Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury

Abstract

Background: Exosomes derived from mesenchymal stem cells (MSC-exos) have been demonstrated with great potential in the treatment of multiple human diseases including acute kidney injury (AKI) by virtue of their intrinsic cargoes. However, there are major challenges of low yield and the lack of an established biomanufacturing platform to efficiently produce MSC-exos, thereby limiting their therapeutic application. Here, we aimed to establish a novel strategy to produce MSC-exos with a hollow fiber bioreactor-based three-dimensional (3D) culture system and evaluate the therapeutic efficacy of 3D-exosomes (3D-exos) on AKI. Methods: Mesenchymal stem cells (MSCs) were isolated from fresh human umbilical cord and cultured in twodimensional (2D) flasks. 2 × 10 8 MSCs were inoculated into the hollow fiber bioreactor for 3D culture. The culture supernatants were collected every 1 or 2 days for isolating exosomes. Exosomes from 2D (2D-exos) and 3D cultures were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting analysis of exosome markers. The yield of exosomes from 2 × 10 8 MSCs seeded in 2D and 3D culture system was compared, based on protein quantification. The therapeutic efficacy of 2D-exos and 3D-exos was investigated in a murine model of cisplatin-induced AKI in vivo and in vitro. Results: 3D culture did not significantly change the surface markers of MSCs, as well as the morphology, size, and exosomal markers of 3D-exos when compared to those of 2D-exos. Compared with conventional 2D culture, the 3D culture system increased total exosome production up to 19.4-fold. 3D-exos were more concentrated in the harvested supernatants (15.5-fold) than 2D-exos, which led to a higher exosome collection efficiency of 3D culture system. In vivo, both 2D-exos and 3D-exos significantly alleviated cisplatin-induced murine AKI evidenced by improved renal function, attenuated pathological changes of renal tubules, reduced inflammatory factors, and repressed T cell and macrophage infiltration. Impressively, 3D-exos were more effective than 2D-exos. Moreover, 3D-exos were taken up by tubular epithelial cells (TECs) with improved efficiency, thereby exhibiting superior antiinflammatory effect and improved viability of TECs in vitro.

Exosomal miR-125b-5p deriving from mesenchymal stem cells promotes tubular repair by suppression of p53 in ischemic acute kidney injury

“…Human umbilical cord mesenchymal stem cells (hucMSCs, donated by Wingor Biotechnology Co, Ltd) were cultured with media mixed with mesenchymal stem cell medium (ScienCell) and DMEM/F12 (Gibco), supplemented with 5% fetal bovine serum (FBS; ScienCell). The identification of hucMSCs was determined as previously described 16 . To prepare MSC-exos, hucMSCs (Passage 2-6) were cultured with FBS-free media for 48 h, and the supernatants were subsequently collected for MSC-exos extraction.…”

“…It is worth noting that EVs derived from MSCs have been proposed as an alternative to MSC-based therapy for several diseases 15 . In the previous study, we found that exosomes derived from MSCs (MSC-exos) could significantly attenuate cisplatin-induced murine AKI through inhibiting inflammation 16 . Although the exact mechanisms are largely assumptive, anti-inflammation, anti-apoptosis, immunomodulation, and promoting proliferation are all conducive to the treatment of AKI 17 .…”

Small Extracellular Vesicles From Human Adipose-derived Stem Cells: A Potential Promoter of Keeping Fat Graft Survival

“…19.4 times [42,43]. Moreover, compared with 2D-sEVs, 3D-sEVs has no signi cant differences in surface markers, size, and shape.…”

Therapeutic potential of mesenchymal stem/stromal cells-derived exosomes in acute respiratory distress syndrome and COVID-19

“…And in combination with the conventional differential ultracentrifugation, threedimensional (3D) culture based on scalable microcarrier yields 20-fold more exosomes. Similarly, Cao et al investigated a strategy to increase total MSC-exosome production to 19.4-fold with a hollow fiber bioreactor-based 3D culture system and evaluated the therapeutic efficacy of 3D-exosomes om AKI [90]. They reported that 3D culture did not significantly change the surface markers of MSCs, as well as the morphology, size, and exosomal markers.…”