By Holly Ober

Cell-based therapies for tissue regeneration and repair require new approaches to avoid the catastrophic loss of cells at implantation due to low oxygen and separation from their natural extracellular matrix. Mesenchymal stem cells (MSCs) are a promising cell population for use in cell-based therapies due to their natural production of molecules that promote blood vessel formation and modulate the defect site.  Compared to individual cells, MSCs have better overall function and survival if formed into spheres prior to transplantation. Since individual MSCs produce more of these factors when cultured in low oxygen environments, many have speculated that a lack of oxygen (hypoxia) in the spheroid’s core “pre-programs” the cells for improved survival and increased production of these regenerative factors. A new study published in Journal of the Royal Society Interface by Kent Leach’s group at UC Davis has now shown that MSC sphere cores have at most only 10% less oxygen than the surrounding air. Hypoxia does not give MSC spheres superior survival and function. Sphere size and the ability of MSCs to adapt their natural extracellular matrix production modulates function, not a hypoxic core.

Oxygen tensions were measured (left) and modeled (center) within MSC spheroids of increasing size. MSC spheroids exhibit cavities within the aggregates (right), providing a path for nutrients to travel throughout the aggregate and maintain cell function.

The group formed human bone marrow-derived MSCs into spheroids of increasing diameters within 25 μl droplets. They used microsensors to measure the oxygen tension as a function of position from the outer surface to the innermost core, then used the data to form mathematical simulations of oxygen transfer. They also visualized regions of hypoxia within the balls under a microscope using dyes to detect oxygen levels. Even at the largest diameter, the MSC spheroids exhibited less than a 10% decrease in oxygen tension between the outer layer of cells and the inner core, demonstrating no evidence of hypoxia.

“Engineering students learn that cells need to be within 100-200 μm of a capillary for diffusion to effectively delivery necessary oxygen and sustain life. We were shocked to see that a large oxygen gradient was not evident in MSC spheroids with diameters exceeding the diffusive limit,” says Dr. Leach.

MSC spheroids adapted their packing density as a function of size, with larger spheroids exhibiting reduced packing density to enable oxygen transport to the core and avoid the dangers of limited nutrients. Smaller spheres consumed up to 4 times more nutrients than the largest spheres, suggesting that the cells controlled their nutrient intake to maintain their viability.

Dr. Leach says “These data remind us that MSCs adapt to their environment to carry out their function. Our discovery that MSCs change their ECM deposition to provide space for nutrient diffusion, evidenced by the existence of cavities within the spheroids, represents a previously unknown method for spheroids to function.”

Collectively, these findings refute the hypothesis that increased production of bioactive factors by MSC spheroids is due to large oxygen gradients formed within the aggregates, thereby supporting the translational potential of spheroids in cell-based therapies for tissue engineering and regeneration.

Murphy KC, Hung BP, Browne-Bourne S, Zhou D, Yeung J, Genetos DC, Leach JK. 2017 Measurement of oxygen tension within mesenchymal stem cell spheroids. J. R. Soc. Interface 14: 20160851.