Protection of human bone marrow MSC from room air oxygen during isolation accelerates cell growth

The role of mesenchymal stromal/stem cells (MSC) in the cancer microenvironment is of great interest, not only for the fundamental roles that MSC play in inflammation, organoids, and regenerative medicine, but also as producers of exosomes. These extracellular vesicles, which can deliver cargo to other cells, are gaining interest as tumor-homing vehicles for therapeutic agents. However, the solid tumor environment is hypoxic, and even normal tissues reside at oxygen levels far below that of room air, where MSC are grown for exosome production. Exposure to supraphysioxic room air changes MSC, modulating HIF-1a which is upstream of signaling pathways related to cell proliferation, differentiation, and cell death. We previously showed that eliminating exposure of human bone marrow MSC to room air during cell culture and handling can prevent HIF-1a modulation, improve cell division rates, and reduce cell senescence in later passages. Here we extend those findings, isolating cells from cadaver bones that were sourced from a licensed organ procurement agency, under constant physioxia (5%O2/ 5%CO2) in an Xvivo System. Flow cytometric analyses and differentiation of the plastic-adherent bone marrow cells into adipocytes, chondrocytes, and osteocytes confirmed the bone marrow MSC phenotype. Having never experienced room air, these MSC exhibited faster cell growth at passage 8 (mean doubling time 43.2 +/- 1.2 hrs, n = 3 separate cultures) than commercially sourced MSC that had been isolated, expanded, and cryopreserved under traditional laboratory conditions (mean doubling time 128 +/- 15.2 hrs, n = 2 separate cultures). We concluded that protecting MSC from room air exposure, even during isolation, can improve human bone marrow MSC growth kinetics for studies of the tumor microenvironment, inflammation, and tumor-homing exosomes.