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Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media

BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply wit...

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Autores principales: Agostini, Francesco, Rossi, Francesca Maria, Aldinucci, Donatella, Battiston, Monica, Lombardi, Elisabetta, Zanolin, Stefania, Massarut, Samuele, Parodi, Pier Camillo, Da Ponte, Alessandro, Tessitori, Giovanni, Pivetta, Barbara, Durante, Cristina, Mazzucato, Mario
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948766/
https://www.ncbi.nlm.nih.gov/pubmed/29751821
http://dx.doi.org/10.1186/s13287-018-0886-1
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author Agostini, Francesco
Rossi, Francesca Maria
Aldinucci, Donatella
Battiston, Monica
Lombardi, Elisabetta
Zanolin, Stefania
Massarut, Samuele
Parodi, Pier Camillo
Da Ponte, Alessandro
Tessitori, Giovanni
Pivetta, Barbara
Durante, Cristina
Mazzucato, Mario
author_facet Agostini, Francesco
Rossi, Francesca Maria
Aldinucci, Donatella
Battiston, Monica
Lombardi, Elisabetta
Zanolin, Stefania
Massarut, Samuele
Parodi, Pier Camillo
Da Ponte, Alessandro
Tessitori, Giovanni
Pivetta, Barbara
Durante, Cristina
Mazzucato, Mario
author_sort Agostini, Francesco
collection PubMed
description BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. METHODS: Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. RESULTS: We demonstrated that SVF can be obtained with a consistent yield (about 185 × 10(3) cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (−11.6%). The relative abundance of ASC (CD34(+)CD45(−)CD31(–)) and endothelial precursors (CD34(+)CD45(−)CD31(+)) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 10(6) cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS: This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.
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spelling pubmed-59487662018-05-17 Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media Agostini, Francesco Rossi, Francesca Maria Aldinucci, Donatella Battiston, Monica Lombardi, Elisabetta Zanolin, Stefania Massarut, Samuele Parodi, Pier Camillo Da Ponte, Alessandro Tessitori, Giovanni Pivetta, Barbara Durante, Cristina Mazzucato, Mario Stem Cell Res Ther Method BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. METHODS: Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. RESULTS: We demonstrated that SVF can be obtained with a consistent yield (about 185 × 10(3) cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (−11.6%). The relative abundance of ASC (CD34(+)CD45(−)CD31(–)) and endothelial precursors (CD34(+)CD45(−)CD31(+)) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 10(6) cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS: This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported. BioMed Central 2018-05-11 /pmc/articles/PMC5948766/ /pubmed/29751821 http://dx.doi.org/10.1186/s13287-018-0886-1 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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 Method
Agostini, Francesco
Rossi, Francesca Maria
Aldinucci, Donatella
Battiston, Monica
Lombardi, Elisabetta
Zanolin, Stefania
Massarut, Samuele
Parodi, Pier Camillo
Da Ponte, Alessandro
Tessitori, Giovanni
Pivetta, Barbara
Durante, Cristina
Mazzucato, Mario
Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media
title Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media
title_full Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media
title_fullStr Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media
title_full_unstemmed Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media
title_short Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media
title_sort improved gmp compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948766/
https://www.ncbi.nlm.nih.gov/pubmed/29751821
http://dx.doi.org/10.1186/s13287-018-0886-1
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