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, lactate and carbon dioxide. Furthermore, the rate of oxidative phosphorylation (OXPHOS) can be used as a probe to evaluate mitochondira activity and carcinomatosis degree. Normal cells produce most ATP molecules 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 and an open chamber slab was easily used for the cell operation such as cell seeding and medium replacement. The position-raised microchannel can reduce the effect of shear force on the cell attachment during perform medium replacement. In this presentation, the different cell-based chips are used to construct a platform to estimate the cellular respiratory activity.Fast Measuring Cellular ActivityAdipocyte 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) detectors of three-electrode electrochemical system was developed for the measurement of DO around the cultivated cells. Adipocytes were estimated with the stimulation of different glucose concentration, insulin and mitochondria activity-controlled drugs, such as oligomycin, FCCP, rotenone and antimycin A. Method The microfluidic chip comprises a polymethylmethacrylate (PMMA) slab containing a position-raised channel-connected container and a glass substrate containing gold ultramicroelectrode to form a three-electrode electrochemical detector. The PMMA slab can be ablated to form a channel with a laser cutter, adhered with a 3M adhesive tape on the channel-side surface, and then cut to form three holes, which were acted as two containers for cell cultivation and reference-electrode solution and one outlet of channel. 50-nm Ti and 200-nm Au thin films were patterned on the glass substrate as the working electrodes, reference electrode and counter electrode. Subsequently, a 10-mm thick negative photoresist, SU8-3010, was employed to define the sensitive area of electrodes. The open container-containing PMMA slab was bond with the electrode-containing glass substrate to form the cell-based microfluidic chip. The cell suspension can be directly dripping in the working electrode container for cell seeding and cultivation. The medium replacement can be performed via the position-raised channel. The DO detector applied by a −0.8 V potential for oxygen reduction was used to estimate the oxygen consumption of cells. Results and Conclusions The result of optical images shows the adipocytes after 1-h cultivation were not removed when replacing the culture medium via the position-raised microchannel. Potential-pulse amperometry was used for the measurement of DO concentration at −0.8 V for 120 s in each measurement. The reductive current of DO is proportional to DO concentration. When applying different mitochondria activity-modulated drugs, the cell-based microfluidic chip can obtain good correlation between the oxygen consumption rate and the cellular metabolic response. 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 microfluidic chip has a great promise to fast estimate the effect of drugs on the cellular metabolic behavior in several tens of minutes cultivation, which is useful for drug screening in a short time.
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Nov 12, 2024
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