Compartmentalized organ-on-a-chip structure for spatiotemporal control of oxygen microenvironments

Hypoxia is a condition where tissue oxygen levels fall below normal levels. In locally induced hypoxia due to blood vessel blockage, oxygen delivery becomes compromised. The site where blood flow is diminished the most forms a zero-oxygen core, and different oxygenation zones form around this core with varying oxygen concentrations. Naturally, these differing oxygen microenvironments drive cells to respond according to their oxygenation status. To study these cellular processes in laboratory settings, the cellular gas microenvironments should be controlled rapidly and precisely. In this study, we propose an organ-on-a-chip device that provides control over the oxygen environments in three separate compartments as well as the possibility of rapidly changing the corresponding oxygen concentrations. The proposed device includes a microfluidic channel structure with three separate arrays of narrow microchannels that guide gas mixtures with desired oxygen concentrations to diffuse through a thin gas-permeable membrane into cell culture areas. The proposed microfluidic channel structure is characterized using a 2D ratiometric oxygen imaging system, and the measurements confirm that the oxygen concentrations at the cell culture surface can be modulated in a few minutes. The structure is capable of creating hypoxic oxygen tension, and distinct oxygen environments can be generated simultaneously in the three compartments. By combining the microfluidic channel structure with an open-well coculture device, multicellular cultures can be established together with compartmentalized oxygen environment modulation. We demonstrate that the proposed compartmentalized organ-on-a-chip structure is suitable for cell culture. Supplementary Information The online version contains supplementary material available at 10.1007/s10544-022-00634-y.


Structural dimension characterization
Table S1 Microfluidic channel heights in the multilayer SU-8 mold measured with a contact profilometer. Four different positions from the mold were measured (n = 4), with the table representing the averages and standard deviations of these measurements.

Average height (µm)
Standard deviation (µm) Microchannel array 2.14 0.07 Inlet and outlet channels together with bypass channels 104.59 0.69 Fig. S1 PDMS microchannel array and (a) acquired SEM image of the 2 µm wide channels with 2 µm spacing (indicated as white bars)

Numerical simulations
In cell culture experiments, the developed microfluidic gas channel structure is attached below an opencompartment coculture device (see Fig. 2b). The device has three compartments connected to each other with microtunnels (Ristola et al. 2019). In cell culture experiments, the compartments are filled with cell culture medium and the cells grow at the bottom of the compartments on a 20-µm thick Silpuran membrane. The microfluidic gas channel structure is located below the membrane. To study how the microtunnels between the compartments affect oxygen profiles in each compartment, we developed a 2D finite-element model using COMSOL Multiphysics (Version 6.0 COMSOL, Inc., Burlington, USA). In the simulations, we applied The Transport of Diluted Species interface and used Fick's law as the governing equation to calculate the oxygen concentration profiles with and without the microtunnel structure.
In the device, oxygen is stored in gas, liquid, and solid (PDMS in this case) phases. For this, mass balance, oxygen concentration, and flux in the three phases (gas, liquid, and PDMS denoted with g, l and p, respectively) are calculated using the following equations. Similar modeling approach has been used earlier to model carbon dioxide concentrations and fluxes (Mäki et al., 2015;Mäki 2018). By assuming no oxygen consumption nor convection in the system, the following mass transportation equations based purely on diffusion can be used for describing a mass balance in the system where c and D are the concentrations and diffusion coefficients of oxygen in different phases.
Between the interfaces of the three phases, the rate constants of oxygen mass transport are denoted as follows.
⇋ , ⇋ , ⇋ 2 where subscripts define source and destination phases; for example, gl refers to the rate constant from gas phase to liquid phase, whereas lg refers the opposite direction.
In a steady state, the dimensionless partition coefficients between two phases, Kp, are calculated using the saturated oxygen concentration values in each phase (Shiku et al., 2006;Skolimowski et al., 2010) Oxygen fluxes Fl between two phases can be described as (Shiku et al., 2006;Skolimowski et al., 2010 Simulations with and without microtunnels were performed using parameters presented in Table S2. The results, presented in the following Figures S2-S4, indicate that microtunnels are only minimally affecting oxygen concentrations.   Figure S3. Blue lines indicate boundary conditions applied in the model. In the simulation, 19% oxygen was applied to the microfluidic gas channel below Compartment B and 0% oxygen below Compartment A, water is used as the liquid, ambient air as the gas and PDMS as the solid