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Researchers Submit Patent Application, "Microfluidic Array Platform for Simultaneous Cell Culture under Oxygen Tensions", for Approval

June 12, 2014

By a News Reporter-Staff News Editor at Politics & Government Week -- From Washington, D.C., VerticalNews journalists report that a patent application by the inventors Tung, Yi-Chung (Taipei, TW); Peng, Chien-Chung (Taipei, TW); Shih, Hsiu-Chen (Taipei, TW); Liao, Wei-Hao (Taipei, TW), filed on October 21, 2013, was made available online on May 29, 2014.

The patent's assignee is Academia Sinica.

News editors obtained the following quote from the background information supplied by the inventors: "Oxygen plays an essential role in biological systems, and modulates cellular functions in vivo. To date, in vitro cell culture studies have been primarily accomplished under atmospheric conditions of approximately 20% oxygen (O.sub.2). However, in a human body, cells respond to a wide range of oxygen tensions. (Decaris et al., Angiogenesis, 12:303, 2009.) For instance, normal brain oxygen levels range from 5% to 10%, and oxygen level in alveolar is about 14%. (McCord et al., Mol. Cancer. Res., 7:489, 2009.) Oxygen affects cellular response in various ways, including metabolic pathways and plasma membrane integrity. Therefore, oxygen tension plays an important role in regulating various cellular functions in both normal physiology and disease states. (Allen & Bhatia, Biotechnol. Bioeng., 82:253-262, 2003.) For example, hypoxiainducible genes regulate several biological processes, including cell proliferation, angiogenesis, metabolism, apoptosis, immortalization, and migration. Hypoxia-inducible factor-1 (HIF-1) is required in normal embryogenesis for normal vascular development at an early stage. (Harris, Nat. Rev. Cancer, 2:38, 2002.) Angiogenesis has been recognized as an essential element in tumor growth. The expression of the potent angiogenesis stimulator, vascular endothelial growth factor (VEGF), is upregulated by hypoxia. Given the above, oxygen gradient is a key factor in tumor growth and progression. (Rice & Huang, Cancer Manage. Res., 3: 9, 2011; Giordano & Johnson, Curr. Opin. Genet. Dev., 11:35, 2001.)

"Conventionally, the oxygen tensions for cell culture were controlled by direct bubbling of oxygen or nitrogen gas into the culture medium to create environments with various oxygen tensions. (Allen & Bhatia, Biotechnol. Bioeng., 82:253-262, 2003.) However, the methods often require complicated instrumentation and a large volume of gas supply. Then, a direct addition of an oxygen scavenging agent into cell culture media was utilized for oxygen tension control due to its simplicity and efficiency. But, the chemical addition would alter the medium compositions, and further affect cellular responses. (Reist et al., J. Neurochem., 71: 2431, 1998.) The aforementioned conventional methods cannot achieve oxygen gradient generation with high spatial resolution. To generate desired oxygen gradients for cell culture, a number of microfluidic devices have been developed. For example, an elastomer bioreactor capable of generating axial oxygen gradient caused by the uptake of oxygen by cells inside microfluidic channels was developed. (Mehta et al., Biomed. Microdevices, 9:123, 2007.) Some polymers with low oxygen diffusivities were exploited to construct hard top soft bottom microfluidic devices to further deplete the oxygen inside the microfluidic channels. (Mehta et al., Anal. Chem., 81:3714, 2009.) Similarly, impermeable capillaries were employed to generate mass-transfer gradients, including oxygen, inside the elastomer microfluidic device. (Pinelis et al., Biomed. Microdevices, 10:807, 2008.) To dynamically control oxygen profiles, an oxygen microgradient array (OMA) device using water electrolysis controlled by electrode patterns was developed. (Park et al., Lab Chip, 6:611, 2006.) In addition, a multi-layer microfluidic device was constructed using a gas-permeable membrane and a computer-controlled multi-channel gas mixer. The device can generate oxygen gradients with arbitrary shapes in a microfluidic device. (Adler et al., Lab Chip, 10:388, 2010.) Recently, it was first reported that oxygen scavenging liquids with a gas permeable membrane were used to control the oxygen gradients without altering cell culture medium compositions. (Skolimowski et al., Lab Chip, 10:2162, 2010.) However, the existing microfluidic cell culture devices with oxygen gradients face several challenges that hinder their practical usage in biological labs. For example, to use oxygen and nitrogen gases for oxygen gradient generation requires precise flow control instruments, tedious interconnections, and bulky gas cylinders to store compressed gases. Because the gas can easily penetrate through the permeable membrane that may cause medium evaporation and bubble generation inside the cell culture channel, the entire setup is unreliable for long-term studies, and cannot be directly implemented into the conventional cell incubators.

"A microfluidic device with a single-layer pattern to generate oxygen gradients across a microfluidic channel for cell culture was developed. (Chen et al., Lab Chip, 11: 3626-3633, 2011) The single-layer construction makes cellular microscopic observation straightforward and device fabrication easier. By confining the areas for chemical reactions, the device can control the oxygen tensions efficiently using minimal chemicals without altering the surrounding gaseous compositions. The device takes advantage of the spatially confined chemical reaction method to simultaneously generate multiple oxygen tensions for cell culture. The localized chemical reactions eliminates the crosstalk between cell culture sites, and provides the device having great compatibility of a cell incubator.

"However, it is still desirable to develop a device for simultaneous performing cell culture under various oxygen tensions."

As a supplement to the background information on this patent application, VerticalNews correspondents also obtained the inventors' summary information for this patent application: "In the present invention, a new microfluidic array platform is provided, which is capable of simultaneously performing cell culture under various oxygen tensions.

"Accordingly, in one aspect, the invention provides a microfluidic array platform for simultaneous cell culture under oxygen tensions. The microfluidic array platform comprises:

"a substrate; a membrane; two layers on the substrate, including a top layer for cell culture on the membrane, and a bottom layer for oxygen tension control underneath the membrane; wherein the membrane is sandwiched between the top layer and the bottom layer; a plurality of cell culture wells constructed in the top layer; one or more microfluidic channels constructed in the bottom layer, which are exploited for oxygen scavenging reactions or/and oxygen generating reactions to control the oxygen tensions in the cell culture wells, wherein each of the microfluidic channels has two or more separate inlets for introducing chemicals for oxygen scavenging reactions or/and oxygen generating reactions.

"In the other aspect, the present invention provides a method for simultaneous cell culture under oxygen tensions using the microfluidic array platform according to the invention.


"For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the preferred embodiments shown.

"In the drawings:

"FIG. 1(a) provides an image showing the schematic of the platform with PDMS layers of the microfluidic cell culture array according to the invention.

"FIG. 1(b) provides an image showing the fabricated platform filled with food dyes, including the chemical reactants mixing areas and the gas exchange areas; wherein the gas exchange areas with different food dye colors represents cell culture wells with different oxygen tensions.

"FIG. 2(a) shows the experimental setup for oxygen tension characterization based on the relative fluorescence lifetime measurement of an oxygen sensitive dye.

"FIG. 2(b) shows the experimentally estimated oxygen tensions generated inside a cell culture well at the surface on which cells are cultured using 1M NaOH and pyrogallol at flow rates of 20 .mu.l min.sup.-1 at various concentrations.

"FIG. 3(a) provides an image showing the fabricated microfluidic array platform after cell culture with drug treatments and PrestoBlue cell viability assay under various oxygen tensions on a single chip.

"FIG. 3(b) shows the relative A549 cell viabilities (normalized to the cell cultured in the growth medium under normoxia for 12 hours) after 12 hours hypoxia-activated anti-cancer drug (TPZ) treatments under various oxygen tensions using the array platform according to the invention, wherein the data were expressed as the mean.+-.SD.

"FIG. 4 shows a comparison of cell viabilities under various drug treatments in normoxia and hypoxia using the cell culture arrays according to the invention and conventional 96-well plate, wherein the data are expressed as the mean.+-.s.e.m. (n=4 for the device experiments; n=8 for the well plate experiments).

"FIG. 5 shows the results of the normalized cell viability (PrestoBlue) experiments of the cells under various drug treatment and oxy using the device according to the invention (control and drug treatments under multiple oxygen tensions), which shows Presto color variation after reacting with cell culture medium with different living cell populations."

For additional information on this patent application, see: Tung, Yi-Chung; Peng, Chien-Chung; Shih, Hsiu-Chen; Liao, Wei-Hao. Microfluidic Array Platform for Simultaneous Cell Culture under Oxygen Tensions. Filed October 21, 2013 and posted May 29, 2014. Patent URL:

Keywords for this news article include: Cancer, Genetics, Oncology, Chemicals, Chemistry, Chalcogens, Academia Sinica.

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Source: Politics & Government Week

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