Construction Of Oxygen And Chemical Double Concentration Gradient Microfluidics And Its Application In Tumor Cell Analysis

Hypoxia is a pathological condition in which the body as a whole （generalized hypoxia）or a region of the body （tissue hypoxia） is deprived of adequate oxygen supply. A greatmany physiological and pathphysiological conditions are accompanied with hypoxia, suchas high latitude, intensive exercises, aging and tumor generation. However, in the majorityof in vitro studies nowadays, cells are cultured in incubators maintaining the internaloxygen level essentially at atmospheric oxygen levels （atmos O₂,21%O₂,）. As awell-established mediator in biological processes, oxygen is required in a wide variety ofmolecular pathways and modulates cellular function in vivo. Therefore the different oxygenconditions between in vivo and in vitro studies will result in different research results.What’s more, under physiological condition, cells are subjected to more than one kind ofconcentration. To conduct research under better simulated physiological microenvironments,it is necessary to develop reliable devices.Conventional method of setting the oxygen content is to perfuse the culture mediumwith a gas mixture of the desired concentration, but several instrumentations, such as gascylinders and oxygen analyzers are usually needed. Oxygen scavenger, such as cobaltchloride and sodium hyposulfite, are also exploited for the establishment of hypoxiacondition. At the same time, these agents have heavy metal toxicity or produce toxicproduct. Recently, Micofluidic device is increasingly served as a new platform due to bettersimulation of in vivo condition and high level integration. PDMS microfluidic devices byflowing gas mixture, gas pre-equilibrated aqueous solution or chemical solution andelectrochemically generating gases into culture chamber have been developed forgenerating oxygen gradients. However, all these developed methods can only provide onekind of concentration. In this study, we designed and fabricated a microfluidic device thatcan generation oxygen concentration gradients in culture channel by aerial oxygen andbiologically compatible oxygen scavenger Na₂SO₃. Moreover, our two circle design can create chemical concentration gradients. Therefore, this approach can simultaneouslygenerate both oxygen and chemical concentrations.In present study, we presented a novel single layer PDMS microfluidic device and amethod capable of controlling the oxygen gradients, and systematically studied theinfluence of several controllable factors （the channel geometry, operating velocities and etc.）on the formation of oxygen concentration. At the same time, the chemical concentrationgradient was established by repeated splitting-and-mixing of the source solutions in a twocircle network. So a microfluidic double concentration gradient was numericallycharacterized. Furthermore, double gradients were applied for two kinds of anticancer drugs（TPZ and BLM） induced cell death （A549and HeLa） assay. Numerical oxygenconcentration analysis indicates that a whole low oxygen concentration range from0%to21%O₂was steadily generated and oxygen concentration gradients profile can be regulatedby several controllable factors （channel geometry, operating velocities and etc）. Doubleconcentration gradients assay demonstrated that device is capable to create the same kind oflow oxygen concentration profile in each cell culture chambers and generate five differentpredictable concentrations in eight cell culture chambers. The evaluated performances ofthis system demonstrate the concentration-depended anti-cancer drugs induced cell deathand the hypoxia-induced cytotoxicity of TPZ. This indicates double gradients generationdevice can be provided for cell study.