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Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function

BACKGROUND: Cardiovascular cell therapy represents a promising field, with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (“Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease”) consist...

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Autores principales: Radrizzani, Marina, Lo Cicero, Viviana, Soncin, Sabrina, Bolis, Sara, Sürder, Daniel, Torre, Tiziano, Siclari, Francesco, Moccetti, Tiziano, Vassalli, Giuseppe, Turchetto, Lucia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189603/
https://www.ncbi.nlm.nih.gov/pubmed/25260977
http://dx.doi.org/10.1186/s12967-014-0276-0
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author Radrizzani, Marina
Lo Cicero, Viviana
Soncin, Sabrina
Bolis, Sara
Sürder, Daniel
Torre, Tiziano
Siclari, Francesco
Moccetti, Tiziano
Vassalli, Giuseppe
Turchetto, Lucia
author_facet Radrizzani, Marina
Lo Cicero, Viviana
Soncin, Sabrina
Bolis, Sara
Sürder, Daniel
Torre, Tiziano
Siclari, Francesco
Moccetti, Tiziano
Vassalli, Giuseppe
Turchetto, Lucia
author_sort Radrizzani, Marina
collection PubMed
description BACKGROUND: Cardiovascular cell therapy represents a promising field, with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (“Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease”) consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility, endotoxin), identity/potency (cell count, CD45/CD34/CD133, viability) and purity (contaminant granulocytes and platelets). The aims of the present work were (1) to optimize the cell manufacturing process in order to reduce contaminants and (2) to implement additional assays in order to improve product characterization and evaluate product stability. METHODS: BM-MNC were isolated by density gradient centrifugation on Ficoll-Paque. The following process parameters were optimized throughout the study: gradient medium density; gradient centrifugation speed and duration; washing conditions. Differential cell count was performed by an automated hematology cell analyzer. Immunophenotype and cell viability were determined by flow cytometry. Functional hematopoietic and mesenchymal precursors and cells with angiogenic potential were assessed by colony-forming assays, cell invasion capacity by a fluorimetric assay. Sterility was tested using an automated microbial detection system, endotoxin by a kinetic chromogenic Limulus amebocyte lysate test. T-test was used for statistical analysis. RESULTS: A new manufacturing method was set up, based on gradient centrifugation on low density Ficoll-Paque, followed by 2 washing steps, of which the second one at low speed. It led to significantly higher removal of contaminant granulocytes and platelets, improving product purity; the frequencies of CD34(+) cells, CD133(+) cells and functional hematopoietic and mesenchymal precursors were significantly increased. The process was successfully validated according to Good Manufacturing Practices. The resulting ATMP mainly consisted of viable MNC including CD34(+) and CD133(+) cell subsets (2.98% ± 1.90% and 0.83% ± 1.32%, respectively), CD184/CXCR4(+) cells (34% ± 15%), CD34(+)/CD133(+)/CD309(+) endothelial precursors (44 ± 21 in 10(6) total cells), cells with invasion capacity, functional hematopoietic and mesenchymal precursors, cells with angiogenic potential; it was stable for 20 hours at 10°C. CONCLUSIONS: The methodological optimization described here resulted in a significant improvement of ATMP quality, a crucial issue to clinical applications in cardiovascular cell therapy.
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spelling pubmed-41896032014-10-23 Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function Radrizzani, Marina Lo Cicero, Viviana Soncin, Sabrina Bolis, Sara Sürder, Daniel Torre, Tiziano Siclari, Francesco Moccetti, Tiziano Vassalli, Giuseppe Turchetto, Lucia J Transl Med Methodology BACKGROUND: Cardiovascular cell therapy represents a promising field, with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (“Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease”) consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility, endotoxin), identity/potency (cell count, CD45/CD34/CD133, viability) and purity (contaminant granulocytes and platelets). The aims of the present work were (1) to optimize the cell manufacturing process in order to reduce contaminants and (2) to implement additional assays in order to improve product characterization and evaluate product stability. METHODS: BM-MNC were isolated by density gradient centrifugation on Ficoll-Paque. The following process parameters were optimized throughout the study: gradient medium density; gradient centrifugation speed and duration; washing conditions. Differential cell count was performed by an automated hematology cell analyzer. Immunophenotype and cell viability were determined by flow cytometry. Functional hematopoietic and mesenchymal precursors and cells with angiogenic potential were assessed by colony-forming assays, cell invasion capacity by a fluorimetric assay. Sterility was tested using an automated microbial detection system, endotoxin by a kinetic chromogenic Limulus amebocyte lysate test. T-test was used for statistical analysis. RESULTS: A new manufacturing method was set up, based on gradient centrifugation on low density Ficoll-Paque, followed by 2 washing steps, of which the second one at low speed. It led to significantly higher removal of contaminant granulocytes and platelets, improving product purity; the frequencies of CD34(+) cells, CD133(+) cells and functional hematopoietic and mesenchymal precursors were significantly increased. The process was successfully validated according to Good Manufacturing Practices. The resulting ATMP mainly consisted of viable MNC including CD34(+) and CD133(+) cell subsets (2.98% ± 1.90% and 0.83% ± 1.32%, respectively), CD184/CXCR4(+) cells (34% ± 15%), CD34(+)/CD133(+)/CD309(+) endothelial precursors (44 ± 21 in 10(6) total cells), cells with invasion capacity, functional hematopoietic and mesenchymal precursors, cells with angiogenic potential; it was stable for 20 hours at 10°C. CONCLUSIONS: The methodological optimization described here resulted in a significant improvement of ATMP quality, a crucial issue to clinical applications in cardiovascular cell therapy. BioMed Central 2014-09-27 /pmc/articles/PMC4189603/ /pubmed/25260977 http://dx.doi.org/10.1186/s12967-014-0276-0 Text en © Radrizzani et al.; licensee BioMed Central Ltd. 2014 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology
Radrizzani, Marina
Lo Cicero, Viviana
Soncin, Sabrina
Bolis, Sara
Sürder, Daniel
Torre, Tiziano
Siclari, Francesco
Moccetti, Tiziano
Vassalli, Giuseppe
Turchetto, Lucia
Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function
title Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function
title_full Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function
title_fullStr Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function
title_full_unstemmed Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function
title_short Bone marrow-derived cells for cardiovascular cell therapy: an optimized GMP method based on low-density gradient improves cell purity and function
title_sort bone marrow-derived cells for cardiovascular cell therapy: an optimized gmp method based on low-density gradient improves cell purity and function
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189603/
https://www.ncbi.nlm.nih.gov/pubmed/25260977
http://dx.doi.org/10.1186/s12967-014-0276-0
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