{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T01:27:45Z","timestamp":1760059665761,"version":"build-2065373602"},"reference-count":45,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2025,6,27]],"date-time":"2025-06-27T00:00:00Z","timestamp":1750982400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001468","name":"Singapore Eye Research Institute","doi-asserted-by":"publisher","award":["TLF 0322-4","MOH-001232-00"],"award-info":[{"award-number":["TLF 0322-4","MOH-001232-00"]}],"id":[{"id":"10.13039\/501100001468","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001349","name":"National Medical Research Council","doi-asserted-by":"publisher","award":["TLF 0322-4","MOH-001232-00"],"award-info":[{"award-number":["TLF 0322-4","MOH-001232-00"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Birmingham Health Partners","award":["TLF 0322-4","MOH-001232-00"],"award-info":[{"award-number":["TLF 0322-4","MOH-001232-00"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Human corneal endothelial cell therapy has recently emerged as a novel solution to treat corneal endothelial diseases. We previously demonstrated the potential of utilizing non-cultured primary corneal endothelial cells (CEnCs) isolated from donor corneas with low endothelial cell density for simple non-cultured endothelial cell injection (SNEC-I) therapy. This study aimed to develop a robust and semi-automated approach for cell counting, characterize the extent of cellular manipulation, and evaluate the translational workflow. To address this, we evaluated manual and automated cell counting approaches and characterized the extent of manipulation of CEnCs through the analysis of cell cycle status, gene expressions, and transcriptomic profiles with single-cell RNA-sequencing. The translational feasibility and functionality of SNEC-I therapy were examined using an established rabbit model of bullous keratopathy. Manual hemocytometry and automated cell-counters exhibited comparable accuracy and reproducibility. Analysis of cell cycle status, cell cycle genes (n = 11), and transcriptomic profiles revealed close resemblance between the native corneal endothelium and its donor-matched SNEC-I-harvested cells. Successful resolution of bullous keratoplasty in the pre-clinical model supports the feasibility, efficacy, and safety of SNEC-I therapy. In conclusion, SNEC-I therapy serves as an attractive corneal endothelial therapeutic approach (from a regulatory standpoint) in view of the minimal extent of cellular manipulation.<\/jats:p>","DOI":"10.3390\/cells14130986","type":"journal-article","created":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T07:55:43Z","timestamp":1751442943000},"page":"986","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cell Counting and Cell Cycle Analysis of Simple Non-Cultured Endothelial Cell Injection (SNEC-I) Therapy: Characterization for Clinical Translation"],"prefix":"10.3390","volume":"14","author":[{"given":"Darren S. J.","family":"Ting","sequence":"first","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Ophthalmology & Visual Sciences Academic Clinical Programme (EYE-ACP), Duke-NUS Graduate Medical School, Singapore 169857, Singapore"},{"name":"Academic Unit of Ophthalmology, Department of Inflammation and Ageing, College of Medicine and Health, University of Birmingham, Birmingham B15 2TT, UK"},{"name":"Birmingham and Midland Eye Centre, Sandwell and West Birmingham NHS Trust, Birmingham B18 7QH, UK"},{"name":"Academic Ophthalmology, School of Medicine, University of Nottingham, Nottingham NG7 2RD, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gary S. L.","family":"Peh","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Ophthalmology & Visual Sciences Academic Clinical Programme (EYE-ACP), Duke-NUS Graduate Medical School, Singapore 169857, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0665-4024","authenticated-orcid":false,"given":"Dawn J. H.","family":"Neo","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiao Yu","family":"Ng","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Belinda Y. L.","family":"Tan","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8092-9455","authenticated-orcid":false,"given":"Raymond C. B.","family":"Wong","sequence":"additional","affiliation":[{"name":"Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC 3002, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5475-8712","authenticated-orcid":false,"given":"Hon Shing","family":"Ong","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Ophthalmology & Visual Sciences Academic Clinical Programme (EYE-ACP), Duke-NUS Graduate Medical School, Singapore 169857, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jodhbir S.","family":"Mehta","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore"},{"name":"Ophthalmology & Visual Sciences Academic Clinical Programme (EYE-ACP), Duke-NUS Graduate Medical School, Singapore 169857, Singapore"},{"name":"Corneal & External Eye Disease, Cataract & Comprehensive Ophthalmology, Refractive Surgery, Singapore National Eye Centre, Singapore 168751, Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1038\/eye.2013.15","article-title":"Corneal endothelium: Developmental strategies for regeneration","volume":"27","author":"Zavala","year":"2013","journal-title":"Eye"},{"key":"ref_2","first-page":"779","article-title":"Central corneal endothelial cell changes over a ten-year period","volume":"38","author":"Bourne","year":"1997","journal-title":"Investig. 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