Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome
Background: Hematopoietic acute radiation syndrome (H-ARS) occurring after exposure to ionizing radiation damages bone marrow (BM) causing cytopenias, increasing susceptibility to infections and death. We and others have shown that cellular therapies like human mesenchymal stromal cells (MSCs), or monocytes/macrophages educated ex-vivo with extracellular vesicles (EVs) from MSCs were effective in a lethal H-ARS mouse model. However, given the complexity of generating cellular therapies and the potential risks of using allogeneic products, development of an “off-the-shelf” cell-free alternative like EVs may have utility in conditions like H-ARS that require rapid deployment of available therapeutics. The purpose of this study was to determine the feasibility of producing MSC-derived EVs at large scale using a bioreactor and assess critical quality control attributes like identity, sterility, and potency in educating monocytes and promoting survival in a lethal H-ARS mouse model. Methods: EVs were isolated by ultracentrifugation from unprimed and liposaccharide (LPS)-primed MSCs grown at large scale using a hollow fiber bioreactor and compared to a small scale system using flasks. The physical identity of EVs included a time course assessment of particle diameter, yield, protein content and surface marker profile by flow-cytometry. Comparison of the RNA cargo in EVs was determined by RNA-seq. Capacity of EVs to generate exosome educated monocytes (EEMos) was determined by qPCR and flow cytometry, and potency was assessed in vivo using a lethal ARS model with NSG mice. Results: Physical identity of EVs at both scales were similar but yields by volume were up to 38-fold more using a large-scale bioreactor system. RNA-seq indicated that flask EVs showed upregulated let-7 family and miR-143 micro-RNAs. EEMos educated with LPS-EVs at each scale were similar, showing increased gene expression of IL-6, IDO, FGF-2, IL-7, IL-10, and IL-15 and immunophenotyping consistent with a PD-L1 high, CD16 low, and CD86 low cell surface expression. Treatment with LPS-EVs manufactured at both scales were effective in the ARS model, improving survival and clinical scores through improved hematopoietic recovery. EVs from unprimed MSCs were less effective than LPS-EVs, with flask EVs providing some improved survival while bioreactor EVs provide no survival benefit. Conclusions: LPS-EVs as an effective treatment for H-ARS can be produced using a scale-up development manufacturing process, representing an attractive off-the-shelf, cell-free therapy. Overall design: RNA-seq was performed on flask-scale EVs isolated from six human MSCs isolates (Flask_Replicate1 to Flask_Replicate6) and LPSEVs from subset of 3 MSC isolates (Bioreactor_Replicate1 to Bioreactor_Replicate3) of that group. RNA-seq was also performed on three technical replicates of the bioreactor-scale EVs (96H Bioreactor) from MSCs (isolate B). RNA sequencing (RNA-seq) was performed by Systems Biosciences (Palo Alto, CA USA).