A new standardized immunofluorescence method for potency quantification (SMPQ) of human conjunctival cell cultures
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A new standardized immunofluorescence method for potency quantification (SMPQ) of human conjunctival cell cultures Marina Bertolin . Claudia Breda . Stefano Ferrari . Mattia Lamon . Diego Ponzin . Barbara Ferrari . Vanessa Barbaro
Received: 31 July 2020 / Accepted: 4 October 2020 Ó Springer Nature B.V. 2020
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 immunofluorescencebased 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 DNp63a (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
M. Bertolin (&) C. Breda S. Ferrari M. Lamon D. Ponzin B. Ferrari V. Barbaro Fondazione Banca degli Occhi del Veneto, Padiglione G. Rama - Via Paccagnella 11, 30174 Venice, Italy e-mail: [email protected] C. Breda e-mail: [email protected] S. Ferrari e-mail: [email protected]
value of DNp63a. The mean intensity value of limbal hTERT cells ranging between 15 and 20 lm in diameter was used to distinguish between DNp63a bright and not bright cells. As DNp63a bright expression was mainly seen in the smaller cell size group (10–15 lm), we defined as conjunctival stem cells (= potency) those cells which were bright and with sizes between 10 and 15 lm. 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. Keywords Conjunctiva Stem cells Cell therapy Potency ATMP
M. Lamon e-mail: [email protected] D. Ponzin e-mail: [email protected] B. Ferrari e-mail: [email protected] V. Barbaro e-mail: [email protected]
Introduction Over the past three decades, cell therapy has shown to be a valuable therapeutic tool. In 1980s, pioneering experiments by Howard Green and colleagues
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demonstrated the potential of cultured epithelial adult stem cells to be used in clinical studies of cell therapy. Human epithelium was successfully grown in vitro and transplanted onto patients to reconstitute a functional epidermis (O’connor et al. 1981). Since then, successful cultures of other types of epithelial cells were reported including
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