{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,23]],"date-time":"2026-07-23T02:50:36Z","timestamp":1784775036028,"version":"3.55.0"},"reference-count":68,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2023,2,3]],"date-time":"2023-02-03T00:00:00Z","timestamp":1675382400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cell injection therapy is emerging as an alternative to treat corneal endothelial dysfunction (CED) and to avoid corneal scarring due to bullous keratopathy. However, establishing a standardized culture procedure that provides appropriate cell yield while retaining functional features remains a challenge. Here, we describe a detailed framework obtained from in vitro culture of human corneal endothelial cells (HCECs) and comparative in vivo experimental models for CED treatment with a new cell tracking approach. Two digestion methods were compared regarding HCEC morphology and adhesion. The effect of Y-27632 (ROCKi) supplementation on final cell yield was also assessed. Cell adhesion efficacy with two cell delivery systems (superparamagnetic embedding and cell suspension) was evaluated in an ex vivo human cornea model and in an in vivo rabbit CED model. The injection of supplemented culture medium or balanced salt solution (BSS) was used for the positive and negative controls, respectively. HCEC isolation with collagenase resulted in better morphology and adhesion of cultured HCEC when compared to EDTA. Y-27632 supplementation resulted in a 2.6-fold increase in final cell yield compared to the control. Ex vivo and in vivo adhesion with both cell delivery systems was confirmed by cell tracker fluorescence detection. Corneal edema and opacity improved in both animal groups treated with cultured HCEC. The corneas in the control groups remained opaque. Both HCEC delivery systems seemed comparable as treatments for CED and for the prevention of corneal scarring.<\/jats:p>","DOI":"10.3390\/ijms24032982","type":"journal-article","created":{"date-parts":[[2023,2,3]],"date-time":"2023-02-03T03:04:23Z","timestamp":1675393463000},"page":"2982","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7298-4315","authenticated-orcid":false,"given":"Francisco","family":"Bandeira","sequence":"first","affiliation":[{"name":"Department of Ophthalmology, Federal University of S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"},{"name":"Medicine School, Barcelona University, 08007 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gustavo Teixeira","family":"Grottone","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Federal University of S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joyce Luciana","family":"Covre","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Federal University of S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Priscila Cardoso","family":"Cristovam","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Federal University of S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Renata Ruoco","family":"Loureiro","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Federal University of S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8879-3997","authenticated-orcid":false,"given":"Francisco Irochima","family":"Pinheiro","sequence":"additional","affiliation":[{"name":"Biotechnology Post-Graduate Program, Potiguar University, Natal 59082-902, Brazil"},{"name":"Department of Surgery, Federal University of Rio Grande do Norte, Natal 59078-970, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1812-9323","authenticated-orcid":false,"given":"Ricardo Pedro","family":"Casaroli-Marano","sequence":"additional","affiliation":[{"name":"Medicine School, Barcelona University, 08007 Barcelona, Spain"},{"name":"Barcelona Tissue Bank, 08005 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Waleska","family":"Donato","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Federal University of S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jos\u00e9 \u00c1lvaro Pereira","family":"Gomes","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Federal University of S\u00e3o Paulo, S\u00e3o Paulo 04023-062, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"108068","DOI":"10.1016\/j.exer.2020.108068","article-title":"Endothelial pump and barrier function","volume":"198","author":"Klyce","year":"2020","journal-title":"Exp. 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