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 language | English |
|---|---|
| Article number | 130 |
| Journal | Stem Cell Research and Therapy |
| Volume | 9 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - May 11 2018 |
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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 journal › Article
}
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
UR - http://www.scopus.com/inward/record.url?scp=85046955387&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85046955387&partnerID=8YFLogxK
U2 - 10.1186/s13287-018-0886-1
DO - 10.1186/s13287-018-0886-1
M3 - Article
VL - 9
JO - Stem Cell Research and Therapy
JF - Stem Cell Research and Therapy
SN - 1757-6512
IS - 1
M1 - 130
ER -