Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods

Abstract

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

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

“…Indeed, the enzymatic digestion automatically performed by the Celution device is shorter than the SSCF digestion and is happening at room temperature whereas the optimal temperature for enzyme activity is 37 °C [ 23 ]. Since 2020, the AP-HM has changed its SVF production protocol for a more efficient manual process [ 21 ]. Furthermore, the majority of patients (sixty-two patients) included in the AP-HM cohort presented a chronic inflammatory disease such as systemic scleroderma or Crohn disease; the SVF may be impacted by this inflammatory signature and that could also explain the lower viability and recovery yield observed in the AP-HM cohort.…”

“…In order to be able to reliably compare the flow cytometry data generated by each center, we first verified that the difference in the antibodies panel used by the two facilities did not hamper the comparability of data when a common gating strategy is used. To that aim, 14 SVF batches manufactured in the two centers using a similar manual digestion protocol [ 21 ] were immunolabeled using the SSCF antibodies panel and the AP-HM antibodies panel. Samples were then analyzed by the same operator using the same defined gating strategy using Kaluza software as follows (Fig.…”

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

“…Indeed, the enzymatic digestion automatically performed by the Celution device is shorter than the SSCF digestion and is happening at room temperature whereas the optimal temperature for enzyme activity is 37°C [23]. Since 2020, the AP-HM has changed its SVF production protocol for a more e cient manual process [21]. Furthermore, the majority of patients (sixtytwo patients) included in the AP-HM cohort presented a chronic in ammatory disease such as systemic scleroderma or Crohn disease, the SVF may be impacted by this in ammatory signature and that could also explain the lower viability and recovery yield observed in the AP-HM cohort.…”

“…In order to be able to reliably compare the ow cytometry data generated by each center, we rst veri ed that the difference in the antibodies panel used by the two facilities did not hamper the comparability of data when a common gating strategy is used. To that aim, 14 SVF batches manufactured in the two centers using a similar manual digestion protocol [21] were immunolabelled using the SSCF antibodies panel and the AP-HM antibodies panel. Samples were then analyzed by the same operator using the same de ned gating strategy using Kaluza software as follows ( gure1):…”

Adipose Tissue: Understanding the Heterogeneity of Stem Cells for Regenerative Medicine

“…To comply with current good manufacturing practices (cGMPs), closed, sterile, and safe isolation processes have been developed. They can involve enzymatic or mechanical procedures to release the cellular components using an automated closed device [20] or a cost-effective protocol alternative to automated methods [21].…”