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. 2018 May 11;9(1):130.
doi: 10.1186/s13287-018-0886-1.

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

Francesco Agostini  1 , Francesca Maria Rossi  2 , Donatella Aldinucci  3 , Monica Battiston  1 , Elisabetta Lombardi  1 , Stefania Zanolin  1 , Samuele Massarut  4 , Pier Camillo Parodi  5 , Alessandro Da Ponte  1 , Giovanni Tessitori  6 , Barbara Pivetta  6 , Cristina Durante  1 , Mario Mazzucato  7
Affiliations

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

Francesco Agostini et al. Stem Cell Res Ther. .

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.

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

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Conflict of interest statement

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the CRO Aviano National Cancer Institute (protocol number: CRO-2016-30), and it was performed in accordance with the Declaration of Helsinki (2004). Signed informed consent was collected from patients.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
a Distinct stratified phases are clearly evident after 1 h of lipoaspirate treatment with collagenase solution at 37 °C. Stromal vascular fraction cells are contained in the lower clear phase; digested adipocytes as well as released oily lipids are stratified in the two upper phases. The image is representative of a completely accomplished digestion process. b Images of diluted stromal vascular fraction cells cytospun on a glass slide. Cells were differentially stained by May-Grunwald/Giemsa protocol. Scale bar = 10 μm
Fig. 2
Fig. 2
a Impact of red blood cell (RBC) lysis on percent nucleated cell (NC) viability measures in SVF. b Impact of overnight lipoaspirate storage at +4 °C on percent NC viability measured by Trypan blue dye exclusion test (without red blood cell lysis) in fresh SVF samples. *p < 0.01, vs without RBC lysis (Student’s t test for paired data); §p < 0.05, vs immediate manipulation (Student’s t test for unpaired data)
Fig. 3
Fig. 3
Representative flow cytometry immunophenotype analysis of SVF cells evaluated before freezing. a Gating strategy identifying three main populations in the SVF: CD34+CD31−CD45− subset (ASC, red), CD45−CD34+CD31+ subset (EPC, green), and CD34–CD45+ subset (hematopoietic cells, blue). Dead cells (7AAD+) were excluded. b A detailed CD34+ cell characterization, showing expression of CD13, CD105, CD73, and CD90 in ASC and EPC. Pericytes were identified as CD34−CD45−CD31−CD146+ population (in violet). Lymphocytes are showed as reference (dark blue)
Fig. 4
Fig. 4
a Impact of different cryopreservation solutions on percent nucleated cell (NC) viability in thawed SVF products after 2 months of storage in liquid nitrogen. A, B, C and D are different cryopreservation solutions: solution A (10% Albital, 5% ACD-A, 10% DMSO, 75% saline solution), solution B (50% human serum, 5% Albital, 2.5% ACD-A, 10% DMSO, 32.5% saline solution), solution C (90% human serum, 10% DMSO), solution D (95% human serum, 5% DMSO). When compared with solutions C and D, the viability of NC stored for 2 months by solutions A and B was significantly lower. *p < 0.01, vs C and D; NSa, not significantly different vs C and vs Pre (one-way ANOVA for independent samples). b Impact of total NC concentration on SVF freezing on post-thaw cell viability. In the high NC concentration group (High), cell viability measured after thawing was significantly higher than in the low concentration (Low) group. §p < 0.05, vs High Group (Student’s t test for unpaired data). c Impact of longer term cryostorage on SVF samples. NC viability measured after 1 year of freezing was not significantly different when compared with results obtained after 2 months storage. *p < 0.01, vs Pre (one-way ANOVA for independent samples). NSb, not significantly different vs 2 months (one-way ANOVA for repeated measures). Tukey’s honestly different significance with Bonferroni’s correction as post-hoc test
Fig. 5
Fig. 5
Representative images of osteogenic, adipogenic, and chondrogenic differentiation assays as well as colony forming unit-fibroblast (CFU-F) assays performed on stromal vascular fraction (SVF) cells before freezing or after 2 months of storage in the presence of cryopreservation solutions (Sol.) C and D. Differentiation was induced (Stim.) by the addition of commercially available osteogenic, adipogenic, and chondrogenic differentiation media to cells at passage P1. Unstimulated cells (Unst.; control) were cultured with 10% FBS medium. Within the chondrogenesis assay, spheroids failed to be obtained from unstimulated cells. Differentiated cells as well as unstimulated samples were stained with Alizarin Red, Oil Red-O, and Safranin-O to detect osteocytes, adipocytes, and chondrocytes, respectively. The differentiation degree was quantified by image analysis of cell staining (adipogenesis and osteogenesis) or by morphometric analysis of spheroids (chondrogenesis); results are reported in histograms. Sample storage in the presence of solutions C and D did not significantly affect cell differentiation potential. Scale bar = 100 μm. C.A., covered area; Vol., volume
Fig. 6
Fig. 6
a Growth curves (logarithmic scale) of adipose tissue-derived stem cells (ASC) seeded at different cell densities (from 1 × 102 to 1 × 104 cells/cm2) in culture medium containing 10% fetal bovine serum (FBS) or 5% supernatant rich in growth factors (SRGF). The presence of SRGF in the cell culture induced a significantly higher growth rate when compared with FBS. b Plastic-adhering cells at P0 and after short-term (Low passage) or longer-term (High passage) expansion in the presence of 10% FBS or 5% SRGF in the cell culture medium. Scale bars = 100 μm. c Cell morphometric analysis. ASC expanded in 5% SRGF medium were smaller than those expanded in 10% FBS medium. The cell area of ASC cultured in 5% SRGF medium was greater at high passage when compared with P0 and low-passage cells. ASC expanded in 5% SRGF medium were more elongated than ASC expanded in 10% FBS medium during all culture phases. Linearity of growth curves was tested by calculating R2 as a measure of goodness of fit of linear regression. Differences between regression coefficients (slopes) of growth curves were tested by a Regression Model Analysis Test. *p < 0.01, vs FBS. Error bars of ASC growth curves could not be graphically reported in the diagram (logarithmic scale of y axis); the coefficient of variation regarding each plotted (mean) value of theoretical cell yield was below 10%. Connectors in c link significantly different means (p < 0.001, ANOVA for independent samples with interaction with Tukey’s HSD with Bonferroni’s correction as post-hoc analysis). MEM, minimum essential medium; SVF, stromal vascular fraction
Fig. 7
Fig. 7
Representative images obtained from osteogenic, adipogenic, and chondrogenic differentiation assays performed on ASC after short-term or longer-term expansion at 1 × 103 cells/cm2 in the presence of 10% fetal bovine serum (FBS) or 5% supernatant rich in growth factors (SRGF) in the cell culture medium. The differentiation degree was quantified by image analysis of cell staining (adipogenesis and osteogenesis) or by morphometric analysis of spheroids (chondrogenesis); results are reported in histograms. The differentiation potential was shown to be not significantly affected when comparing ASC expanded in 10% FBS and in 5% SRGF-containing media, both at high and low passages. Scale bar = 100 μm. C.A., covered Area; MEM, minimum essential medium; Vol., volume; Unst., unstimulated
Fig. 8
Fig. 8
a Representative karyotypes of adipose tissue-derived stem cells (ASC) expanded at high passages in 10% fetal bovine serum (FBS)- or 5% supernatant rich in growth factors (SRGF)-containing medium. At least 20 metaphases were analyzed and no clonal or recurrent chromosomal alterations could be identified. b Displays images taken from colony formation assays in methylcellulose medium performed on high-passage ASC cultured in 5% SRGF- or 10% FBS-containing medium. ASC expanded utilizing both cell culture media failed to display colony formation. HT1080 fibrosarcoma cells were used as positive control (c+). Scale bar = 100 μm. MEM, minimum essential medium
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