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

Pauline François,Benjamin Guillet,Baptiste Bertrand,Laurent Arnaud,Jérémy Magalon,Florence Sabatier,Luc Lyonnet,Fanny Grimaud,Chloé Dumoulin,Laurent Giraudo,Stéphanie Simoncini,Houssein Aboudou,Marie Vogtensperger,Mélanie Velier,Julie Veran,Françoise Dignat-George

doi:10.3390/cells9102158

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.

Highlights

Adipose tissue (AT) has long been known for its filling ability, and autologous fat transfer, called lipofilling, is performed daily to correct soft tissue defects [1,2]
0.25 U/mL AT for 15 or 30 min at 37 ◦ C yielded stromal vascular fraction (SVF) with median cell viabilities of 70.50%, 71.50%, and 74.00%, respectively, which are below the 80% threshold, which is a value approved in several investigational medicinal product dossiers by the French national medecines safety agency
A non-significant difference was observed for leukocytes, with 40.60% (29.00–45.90%) vs. 25.20% (20.50–34.00%) for the LG and Celution protocols, respectively. These results showed that SVF manufactured according to the LG process contains similar proportions of regenerative cells compared to the reference

Summary

Introduction

Adipose tissue (AT) has long been known for its filling ability, and autologous fat transfer, called lipofilling, is performed daily to correct soft tissue defects [1,2]. AT is a major source of multipotent mesenchymal stem/stromal cells (MSCs) [3,4]. These adipose-derived stem/stromal cells (ASCs) are used for fat engraftment but are considered a promising product in the field of cell-based therapies. The therapeutic use of ASCs requires an ex vivo expansion step that takes several weeks [14]. This limitation can be circumvented by using the stromal vascular fraction (SVF), which is a heterogeneous cell population containing 15–30% ASCs [15]

Objectives

Methods

Results

Conclusion