Abstract
The aim of this study is to set up a standardized and reproducible method to determine the potency (= stem cell content) of human conjunctival cell cultures by means of immunofluorescence-based analyses. This will help the development of new Advanced Therapy Medicinal Products (ATMPs) to use in future cell therapy clinical studies when fewer cells are available to perform the quality controls. To achieve this purpose, a reference standard was investigated and the expression levels of ΔNp63α (considered as a marker of conjunctival stem cells) was correlated to cell size. The limbal hTERT cells were used as reference standard to define the expression value of ΔNp63α. The mean intensity value of limbal hTERT cells ranging between 15 and 20 µm in diameter was used to distinguish between ΔNp63α bright and not bright cells. As ΔNp63α bright expression was mainly seen in the smaller cell size group (10–15 µm), we defined as conjunctival stem cells (= potency) those cells which were bright and with sizes between 10 and 15 µm. Assays on cells from clonal analyses were used to validate the method, as they do allow to observe a decrease in potency (Holoclones > Meroclones > Paraclones). The stem cell content of conjunctival grafts was found to be 11.3% ± 5.0 compared to 21.9% ± 0.6, 9.0% ± 8.1 and 0% from Holoclones, Meroclones and Paraclones, respectively. This new method, here named as Standardized Method for Potency Quantification, will allow to detect the potency in conjunctival cell cultures, thus obtaining a quality control assay responding to the GMP standards required for ATMP release.
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Acknowledgments
The authors would like to thank Dr Sandro Scalet (The Veneto Eye Bank Foundation, Venice, Italy) for his help in harvesting the conjunctival biopsies and Prof. Daniel Aberdam, PhD (INSERM and Université de Paris) for providing the Limbal hTERT cell line. This work was supported (1) by the 5 × 1000 funds from the Italian Ministry of Health and the Italian Ministry of University and Research and (2) by Fiorenzato M.C. s.r.l (Venice, Italy) through a grant to Mattia Lamon.
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This work was partially supported through the 5 × 1000 funds from the Italian Ministry of Health and the Italian Ministry of University and Research and by Fiorenzato M.C. s.r.l. (Venice, Italy) through a grant to Mattia Lamon.
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Marina Bertolin, Claudia Breda, Mattia Lamon and Barbara Ferrari, performed the experiments and collected the data. Stefano Ferrari, Diego Ponzin and Vanessa Barbaro contributed to the revision and expert opinion.
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This article does not contain any studies with human participants or animals performed by any of the authors. Human corneas unsuitable for transplantation and duly consented for research by the donor’s next of kin were obtained by Fondazione Banca degli Occhi del Veneto, Italy. Forms to obtain consent and use of corneal tissues followed the standards set by the Italian National Transplant Service (Centro Nazionale Trapianti).
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Bertolin, M., Breda, C., Ferrari, S. et al. A new standardized immunofluorescence method for potency quantification (SMPQ) of human conjunctival cell cultures. Cell Tissue Bank 22, 145–159 (2021). https://doi.org/10.1007/s10561-020-09874-9
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DOI: https://doi.org/10.1007/s10561-020-09874-9