CN110923138A - Cell co-culture micro-fluidic chip capable of realizing oxygen concentration gradient and application thereof - Google Patents
Cell co-culture micro-fluidic chip capable of realizing oxygen concentration gradient and application thereof Download PDFInfo
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Abstract
The invention discloses a cell co-culture micro-fluidic chip capable of realizing oxygen concentration gradient, which comprises an oxygen-deficient channel for generating oxygen gradient and a cell culture unit consisting of five parallel channels; the oxygen-poor channel is provided with two liquid inlets and one liquid outlet, and oxygen in the channel is absorbed through chemical reaction generated by liquid mixing, so that an oxygen gradient is generated in the cell culture unit; the hexagonal micro-column array is arranged between the cell culture channels, and the liquid flowing through the hexagonal micro-column array can generate surface tension without leaking to the adjacent channels, so that a plurality of cells can be cultured in different channels in the chip. The invention also provides an application of the microfluidic chip. The invention can construct a rapid and stable oxygen concentration gradient in vitro, and co-culture different cells in parallel channels in a chip for simulating an in vitro tumor hypoxia microenvironment.
Description
Technical Field
The invention relates to the field of microfluidic devices, in particular to a microfluidic chip for realizing oxygen concentration gradient and cell co-culture.
Background
Tumors grow in a complex tissue environment. Including tumor cells, stromal cells, various growth factors infiltrated therein, and complex physicochemical factors such as hypoxia, low pH, etc. The complex microenvironment of a tumor plays a crucial role in its development, invasion, and metastasis. The rapid proliferation of tumor cells leads to ischemia inside the tissue, and thus to hypoxia, the degree of which is related to the distance of the tumor cells from the blood vessels, and therefore, the oxygen concentration inside the tumor tends to be in a linear gradient. However, most in vitro tumor culture models ignore such hypoxic microenvironments, and some models, such as by designing hypoxic chambers, anaerobic incubators, or adding substances capable of absorbing oxygen, such as sodium dithionite, sodium cobaltous chloride, etc., to the cell culture medium, can maintain the tumor cells in a hypoxic state, but cannot form a stable oxygen gradient, so that the in vitro model is not accurate.
The microfluidic technology has the characteristics of integration, flexible design, high flux, low sample consumption and the like, and meanwhile, the PDMS material commonly used for manufacturing the chip has good air permeability, high transparency and good biocompatibility, so that the micro-fluidic chip is widely applied to the fields of cell biology and medicine. In addition, the micro-fluidic technology can accurately control external physical and chemical parameters, and the cells are subjected to patterned culture through design, so that the real spatial arrangement of in-vivo tumors is more approximate, and a more accurate in-vitro tumor model is established. At present, there is a report of generating an oxygen concentration gradient based on a microfluidic technology so as to simulate an in vitro tumor hypoxia microenvironment. However, most of the current microfluidic chips need to use a heavy gas steel cylinder to generate oxygen gradient or manufacture a complex multilayer chip for operation; and most hypoxic chips only culture tumor cells in the chip, ignoring stromal cells that play a critical role in the tumor microenvironment.
Therefore, it is necessary to combine oxygen gradient with cell co-culture technology in a microfluidic chip to establish an in vitro culture model closer to the tumor microenvironment.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a microfluidic chip capable of realizing oxygen concentration gradient and cell co-culture. The invention also provides an application of the microfluidic chip.
The invention has the technical scheme that the cell co-culture micro-fluidic chip capable of realizing oxygen gradient comprises an oxygen-deficient channel 1 for generating oxygen gradient and a cell culture unit 2 arranged on one side of the oxygen-deficient channel 1;
the anoxic channel 1 comprises two liquid inlets 3 and a liquid outlet 4, and the two liquid inlets are converged into a pipeline to be communicated with a main pipeline of the anoxic channel;
the cell culture unit 2 comprises a plurality of parallel channels which are arranged in parallel and are formed by a plurality of micro-column arrays at intervals; two ends of the parallel channel are respectively connected with a liquid storage tank 6 through necessary pipelines.
The oxygen-poor channel is provided with two liquid inlets 3 and a liquid outlet 4, and oxygen in the channel is absorbed through chemical reaction generated by liquid mixing; i.e. one parallel channel with two reservoirs. The pipeline of the parallel channel and the liquid storage tank is folded at a certain angle relative to the parallel channel.
According to the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient, preferably, the number of the parallel channels of the cell culture unit 2 is 4-6; the cross section of each micro-column in the micro-column array is polygonal.
Further, the cross section of the micro-column array is a regular polygon. Further, the shape is a regular hexagon.
According to the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient, the material of the chip is preferably PDMS, and the height of the chip is 80-120 μm.
According to the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient, an S-shaped folding buffer pipeline area is preferably arranged between a liquid inlet and a main pipeline of an oxygen-deficient channel in the oxygen-deficient channel. Its function is to promote thorough mixing of the liquids.
According to the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient, preferably, in the oxygen-deficient channel, the width of a liquid inlet channel is 200-400 μm; each channel of the cell culture unit is 100-800 μm wide. The width of the liquid inlet channel is 200-400 μm, and the improved scheme can make the mixing more uniform; the width of each channel of the cell culture unit is 100-800 μm, and the improved scheme can transfer the oxygen gradient to one channel but not affect all the channels.
According to the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient, preferably, in the oxygen-deficient channel, two liquid inlets are converged into a pipeline, and a triangular baffle plate array 7 is arranged at the pipeline.
The triangular baffle plate array is composed of a plurality of triangular baffle plates, and the distance between every two baffle plates is 50-150 mu m.
After the two fluids are converged, the two fluids flow through the triangular baffle plate array to generate vortex, so that the mixing of the fluids is promoted. The improved scheme can improve the mixing efficiency of the two reactants, thereby enlarging the range of the oxygen gradient.
Further, the triangular baffle is in a regular triangle shape.
According to the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient, a PDMS film is preferably arranged between the oxygen-deficient channel and the cell culture unit. This arrangement prevents the reaction solution of the anoxic channel from entering the cell culture unit and facilitates the diffusion of oxygen.
Because pyrogallol can absorb ambient oxygen under alkaline conditions, an alkaline environment is made by using sodium hydroxide, and PDMS has good air permeability, oxygen in a cell culture channel is consumed by the pyrogallol in an adjacent anoxic channel through the PDMS, so that an oxygen gradient is formed.
Further, the thickness of the PDMS film is 80-120 μm.
According to the cell co-culture microfluidic chip capable of realizing the oxygen gradient, the side length of the micro-columns in the micro-column array in the cell culture unit is preferably 80-120 microns, and each micro-column is 80-120 microns apart. The improved scheme increases the side length of the regular hexagon before optimization from 75 mu m to about 100 mu m, and avoids flowing fluid from flowing into an adjacent channel.
According to the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient, the liquid storage tank of the cell culture unit is preferably a cylinder, and the height of the liquid storage tank is equal to the thickness of the chip.
Further, the diameter of the circle on the bottom surface of the cylinder is 2-4 mm.
The invention also provides the application of the cell co-culture micro-fluidic chip capable of realizing the oxygen gradient in the aspect of tumor cell drug resistance under the anoxic condition. In this case, the two liquids at the two liquid inlets are sodium hydroxide and pyrogallol, respectively.
The invention has the beneficial effects that:
the invention optimizes the positions of the liquid inlet and the liquid outlet of the anoxic channel, so that the catheter is easier to insert, and the stability of an anoxic experiment is ensured.
The micro-fluidic chip provided by the invention uses the mixture of two chemical substances, so that a stable oxygen gradient in time and space is manufactured in the micro-fluidic chip, and the micro-fluidic chip can be used for co-culturing cancer cells and stromal cells in channels, researching the paracrine action of cells and simulating the tumor hypoxia microenvironment more accurately.
Compared with the prior art, the scheme does not need heavy instruments and equipment, has relatively simple operation and good repeatability, and can quickly form a stable oxygen gradient; the patterned parallel cell culture unit provides various cell co-culture conditions, the culture modes are various, and the chip model can realize various functions such as drug screening, cell migration, cell interaction research, three-dimensional cell culture and the like.
Drawings
FIG. 1 is a two-dimensional top view of a microfluidic chip according to example 1 of the present invention;
FIG. 2 is a diagram of an anoxic channel of a microfluidic chip according to example 1 of the present invention;
FIG. 3 is a line graph of pyrogallol concentration versus flow rate versus oxygen concentration for example 2 of the present invention;
FIG. 4 is an inverted microscope photograph of co-culture of two cells in example 3 of the present invention;
FIG. 5 is a fluorescence micrograph of two stains of living cells according to example 3 of the present invention.
1-anoxic channel, 2-cell culture unit, 3-liquid inlet, 4-liquid outlet, 5-micro-column array, 6-liquid storage tank and 7-triangular baffle array.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
Example 1
As shown in fig. 1 and 2, a microfluidic chip capable of realizing oxygen concentration gradient cell co-culture includes a substrate made of PDMS polymer and glass material, where the PDMS layer is composed of an oxygen-deficient channel and a cell culture unit; the oxygen-deficient channel comprises two liquid inlets and a liquid outlet, the liquid inlets are used for mixing sodium hydroxide and pyrogallol, an oxygen gradient is generated in the area around the channel, and the residual liquid is discharged from the liquid outlet; the cell culture unit is composed of five parallel and communicated channels, two liquid storage tanks with the diameter of 3mm are arranged at two ends of each channel, the total number of ten channels are used for storing cell culture liquid, a regular hexagonal micro-column array 5 is arranged between the channels, the side length of the micro-column array is 75 micrometers, the micro-column array is used for enabling liquid flowing through to generate surface tension, and therefore the micro-column array can be kept in one channel, but the liquid can be manually injected into the liquid storage tanks when needed, and therefore the channels are communicated.
Example 2
The trend of the oxygen concentration was determined when pyrogallol was mixed with sodium hydroxide at different flow rates.
(1) Oxygen content in the chip was determined using fluorescence: the tris (4, 7-biphenyl-1, 10-phenanthroline) ruthenium dichloride is used as an indicator, the fluorescence of the substance is quenched due to the existence of oxygen, and the quenching degree is higher as the oxygen concentration is higher, so that the substance can be used as the indicator for detecting the oxygen content in the chip. The maximum absorption wavelength of the indicator is 455nm and the maximum emission wavelength is 613 nm.
The indicator was dissolved in absolute ethanol, prepared as a 200 μ M solution, injected into the cell culture unit using a pipette gun, and the fluorescence intensity thereof was photographed using a fluorescence microscope.
(2) Calculation of oxygen concentration and fluorescence intensity: calculated using the Stern-Volmer formula: I0/I ═ 1+ Kq [ O2], where I0 represents the fluorescence intensity in the absence of oxygen, I represents the fluorescence intensity to be measured, and Kq is the quenching coefficient. Therefore, pure nitrogen and pure oxygen are used for simulating the conditions of oxygen concentration of 0% and 100%, Kq is calculated, experiments are repeated in air, the oxygen concentration of the air is set to be 21%, and the accuracy of Kq is verified by substituting the formula.
(3) Effect of pyrogallol concentration and flow rate on oxygen concentration: phloroglucinol (0.1-0.6mg/mL, 6 groups) and 1M sodium hydroxide with different concentrations are respectively pumped into liquid inlets 3 and 4 by using a micro-flow pump, the flow rates are respectively 5, 10, 20, 50 and 100 mu L/min, after the fluorescence intensity is stable, the fluorescence intensity in a channel is shot by using a fluorescence microscope, in order to examine the maximum hypoxia concentration caused by the chip, the fluorescence intensity at the leftmost end of an L1 channel, namely the position closest to the hypoxia channel, is selected and recorded, and then the fluorescence intensity is converted into the oxygen concentration according to a formula, and the result is shown in figure 3. The results show that both the flow rate and the pyrogallol concentration have a greater effect on the range of oxygen deficiency.
Example 3:
co-culture of mouse hepatoma cells and hepatic stellate cells in the chip: before the experiment, the chip was pretreated, i.e., the sterilized chip was placed in a clean bench, and L1, M and R2 channels of the chip were coated with 10. mu.g/mL fibronectin for 1 hour to promote cell adhesion and growth. Digesting mouse hepatoma cell Hepa1-6 with cell density of 80-90% in pancreatin and culture flask, placing in 1.5mL centrifuge tube, and resuspending the cells in high speed centrifuge, and making into 3 × 106Suspension of one/mL, sameIn addition, the mouse hepatic stellate cell JS-1 is configured to be 2 x 106Suspension per mL. Using a pipette gun to inoculate resuspended Hepa1-6 cells in L1 and R2 channels, inoculating JS-1 cells in M channel, placing the chip in an incubator for 4h, removing redundant cells in the liquid storage tank after the cells adhere to the wall, adding a fresh culture medium to enable the cells to normally grow, and as shown in FIG. 4, the left side is Hepa1-6 cells, and the right side is JS-1 cells, and due to fluid resistance, the two cells can independently grow in the respective channels. After the cells had grown for 2 days, live cell staining was performed on both cells using the live cell stains CMFDA and CMTPX, respectively, where Hepa1-6 cells, JS-1 cells are shown in FIG. 5. The result shows that the chip can carry out the cell co-culture of the channels and is used for the research of the paracrine action of the cells and the influence of the microenvironment.
The invention realizes the oxygen concentration gradient in the chip through chemical reaction, realizes the co-culture of tumor cells and stromal cells, is close to a tumor microenvironment, and provides the micro-fluidic chip capable of realizing the oxygen concentration gradient and the cell co-culture.
Claims (10)
1. A cell co-culture micro-fluidic chip capable of realizing oxygen gradient is characterized in that: the micro-fluidic chip comprises an oxygen-deficient channel (1) for generating oxygen gradient and a cell culture unit (2) arranged on one side of the oxygen-deficient channel (1);
the anoxic channel (1) comprises two liquid inlets (3) and a liquid outlet (4), and the two liquid inlets are converged into a pipeline to be communicated with a main pipeline of the anoxic channel;
the cell culture unit (2) comprises a plurality of parallel channels which are arranged in parallel, and the parallel channels are formed by a plurality of micro-column arrays (5) at intervals; two ends of the parallel channel are respectively connected with a liquid storage tank (6) through necessary pipelines.
2. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: the number of parallel channels of the cell culture unit (2) is 4-6; the cross section of each micro-column in the micro-column array is polygonal.
3. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: the chip is made of PDMS, and the height of the chip is 80-120 μm.
4. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: in the oxygen-poor passage, an S-shaped folding buffer pipeline area is arranged between the liquid inlet and the main pipeline of the oxygen-poor passage.
5. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: in the oxygen-deficient channel, the width of a liquid inlet channel is 200-400 μm; each parallel channel of the cell culture unit is 100-800 μm wide.
6. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: in the anoxic channel, two liquid inlets are converged into a pipeline, and a triangular baffle plate array (7) is arranged at the pipeline.
7. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: and a PDMS film is arranged between the oxygen-deficient channel and the cell culture unit.
8. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: the side length of the micro-columns in the micro-column array in the cell culture unit is 80-120 mu m, and the interval of each micro-column is 80-120 mu m.
9. The oxygen gradient-realizable cell co-culture microfluidic chip according to claim 1, characterized in that: the liquid storage tank of the cell culture unit is a hollow cylinder, and the height of the liquid storage tank is the same as the thickness of the chip.
10. The use of the oxygen gradient-enabling cell co-culture microfluidic chip of claim 1 in tumor cell in vitro tumor cell culture and drug resistance under hypoxic conditions.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111575184A (en) * | 2020-05-21 | 2020-08-25 | 上海理工大学 | Micro-fluidic chip capable of controlling dissolved oxygen level of cell culture environment and manufacturing method |
| CN112403542A (en) * | 2020-11-05 | 2021-02-26 | 清华大学 | Micro-fluidic chip for quantitatively researching biofilm and manufacturing method thereof |
| CN114317269A (en) * | 2022-03-09 | 2022-04-12 | 苏州大学 | Multi-organ chip and application thereof in drug evaluation |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575184A (en) * | 2020-05-21 | 2020-08-25 | 上海理工大学 | Micro-fluidic chip capable of controlling dissolved oxygen level of cell culture environment and manufacturing method |
| CN111575184B (en) * | 2020-05-21 | 2022-11-15 | 上海理工大学 | Micro-fluidic chip capable of controlling dissolved oxygen level of cell culture environment and manufacturing method |
| CN112403542A (en) * | 2020-11-05 | 2021-02-26 | 清华大学 | Micro-fluidic chip for quantitatively researching biofilm and manufacturing method thereof |
| CN112403542B (en) * | 2020-11-05 | 2021-09-28 | 清华大学 | Micro-fluidic chip for quantitatively researching biofilm and manufacturing method thereof |
| CN114317269A (en) * | 2022-03-09 | 2022-04-12 | 苏州大学 | Multi-organ chip and application thereof in drug evaluation |
| CN114317269B (en) * | 2022-03-09 | 2022-05-31 | 苏州大学 | Multi-organ chip and application thereof in drug evaluation |
| WO2023168857A1 (en) * | 2022-03-09 | 2023-09-14 | 苏州大学 | Multi-organ-on-chip and application thereof in drug evaluation |
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Application publication date: 20200327 |