Inter-center comparison of good manufacturing practices-compliant stromal vascular fraction and proposal for release acceptance criteria: a review of 364 productions.

BACKGROUND: Even though the manufacturing processes of the stromal vascular fraction for clinical use are performed in compliance with the good manufacturing practices applying to advanced therapy medicinal products, specifications related to stromal vascular fraction quality remain poorly defined. We analyzed stromal vascular fraction clinical batches from two independent good manufacturing practices-compliant manufacturing facilities, the Swiss Stem Cell Foundation (SSCF) and Marseille University Hospitals (AP-HM), with the goal of defining appropriate and harmonized release acceptance criteria. METHODS: This retrospective analysis reviewed the biological characteristics of 364 batches of clinical-grade stromal vascular fraction. Collected data included cell viability, recovery yield, cell subset distribution of stromal vascular fraction, and microbiological quality. RESULTS: Stromal vascular fraction from SSCF cohort demonstrated a higher viability (89.33% ± 4.30%) and recovery yield (2.54 × 105 ± 1.22 × 105 viable nucleated cells (VNCs) per mL of adipose tissue) than stromal vascular fraction from AP-HM (84.20% ± 5.96% and 2.25 × 105 ± 1.11 × 105 VNCs per mL). AP-HM batches were significantly less contaminated (95.71% of sterile batches versus 74.15% for SSCF batches). The cell subset distribution was significantly different (higher proportion of endothelial cells and lower proportion of leukocytes and pericytes in SSCF cohort). CONCLUSIONS: Both centers agreed that a good manufacturing practices-compliant stromal vascular fraction batch should exert a viability equal or superior to 80%, a minimum recovery yield of 1.50 × 105 VNCs per mL of adipose tissue, a proportion of adipose-derived stromal cells at least equal to 20%, and a proportion of leukocytes under 50%. In addition, a multiparameter gating strategy for stromal vascular fraction analysis is proposed.

Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs.

Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFß-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFß3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.

Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate.

In recent years, there has been a shift toward tissue-engineering strategies using stem cells for plastic and reconstructive surgical procedures. Therefore, it is important to develop safe and reproducible protocols for the extraction of adipose-derived stromal cells (ASCs) to allow cells to be stored in liquid nitrogen for future needs. The aspirated liposuction obtained from healthy donors were immediately processed after the suction using a protocol developed in our laboratory. The resulting stromal vascular fraction (SVF) was then characterized by the presence of adipose-derived stromal cells, at later stage frozen in liquid nitrogen. After that, cells were thawed and again characterized by adipose-derived stromal cells, cellular survival, differentiation ability and Colony Forming Unit-Fibroblast like colonies (CFU-F). Extraction and freezing of cells contained in the stromal vascular fraction demonstrate that thawed cells maintain the full capability to grow and differentiate in culture. The advent of adipose-derived stromal cells use in tissue engineering will assume a wide role in esthetic restoration in plastic surgery. It is thus important to develop clinically translatable protocols for the preparation and storage of adipose-derived stromal cells. Our results show that adipose-derived stromal cells in serum free can easily be frozen and stored in liquid nitrogen with retention of 85% of cell viability and 180,890 cell/g yield plus normal proliferative capacity and differentiation potential compared with fresh controls. These observations set the basis for adipose-derived stromal cells banking.