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Title: Engineering Extracellular Matrix‐Bound Nanovesicles Secreted by Three‐Dimensional Human Mesenchymal Stem Cells
Abstract Extracellular matrix (ECM) in the human tissue contains vesicles, which are defined as matrix‐bound nanovesicles (MBVs). MBVs serve as one of the functional components in ECM, recapitulating part of the regulatory roles and in vivo microenvironment. In this study, extracellular vesicles from culture supernatants (SuEVs) and MBVs are isolated from the conditioned medium or ECM, respectively, of 3D human mesenchymal stem cells. Nanoparticle tracking analysis shows that MBVs are smaller than SuEVs (100–150 nm). Transmission electron microscopy captures the typical cup shape morphology for both SuEVs and MBVs. Western blot reveals that MBVs have low detection of some SuEV markers such as syntenin‐1. miRNA analysis of MBVs shows that 3D microenvironment enhances the expression of miRNAs such as miR‐19a and miR‐21. In vitro functional analysis shows that MBVs can facilitate human pluripotent stem cell‐derived forebrain organoid recovery after starvation and promote high passage fibroblast proliferation. In macrophage polarization, 2D MBVs tend to suppress the pro‐inflammatory cytokine IL‐12 β , while 3D MBVs tend to enhance the anti‐inflammatory cytokine IL‐10. This study has the significance in advancing the understanding of the bio‐interface of nanovesicles with human tissue and the design of cell‐free therapy for treating neurological disorders such as ischemic stroke.  more » « less
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Advanced Healthcare Materials
Medium: X
Sponsoring Org:
National Science Foundation
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    Basic Protocol 1: Isolating and culturing human corneal keratocytes and fibroblasts

    Basic Protocol 2: Isolating and culturing human corneal epithelial cells

    Basic Protocol 3: Isolating and culturing human corneal endothelial cells

    Basic Protocol 4: 3D corneal stromal construct assembly

    Basic Protocol 5: 3D corneal epithelial‐stromal construct assembly

    Basic Protocol 6: 3D corneal endothelial‐stromal construct assembly

    Basic Protocol 7: Isolating extracellular vesicles from corneal cell conditioned medium

    Support Protocol: Cryopreserving human corneal fibroblasts, corneal epithelial cells, and corneal endothelial cells

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