Established clinical guidelines for treatment of severe traumatic brain injury aim at maintaining intracranial and cerebral perfusion pressures. Recently, it has been shown that additional regulation of cerebral oxygen delivery helps to decrease patient mortality and leads to improved 6-month quality-of-life scores. However, eubaric oxygen-guided therapy is still controversial since it is well known that hyperoxia can cause unwanted secondary brain injury. Research studies are warranted to better understand the range of oxygen pressures that positively influence brain cell behavior. We perform such studies using a two-enzyme in vitro system that allows exposing tissue culture cells to various steady-state, or rapidly changing, oxygen pressures. Here, we present a mathematical model of the system and its validation by real-time monitoring of oxygen tensions. We additionally present preliminary evidence that human brain macrophages have a different oxygen tolerance compared to systemic macrophages and propose improvements to our in vitro system to make it applicable for data collection that aim at refining oxygen-guided therapy for patients with traumatic brain injury.