Measuring and regulating oxygen levels in microphysiological systems: design, material, and sensor considerations

Abstract

As microfabrication techniques and tissue engineering methods improve, microphysiological systems (MPS) are being engineered that recapitulate complex physiological and pathophysiological states to supplement and challenge traditional animal models. Although MPS provide unique microenvironments that transcend common 2D cell culture, without proper regulation of oxygen content, MPS often fail to provide the biomimetic environment necessary to activate and investigate fundamental pathways of cellular metabolism and sub-cellular level. Oxygen exists in the human body in various concentrations and partial pressures; moreover, it fluctuates dramatically depending on fasting, exercise, and sleep patterns. Regulating oxygen content inside MPS necessitates a sensitive biological sensor to quantify oxygen content in real-time. Measuring oxygen in a microdevice is a non-trivial requirement for studies focused on understanding how oxygen impacts cellular processes, including angiogenesis and tumorigenesis. Quantifying oxygen inside a microdevice can be achieved via an array of technologies, with each method having benefits and limitations in terms of sensitivity, limits of detection, and invasiveness that must be considered and optimized. This article will review oxygen physiology in organ systems and offer comparisons of organ-specific MPS that do and do not consider oxygen microenvironments. Materials used in microphysiological models will also be analyzed in terms of their ability to control oxygen. Finally, oxygen sensor technologies are critically compared and evaluated for use in MPS.

Fig.1

Schematic representation of oxygen content inside organs. The human body is composed of numerous oxygen gradients and organ-specific microenvironments that require microphysiological systems to study oxygen exchange in a tunable and controllable manner. The brain contains neurons, astrocytes, and pericytes situated above and within the basal lamina. Oxygen crosses the endothelium to reach brain cells via the capillary lumen. A tumor creates new vasculature as it grows (neoangiogenesis), while the core of the tumor continues to become more hypoxic. The lung receives oxygen in air, which then binds to hemoglobin molecules after crossing through the epithelium and interstitial space. For the liver, oxygen rich blood travels from the portal vein toward the central vein, providing nutrients to sinusoidal endothelial cells, stellate cells in the perisinusoidal space, and hepatocytes. The kidney contains oxygen gradients within each renal medulla and from the inner papilla to the renal cortex. The intestine has a steep oxygen gradient from the microvasculature underneath the crypt-villus axis to the bacteria populated lumen. The skin is an oxygen barrier with the epidermis at very low oxygen. The heart contains numerous levels of oxygen as oxygen-poor blood flows into the vena cava and oxygen-rich blood exits via the aorta.

Fig.2

Methods for controlling oxygen in microphysiological systems (MPS) to study cellular responses. (a) Gas diffusion used to generate an oxygen gradient across a layer of Madin-Darby Canine Kidney (MDCK) cells. Reproduced from Ref. 27 with permission from The Royal Society of Chemistry, copyright 2010. (b) Gas filled channels create an oxygen gradient from 0% to 20% oxygen. Reproduced from Ref. with permission from The Royal Society of Chemistry, copyright 2012. (c) Oxygen scavengers in leftmost chemical reaction channel remove oxygen, while oxygen generators in the rightmost channel create a gradient of oxygen between the chemical reaction channels, inside the cell culture channel. Reproduced from Ref. 22 with permission from The Royal Society of Chemistry, copyright 2011. (d) Oxygen diffused through collagen hydrogels of various height and fiber density to create oxygen gradients. Reproduced from Ref. 85 with permission from The Royal Society of Chemistry, copyright 2017. (e) Cells consume oxygen inside the liver MPS to create oxygen-limited environment. Reproduced from Ref. 49 with permission from The Royal Society of Chemistry, copyright 2010. (f) Transwell insert contains Parylene coating to prevent environmental oxygen from contaminating inner hypoxic device. Reproduced from Ref. 115 with permission from The Royal Society of Chemistry, copyright 2010.

Fig.3

Measuring oxygen in microdevices. (a) Schematic of non-Clark type amperometric oxygen sensing. An optional membrane or coating can be applied over the electrodes to limit fouling or cell-electrode interactions. An oxygen-selective membrane can also be integrated to achieve a Clark-type system, i.e. using only an anode and cathode. (b) An example of amperometric oxygen sensing. (Top) Schematic of the fabricated microfluidic device. (Bottom, left) Fabricated microfluidic device with integrated electrochemical O₂ sensors. (Bottom, right) Amperometric continuous oxygen measurement. Reproduced from Ref. 123 with permission from The Royal Society of Chemistry, copyright 2014. (c) Schematic of optical oxygen sensing. (d) An example of optical oxygen sensing. (Top, left) Schematic of the fabricated microfluidic device. (Top, right) Fabricated microfluidic device with integrated optical O2 sensors. (Bottom) Fluorescent images of the microbeads doped with two dyes, oxygen sensitive ruthenium dye and oxygen-irresponsive Nile blue, to 21% and 7% oxygen concentrations. Reproduced from Ref. 243 with permission from The Royal Society of Chemistry, copyright 2015.

Fig. 4

Common oxygen sensing via luminescent optical dyes. (a-d) Chemical structures of Ru(II) complexes and metalloporphyrins. M stands for Pt(II) or Pd(II) metal ions.