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

Francesco Agostini, Francesca Maria Rossi, Donatella Aldinucci, Monica Battiston, Elisabetta Lombardi, Stefania Zanolin, Samuele Massarut, Pier Camillo Parodi, Alessandro Da Ponte, Giovanni Tessitori, Barbara Pivetta, Cristina Durante, Mario Mazzucato

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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 × 103 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 × 106 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.

Original languageEnglish
Article number130
JournalStem Cell Research and Therapy
Volume9
Issue number1
DOIs
Publication statusPublished - May 11 2018

Fingerprint

Stem cells
Stromal Cells
Blood Vessels
Stem Cells
Intercellular Signaling Peptides and Proteins
Cell Survival
Cells
Freezing
Flow cytometry
Serum
Platelets
Dimethyl Sulfoxide
Adipose Tissue
Flow Cytometry
Blood Platelets
Tissue
Thawing
Trypan Blue
Osteocytes
Fibroblasts

Keywords

  • Adipose stem/stromal stem cells
  • Adipose tissue
  • Advanced therapy medicinal product
  • Anchorage independent growth
  • Cell morphology
  • Cell viability
  • CFU-F
  • Differentiation potential
  • Freezing protocol
  • Good manufacturing practice
  • Growth rate
  • Immunophenotype characterization
  • Karyotype
  • Stromal vascular fraction

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Molecular Medicine
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Cell Biology

Cite this

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.

In: Stem Cell Research and Therapy, Vol. 9, No. 1, 130, 11.05.2018.

Research output: Contribution to journalArticle

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. In: Stem Cell Research and Therapy. 2018 ; Vol. 9, No. 1.
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abstract = "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 × 103 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 × 106 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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author = "Francesco Agostini and Rossi, {Francesca Maria} and Donatella Aldinucci and Monica Battiston and Elisabetta Lombardi and Stefania Zanolin and Samuele Massarut and Parodi, {Pier Camillo} and {Da Ponte}, Alessandro and Giovanni Tessitori and Barbara Pivetta and Cristina Durante and Mario Mazzucato",
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TY - JOUR

T1 - Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media

AU - Agostini, Francesco

AU - Rossi, Francesca Maria

AU - Aldinucci, Donatella

AU - Battiston, Monica

AU - Lombardi, Elisabetta

AU - Zanolin, Stefania

AU - Massarut, Samuele

AU - Parodi, Pier Camillo

AU - Da Ponte, Alessandro

AU - Tessitori, Giovanni

AU - Pivetta, Barbara

AU - Durante, Cristina

AU - Mazzucato, Mario

PY - 2018/5/11

Y1 - 2018/5/11

N2 - 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 × 103 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 × 106 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.

AB - 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 × 103 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 × 106 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.

KW - Adipose stem/stromal stem cells

KW - Adipose tissue

KW - Advanced therapy medicinal product

KW - Anchorage independent growth

KW - Cell morphology

KW - Cell viability

KW - CFU-F

KW - Differentiation potential

KW - Freezing protocol

KW - Good manufacturing practice

KW - Growth rate

KW - Immunophenotype characterization

KW - Karyotype

KW - Stromal vascular fraction

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