Development of Autologous Platelet-Rich Plasma Mixed-Microfat as an Advanced Therapy Medicinal Product for Intra-Articular Injection of Radio-Carpal Osteoarthritis: From Validation Data to Preliminary Clinical Results.

Wrist osteoarthritis (OA) is one of the most common conditions encountered by hand surgeons with limited efficacy of non-surgical treatments. The purpose of this study is to describe the Platelet-Rich Plasma (PRP) mixed-microfat biological characteristics of an experimental Advanced Therapy Medicinal Product (ATMP) needed for clinical trial authorization and describe the clinical results obtained from our first three patients 12 months after treatment (NCT03164122). Biological characterization of microfat, PRP and mixture were analysed in vitro according to validated methods. Patients with stage four OA according to the Kellgren Lawrence classification, with failure to conservative treatment and a persistent daily painful condition >40 mm according to the visual analog scale (VAS) were treated. Microfat-PRP ATMP is a product with high platelet purity, conserved viability of stromal vascular fraction cells, chondrogenic differentiation capacity in vitro and high secretion of IL-1Ra anti-inflammatory cytokine. For patients, the only side effect was pain at the adipose tissue harvesting sites. Potential efficacy was observed with a pain decrease of over 50% (per VAS score) and the achievement of minimal clinically important differences for DASH and PRWE functional scores at one year in all three patients. Microfat-PRP ATMP presented a good safety profile after an injection in wrist OA. Efficacy trials are necessary to assess whether this innovative strategy could delay the necessity to perform non-conservative surgery.

Human nasal olfactory stem cells, purified as advanced therapy medicinal products, improve neuronal differentiation.

Background: Olfactory ecto-mesenchymal stem cells (OE-MSC) are mesenchymal stem cells derived from the lamina propria of the nasal mucosa. They display neurogenic and immunomodulatory properties and were shown to induce recovery in animal models of spinal cord trauma, hearing loss, Parkinsons's disease, amnesia, and peripheral nerve injury. As a step toward clinical practice, we sought to (i) devise a culture protocol that meets the requirements set by human health agencies and (ii) assess the efficacy of stem cells on neuron differentiation. Methods: Nasal olfactory mucosa biopsies from three donors were used to design and validate the good manufacturing process for purifying stem cells. All processes and procedures were performed by expert staff from the cell therapy laboratory of the public hospital of Marseille (AP-HM), according to aseptic handling manipulations. Premises, materials and air were kept clean at all times to avoid cross-contamination, accidents, or even fatalities. Purified stem cells were cultivated for 24 or 48 h and conditioned media were collected before being added to the culture medium of the neuroblastoma cell line Neuro2a. Results: Compared to the explant culture-based protocol, enzymatic digestion provides higher cell numbers more rapidly and is less prone to contamination. The use of platelet lysate in place of fetal calf serum is effective in promoting higher cell proliferation (the percentage of CFU-F progenitors is 15.5%), with the optimal percentage of platelet lysate being 10%. Cultured OE-MSCs do not show chromosomal rearrangement and, as expected, express the usual phenotypic markers of mesenchymal stem cells. When incorporated in standard culture medium, the conditioned medium of purified OE-MSCs promotes cell differentiation of Neuro2a neuroblastoma cells. Conclusion: We developed a safer and more efficient manufacturing process for clinical grade olfactory stem cells. With this protocol, human OE-MSCs will soon be used in a Phase I clinical based on their autologous transplantation in digital nerves with a neglected injury. However, further studies are required to unveil the underlying mechanisms of action.

The Use of Higher Proportions of Platelet-Rich Plasma to Enrich Microfat Has Negative Effects: A Preclinical Study.

BACKGROUND: Platelet-rich plasma improves engraftment after fat transfer. However, the effects of platelet dose have never been investigated. The authors used magnetic resonance imaging to compare surviving graft volumes in mice after administration of four different formulations (microfat alone, and three platelet-rich plasma-enriched microfat mixes). METHODS: The authors used a random, double-blinded, fat transfer protocol using three different platelet levels: 1 million (low-dose), 500 million (medium-dose), and 1000 million (high-dose) platelets/ml, and fat alone (control). The authors grafted 0.4 ml of the 70/30 platelet-rich plasma-enriched microfat mixtures (0.4 million, 200 million, and 400 million platelets per 0.12 ml for the low-dose, medium-dose, and high-dose mixtures, respectively) or 0.4 ml of microfat alone into 22 nude mice and monitored surviving graft volumes every month for 3 months. Then, the authors histologically analyzed all grafts to assess neoangiogenesis status and fat integrity. RESULTS: Three-dimensional magnetic resonance imaging showed that the median surviving graft volumes at 3 months were 9.5 percent (interquartile range, 0 to 25 percent; p = 0.003) (high-dose), 4.1 percent (interquartile range, 0 to 18 percent; p = 0.001) (medium-dose), and 18 percent (interquartile range, 8 to 38 percent; p = 0.41) (low-dose) compared to 36 percent (interquartile range, 28 to 53 percent) for the control value. The histologic integrity of microfat-alone grafts was significantly better than those of the other grafts, although the high-dose and low-dose grafts exhibited higher levels of neoangiogenesis. CONCLUSION: Higher platelet levels in microfat grafts were associated with poor graft survival in nude mice; a clinical review would be appropriate.

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.