Abstract
Controlling oxygen concentration at a microscale level can benefit experimental investigations involving oxidative stress, ischemia, and reactive oxygen species (ROS) mediated cellular pathways. Here, we report the application of microfluidic gradient generation in an open-well culture model, in which a gradient of gas is delivered via diffusion through a gas permeable substrate that separates cells from the gas microchannels below. By using diffusion to localize oxygen delivery, microgradients of oxygen concentrations can be rapidly and controllably applied without exposing cells to mechanical stresses or reducing culture volumes inside microfluidic culture chambers. Furthermore, we demonstrate the modulation of intracellular ROS levels in Madin-Darby Canine Kidney (MDCK) cells by applying these oxygen microgradients. Increases in ROS levels consistent with both oxidative stress and hypoxic exposures were observed in MDCK cells. The measured ROS increases were comparable to 100 microM hydrogen peroxide exposure in a control comparison, which is within the range of standard ROS induction methods. Incubation with 200 microM vitamin C was able to demodulate the ROS response at both hypoxic and hyperoxic exposures. By providing microfluidic controlled gradients, constant ROS exposure, and a shear-free open well design, the devices introduced here greatly improve upon standard oxygen-based culturing methods.
Highlights
Molecular oxygen is critical in many cellular pathways involving careful homeostatic balance in order to maintain growth, proliferation, and controlled cell death
The range of oxygen concentrations provided by the parallel channel Microgradient Cell Culture Platform (MCCP) was 7–97%
In addition to the linear and sigmoidal profiles achieved in these devices, future modifications can be made to the channel flow [22] and resistances [28] to provide a number of distinct oxygen profiles
Summary
Molecular oxygen is critical in many cellular pathways involving careful homeostatic balance in order to maintain growth, proliferation, and controlled cell death. ROS is implicated in stabilizing HIF-1α during hypoxia [6], as well as in providing deleterious radicals in inflammatory and hyperoxic conditions [7,8,9] Because these oxidative mechanisms overlap both hypoxic and hyperoxic regimes, a gradient-based assay is required to probe oxygen as a controlled and dosedependent variable. To illustrate such an assay, we applied oxygen microgradients to systematically modulate the ROS levels in a cell culture-based platform without using exogenous chemicals such as hydrogen peroxide, which is the current gold standard for these types of applications. This gradient-based approach enables new experimental protocols previously impossible or very difficult to implement with standard cell culture methods
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