Precise and fast control of the dissolved oxygen level for tumor-on-chip.

ABSTRACT

In vitro cell cultures are most often performed in unphysiological hyperoxia since the oxygen partial pressure of conventional incubators is set at 141 mmHg (18.6%, close to ambient air oxygen 20.1%). This value is higher than human tissue oxygen levels, as the in vivo oxygen partial pressures range from 104 mmHg (lung alveoli) to 8 mmHg (skin epidermis). Importantly, under pathological conditions such as cancer, cells can experience oxygen pressure lower than the healthy tissue. Although hypoxic incubators can regulate gas oxygen, they do not take into account the dissolved oxygen concentration in the cell culture medium. In the context of organ on chip and micro-physiological system development, we present here a new system, called Oxalis (OXygen ALImentation System) that allows fine control of the dissolved oxygen level in the cell culture medium. Oxalis regulates simultaneously the gas composition and the inlet reservoir pressure by modulating the pneumatic valve opening. This dual regulation allows both the pressure driven liquid flowrate and the level of oxygen dissolved in the chip to be controlled independently. Oxalis offers unprecedented features such as an oxygen equilibration time lower than 3 minutes and an accuracy of 3 mmHg. These performances can be reached for chip perfusion flow as low as 1 µL min-1. This low flow rate allows the shear stress experienced by the cells in the chip to be accurately controlled. In addition, the system enables modulation of the pH in the cell culture medium through the modulation of CO2. The fine control and monitoring of both O2 and pH pave the way for new precise investigations on physiological and pathological biological processes. Using Oxalis in the context of tumor-on-chip, we demonstrate the capacity of the system to recapitulate hypoxia-induced gene expression, offering an innovative strategy for future studies on the role of hypoxia in malignant progression and drug resistance.