Fine temporal control of the medium gas content and acidity and on-chip generation of series of oxygen concentrations for cell cultures.
We describe the design, operation, and applications of two microfluidic devices that generate series of concentrations of oxygen, [O(2)], by on-chip gas mixing. Both devices are made of polydimethylsiloxane (PDMS) and have two layers of channels, the flow layer and the gas layer. By using in-situ measurements of [O(2)] with an oxygen-sensitive fluorescent dye, we show that gas diffusion through PDMS leads to equilibration of [O(2)] in an aqueous solution in the flow layer with [O(2)] in a gas injected into the gas layer on a time scale of approximately 1 sec. Injection of carbon dioxide into the gas layer causes the pH in the flow layer to drop within approximately 0.5 sec. Gas-mixing channel networks of both devices generate series of 9 gas mixtures with different [O(2)] from two gases fed to the inlets, thus creating regions with 9 different [O(2)] in the flow layer. The first device generates nitrogen-oxygen mixtures with [O(2)] varying linearly between 0 and 100%. The second device generates nitrogen-air mixtures with [O(2)] varying exponentially between 0 and 20.9%. The flow layers of the devices are designed for culturing bacteria in semi-permeable microchambers, and the second device is used to measure growth curves of E. coli colonies at 9 different [O(2)] in a single experiment. The cell division rates at [O(2)] of 0, 0.2, and 0.5% are found to be significantly different, further validating the capacity of the device to set [O(2)] in the flow layer with high precision and resolution. The degree of control of [O(2)] achieved in the devices and the robustness with respect to oxygen consumption due to respiration would be difficult to match in a traditional large-scale culture. The proposed devices and technology can be used in research on bacteria and yeast under microaerobic conditions and on mammalian cells under hypoxia.
Department of Physics, University of California, San Diego, , 9500 Gilman Drive, MC 0374, La Jolla, CA 92093, USA.
Mark
M
Polinkovsky
Edgar
E
Gutierrez
Andre
A
Levchenko
Alex
A
Groisman
D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D003470:Culture Media; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D009584:Nitrogen / Q000032:analysis; D010100:Oxygen / Q000032:analysis*; D012996:Solutions
D003470:Culture Media; D004129:Dimethylpolysiloxanes; D012996:Solutions; C013830:baysilon; D009584:Nitrogen; D010100:Oxygen