Microfabrication of cell culture microenvironments with spatially controlled oxygen levels

In vivo cell microenvironments are characterized by gradients of soluble molecules, particularly oxygen, whose levels depend on the distance from blood vessels and cellular metabolic activity. In pathological conditions, oxygen levels often decrease, leading to hypoxia and steeper concentration gradients. Standard in vitro cultures provide homogeneous conditions that poorly replicate the in vivo environment. This work presents the microfabrication of devices that generate cell culture microenvironments characterized by three-dimensional oxygen gradients that resemble those found in vivo. We patterned the material as a hydrogel to create oxygen gradients, thanks to immobilized proteins that regulate oxygen. We developed a digital inkjet printing procedure based on two reactive inks to generate patterns. The resulting spatial oxygen concentrations were quantified using threedimensional electrochemical mapping. Living cells were grown and observed in the in vivo-mimetic oxygen gradients, and the growth of malignant cells was consistent with adaptation to hypoxic conditions.