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Cytotherapy Corner October 2022

Cytotherapy Corner
October 2022

Donald G. Phinney, PhD

Cytotherapy Senior Editor
Department of Molecular Medicine
UF Scripps Biomedical Research
Jupiter, FL, USA


            In this issue of Cytotherapy Corner I want to highlight a manuscript published by Malvicini et al. (October 2022 issue). It standardizes an in vitro assay to quantify the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs), based on their ability to prevent the acquisition of the M1 phenotype in lipopolysaccharide (LPS)-stimulated macrophages. This manuscript is timely owing to the current focus on developing EV-based therapeutics for clinical use. These efforts are hindered by the fact that no standardized methods exist for isolating EVs even though evidence suggests that culture conditions and isolation methods impact cargo content. Consequently, development of assays that predict potency is essential for clinical advancement of these products. Indeed, the lack of such assays severely hindered the clinical development of MSC-based products, and since MSCs are widely used to source EVs, it is not surprising that EV researchers are adapting lessons learned from the MSC field to prevent similar setbacks.   

            In this study, EVs were purified from media conditioned by human umbilical cord MSCs via anion exchange chromatography using CD63 expression as a read out for protein elution. The concentration and size distribution of eluted EVs was quantified by nanoparticle tracking analysis, TEM, and flow cytometric analysis. In total, six independent EV preparations were produced for downstream analysis. The authors then demonstrated that nitric oxide (NO) release by macrophages exposed to LPS provided a reliable and reproducible readout of polarization to the M1 phenotype, which was validated by quantification of inflammatory cytokine gene expression and protein secretion over a time course of LPS exposure. Studies further confirmed that LPS stimulated macrophages failed to express markers of M2 polarization. The authors then present a series of experiments examining effects of LPS dose, cell seeding density, and time course on nitrite release from macrophages, and describe optimization of a spectrophotometric assay to quantify nitrite levels in conditioned media. The latter was necessary to ensure that the dynamic range of the assay was sufficient to read out the inhibitory activity of EVs. Once optimized, the authors demonstrated that EVs inhibit macrophage polarization to the M1 phenotype in a dose dependent manner, and the assay was sensitive enough to read out significant differences in potency between EV preparations. The authors cross validated the assay performance by reading out additional M1 markers, and then validated the assay in terms of linearity, repeatability, precision, and reproducibility according to FDA guidelines. Overall, the assay provides a rapid, high throughput process to quantify impacts of tissue source, isolation process, storage, etc. on EV bioactivity and as such represents an important advance in the field.  One limitation of this assay is that the authors employed a mouse macrophage cell line to assess the anti-inflammatory of EVs derived from human MSCs. Therefore, while the FDA allows use of xenogeneic models for potency assay development, objectively the results would be strengthened if they were extended to human macrophages. 

I also want to highlight a manuscript by Sachetti et al. (November 2022 issue) that provides a descriptive analysis of the public investments made by the Brazilian government toward research and development of Advanced Therapy Medicinal Products (ATMPs). The analysis evaluates overall support provided for scientific, technological, and innovation research and is stratified based on ATMP type, year of funding, amount invested, and disease target. The analysis is interesting owing to the immense increase in available funding for ATMPs over the last decade from both government and private entities and provides a unique look into how developing countries are attempting to capitalize these advances while balancing other critical health sector needs.   

In this manuscript, the authors outline efforts by the Brazilian government over the last several decades to establish funding for research aimed at improving public health and drive innovation in science and biotechnology. The authors provide a concise but highly informative summary of these efforts, which include establishment of a regulatory framework for ATMP production under GMP guidelines in 2018 by the Brazilian Health Regulatory Agency (Anvisa), creation of several Cell Technology Centers (CTCs) to produce ATMPs, and the launch in 2020 of the Brazilian Program of Genomics and Precision Health that including an initial 4-year investment of ~ 300 million dollars. The authors then detail government funding in ATMPs spanning 2004 to 2020, which included a total of 282 funded research projects. Interestingly, most of these projects were directed toward cell therapy (~76%), with tissue engineering (~14%) and gene therapy (~9%) rounding out the top three.  Moreover, early-stage projects including basic and pre-clinical studies represented ~78% of all funded projects and ~45% of the total investment. Clinical trials represented ~14% of funded research and ~22% of the total budget, and ~8% of projects were directed toward research infrastructure for cell therapy.  Clinical trials funded by these efforts covered a wide range of disease areas with Cardiology (29%) and Neurology (21%) accounting for half of trials. Other disease areas that received funding included Orthopedics (12%), Hematology (8%), Pulmonology (8%), and Oncology (6%). As evidenced by these statistics, cell therapy was considered a strategic area for public health investment in Brazil and these investments have allowed the country to become a leader in cell-based therapy research in Latin America.


Happy reading.