Continuous, non-invasive monitoring of oxygen consumption in a parallelized microfluidic in vitro system provides novel insight into the response to nutrients and drugs of primary human hepatocytes

Abstract

Oxygen plays a fundamental role in cellular energy metabolism, differentiation and cell biology in general. Consequently, in vitro oxygen sensing can be used to assess cell vitality and detect specific mechanisms of toxicity. In 2D in vitro models currently used, the oxygen supply provided by diffusion is generally too low, especially for cells having a high oxygen demand. In organ-on-chip systems, a more physiologic oxygen supply can be generated by establishing unidirectional perfusion. We established oxygen sensors in an easy-to-use and parallelized organ-on-chip system. We demonstrated the applicability of this system by analyzing the influence of fructose (40 mM, 80 mM), ammonium chloride (100 mM) and Na-diclofenac (50 µM, 150 µM, 450 µM, 1500 µM) on primary human hepatocytes (PHH). Fructose treatment for two hours showed an immediate drop of oxygen consumption (OC) with subsequent increase to nearly initial levels. Treatment with 80 mM glucose, 20 mM lactate or 20 mM glycerol did not result in any changes in OC which demonstrates a specific effect of fructose. Application of ammonium chloride for two hours did not show any immediate effects on OC, but qualitatively changed the cellular response to FCCP treatment. Na-diclofenac treatment for 24 hours led to a decrease of the maximal respiration and reserve capacity. We also demonstrated the stability of our system by repeatedly treating cells with 40 mM fructose, which led to similar cell responses on the same day as well as on subsequent days. In conclusion, our system enables in depth analysis of cellular respiration after substrate treatment in an unidirectional perfused organ-on-chip system.

On-chip analysis of glycolysis and mitochondrial respiration in human induced pluripotent stem cells

“…The chip was placed in a custom-made holder in the incubator (37 ​°C, 0% CO 2 ) to align 2 ​m polished plastic optical fibers (1/2.2 ​mm) with the integrated sensor spots. 32 optical fibres guided the excitation light and collected the emitted light to the 48-channel phase fluorometer (PyroScience) as a readout [ 17 ].…”

“…The sensing elements (dye and support matrix) can be miniaturized to fit microfluidic channels allowing seamless integration into the devices without interfering with the tissue [ 15 ]. Optical oxygen and pH sensors have already shown versatility within microfluidic chips [ [16] , [17] , [18] ]. They have been used to assess the OCR and ECAR of cultured cells inside microfluidic systems and the influence of drugs or nanoparticles on these parameters [ 10 , 16 ].…”

The Modern Day Heracles

“…Like our HL-HFM bioreactor, these systems are designed to include media perfusion, which has been suggested to provide oxygen levels and gradients similar to what hepatocytes encounter in vivo. [131][132][133] Hepatocytes have a high demand for oxygen to maintain their metabolic functions and different oxygen concentrations in vitro have been shown to promote or inhibit hepatocyte maturation. [131][132][133] However, microfluidic liver-on-a-chip systems offer limited oxygen availability and control compared to conventional culture vessels, due to low ratios of surface-to-volume and thus cells-to-medium.…”

“…[131][132][133] Hepatocytes have a high demand for oxygen to maintain their metabolic functions and different oxygen concentrations in vitro have been shown to promote or inhibit hepatocyte maturation. [131][132][133] However, microfluidic liver-on-a-chip systems offer limited oxygen availability and control compared to conventional culture vessels, due to low ratios of surface-to-volume and thus cells-to-medium. 134,135 Our larger scale HL-HFM system is unlikely to encounter this limitation.…”