Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells

Exposure to ionizing radiation can have devastating effects on the body, particularly on the bone marrow, leading to a condition known as hematopoietic acute radiation syndrome (H-ARS). This syndrome damages the bone marrow, resulting in low blood cell counts, increased susceptibility to infections, and even death. While cellular therapies like mesenchymal stromal cells (MSCs) have shown promise in treating H-ARS, their complex generation process and potential risks associated with allogeneic products have spurred researchers to explore alternative treatments.

Researchers at the University of Wisconsin, Madison have investigated the feasibility of using extracellular vesicles (EVs) derived from MSCs as a cell-free alternative for treating H-ARS. EVs are small vesicles released by cells that contain various molecules, including proteins and nucleic acids, which can modulate cellular functions and communication. The researchers aimed to produce MSC-derived EVs at a large scale using a bioreactor and assess their quality and efficacy in treating H-ARS.

EVs were isolated from both unprimed MSCs and MSCs primed with lipopolysaccharide (LPS) using a hollow fiber bioreactor and compared to those isolated from cells grown in traditional flasks. The physical characteristics of the EVs, including size, yield, protein content, and surface marker profile, were assessed. Additionally, the RNA content of the EVs was analyzed using RNA sequencing.

Proposed GMP manufacturing platform for MSC-EV production

Fig. 1

A Mesenchymal stromal cells (MSCs) isolated from bone marrow (BM) should be characterized and qualified before B making a master cell bank (MCB), C followed by an expansion to generate multiple working cell banks (WCB). D Early expansion in flasks (Passage P0–P1) is followed by E expansion in a closed system bioreactor (P2) in serum free media. F EVs may be isolated directly with differential ultracentrifugation steps or concentrated beforehand using tangential flow filtration (TFF). G Resuspension of the EV pellet followed by sterile filtration (0.22 u) possibly with endotoxin removing capability. H The final EV testing and monitored storage with a consistent quality control (QC) strategy is needed to fulfill regulatory requirements for product release for I in vivo or ex vivo clinical testing. 

The results showed that EVs produced using the bioreactor system had similar physical characteristics to those produced in flasks but yielded significantly higher quantities. RNA analysis revealed differences in the RNA cargo of EVs from flasks, with upregulated expression of specific microRNAs. Furthermore, EVs from LPS-primed MSCs were found to be more potent in educating monocytes and promoting survival in a lethal H-ARS mouse model compared to EVs from unprimed MSCs.

Monocytes educated with LPS-primed EVs showed increased expression of genes associated with immune regulation and immunosuppression. Treatment with LPS-primed EVs resulted in improved survival and hematopoietic recovery in the H-ARS mouse model, with those produced at a larger scale showing greater efficacy.

Overall, this study highlights the potential of MSC-derived EVs as a promising therapy for H-ARS. The ability to produce EVs at a large scale using a bioreactor system offers a more feasible and accessible approach for therapeutic applications. Further research in this area may lead to the development of novel treatments for radiation injury and other related conditions, ultimately improving patient outcomes and quality of life.

Kink JA, Bellio MA, Forsberg MH, Lobo A, Thickens AS, Lewis BM, Ong IM, Khan A, Capitini CM, Hematti P. (2024) Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome. Stem Cell Res Ther 15(1):72. [article]

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