The Art of Inducing Hypoxia

Many cells in the human body strongly react on decreased oxygen concentrations, generally defined as hypoxia. Therefore, inducing hypoxia in vitro is essential for research. Classically, hypoxia is induced using a hypoxia chamber, but alternative methods exist that do not require special equipment. Here, we compared three different methods to induce hypoxia without a hypoxia chamber: the chemical stabilization of HIF-1α by CoCl2, the decrease in pericellular oxygen concentrations by increased media height, and the consumption of oxygen by an enzymatic system. Hypoxia induction was further analyzed within three different cell culture systems: 2D (adherent) osteoprogenitor cells, monocytic (suspension) cells, and in a 3D in vitro fracture hematoma model. The different methods were analyzed within the scope of fracture healing regarding inflammation and differentiation. We could show that all three induction methods were feasible for hypoxia induction within adherent cells. Increased media heights did not stimulate a hypoxic response within suspension cells and in the 3D system. Chemical stabilization of HIF-1α showed limitations when looking at the expression of cytokines in osteoprogenitors and monocytes. Enzymatic reduction of oxygen proofed to be most effective within all three systems inducing inflammation and differentiation.