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A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach

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 chal...

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Autores principales: Bandeira, Francisco, Grottone, Gustavo Teixeira, Covre, Joyce Luciana, Cristovam, Priscila Cardoso, Loureiro, Renata Ruoco, Pinheiro, Francisco Irochima, Casaroli-Marano, Ricardo Pedro, Donato, Waleska, Gomes, José Álvaro Pereira
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917640/
https://www.ncbi.nlm.nih.gov/pubmed/36769303
http://dx.doi.org/10.3390/ijms24032982
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author Bandeira, Francisco
Grottone, Gustavo Teixeira
Covre, Joyce Luciana
Cristovam, Priscila Cardoso
Loureiro, Renata Ruoco
Pinheiro, Francisco Irochima
Casaroli-Marano, Ricardo Pedro
Donato, Waleska
Gomes, José Álvaro Pereira
author_facet Bandeira, Francisco
Grottone, Gustavo Teixeira
Covre, Joyce Luciana
Cristovam, Priscila Cardoso
Loureiro, Renata Ruoco
Pinheiro, Francisco Irochima
Casaroli-Marano, Ricardo Pedro
Donato, Waleska
Gomes, José Álvaro Pereira
author_sort Bandeira, Francisco
collection PubMed
description 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.
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spelling pubmed-99176402023-02-11 A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach Bandeira, Francisco Grottone, Gustavo Teixeira Covre, Joyce Luciana Cristovam, Priscila Cardoso Loureiro, Renata Ruoco Pinheiro, Francisco Irochima Casaroli-Marano, Ricardo Pedro Donato, Waleska Gomes, José Álvaro Pereira Int J Mol Sci Article 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. MDPI 2023-02-03 /pmc/articles/PMC9917640/ /pubmed/36769303 http://dx.doi.org/10.3390/ijms24032982 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bandeira, Francisco
Grottone, Gustavo Teixeira
Covre, Joyce Luciana
Cristovam, Priscila Cardoso
Loureiro, Renata Ruoco
Pinheiro, Francisco Irochima
Casaroli-Marano, Ricardo Pedro
Donato, Waleska
Gomes, José Álvaro Pereira
A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach
title A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach
title_full A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach
title_fullStr A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach
title_full_unstemmed A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach
title_short A Framework for Human Corneal Endothelial Cell Culture and Preliminary Wound Model Experiments with a New Cell Tracking Approach
title_sort framework for human corneal endothelial cell culture and preliminary wound model experiments with a new cell tracking approach
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917640/
https://www.ncbi.nlm.nih.gov/pubmed/36769303
http://dx.doi.org/10.3390/ijms24032982
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