Culture enhanced mesenchymal stromal cells with increased potency

Robb, Kevin (1, 2), Gómez-Aristizábal, Alejandro (1, 2), Gandhi, Rajiv (1, 3), Viswanathan, Sowmya (1, 2, 4, 5)

(1) The Arthritis Program, Krembil Research Institute, University Health Network, Toronto

(2) Institute of Biomaterials and Biomedical Engineering, University of Toronto

(3) Division of Orthopaedic Surgery, Toronto Western Hospital, University of Toronto

(4) Cell Therapy Program, University Health Network, Toronto

(5) Division of Hematology, Department of Medicine, University of Toronto

Background: Osteoarthritis (OA) is a debilitating disease associated with joint inflammation and cartilage degradation. Mesenchymal stromal cells (MSCs) have been investigated as a promising therapy but insufficient in vivo potency and donor variability can limit their efficacy. To address this, the Viswanathan lab has pioneered a proprietary strategy to culture MSCs in 3D aggregates to augment their anti-inflammatory functions (US62/397,572). The objective of this work is to assess the ability of 3D MSCs to reduce inflammation, fibrosis, and cartilage degradation relative to conventional 2D culture controls within in vitro and in vivo OA models. Comparative analysis will be performed with hypoxic MSC cultures and statistical modeling will be used to select an optimal culture method based on cost, scalability, and potency attributes.

Methods: MSCs were isolated from human bone marrow or adipose tissue and subject to a serum-free 3D cell aggregation protocol using a proprietary cytokine cocktail or small molecule inhibitor. 2D culture in serum-free medium or with 10% fetal bovine serum served as controls. 24 h MSC licensing experiments were performed with tumour necrosis factor-α (TNFα) or interferon-γ (IFNγ). Co-cultures of MSCs with human peripheral blood-derived monocyte/macrophages were performed over 48 h.

Results: MSCs expressed a curated panel of surface markers based on international MSC characterization guidelines. TNFα-licensed 3D MSCs displayed highest gene expression of the anti-inflammatory potency marker TNFα-stimulated gene 6 (TSG6) versus controls. IFNγ-licensed 3D MSCs displayed reduced transcript levels of pro-inflammatory markers chemokine (C-X-C motif) ligand 8 (CXCL8) and cyclooxygenase-2 (COX2), as well as the pro-fibrotic marker transforming growth factor-β (TGFβ) versus controls. A trend toward increased expression of the homeostatic macrophage marker CD163 was observed in co-cultures with 3D MSCs versus 2D controls.

Conclusion: These results demonstrate that 3D MSCs may have improved immunomodulatory capacity and are less fibrotic and inflammatory relative to MSCs cultured in 2D. Ongoing studies will investigate 3D MSCs relative to hypoxic cultured MSCs and a statistics-based decision-making hierarchy will be developed to provide insight into the efficacy and clinical translatability of the different culture methods. Ultimately, this work will aid in developing MSC therapies with increased potency for treatment of osteoarthritis.