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Cell co-culture microfluidic chip capable of realizing oxygen concentration gradient, and application thereof

A microfluidic chip, cell culture technology, applied in tissue cell/virus culture devices, specific-purpose bioreactors/fermenters, enzymology/microbiology devices, etc., can solve the problem of neglecting stromal cells, etc. Various cultivation methods, simple operation and good repeatability

Inactive Publication Date: 2020-03-27
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most microfluidic chips currently need to use bulky gas cylinders to generate oxygen gradients, or manufacture complex multi-layer chips for operation; and most hypoxic chips only cultivate tumor cells in the chip, ignoring the role played by the tumor microenvironment. stromal cells

Method used

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  • Cell co-culture microfluidic chip capable of realizing oxygen concentration gradient, and application thereof
  • Cell co-culture microfluidic chip capable of realizing oxygen concentration gradient, and application thereof
  • Cell co-culture microfluidic chip capable of realizing oxygen concentration gradient, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Such as figure 1 , 2 As shown, a microfluidic chip that can realize oxygen concentration gradient level cell co-cultivation includes a layer of substrate made of PDMS polymer and glass material, and the PDMS layer is composed of hypoxic channels and cell culture units; hypoxic channels It includes two liquid inlets and one liquid outlet, the liquid inlet is used to mix sodium hydroxide and pyrogallol, an oxygen gradient is generated in the area around the channel, and the remaining liquid is discharged from the liquid outlet; the cell culture unit consists of five parallel and Composed of connected channels, there are two reservoirs with a diameter of 3mm at both ends of each channel, a total of ten, used to store cell culture fluid, between the channels is a regular hexagonal micro-column array 5 with a side length of 75 μm , used to create surface tension in the passing liquid so that it can be kept in a channel, but liquid can be manually injected into the reservoir...

Embodiment 2

[0040] Determination of the change trend of oxygen concentration when different concentrations of pyrogallol are mixed with sodium hydroxide at different flow rates.

[0041] (1) Use the fluorescence method to measure the oxygen content in the chip: use tris(4,7-biphenyl-1,10-phenanthroline) ruthenium dichloride as the indicator, the fluorescence of this substance will be reduced due to the presence of oxygen Quenching, and the higher the oxygen concentration, the higher the quenching degree, so it can be used as an indicator for detecting the oxygen content in the chip. The maximum absorption wavelength of the indicator is 455nm, and the maximum emission wavelength is 613nm.

[0042] Dissolve the indicator in absolute ethanol to make a 200 μM solution, use a pipette gun to inject into the cell culture unit, and use a fluorescence microscope to photograph its fluorescence intensity.

[0043](2) Calculation of oxygen concentration and fluorescence intensity: use the Stern-Volm...

Embodiment 3

[0046] Co-cultivation of mouse hepatoma cells and hepatic stellate cells in the chip: Before the experiment, the chip was pretreated, that is, the sterilized chip was placed in an ultra-clean bench, and the chip was coated with 10 μg / mL fibronectin. L1, M and R2 channels for 1 hour to promote cell attachment and growth. Digest mouse hepatoma cells Hepa1-6 with a cell density of 80%-90% in a culture flask using trypsin, place in a 1.5mL centrifuge tube, and resuspend the cells in a high-speed centrifuge to make a volume of 3×10 6 cells / mL suspension, similarly, mouse hepatic stellate cell JS-1 was configured into 2×10 6 individual / mL suspension. Use a pipette gun to inoculate the resuspended Hepa1-6 cells in the L1 and R2 channels, inoculate the JS-1 cells in the M channel, and place the chip in the incubator for 4 hours. Cells are removed and fresh medium is added to allow the cells to grow normally, e.g. Figure 4 As shown, Hepa1-6 cells are on the left, and JS-1 cells are...

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Abstract

The invention discloses a cell co-culture microfluidic chip capable of realizing an oxygen concentration gradient. The cell co-culture microfluidic chip comprises a hypoxia channel generating an oxygen gradient, and a cell culture unit composed of five parallel channels, wherein the hypoxia channel has two liquid inlets and one liquid outlet, oxygen in the channels is absorbed by a chemical reaction generated by liquid mixing, and thereby the oxygen gradient is generated in the cell culture unit; and hexagonal micro-pillar arrays are arranged between the cell culture channels, a liquid flowingthrough can produce surface tension and cannot leak to adjacent channels, and thereby multiple cells can be cultured in different channels within the chip. The invention also provides an applicationof the microfluidic chip. The microfluidic chip can construct a rapid and stable oxygen concentration gradient in vitro, different cells are co-cultured in the parallel channels in the chip, and the microfluidic chip is used for simulation of tumor hypoxia micro-environment in vitro.

Description

technical field [0001] The invention relates to the field of microfluidic devices, in particular to a microfluidic chip for realizing oxygen concentration gradient and cell co-cultivation. Background technique [0002] Tumors grow in complex tissue environments. Including tumor cells, stromal cells, a variety of growth factors infiltrated in them, and complex physical and chemical factors, such as hypoxia, low pH, etc. The complex microenvironment of tumors plays a vital role in its development, invasion, and metastasis. The rapid proliferation of tumor cells will lead to ischemia inside the tissue, and then lead to hypoxia, and the degree of this hypoxia is related to the distance between tumor cells and blood vessels. Therefore, the oxygen concentration inside the tumor often presents a linear gradient. However, most in vitro tumor culture models ignore this hypoxic microenvironment, and some models, such as designing hypoxic chambers, anaerobic incubators, or adding sub...

Claims

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Application Information

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IPC IPC(8): C12M3/00C12M1/00
CPCC12M23/16C12M23/58
Inventor 胡坪孙威陈雨晴康璐王月荣张敏章弘扬
Owner EAST CHINA UNIV OF SCI & TECH
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