Engineering mesenchymal stem cell paracrine activity with 3D culture

Abstract

Background & Aim Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine effects, i.e. cytokines, chemokines, and extracellular vesicles (EVs). Consequently, there is growing interest in the use of these factors, rather than whole cells, in regenerative medicine. Typical 2D cultivation of stem cells on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. We seek to determine the biological implications of EV production in 3D culture and investigate their anti-fibrotic activity. Methods, Results & Conclusion Bone marrow MSCs were maintained and passaged on tissue culture plastic using DMEM//10% FBS. MSCs were aggregated into 3D spheroids using non-adherent 96-well plates. Both 2D and 3D MSCs were then maintained in serum free media for 4 days. EVs were isolated by ultracentrifugation and characterised on the ExoView platform which allows simultaneous detection of particle size and expression of EV markers CD81/CD63/CD9/CD105. miRNAs were isolated from cell lysates and qRT-PCR was performed to analyse expression of candidate miRNAs. To model the progress of pulmonary fibrosis, human lung fibroblasts (HLFs) were cultured with TGF-β1 to induce fibroblast activation, subsequently exposed to 2D and 3D MSC-EVs, and collagen production by HLF was measured. Further, 2D and 3D MSC-EVs were added into human lung MSCs isolated from healthy and IPF patients and cell proliferation was assessed using MTS assay. The yield and characteristics of EVs derived from bone marrow MSCs were unaffected by 3D culture. The particles showed expression of CD81/CD63/CD9/CD105 and average diameter of