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

Abstract

BackgroundThe 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.MethodsThroughout 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.ResultsWe 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.ConclusionsThis 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.

1H-NMR and MALDI-TOF MS as metabolomic quality control tests to classify platelet derived medium additives for GMP compliant cell expansion procedures

“…ASC were chosen as model of in vitro proliferating cells: the experimental protocol was approved by the Ethics Committee of the CRO Aviano National Cancer Institute (protocol number: CRO-2016-30) and it complied with the Declaration of Helsinki (2004). Cells were obtained as previously published [ 4 ] from n = 3 separate donors and they were seeded at passage P4 in 96-well plates (BD Biosciences, Bedford, MA; US) at 3000 cells/cm 2 in complete Minimum Essential Medium Eagle - Alpha Modification (Alpha-MEM - Lonza; Basel, Switzerland)) containing 100 IU/ml of Penicillin, 100 microg/ml of Streptomycin (both from Sigma, St. Louis, MO; USA) and 5% (vol/vol)SRGF. After 4-6 hours, adherent cells were washed and medium was replaced with Alpha-MEM containing antibiotics and 5% (vol/vol) A, B, C and D (pools) each in four technical replicate wells.…”

“…In a previously published work, we demonstrated that such GMP compliant medium additive can efficiently stimulate stromal cell proliferation in culture [ 3 ]. Moreover, in a recent paper [ 4 ] we demonstrated that SRGF can strongly promote growth of adipose mesenchymal stromal stem cells (ASC) by direct analysis of cell proliferation and evaluating cell morphology. Rapidly dividing mesenchymal stem cells are, in fact, known to be elongated and spindle shaped [ 5 , 6 ].…”

“…Rapidly dividing mesenchymal stem cells are, in fact, known to be elongated and spindle shaped [ 5 , 6 ]. Interindividual differences between platelet donors could affect the capability to stimulate cell growth in vitro [ 7 , 8 ]: we demonstrated that, pooling together n = 16 SRGF products from single donors, standardized batches of medium additive can be obtained for applications in academic GMP facilities [ 4 , 9 ]. Nevertheless, we previously demonstrated that also the production method can affect medium additive capacity to stimulate cell growth in culture [ 10 ].Growth factors can, in fact, be extracted from platelets treating PRP by the so called freeze-and-thaw method [ 2 , 11 ], and we previously demonstrated that medium additives obtained by recalcification of PRP can promote a faster cell proliferation rate [ 10 ].…”

Lipoaspirate Storage Time and Temperature: Effects on Stromal Vascular Fraction Quality and Cell Composition

“…The ASCs and adipose-derived stem/progenitor cells are thought to have overlapping paracrine and immunosuppressive functions and the bulk of the clinical effects are thought to be caused by the ASCs [Viswanathan et al, 2019]. The reported percentage of adipose-derived stem/progenitor cells in the SVF ranges from < 1%, as determined according to a narrow definition of colony-forming units (CFU-F), whereas ASCs seem to make up more than 20% when defined according to surface markers only [Bourin et al, 2013;Agostini et al, 2018].…”

Intraoperative Strategies for Minimal Manipulation of Autologous Adipose Tissue for Cell- and Tissue-Based Therapies: Concise Review

“…In addition, different digestion conditions may affect phenotypical and functional characteristics of the isolated cells . Moreover, the procedure is generally considered expensive, because clinical grade products must be used to avoid xenogenic contamination . More importantly, collagenase digestion to isolate SVF falls outside the “minimal manipulation” guidelines set by regulatory agencies since the procedure alters substantially the original characteristic of the adipose tissue.…”