(Invited) Development of Cell-Based Microfluidic Chip for the Estimation of Cell Activity
Authors: Wu Ching-Chou , Wang Chieh-Jen , Yan Jia-De , Chang Lee-Tian , Chang Jui-Chih 
Published: May 2020

Introduction

Cells intake nutrients and oxygen to produce cellular energy (adenosine triphosphate, ATP) and simultaneously excrete acidic waste and carbon dioxide. Cell activity can be estimated by measuring cellular metabolism-related molecules, such as oxygen and proton. Furthermore, the rate of oxidative phosphorylation (OXPHOS) can be used as a probe to evaluate carcinomatosis degree. Normal cells produce ATP via OXPHOS, but cancer cells mainly produce ATP via cytosol glycolysis, which consumes less oxygen and produces more protons. Therefore, to measure the consuming rate of oxygen and the change in extracellular pH values is meaningful for the estimation of cell activity and carcinomatosis degree. In our previous studies [1-4], Clark and non-Clark oxygen sensing chips were designed and fabricated for estimating the respiratory activity of mammalian cells, such as HeLa cells [3]. The time-lapse monitoring shows that the respiratory activity of HeLa cells increases with the cultivation time. Moreover, an oxygen electrode array integrated with a microfluidic channel and microstructures can be constructed to measure the oxygen consumption rate of single bovine embryo for the estimation of embryo development [2]. It is worth noting that the microfluidic flow of cell-based chip may induce a shear force or a viscous force to move the attached cell away from the detecting electrodes during medium replacement. In our previous study, a cell-based chip consisting of position-raised microchannel, an open chamber slab and a IrOx-deposited microelectrode-containing substrate was developed for the measurement of extracellular acidification. The position-raised microchannel can reduce the effect of shear force on the cell attachment during perform medium replacement. The design of cell-based chip was used to construct a platform to estimate the mitochondria activity of adipocytes and monocytes. In this presentation, I will introduce the recent progress of cell-based chip in the applications of estimating mitochondria activity.

Measuring the activity of low cell-matrix adhesion-type cells

Adipocyte activity determines the metabolism of carbohydrate and fatty acid of human beings, related to the formation of diabetes. Evaluation of adipocyte activity allows the researchers to realize the causes of type II diabetes and therapeutic methods. In the study, a microfluidic chip containing dissolved oxygen (DO) sensors of three-electrode electrochemical system was developed for the measurement of DO around the cultivated adipocytes. Moreover, the iridium oxide (IrOx) layer was electrodeposited on a gold electrode as the reference electrode. Adipocytes were estimated with the stimulation of different glucose concentration, insulin and mitochondria activity-controlled drugs, such as oligomycin, FCCP, rotenone .

Method

The microfluidic chip comprises a poly(dimethylsiloxane) (PDMS) slab containing a microchannel-connected container and a glass substrate containing IrOx-electrodeposited gold ultramicroelectrode. The schematic cross-section structure of chip is shown in Fig. 1. The PDMS slab is constructed for the purpose of cell culture and fluidic control of medium by means of the open container and the position-raised microchannel, respectively. A 50-mm thick PDMS membrane prepared by spin-coating on polyacrylate substrate was irreversibly bonded with the bottom side of PDMS microchannel replica using oxygen plasma treatment to act as a spacer for raising the position of the microchannel. Furthermore, the PDMS spacer locating between the open container and the reference electrode setting container can increase the liquid-junction area between the DO electrodes and the IrOx reference electrode. Finally, a Teflon tubing was connected to the outlet of position-raised microchannel and the other end of tubing was connected with a pump. 50-nm Ti and 250-nm Au thin films were patterned on the glass substrate as the working electrodes. Subsequently, a 10-mm thick negative photoresist, SU8-3010, was employed to define the sensitive area of electrodes. Furthermore, a self-made plexiglass clamp was used to enhance the fixation between the PDMS slab and the glass substrate.

Results and Conclusions

Figure 2 shows the optical images of adipocytes before and after medium replacement. The result shows the low-adhesion-type adipocyte don’t move during the fluidic replacement via the position-raised microchannel. A pulse amperometry was used for the measurement of DO concentration at -0.7 V for 120 s with 5 min time interval, shown in Fig. 3. The reductive current of DO is proportional to DO concentration. When applying different mitochondria activity-modulated drugs, the oxygen consumption rate was good correlation with cellular physiological behavior. For example, oligomycin is a ATPase inhibitor, which decreased the oxygen consumption rate (OCR). FCCP is an uncoupling protein, dissipating proton gradient, to increase mitochondria activity, which increases OCR. The DO microfluidic chip has a great promise in the application of estimating the effect of drugs on the cellular physiological behavior to replace animal and clinical experiments, and estimate the mitochondria activity to realize the degree and metabolic aging.

References

[1] C.-C.Wu, T. Yasukawa, H. Shiku, T. Matsue, Fabrication of miniature Clark oxygen sensor integrated with microstructure, Sensors and Actuators B 110 (2005) 342–349. doi.org/10.1016/j.snb.2005.02.014.
[2] C.-C.Wu, T. Saito, T. Yasukawa, H. Shiku, H. Abe, H. Hoshi, T. Matsue, Microfluidic chip integrated with amperometric detector array for in situ estimating oxygen consumption characteristics of single bovine embryos, Sensors and Actuators B 125 (2007) 680–687. doi.org/10.1016/j.snb.2007.03.017.
[3] C.-C. Wu, H.-N. Luk, Y.-T. Tsai Lin, C.-Y. Yuan, A Clark-type oxygen chip for in situ estimation of the respiratory activity of adhering cells, Talanta 81 (2010) 228–234. doi: 10.1016/j.talanta.2009.11.062
[4] C.-C.Wu, W.-C. Lin, S.-Y. Fu, The open container-used microfluidic chip using IrOx ultramicroelectrodes for the in situ measurement of extracellular acidification, Biosensors and Bioelectronics 26 (2011) 4191–4197. doi.org/10.1016/j.bios.2011.04.034

Figure 1