shing","family":"Ong","sequence":"first","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Eye-Academic Clinical Program (ACP), Duke-National University of Singapore (NUS), Graduate Medical School, Singapore 169857, Singapore"},{"name":"Corneal and External Diseases Department, Singapore National Eye Centre, Singapore 168751, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gary","family":"Peh","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Eye-Academic Clinical Program (ACP), Duke-National University of Singapore (NUS), Graduate Medical School, Singapore 169857, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dawn Jin Hui","family":"Neo","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heng-Pei","family":"Ang","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Khadijah","family":"Adnan","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chan Lwin","family":"Nyein","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4836-6330","authenticated-orcid":false,"given":"Fernando","family":"Morales-Wong","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maninder","family":"Bhogal","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Cornea Unit, Guy\u2019s &amp; St Thomas\u2019 Hospital, London SE1 7EH, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3044-5057","authenticated-orcid":false,"given":"Viridiana","family":"Kocaba","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Netherlands Institute for Innovative Ocular Surgery, Melles Cornea Clinic, Amnitrans EyeBank Rotterdam, 3071 AA Rotterdam, The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6362-4898","authenticated-orcid":false,"given":"Jodhbir S.","family":"Mehta","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Eye-Academic Clinical Program (ACP), Duke-National University of Singapore (NUS), Graduate Medical School, Singapore 169857, Singapore"},{"name":"Corneal and External Diseases Department, Singapore National Eye Centre, Singapore 168751, Singapore"},{"name":"School of Material Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,6,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1136\/bjophthalmol-2011-300539","article-title":"Global estimates of visual impairment: 2010","volume":"96","author":"Pascolini","year":"2012","journal-title":"Br. 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