Regulatory Framework, Challenges, and Initial Strategic Planning for Advanced Therapy Products (PTAs) Development in Brazil.

Advanced Therapies are a class of innovative complex biological products used for therapeutic purposes, encompassing cell therapy, tissue engineering, and gene therapy products. These are promising therapeutic strategies for several complex diseases with low or non-existent therapeutic alternatives. The proper transposition of basic research in this area into medicinal products must comply with regulatory requirements. Here we review the main regulatory recommendations, emphasizing on the Brazilian regulation. The critical points are the manufacturing process, challenges in characterizing the product, development of non-clinical trials, lack of adequate animal models representative of the clinical situation, and absence of valid and measurable therapeutic endpoints. Based on that, we propose a framework for strategic planning of pre-clinical studies in this field. The detailed example involves producing a nonviral vector-based gene editing product, but the regulations and methods may be extrapolated for developing different types of advanced therapies.

A novel method to pack cryopreserved amniotic membrane for wound dressing-the pathway through validation of a new biological product.

Amniotic membrane (AM) has been widely used as a biological dressing for many pathologies and illnesses worldwide, and products derived from this tissue have been commercially available in several countries. In Brazil, regulatory agencies have recently authorized its clinical use as a non-experimental therapy for burns, diabetic and venous stasis ulcers, and intrauterine adhesions. In this study, we present our pathway through validating the first available service in the country of AM cryopreservation, with a protocol for long-term storage in high-efficiency nitrogen cryogenic freezers and a specific way of packing the tissue for optimal clinical handling and efficient storage space utilization while preserving live cells and the tissue's biological properties. Using gauze as support, cryoprotectant dimethyl sulfoxide and product presentation as a multilayer roll exhibited the best cell viability results and maintained the tissue integrity and presence of stem/progenitor cells. Essential proteins involved in tissue regeneration and immune and antimicrobial control were detected from the secretome of cryopreserved tissue similar to fresh tissue. Furthermore, immunogenic markers, such as human leukocyte antigens, were detected at very low levels in the tissue, confirming their low immunogenicity. Finally, we demonstrate that the tissue can be kept under refrigerated conditions for up to 7 d for further use, maintaining sterility and considerable cell viability. Our cryopreservation and storage protocol kept the AM viable for at least 20 months. In conclusion, this study enabled us to determine a novel efficient protocol for long-term AM preservation for future clinical applications.

Cell Therapy Improves Cardiac Function in Anthracycline-Induced Cardiomyopathy Preclinical Models: A Systematic Review and Meta-Analysis.

Although anthracycline (ANT)-based treatment strongly contributes to cancer survivorship, the use of these agents is limited by the risk of cardiotoxicity. For those patients who evolve to heart failure, myocardial regenerative approaches are of particular interest, and a growing body of preclinical studies has been investigating the use of cell therapy for ANT-induced cardiomyopathy (AIC). However, since animal models and modalities of cell therapy are highly heterogeneous between studies, the efficacy of cell therapy for AIC is not clear. Thus, we conducted a systematic review and meta-analysis of experimental studies reporting the use of cell therapy with mesenchymal stromal cells (MSC) or bone marrow mononuclear cells (BMMNC) in animal models of AIC with regard to global cardiac function. The Medline, EMBASE, and Web of Science databases were searched from inception to November 2019. Two reviewers independently extracted data on study quality and the results of left ventricular ejection fraction (LVEF) and fractional shortening (FS) obtained by echocardiography. The quality of outcomes was assessed using the Cochrane, Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES), and SYRCLE bias risk tools. Pooled random-effects modeling was used to calculate pooled mean differences (MD) and 95% confidence intervals (CIs). Twenty-two studies comprising 381 small animals (rabbits and rodents) were included. A pooled meta-analysis of all treatments showed that cell therapy increased LVEF by 9.87% (95% CI 7.25-12.50, P < 0.00001) and FS by 7.80% (95% CI 5.68-9.92, P < 0.00001) in small animals with AIC. Cell therapy with MSC/BMMNC is effective to mitigate the deleterious effects of ANT on cardiac function in preclinical models. Nevertheless, due to the small number of studies and considerable heterogeneity, future translational studies must be designed to diminish between-study discrepancies and increase similarity to the clinical landscape.

Coronary corium, a new source of equine mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have attracted great attention for therapeutic applications. Since cells derived from different tissues have different properties, using the right tissue source may impact their efficiency in regenerative medicine. This study describes for the first time the isolation and characterization of MSCs derived from the equine coronary corium, which may be useful for treating diseases such as laminitis. Seven coronary corium samples were used for isolation of cells (ccMSCs). Adherent cells were characterized for morphology, immunophenotype, proliferation and differentiation potential, in vitro migration and colony-forming capacity. The cells displayed the characteristic fibroblastoid morphology, with population doubling time increasing until passage 7 and reaching a plateau in passage 10. Cells were negative for CD14 and CD45, and positive for CD73 and CD90. ccMSCs showed chondrogenic and osteogenic, but not adipogenic differentiation, and migrated with nearly total closing of the empty area in 48 h, in the scratch assay. The clonogenic potential was in average 18% to 23%. This study describes for the first time the establishment of mesenchymal stromal cell cultures from the equine coronary corium. The results are similar to MSCs isolated from many other equine tissues, except for restricted differentiation potential. As coronary corium stem cell regulation may contribute to the pathogenesis of equine chronic laminitis, the use of ccMSCs in cell therapy for this significantly debilitating disease should be further investigated.

Induction of Expression of CD271 and CD34 in Mesenchymal Stromal Cells Cultured as Spheroids.

Cultured mesenchymal stromal cells (MSCs) are cells that can be used for tissue engineering or cell therapies owing to their multipotency and ability to secrete immunomodulatory and trophic molecules. Several studies suggest that MSCs can become pericytes when cocultured with endothelial cells (ECs) but failed to use pericyte markers not already expressed by MSCs. We hypothesized ECs could instruct MSCs to express the molecules CD271 or CD34, which are expressed by pericytes in situ but not by MSCs. CD271 is a marker of especial interest because it is associated with multipotency, a characteristic that wanes in MSCs as they are culture expanded. Consequently, surface expression of CD271 and CD34 was detected in roughly half of the MSCs cocultured with ECs as spheroids in the presence of insulin-like growth factor 1 (IGF-1). Conversely, expression of CD271 and CD34 was detected in a similar proportion of MSCs cultured under these conditions without ECs, and expression of these markers was low or absent when no IGF-1 was added. These findings indicate that specific culture conditions including IGF-1 can endow cultured MSCs with expression of CD271 and CD34, which may enhance the multipotency of these cells when they are used for therapeutic purposes.

Mesenchymal stem cell-conditioned medium triggers neuroinflammation and reactive species generation in organotypic cultures of rat hippocampus.

Cell therapy using bone marrow-derived mesenchymal stem cells (MSCs) seems to be a new alternative for the treatment of neurodegenerative diseases. Despite several promising results with their use, possible side effects are still unknown. In a previous work, we have shown that MSC-conditioned medium is toxic to hippocampal slice cultures and aggravates cell death induced by oxygen and glucose deprivation. In this work, we investigated whether the inflammatory response and/or reactive species formation could be involved in that toxicity. Rat organotypic hippocampal cultures were exposed for 24 h to conditioned medium from MSCs isolated from rat bone marrow. A marked glial activation was observed after exposure of cultures to MSC-conditioned medium, as evidenced by glial fibrillary acid protein (GFAP) and isolectin B(4) increase. Tumor necrosis factor-α and interleukin-6 levels were increased in the culture medium, and 2,7-dihydrodichlorofluorescein diacetate oxidation (indicating reactive species generation) and inducible nitric oxide synthase (iNOS) immunocontent were also higher after exposure of cultures to MSC-conditioned medium. Antioxidants (ascorbic acid and TROLOX(®)), N(ω)-nitro-l-arginine methyl ester hydrochloride, and anti-inflammatory drugs (indomethacin and dexamethasone) reduced cell death in hippocampal organotypic cultures after their exposure to MSC-conditioned medium. The results obtained here suggest that MSC-secreted factors trigger reactive species generation and neuroinflammation in organotypic cultures of hippocampus, introducing a note of caution in the use of these cells for neurological application.