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Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

ABSTRACT: Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investigate the possibility to produce a large amount of Good Manufacturing Practice (GMP)-compliant MSCs loaded with the chemotherapeutic drug Paclitaxel (MSCs-PTX), using a closed bioreactor system. Cells were obtained starting from 13 adipose tissue lipoaspirates. All samples were characterized in terms of number/viability, morphology, growth kinetics, and immunophenotype. The ability of MSCs to internalize PTX as well as the antiproliferative activity of the MSCs-PTX in vitro was also assessed. The results demonstrate that our approach allows a large scale expansion of cells within a week; the MSCs-PTX, despite a different morphology from MSCs, displayed the typical features of MSCs in terms of viability, adhesion capacity, and phenotype. In addition, MSCs showed the ability to internalize PTX and finally to kill cancer cells, inhibiting the proliferation of tumor lines in vitro. In summary our results demonstrate for the first time that it is possible to obtain, in a short time, large amounts of MSCs loaded with PTX to be used in clinical trials for the treatment of patients with oncological diseases.

SUBMITTER: Lisini D

PROVIDER: S-EPMC7284468 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

Lisini Daniela D Nava Sara S Frigerio Simona S Pogliani Simona S Maronati Guido G Marcianti Angela A Coccè Valentina V Bondiolotti Gianpietro G Cavicchini Loredana L Paino Francesca F Petrella Francesco F Alessandri Giulio G Parati Eugenio A EA Pessina Augusto A

Pharmaceutics 20200430 5

Mesenchymal stromal cells (MSCs) prepared as advanced therapies medicinal products (ATMPs) have been widely used for the treatment of different diseases. The latest developments concern the possibility to use MSCs as carrier of molecules, including chemotherapeutic drugs. Taking advantage of their intrinsic homing feature, MSCs may improve drugs localization in the disease area. However, for cell therapy applications, a significant number of MSCs loaded with the drug is required. We here investi ...[more]

PMID: 32365861

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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.

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.

Project description:BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

Dataset Information

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

Publications

Automated Large-Scale Production of Paclitaxel Loaded Mesenchymal Stromal Cells for Cell Therapy Applications.

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