Biological characterization of human MSCs secretome and extracellular vesicles (EVs): from 2D-cultures to robotic systems

Human mesenchymal stem cells (hMSCs) are a promising cell therapy candidate for the treatment of several clinical conditions. In recent years, the use of extracellular ves-icles emerges for therapeutic purposes. Most of cells indeed release a wide range of extracellular vesicles of different size, content and functions, which interact with target cells by modifying the phenotype or function. Manufacture of hMSCs requires in vitro isolation and expansion to increase the avail-able number of final products to meet clinical demand. Through Autostem (EU-H2020 proposal) European academic and industrial group worked in collaboration to enable large-scale hMSCs production, at clinical-grade quality, by implementing a robotic auto-mated pipeline for cell isolation and culture. The goal is to develop an automated, closed and good manufacturing practice (GMP) MSC cell technology, a “StromalCellFactory” to produce allogenic hBMSCs to use in therapy. We developed a “donor-to-patient” system for aseptic therapeutic cell manufacturing using a xeno-free medium. We characterized cells, secretome and extracellular vesicles of hBMSCs, isolated in plastic adherence and grown in FBS and in a chemically defined xeno-free medium, compared to hBMSCs isolated in a 3D-bioreactor, demonstrating that our serum-free system allow to select a highly proliferating-hBMSC population with a strong osteogenic potential, both in vitro and in vivo. These cells have also a secretion pattern characterized by biological factors involved in homeostasis, wound healing and angiogenesis, beside showing a stronger extracellular vesicles secretion. We validated also the potential therapeutic benefits of secreted factors, conditioned medium and exosomes isolated from hBMSCs cultured in our culture system, for carti-lage repair. In a damaged tissue, the initial inflammatory response plays a key role triggering tissue repair and homeostasis, but can be detrimental in the long term, causing fibrosis. We observed that under inflammatory condition human Articular Chondrocytes (hACs) are able to internalize and recruit more MSC-derived exosomes, compare to the control chondrocytes. We investigated the effects of conditioned medium and extracellular ves-icles in the activation of different regeneration pathways (IL6, IL8, COX2 and NF-kB). The effect of MSCs-derived conditioned media and exosomes revealed to be regener-ating and protective for the articular cartilage and they may be a possible therapy for osteoarthritis.