A New Approach for On-Demand Generation of Various Oxygen Tensions for In Vitro Hypoxia Models

Abstract

The development of in vitro disease models closely mimicking the functions of human disease has captured increasing attention in recent years. Oxygen tensions and gradients play essential roles in modulating biological systems in both physiologic and pathologic events. Thus, controlling oxygen tension is critical for mimicking physiologically relevant in vivo environments for cell, tissue and organ research. We present a new approach for on-demand generation of various oxygen tensions for in vitro hypoxia models. Proof-of-concept prototypes have been developed for conventional cell culture microplate by immobilizing a novel oxygen-consuming biomaterial on the 3D-printed insert. For the first time, rapid (~3.8 minutes to reach 0.5% O2 from 20.9% O2) and precisely controlled oxygen tensions/gradients (2.68 mmHg per 50 μm distance) were generated by exposing the biocompatible biomaterial to the different depth of cell culture media. In addition, changing the position of 3D-printed inserts with immobilized biomaterials relative to the cultured cells resulted in controllable and rapid changes in oxygen tensions (<130 seconds). Compared to the current technologies, our approach allows enhanced spatiotemporal resolution and accuracy of the oxygen tensions. Additionally, it does not interfere with the testing environment while maintaining ease of use. The elegance of oxygen tension manipulation introduced by our new approach will drastically improve control and lower the technological barrier of entry for hypoxia studies. Since the biomaterials can be immobilized in any devices, including microfluidic devices and 3D-printed tissues or organs, it will serve as the basis for a new generation of experimental models previously impossible or very difficult to implement.

A microfluidic-based lid device for conventional cell culture dishes to automatically control oxygen level

“…Meanwhile, the degradation is prevented under hypoxic conditions [21]. Furthermore, the level of HIF-1 (ranging from 0.75 to 2.88) gradually increased as pO 2 decreased (ranging from 0.186 to 0atm), which is also concordant with the inverse proportion of HIF-1 to oxygen levels observed in other types of hypoxic culture systems (Figure2C) [7,13]. Therefore, the hypoxic culture using the device does not disturb the natural cellular response to hypoxia, and is applicable to hypoxia-related studies, including HIF-1 and its downstream cascade.…”

Enzyme-immobilized hydrogels to create hypoxia for in vitro cancer cell culture

“…This system has been used to induce hypoxia in solutions and in microfluidic devices (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). The use of GOX/CAT is beneficial in that the system provides a rapid onset of hypoxia (usually within a few minutes) (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013;Zitta et al, 2012). One drawback to any GOX system, however, is the production of hydrogen peroxide, a reactive oxygen species (ROS) (Fruehauf and Meyskens, 2007) whose accumulation would not only cause undesired cellular response but also inactivate both GOX and CAT (Hielscher and Gerecht, 2015;Pal et al, 2000;Trachootham et al, 2009;Tse and Gough, 1987).…”

“…Some recent work has started to explore the ability of GOX/CAT reactions to induce hypoxia for in vitro cell culture (Askoxylakis et al, 2011;Baumann et al, 2008;Huang et al, 2013;Li et al, 2016;Millonig et al, 2009;Mueller et al, 2009;Rajan et al, 2013;Sobotta et al, 2013). The GOX/CAT system has also been adapted to 3D printed inserts (Li et al, 2016) where GOX and CAT were coated on printed disks and the degrees of solution hypoxia were controlled by the distance between the enzyme-immobilized disks and the solution in the culture plate.…”

“…The GOX/CAT system has also been adapted to 3D printed inserts (Li et al, 2016) where GOX and CAT were coated on printed disks and the degrees of solution hypoxia were controlled by the distance between the enzyme-immobilized disks and the solution in the culture plate. In that design, hypoxia conditions (between 0 and ~12% O 2 ) were maintained for up to 5 hours and the system was used to induce hypoxic response in peritoneal macrophages.…”

Finite Element Model of Oxygen Transport for the Design of Geometrically Complex Microfluidic Devices Used in Biological Studies

“…Various studies in the literature have developed means of controlling O 2 in microfluidic devices for a variety of applications [12–19], e.g. microfluidic devices for establishing hypoxia in cell cultures [12].…”