Natural Killer (NK) cells, integral to the innate immune system, are notable in cell therapies because for their applicability in allogeneic treatments, distinguishing them from T cells typically employed in conventional cell therapies. However, their limited half-life poses a challenge for therapy. Although attempts to leverage feeder cells are common, safer methods are needed to mitigate the associated risks. In our study, an upregulation in the expression of 4-1BBL in Colo-205 cells under extracellular stresses such as hypoxia and cytochalasin D was observed. This enhanced binding to the 4-1BB receptors on NK cells promotes proliferation in NK cells. Elevated CD56 expression of a marker strongly linked to NK cell proliferation in co-culture further supports this process. Applying extracellular stressors, specifically hypoxia and cytochalasin D, to Colo-205 cells successfully tailored feeder cells, significantly enhancing NK cell proliferation.

Hypoxia is considered an important factor in the proliferation and stemness of mesenchymal stem cells (MSCs). This study aimed to obtain large numbers of mouse clonal MSCs (mc-MSCs) and evaluate the effect of hypoxia on the proliferation and differentiation of mc-MSCs. mc-MSCs were cultured on a Cytodex 3 microcarrier in a spinner flask for a suspension culture to increase mass productivity. To produce the hypoxic conditions, CoCl2 and Na2SO3 were used in a low glucose DMEM medium. The hypoxia environment was established using 0.5g/L Na2SO3 with 10μM or 100 µM CoCl2 for 24 hours. As a result, the proliferation of mc-MSCs under hypoxic conditions was 1.56 times faster than the control group over seven days. The gene expression of HIF-1a and VEGFA increased 4.62 fold and 2.07 fold, respectively. Furthermore, the gene expression of ALP, RUNX2, COL1A, and osteocalcin increased significantly by 9.55, 1.55, 2.29, and 2.53 times, respectively. In contrast, the expression of adipogenic differentiation markers, such as PPAR-γ and FABP4, decreased. These results show that the hypoxia environment produced by these chemicals in a suspension culture increases the proliferation of mc-MSCs and promotes the osteogenic differentiation of mc-MSCs.