Cell counting and cell cycle analysis of Simple Non-cultured Endothelial Cell Injection (SNEC-I) therapy : characterization for clinical translation
Ting, Darren Shu Jeng Peh, Gary S L Neo, Dawn J H Ng, Xiao Yu Tan, Belinda Y L Wong, Raymond C B Ong, Hon Shing Mehta, Jodhbir S
Ting, Darren Shu Jeng
Peh, Gary S L
Neo, Dawn J H
Ng, Xiao Yu
Tan, Belinda Y L
Wong, Raymond C B
Ong, Hon Shing
Mehta, Jodhbir S
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Affiliation
Singapore Eye Research Institute; Duke-NUS Graduate Medical School; Sandwell and West Birmingham NHS Trust; University of Birmingham; et al.
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Publication date
2025-06-27
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Abstract
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.
Citation
Ting DSJ, Peh GSL, Neo DJH, Ng XY, Tan BYL, Wong RCB, Ong HS, Mehta JS. Cell Counting and Cell Cycle Analysis of Simple Non-Cultured Endothelial Cell Injection (SNEC-I) Therapy: Characterization for Clinical Translation. Cells. 2025 Jun 27;14(13):986. doi: 10.3390/cells14130986
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Article