Large-scale bioreactor production of extracellular vesicles from mesenchymal stromal cells for treatment of acute radiation syndrome
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ABSTRACT:
PROVIDER: PRJNA1075818 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27950155.fastq.gz | Fastqsanger.gz | |||
| SRR27950156.fastq.gz | Fastqsanger.gz | |||
| SRR27950157.fastq.gz | Fastqsanger.gz | |||
| SRR27950158.fastq.gz | Fastqsanger.gz | |||
| SRR27950159.fastq.gz | Fastqsanger.gz |
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Project description: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.
2024-02-17 | GSE255642 | GEO
Project description:BackgroundHematopoietic acute radiation syndrome (H-ARS) occurring after exposure to ionizing radiation damages bone marrow 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.MethodsEVs were isolated by ultracentrifugation from unprimed and lipopolysaccharide (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.ResultsPhysical 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.ConclusionsLPS-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.
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Project description:Mesenchymal stromal cells (MSCs) are being tested in numerous clinical trials, yet the limited progression of these trials to advanced stages indicates unresolved translational challenges. Expanding MSCs is a critical step in most therapeutic applications and bioreactor-based culture offers large-scale production compared to monolayer cultures. Nevertheless, since MSCs sense their microenvironment, it is crucial to understand how these systems affect their properties. We expanded human adipose-derived mesenchymal stromal cells (AD-MSCs) from the same donors in both small- and large-scale platforms. Bulk-RNA sequencing and mass-spectrometry analysis demonstrate that small-scale culture had a broader range of differentially expressed genes and proteins within immunomodulatory, cell migration and cell adhesion pathways. In contrast, the large-scale culture showed a lower amount of differentially expressed genes - and proteins were proteinsparticularly associated mainly with extracellular matrix synthesis. Our findings demonstrate that expansion platforms significantly impact MSCs’ properties of MSCs, highlighting the need with optimise expansion conditions to obtain high cell yield without compromising MSCs’ their attributes.
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Project description:Polymersomes have some fundamental advantages compared to their liposomal counterparts. Due to the increased stability of the polymeric membrane, polymersomes are intended to be reasonably applicable as carrier-systems and universal reaction compartments for diverse medical and biotechnological applications. Regardless of the application area, suitable methods to produce large vesicle quantities in a controlled and cost-effective manner have to be developed to put polymersome technology into action at the industrial scale. In this work, the amphiphilic triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 was formed into uniform polymersomes. A recently established production process, based on the use of miniaturized stirred-tank reactors at the milliliter-scale (12 mL), was successfully scaled-up to the liter-scale (1.5 L) based on solid process engineering parameters. Dynamic light scattering measurements show that using standard propeller stirrers with a dimensionless diameter dD-1 ≥0.65 in an unbaffled stirred-tank reactor led to a narrow particle size distribution when providing a Froude number of Fr = 6.52 at the same time. Polymersomes with a mean diameter of 180 nm and a low polydispersity index (PDI<0.2) were generated within about 1 h in one single production step. Thus, this work provides the fundamental basis for further scale-up purposes, regarding polymersome production in stirred-tank reactors at the industrial scale.
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Project description:Various culture media that can rapidly expand bone marrow stromal cells (BMSCs) are currently available. However, the effects of those culture media on the contents of extracellular vesicles released by bone marrow stromal cells have not been fully understood. Using BMSCs from 6 healthy donors were cultured in two different culture media and characterized the small RNA profiles in extracellular vesicles.
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Project description:To date, extracellular vesicles (EVs) have been extensively investigated as potential substitutes for cell therapy. Research has suggested their ability to overcome serious risks associated with the application of these cells. Although, the translation of EVs into clinical practice is hampered by the lack of a cheap reasonable way to obtain a clinically relevant number of EVs, an available method for the large-scale production of EVs ensures vesicles' integrity, preserves their biological activity, and ensures they are well reproducible, providing homogeneity of the product from batch to batch. In this review, advances in the development of methods to increase EVs production are discussed. The existing approaches can be divided into the following: (1) those based on increasing the production of natural EVs by creating and using high capacity "cell factories", (2) those based on the induction of EVs secretion under various cell stressors, and (3) those based on cell fragmentation with the creation of biomimetic vesicles. The aim of this review is to stimulate the introduction of EVs into clinical practice and to draw attention to the development of new methods of EVs production on a large scale.
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Project description:RNA-seq was performed on cultured human induced pluripotent cell derived cardiomyocytes (iPSC-CMs). There are four groups, with three samples per group: H1,H2,H3. Negative control. Healthy, normoxic iPSC-CMs D1,D2,D3. Positive control. iPSCs cultured under hypoxic (0-1% O2) for 48h with 2% v/v PBS as vehicle control EV1,EV2,EV3. Treatment 1. iPSCs cultured under hypoxic (0-1% O2) for 48h, with cardiac stromal cell derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) EV21, EV23, EV24. Treatment 2 iPSCs cultured under hypoxic (0-1% O2) for 48h, with bone marrow mesenchymal stromal cell (BM-MSC) derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) All EVs were isolated from cultured human cells using sequential centrifugation methods. Cells were cultured using commercial EV-depleted FBS to avoid contamination of bovine EVs. EVs were validated for CD81, CD9, ALIX positivity, and visualised by cryoEM. Particle to protein ratios were not different between cardiac and bone marrow EV isolates. Therefore EV doses were standardised so that the same dose by protein (67ng/µl) and EV number (~2,000 EVs per iPSC-CM) were added.
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