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

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.

Feasibility of First Injection of Autologous Adipose Tissue-Derived Stromal Vascular Fraction in Human Scarred Vocal Folds: A Nonrandomized Controlled Trial.

Importance: Patients with scarred vocal folds, whether congenitally or after phonosurgery, often exhibit dysphonia that negatively affects daily life and is difficult to treat. The autologous adipose tissue-derived stromal vascular fraction (ADSVF) is a readily accessible source of cells with angiogenic, anti-inflammatory, immunomodulatory, and regenerative properties. Objective: To evaluate the feasibility and tolerability of local injections of autologous ADSVF in patients with scarred vocal folds. Design, Setting, and Participants: CELLCORDES (Innovative Treatment for Scarred Vocal Cords by Local Injection of Autologous Stromal Vascular Fraction) is a prospective, open-label, single-arm, single-center, nonrandomized controlled trial with a 12-month follow-up and patient enrollment from April 1, 2016, to June 30, 2017. Eight patients with severe dysphonia attributable to vocal fold scarring associated with a congenital malformation or resulting from microsurgical sequelae (voice handicap index score >60 of 120) completed the study. Data analysis was performed from September 1, 2018, to January 1, 2019. Interventions: Injection of ADSVF into 1 or 2 vocal folds. Main Outcomes and Measures: The primary outcomes were feasibility and the number and severity of adverse events associated with ADSVF-based therapy. The secondary outcomes were changes in vocal assessment, videolaryngostroboscopy, self-evaluation of dysphonia, and quality of life at 1, 6, and 12 months after cell therapy. Results: Seven women and 1 man (mean [SD] age, 44.6 [10.4] years) were enrolled in this study. Adverse events associated with liposuction and ADSVF injection occurred; most of them resolved spontaneously. One patient received minor treatment to drain local bruising, and another experienced a minor contour defect at the liposuction site. At 12 months, the voice handicap index score was improved in all patients, with a mean (SD) improvement from baseline of 40.1 (21.5) points. Seven patients (88%) were considered to be responders, defined as improvement by 18 points or more in the voice handicap index score (the minimum clinically important difference). Conclusions and Relevance: The findings suggest that autologous ADSVF injection in scarred vocal folds is feasible and tolerable. The findings require confirmation in a randomized clinical trial with a larger population. Trial Registration: ClinicalTrials.gov Identifier: NCT02622464.

A retrospective analysis of characteristic features of responder patients to autologous serum eye drops in routine care.

PURPOSE: Autologous serum eye drops (ASEDs) are used worldwide to treat dry eye disease (DED). However, the biological composition of ASEDs has not been well investigated, and effectiveness predictive factors remain to be identified. The main objective of this study was to compare the response of patients treated with ASEDs biologically characterized and used for DED routine care. METHODS: This retrospective observational study was conducted in a single university hospital, and included 50 patients (87 eyes) with DED refractory to conventional treatment and resulting from various etiologies with Ocular Surface Disease Index (OSDI) ≥ 20. Each patient used eight drops a day per treated eye with 20% diluted ASEDs. Undiluted serum extensive biological characterizations were performed, and symptoms were recorded before the initiation of ASEDs and closer to the sixth month of treatment. Responders were defined as presenting an improvement from baseline ≥14 points in OSDI and/or ≥1 grade in corneal fluorescence staining for all eyes treated. RESULTS: The OSDI and the Oxford scale were significantly reduced from 68.7 ±â€¯23.2 to 54.8 ±â€¯25.7 and 3.2 ±â€¯1.5 to 2.1 ±â€¯1.3 (p ≤ 0.0001), respectively. A total of 68% of the patients were responders. Nonresponding patients had significantly higher epidermal growth factor concentrations in the serum compared to responders (p = 0.017). CONCLUSIONS: ASED administration resulted in significant clinical improvement in the management of DED. Biological differences observed between responders and nonresponders suggested that a better understanding of the biological activity of ASEDs is still required.

New fat-derived products for treating skin-induced lesions of scleroderma in nude mice.

INTRODUCTION: Scleroderma is characterized by cutaneous manifestations that mainly affect the hands, arms and face. As of today, there is no treatment for fibrotic skin lesions of scleroderma. Previously we generated and validated a model of scleroderma-like skin sclerosis in nude mice, appropriate to inject human derived products. We showed that the subcutaneous injection of micro-fat (MF), purified and injected using small caliber cannulas, have anti-fibrotic and pro-angiogenic effects and appears more suitable for the treatment of skin lesions of scleroderma compared to the gold standard (Coleman's technique or macro-fat). Here we compared the long-term efficacy of micro-fat "enriched" with other therapeutic products including the stromal vascular fraction (SVF) of fat and platelet-rich plasma (PRP) from blood in our murine model of scleroderma. METHODS: We used 72 nude mice in this study. We formed six experimental groups: Macro-fat, MF, SVF, PRP, MF + SVF, MF + PRP. This project has three phases: i) Induction of skin sclerosis by daily subcutaneous injections of bleomycin (BLM) for 4 weeks in nude mice; ii) Purification and injection of the different cell therapy products; iii) Histological analyses done 8 weeks post-injections. RESULTS: MF + SVF and MF + PRP significantly reversed dermal and epidermal sclerosis (P <0.01). Macro-fat, SVF, PRP only corrected the dermal sclerosis (P <0.05). Epidermal sclerosis was reduced in treatments containing MF (P <0.01). MF was more stable. Products containing the SVF were associated with a significant increase of the local vascularization (P <0.01). CONCLUSIONS: All tested substances were effective in treating skin-induced lesions of scleroderma with different levels of fibrosis and vascular improvement; MF derived products are more stable and SVF demonstrated better pro-angiogenic effects. The observed efficacy of this combination of products in the animal model provides a rationale for potential clinical applications to treat human disease.

Characterization and comparison of 5 platelet-rich plasma preparations in a single-donor model.

PURPOSE: The purpose of this study was to compare the biological characteristics of platelet-rich plasma (PRP) obtained from 4 medical devices and a preparation developed in our laboratory using a single-donor model. METHODS: Ten healthy persons donated blood that was processed to produce PRP by use of 4 commercial preparation systems and a protocol developed in our laboratory. Volumes and platelet, white blood cell (WBC), and red blood cell concentrations were recorded. The platelet activation status was assessed by flow cytometry. Enzyme-linked immunosorbent assay was used to determine the concentrations of vascular endothelial growth factor, platelet-derived growth factor AB, epidermal growth factor, and transforming growth factor ß1. We calculated platelet capture efficiency, relative composition, and increase factors from whole blood in platelets and WBC, as well as platelet and growth factor (GF) doses, provided from each preparation. RESULTS: Leukocyte-rich PRP was obtained with RegenPRP (RegenLab, Le Mont-sur-Lausanne, Switzerland) and the Mini GPS III System (Biomet Biology, Warsaw, IN) and provides PRP with higher proportions of red blood cells, WBCs, and neutrophils than leukocyte-poor PRP obtained with the Selphyl System (Selphyl, Bethlehem, PA), Arthrex ACP (Arthrex, Naples, FL), and the preparation developed in our laboratory. The highest platelet and GF concentrations and doses were obtained with the Mini GPS III System and the preparation developed in our laboratory. Different centrifugation protocols did not show differences in the percentages of activated platelets. Finally, a positive correlation between platelet doses and all the GFs studied was found, whereas a positive correlation between WBC doses and GFs was found only for vascular endothelial growth factor and epidermal growth factor. CONCLUSIONS: In a single-donor model, significant biological variations in PRP obtained from different preparation systems were highlighted. The observed differences suggest different results for treated tissue and could explain the large variability in the clinical benefit of PRP reported in the literature. CLINICAL RELEVANCE: Our findings will help clinicians to choose a system that meets their specific needs for a given indication.