Parallel cell culture microfluidic chip, culture method and application

Abstract

The present invention relates to the technical field of microfluidic chips, and specifically discloses a parallel cell culture microfluidic chip, a culture method and an application. The cell culture microfluidic chip of the present invention includes a gas and cell sampling layer, a culture layer and a glass slide; the gas and cell sampling layer includes multiple gas inlets, a gas mixing channel, multiple gas covering channels containing different gases, multiple independent sampling modules and liquid outlet modules; the culture layer includes: a culture fluid inlet, a culture fluid main channel and multiple cell isolation structures, each cell isolation structure includes: a gas-liquid premixing channel and multiple branch culture and observation channels with trap structures; the gas covering channel covers the cell isolation structure; each sampling module is respectively connected to one end of a branch culture and observation channel in each cell isolation structure, and the liquid outlet module is connected to the other end of the branch culture and observation channel. The present invention simplifies the sampling process and can simultaneously culture multiple cells under different gas environments.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a parallel cell culture microfluidic chip, a culture method and an application thereof. Background Art

[0002] Oxygen is an essential molecule for life. It not only plays a central role in cellular energy metabolism but also regulates multiple key cell signaling pathways. Particularly in specific biological environments (such as fermenting wine barrels or tumor microenvironments), changes in oxygen concentration regulate cell signaling pathways, enabling cells to effectively respond to these complex environmental changes. Therefore, establishing a precise oxygen microenvironment is crucial for quantitatively studying oxygen stress responses.

[0003] As a microorganism with both anaerobic and aerobic metabolic pathways, yeast can adapt to a variety of oxygen environments. Experimental operations on yeast are relatively simple, and the cellular processes of yeast are relatively conserved among different species, which makes yeast an ideal model organism for studying oxygen stress response. In the process of exploring the response of yeast to different oxygen environments, the traditional method is to change the gas mixing ratio and mix the mixed gas with the culture medium to achieve an environment with different oxygen concentrations. However, this method has limitations: it takes a long time to reach gas equilibrium, the gas consumption is high, and only a single oxygen concentration environment can be achieved in each experiment. If you want to construct multiple oxygen environments, you cannot take into account both experimental throughput and ease of operation.

[0004] To overcome these limitations, researchers have developed methods based on the natural diffusion of gases within microfluidic chips to create continuous oxygen concentration gradients. However, this approach has drawbacks: high-precision oxygen concentration control requires high chip fabrication process requirements, and nonlinear oxygen concentration gradients cannot be easily constructed. Measuring at precise oxygen concentrations requires extremely precise control of the position of the culture chamber. Furthermore, nonlinear responses are common in biological processes, making nonlinear oxygen concentration variations (such as exponential gradients) highly valuable for research. However, the oxygen concentration gradients constructed using this method are approximately linear, meaning that nonlinear concentration gradients can only be constructed by designing nonlinearly distributed culture chamber positions, which is highly inconvenient. To precisely control the oxygen concentration at each location, researchers have developed a "Christmas Tree" structure. This structure modulates the distribution ratio (i.e., concentration) of the fluid by designing the length of the flow channel. However, applying this structure for high-throughput biological research on oxygen stress responses in yeast cells, an important model organism, remains a major technical challenge, necessitating further research. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a device and method for simultaneously culturing multiple cells under different gas environments.

[0006] In order to achieve this object, the technical solution of the present invention is as follows:

[0007] A parallel cell culture microfluidic chip comprises a gas and cell sampling layer, a culture layer and a glass slide connected in sequence from top to bottom;

[0008] The gas and cell sampling layer includes multiple gas inlets, gas mixing channels, and multiple gas covering channels; the multiple gas inlets respectively introduce different gases, the gas mixing channels mix different gases in different proportions to obtain multiple mixed gases, each of the mixed gases correspondingly introduces into one of the multiple gas covering channels; the gas covering channels have gas outlets;

[0009] The gas and cell sampling layer further comprises a sampling area, wherein the sampling area comprises a plurality of independent sampling modules and liquid outlet modules, wherein the sampling modules comprise a sampling channel and a sampling connection area that are interconnected, and the liquid outlet module comprises a liquid outlet channel and a liquid outlet connection area that are interconnected;

[0010] The culture layer includes: a culture liquid inlet, a culture liquid main channel and a plurality of cell isolation structures connected to the culture liquid main channel, each of the cell isolation structures includes: a gas-liquid premixing channel and a plurality of branch culture and observation channels, and the gas-liquid premixing channel is connected to the plurality of branch culture and observation channels through a fence structure;

[0011] Each of the branch culture and observation channels is a U-shaped structure, with a sample inlet through-hole and a sample outlet through-hole respectively provided at the end points of two sides of the U-shaped structure, the two sides of the U-shaped structure being connected by a plurality of trap structures, and the trap structure can capture at least one cell on a side facing one side of the U-shaped structure having the sample inlet through-hole;

[0012] The gas covering channel covers the cell isolation structure, which can fully exchange the gas between the corresponding gas covering channels and the cell isolation structures above and below; the sampling connection area of ​​each sampling module is respectively connected to the sampling through hole of a branch culture and observation channel in each cell isolation structure to transport a type of cell to be cultured to the corresponding branch culture and observation channel, and the liquid outlet connection area of ​​the liquid outlet module is connected to the sampling through hole of the branch culture and observation channel.

[0013] The parallel cell culture microfluidic chip of the present invention generates different mixed gases (especially capable of achieving nonlinear gas concentration changes) through gas mixing channels. Gas-covered channels then contact the cell isolation structures from above and below, allowing the gases to diffuse through the chip material, enabling sufficient gas exchange between them and providing a gaseous environment for the cells within the cell isolation structures. Because different gas-covered channels contain gases of different compositions, correspondingly, different cell isolation structures also contain different gases, enabling simultaneous culture of cells under different gaseous environments during a single culture.

[0014] For example, if cells are cultured in microenvironments with varying oxygen concentrations, two gas inlets can be provided: one for nitrogen and the other for oxygen. Nitrogen and oxygen are mixed in varying proportions in the gas mixing channel, thereby creating multiple cell culture microenvironments with varying oxygen concentrations. This allows for simultaneous observation of cells under varying oxygen concentrations during a single culture.

[0015] The present invention also greatly simplifies the injection process through the injection through-hole structure and the layered connection structure of the injection connection area.

[0016] In the present invention, the multiple gas covering channels may each have their own gas outlet, or may share one gas outlet.

[0017] In the present invention, the fence structure can prevent cells in the branch culture and observation channels from flowing into the gas-liquid premixing channel.

[0018] In the present invention, the injection area includes a plurality of independent liquid outlet modules, the number of which is consistent with the number of the injection modules, for correspondingly discharging liquid from the branch culture and observation channels connected to each injection module.

[0019] By adjusting the number of independent sample injection modules and their corresponding branched culture and observation channels, the present invention enables simultaneous culture of multiple cell types within the same chip. Furthermore, by combining the different gas concentrations within the branched culture and observation channels, the present invention allows the simultaneous culture of different cells under varying gas concentrations within the same chip.

[0020] When the number of liquid outlet modules corresponds to the number of sample injection modules, multiple independent culture environments consisting of the liquid outlet modules, branch culture and observation channels, and sample injection modules will be formed.

[0021] Preferably, the trap structure is dumbbell-shaped, and a space for capturing cells is provided on one side of the dumbbell-shaped structure facing the U-shaped structure having the sample injection hole.

[0022] More preferably, the trap structure may be an asymmetric dumbbell shape.

[0023] When the cell fluid sample is injected into the present invention, the cell fluid enters the sample injection connection area from the sample injection channel, and then enters one side of the U-shaped structure of the branch culture and observation channel through the sample injection through-hole. When flowing through the trap structure, the cells in the cell fluid are captured in the dumbbell-shaped structure. Then, the uncaptured cells continue to flow into the other side of the U-shaped structure with the remaining cell fluid, and finally flow into the liquid outlet connection area through the sample outlet through-hole, and then are discharged from the chip through the liquid outlet channel and the liquid outlet hole.

[0024] Preferably, a reinforcement structure is provided in the main culture liquid channel and / or the gas-liquid premixing channel to prevent the main culture liquid channel from breaking and / or the gas-liquid premixing channel from collapsing during processing.

[0025] In the parallel cell culture microfluidic chip of the present invention, in each injection module, each injection channel includes an injection hole.

[0026] In each of the liquid outlet modules, each of the liquid outlet channels includes a liquid outlet hole.

[0027] In the parallel cell culture microfluidic chip of the present invention, the sampling module includes one sampling channel and multiple sampling connection areas connected in series through the sampling channel, and each sampling connection area is simultaneously connected to the sampling through holes of multiple branch culture and observation channels.

[0028] The liquid outlet module includes a liquid outlet channel and multiple liquid outlet connection areas connected in series through the liquid outlet channel. Each liquid outlet connection area is simultaneously connected to the sample outlet holes of multiple branch culture and observation channels to facilitate the discharge of liquid.

[0029] When multiple branch culture and observation channels need to be injected with the same cell sample, they can be simultaneously connected to the same injection connection area to facilitate cell injection. Preferably, when multiple branch culture and observation channels need to be injected with the same cell sample, multiple injection connection areas are designed, each of which is connected to a portion of the multiple branch culture and observation channels, and each injection connection area is connected in series through an injection channel. This can not only facilitate cell injection, but also improve the uniformity of cell distribution in each branch culture and observation channel when injecting cells.

[0030] In the parallel cell culture microfluidic chip of the present invention, the gas mixing channel includes two gas inlet channels and K gas distribution units, K is an integer greater than or equal to 1, each gas inlet channel is connected to one gas inlet, the number of the gas inlets is 2, each gas distribution unit includes a horizontal flow channel and multiple downstream flow channels, the gas is distributed through the horizontal flow channel and flows into the multiple downstream flow channels, and the downstream flow channels mix the two gases evenly; each gas inlet channel is connected to the horizontal flow channel of the first gas distribution unit, when K is 1, the multiple downstream flow channels of the first gas distribution unit are respectively connected one-to-one with the multiple gas coverage channels; when K is greater than 1, the K gas distribution units are arranged in sequence along the gas flow direction, the multiple downstream flow channels of the first gas distribution unit are connected to the horizontal flow channel of the second gas distribution unit, the multiple downstream flow channels of the second gas distribution unit are connected to the horizontal flow channel of the third gas distribution unit, and so on, the multiple downstream flow channels of the last gas distribution unit are respectively connected one-to-one with the multiple gas coverage channels.

[0031] In the present invention, the horizontal flow channels in each gas distribution unit are sized to ensure no pressure differential within each horizontal flow channel. Gas flowing from any downstream flow channel in the previous gas distribution unit flows only to the two or three nearest downstream flow channels in the next gas distribution unit (this requirement is achieved by combining the design of the horizontal flow channels with the obstruction of adjacent airflow). The width of these channels is greater than the width of the downstream flow channels. The narrow and long design of the downstream flow channels provides flow resistance, increasing the residence time of the gas in the downstream flow channels and thus achieving sufficient mixing.

[0032] The downstream flow channel is preferably serpentine, which is beneficial for gas mixing and saves chip space.

[0033] As a specific embodiment, the specific steps for designing the gas mixing channel of the present invention are as follows:

[0034] 1. Set the target: Input two initial gases into the chip to generate B gases whose concentrations change along the same gradient (i.e., B mixed gases).

[0035] 2. Determine the shape of the network (the layout of the gas distribution unit and downstream flow channels):

[0036] (1) The gas distribution units are arranged from top to bottom to form a multi-layer structure, and the total number of layers K must meet

[0037] (2) The downstream flow channel (referred to as A) of the upper gas distribution unit inputs the gas into the adjacent lower gas distribution unit, and then enters the downstream flow channel (referred to as B) of the lower gas distribution unit for gas mixing after being distributed through the horizontal flow channel in the lower gas distribution unit.

[0038] Each A has only 2 or 3 nearest neighbors B that receive its gas. Each B has only 1 or 2 A that provide it with gas.

[0039] When A has only three Bs that are its closest neighbors and receive its gas, the air inlet of the middle B among the three Bs corresponds to the air outlet setting of A.

[0040] On the basis of satisfying the above conditions, the shape of the network can be designed.

[0041] 3. Determine the gas concentration c in each downstream flow channel:

[0042] (1) In all gas distribution units, the gas concentration in each downstream flow channel increases from left to right. The gas concentration in each downstream flow channel in the last layer of gas distribution units is the design target in step 1.

[0043] (2) Boundary conditions: The two gas inlet channels are located on the left and right sides of the horizontal flow channel of the first gas distribution unit, respectively. The gas concentration in the first downstream flow channel (starting from the left) of each layer of the gas distribution unit is equal to that in the left gas inlet channel; the gas concentration in the last downstream flow channel (starting from the left) of each layer of the gas distribution unit is equal to that in the right gas inlet channel.

[0044] (3) Two nearest neighbor condition: When A has only two nearest neighbors B that receive its gas, C 下层,左 <C 上层 <C 下层,右 , C 下层,左 is the gas concentration in B, which is the closest to A on the lower left side, and C 上层 is the gas concentration in A, C 下层,右 is the gas concentration in B, which is the closest to A on the lower right side.

[0045] (4) Three-neighbor condition: When A has only three Bs that are its nearest neighbors and receive its gas, the gas concentration of the B among the three nearest neighbors is equal to the gas concentration of the A that is its closest neighbor.

[0046] On the basis of satisfying the above conditions, the concentration of gas in the downstream flow channel is designed.

[0047] The present invention can also be designed in such a way that the final output gas concentration decreases from left to right, as long as the above conditions are adjusted accordingly.

[0048] 4. Determine the volume flow rate q of each downstream flow channel (the volume of gas passing through the downstream flow channel per unit time):

[0049] (1) The volume flow rates of all downstream flow channels of the last layer of gas distribution units are uniformly set to the same fixed value, and then the volume flow rates of the downstream flow channels of the remaining layers of gas distribution units are designed based on this fixed value.

[0050] (2) Two nearest neighbor condition: When A has only two nearest neighbors B that receive its gas, q 上层 =(1-α 下层，左 )·q 下层，左 +α 下层，右 ·q 下层，右 ,q 上层 is the volume flow rate of a downstream flow channel in the upper gas distribution unit, α 下层，左 is the volume flow distribution parameter of the downstream flow channel (abbreviated as D) located on the left side of the lower gas distribution unit closest to the downstream flow channel, α 下层，右 is the volume flow distribution parameter of the downstream flow channel (abbreviated as E) located on the right side of the gas distribution unit in the lower layer that is closest to the downstream flow channel, q 下层，左 is the volume flow rate of D, q 下层，右 is the volume flow rate of E;

[0051] D receives gas from two downstream channels in the upper gas distribution unit. Among the two upper downstream channels corresponding to D, the gas concentration in the downstream channel located on the upper left side of D is c x , the gas concentration in the downstream channel located on the upper right side of D is c y , the gas concentration in D is c z ,

[0052] E also receives gas from two downstream flow channels in the upper gas distribution unit, α 下层，右 Compare α 下层，左 Obtained by calculation.

[0053] (3) Three nearest neighbor condition: When A has only three nearest neighbors B that receive its gas, q 上层 =(1-α 下层，左 )·q 下层，左 +q 下层，中 +α 下层，右 ·q 下层，右 ,q 上层 is the volume flow rate of a downstream flow channel in the upper gas distribution unit, α 下层，左 is the volume flow distribution parameter of the downstream flow channel (abbreviated as D) located on the left side of the lower gas distribution unit closest to the downstream flow channel, α 下层，右 is the volume flow distribution parameter of the downstream flow channel (abbreviated as E) located on the right side of the lower gas distribution unit closest to the downstream flow channel, α 下层，中is the volume flow distribution parameter of the downstream flow channel (abbreviated as F) located directly below the downstream flow channel in the lower gas distribution unit that is closest to the downstream flow channel, q 下层，左 is the volume flow rate of D, q 下层，右 is the volume flow rate of E; q 下层，中 is the volume flow rate of F; α 下层，左 and α 下层，右 The calculation method of is as described above.

[0054] On the basis of satisfying the above conditions, the volume flow rate of each downstream flow channel is solved.

[0055] 5. Determine the length of each downstream flow channel:

[0056] (1) The gas pressures of the multiple downstream flow channels in each gas distribution unit are equal and preset to a constant H. The product of the length of each downstream flow channel in the gas distribution unit and its volume flow rate is H. H in different gas distribution units can be the same or different.

[0057] (2) The length of each downstream flow channel can ensure that the gas can flow along the upper gas distribution unit to the lower gas distribution unit (that is, the flow resistance of each downstream flow channel is much greater than the flow resistance of the horizontal flow channel in the gas distribution unit in which it is located).

[0058] On the basis of satisfying the above conditions, the length of each downstream flow channel can be designed.

[0059] The gas mixing channel structure of the present invention can realize the simultaneous construction of multiple precise gas concentrations, thereby expanding the application range of the chip.

[0060] In the parallel cell culture microfluidic chip of the present invention, the material of the gas and cell sampling layer and the culture layer is polydimethylsiloxane; the sum of the wall thicknesses of the corresponding gas covering channel and the gas-liquid premixing channel in covering contact is 20-80 microns; the sum of the wall thicknesses of the corresponding gas covering channel and the branch culture and observation channel in covering contact is 20-80 microns.

[0061] Preferably, the sum of the wall thicknesses of the gas covering channel and the gas-liquid premixing channel in corresponding covering contact is 50 microns; the sum of the wall thicknesses of the gas covering channel and the branch culture and observation channel in corresponding covering contact is 50 microns.

[0062] In the present invention, the wall thicknesses of the gas covering channel, gas-liquid premixing channel, branch culture and observation channel are within the above-mentioned limits of the present invention, which is conducive to ensuring the effective diffusion of gas from the upper gas covering channel to the lower cell isolation structure.

[0063] The present invention also provides a method for preparing the above-mentioned parallel cell culture microfluidic chip, which comprises:

[0064] Step S1: preparing molds for the gas and cell injection layers and the culture layer respectively;

[0065] Step S2: coating the materials for preparing the cell culture microfluidic chip on the mold and curing them to obtain the gas and cell sampling layers and the culture layer;

[0066] Step S3: Covering the gas and cell sampling layer on the culture layer so that the gas covering channel covers the cell isolation structure, and aligning the sampling connection area with the sampling through hole and the liquid outlet connection area with the sampling through hole, and then bonding them;

[0067] Step S4: After drilling holes at the gas inlet, gas outlet, culture liquid inlet, the sample injection hole on the sample injection channel, and the liquid outlet hole on the liquid outlet channel, the glass slide is bonded under the culture layer.

[0068] The present invention further provides a cell culture method, which uses the above-mentioned parallel cell culture microfluidic chip to culture cells.

[0069] The method of the present invention comprises:

[0070] (1) vacuum treatment of the parallel cell culture microfluidic chip;

[0071] (2) injecting the cell solution into the injection holes on the injection channel of the gas and cell injection layer, so that the cell solution enters each cell isolation structure of the culture layer through the injection holes;

[0072] (3) Close the injection hole and add culture medium through the culture medium inlet;

[0073] (4) After introducing different gases into each gas inlet, cell culture is carried out.

[0074] During cell culture, the present invention continuously discharges gas from the gas inlet into the chip, passes through the gas mixing channel into multiple gas-covered channels, and finally is continuously discharged from the gas outlet, so that the cells are always in the designed gas environment. The culture fluid continuously enters multiple cell isolation structures along the culture fluid main channel from the culture fluid inlet, and after fully contacting the cells therein, it is continuously discharged from the upper liquid outlet module through the sample outlet hole, so that the cells always have sufficient material foundation for growth. The gas introduced into the gas-covered channel in the upper layer enters the lower cell isolation structure by free diffusion, contacts the culture fluid therein, and provides a complete growth environment for the cells.

[0075] The method of the present invention can be used for culture observation of various cells, such as yeast cells or Escherichia coli cells.

[0076] The present invention also provides an application of the above-mentioned parallel cell culture microfluidic chip or method for simultaneously culturing different cells in different gas environments.

[0077] As a specific application, the present invention can construct a variety of oxygen concentration gradients through the gas mixing channel. Through the connection structure of multiple independent sampling holes and the sampling connection area, a variety of yeasts can be directly loaded into the yeast culture area under different oxygen environments through the sampling area, realizing high-throughput quantitative research on the response behavior of yeast under different oxygen environments.

[0078] The beneficial effects of the present invention are at least:

[0079] The parallel cell culture microfluidic chip of the present invention can simultaneously culture different cells in different gas environments, and the injection process is simplified, and the dynamic response behavior of cells in different gas environments can be realized with high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 It is a schematic diagram of the overall structure of the microfluidic chip of the present invention.

[0081] Figure 2 It is a schematic diagram of the structure of the gas and cell sampling layer of the microfluidic chip of the present invention.

[0082] Figure 3 Schematic diagram of the culture layer structure of the microfluidic chip of the present invention; wherein the portion indicated by the dotted line is a partial enlarged view of the branch culture and observation channel.

[0083] Figure 4 It is a schematic diagram of the gas and cell sampling layer and the culture layer of the microfluidic chip of the present invention after superposition and assembly.

[0084] Figure 5 Schematic diagram of the manufacturing method and local structure of the microfluidic chip of the present invention; in the figure, A is a schematic diagram of the manufacturing method flow, B is a schematic diagram of the correspondence between the sampling area of ​​the gas and cell sampling layer and the sampling through-holes of the culture layer, and C is a schematic diagram of the gas exchange between the gas and cell sampling layer and the culture layer.

[0085] Figure 6 This is a partial physical picture of the branch culture and observation channels of the microfluidic chip of the present invention during culture, wherein the portion indicated by the dotted line is a partially enlarged phase contrast image.

[0086] Figure 7 This is a fluorescence timing diagram of the microfluidic chip of the present invention when different yeast cells are loaded simultaneously.

[0087] Figure 8These are the gas concentration detection results when gases are alternately introduced. The left picture shows the detection results in the branch culture and observation channels in 6 different cell isolation structures, where channel 1 is the left gas inlet and channel 2 is the right gas inlet. The right picture is a schematic diagram of the measurement area.

[0088] Figure 9 The gas concentration test results at different positions in the same branch culture and observation channel when nitrogen is introduced are shown in the upper figure (the horizontal axis is the channel length), and the lower figure is a schematic diagram of the position of the channel length value in the channel.

[0089] Figure 10 Comparison of experimental and theoretical gas concentrations in branch culture and observation channels of six different cell isolation structures when nitrogen and air are introduced.

[0090] Reference numerals:

[0091] 1. Gas and cell sampling layer; 11. Gas inlet; 12. Gas mixing channel; 13. Gas covering channel; 131. Gas outlet; 14. Sampling area; 141. Sampling channel; 142. Sampling connection area; 143. Liquid outlet channel; 144. Liquid outlet connection area; 145. Liquid outlet hole; 146. Sampling hole; 2. Culture layer; 21. Culture fluid inlet; 22. Culture fluid main channel; 23. Cell isolation structure; 123. Gas-liquid premixing channel; 124. Fence structure; 125. Branch culture and observation channel; 1251. Sampling through hole; 1252. Sample outlet through hole; 1253. Trap structure; 3. Glass slide; 4. Cells. DETAILED DESCRIPTION

[0092] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0093] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0094] Example 1

[0095] This embodiment provides a high-throughput microfluidic chip for cell culture that can generate microenvironments with different gas concentrations and a method for preparing the same.

[0096] The schematic diagram of the overall structure of the microfluidic chip of the present invention is shown in FIG. Figure 1 , the specific gas and cell sampling layer 1 structure diagram is shown in Figure 2, the schematic diagram of the culture layer 2 is shown in Figure 3 The corresponding schematic diagram of the positions of the gas and cell injection layer and the culture layer after superposition and assembly is shown in Figure 4 (Gas and cell injection layer 1 shown in blue is superimposed on culture layer 2 shown in yellow).

[0097] The structure of a high-throughput cell culture microfluidic chip (parallel cell culture microfluidic chip) that can generate an oxygen concentration gradient microenvironment provided in an embodiment of the present invention includes a gas and cell sampling layer 1, a culture layer 2 and a glass slide 3; the gas and cell sampling layer 1 and the culture layer 2 are made of polydimethylsiloxane (PDMS).

[0098] The gas and cell sampling layer 1 includes two gas inlets 11, a gas mixing channel 12, and six gas covering channels 13. The left gas inlet 11 is used to introduce nitrogen (oxygen concentration is 0%), and the right gas inlet 11 is used to introduce air (oxygen concentration is 20.9%). The gas mixing channel 12 mixes nitrogen and air in different proportions to obtain six gases with different oxygen concentrations (oxygen concentrations are 0%, 0.5%, 1%, 2.5%, 7.5%, and 20.9%). Each gas is introduced into one of the six gas covering channels 13. The six gas covering channels 13 share a gas outlet 131.

[0099] Specifically, the gas mixing channel 12 includes two gas inlet channels and three gas distribution units. Each gas inlet channel is connected to a gas inlet 11. Each gas distribution unit includes a horizontal flow channel and multiple downstream flow channels. The gas is distributed through the horizontal flow channel and flows into multiple downstream flow channels. The downstream flow channels mix the two gases evenly.

[0100] The width of each gas inlet channel and the downstream flow channel is 40 microns and the height is 30 microns, and the width of each horizontal flow channel is 260 microns and the height is 30 microns.

[0101] The two gas inlet channels are located at the left and right ends of the horizontal flow channel of the first gas distribution unit, with the left gas inlet channel being 2 mm long and the right gas inlet channel being 5.6 mm long. The three gas distribution units are arranged in a multi-layer structure from top to bottom along the direction of gas flow.

[0102] The first gas distribution unit has three downstream flow channels (lengths from left to right: 12 mm, 149 mm, and 22.4 mm). The gas in each gas inlet channel is distributed to the two adjacent downstream flow channels in the first gas distribution unit. The horizontal flow channel of the first gas distribution unit is 3.9 mm long.

[0103] The three downstream channels of the first gas distribution unit are connected to the horizontal channels (4.7 mm long) of the second gas distribution unit. The gas in each downstream channel of the first gas distribution unit is distributed to the two adjacent downstream channels of the second gas distribution unit. The second gas distribution unit includes four downstream channels (lengths from left to right: 14.1 mm, 10 mm, 17.6 mm, and 18.1 mm, respectively).

[0104] The four downstream flow channels of the second gas distribution unit are connected to the horizontal flow channels (5.5 mm in length) of the third gas distribution unit. Of these four downstream flow channels, except for the second downstream flow channel (counted from left to right), which is distributed to the three downstream flow channels closest to it in the third gas distribution unit, the gas in the remaining downstream flow channels of the second gas distribution unit is distributed to the two downstream flow channels closest to it in the third gas distribution unit. The third gas distribution unit includes six downstream flow channels of equal length (4.7 mm in length).

[0105] The six downstream flow channels of the third gas distribution unit are connected to the six gas covering channels 13 in a one-to-one correspondence, and the obtained six gases with different oxygen concentrations are respectively introduced into each gas covering channel 13.

[0106] The gas and cell sampling layer 1 further includes a sampling area 14, which includes four independent sampling modules and four liquid outlet modules. Each sampling module includes two sampling connection areas 142 connected in series by a sampling channel 141, and each liquid outlet module includes two liquid outlet connection areas 144 connected in series by a liquid outlet channel 143.

[0107] The height of the sample inlet channel 141 and the liquid outlet channel 143 is 30 microns; the height of each gas covering channel 13, the sample inlet connection area 142 and the liquid outlet connection area 144 is 100 microns.

[0108] The culture layer 2 includes: a culture medium inlet 21, a culture medium main channel 22 and six cell isolation structures 23 connected to the culture medium main channel 22;

[0109] Each cell isolation structure 23 includes: a gas-liquid premixing channel 123 and four branch culture and observation channels 125. The gas-liquid premixing channel 123 is connected to the four branch culture and observation channels 125 through a fence structure 124 to prevent cells from entering the gas-liquid premixing channel 123 and causing contamination.

[0110] Each branch culture and observation channel 125 is a U-shaped structure, and the endpoints of the two sides of the U-shaped structure are respectively provided with a sample inlet hole 1251 and a sample outlet hole 1252. The two sides of the U-shaped structure are connected by 100 trap structures 1253 (the number of trap structures 1253 can be adjusted according to culture requirements). The trap structure 1253 is an asymmetric dumbbell shape, and can capture at least one cell facing the side of the U-shaped structure with the sample inlet hole 1251.

[0111] The flow channels of the gas-liquid premixing channel 123 and the branch culture and observation channel 125 are 20 microns high. The fence structure 124 is 2 microns high. The trap structure 1253 is 7 microns high. The opening on the side of the U-shaped structure facing the sample inlet 1251 is 7 microns wide, with a narrowest point of 2 microns, used to trap cells. The sample inlet 1251 and the sample outlet 1252 are 70 microns high.

[0112] Six gas-covering channels 13 cover the gas-liquid premixing channel 123 and the branch culture and observation channel 125, allowing for sufficient gas exchange between the gas-covering channels 13, the gas-liquid premixing channel 123, and the branch culture and observation channel 125, thereby creating different oxygen concentration environments within the six cell isolation structures 23. The combined wall thickness of the corresponding gas-covering channels 13 and the gas-liquid premixing channels 123 in contact with each other is 50 microns; the combined wall thickness of the corresponding gas-covering channels 13 and the branch culture and observation channels 125 in contact with each other is also 50 microns, allowing gas to diffuse through the 50-micron PDMS material.

[0113] The sampling connection area 142 of each sampling module is respectively connected to the sampling through-hole 1251 of one branch culture and observation channel 125 in each cell isolation structure 23 (that is, the sampling connection area 142 of each sampling module is connected to the sampling through-holes 1251 of four branch culture and observation channels 125 in total, and the four branch culture and observation channels 125 belong to six different cell isolation structures 23) to transport a type of cell to be cultured to the corresponding branch culture and observation channel 125. Different sampling modules are connected to the sampling through-holes 1251 of different branch culture and observation channels 125 in each cell isolation structure 23 through the two sampling connection areas 142 they contain (that is, each sampling module includes two sampling connection areas 142, and each sampling connection area 142 is connected to the sampling through-holes 1251 of two of the four branch culture and observation channels 125 corresponding to it). Different liquid outlet modules are connected to the sample outlet holes 1252 of different branch culture and observation channels 125 in each cell isolation structure 23 through the two liquid outlet connection areas 144 they contain (that is, each liquid outlet module includes two liquid outlet connection areas 144, and each liquid outlet connection area 144 is connected to the sample outlet holes 1252 of two of the four corresponding branch culture and observation channels 125), thereby forming four culture spaces with different sample sources in each cell isolation structure 23, which can culture four different cells at the same time. The corresponding relationship between the sample injection connection area 142 and the sample injection hole 1251 is shown in FIG. Figure 5 B in.

[0114] The culture fluid is injected from the culture fluid inlet 21, and flows in sequence through the culture fluid main channel 22, the gas-liquid premixing channel 123, the branch culture and observation channel 125, the sample outlet through hole 1252, the liquid outlet connection area 144, the liquid outlet channel 143, and finally flows out from the liquid outlet hole 145.

[0115] The cell fluid sample is injected from the injection hole 146, flows through the injection channel 141, the injection connection area 142, the injection through hole 1251 in sequence, and enters the branch culture and observation channel 125. After the cells are captured by each trap structure 1253, the remaining cell fluid sample flows out from the sample outlet through hole 1252, the liquid outlet connection area 144, the liquid outlet channel 143, and finally out of the liquid outlet 145.

[0116] The gas enters from the two gas inlets 11 , passes through the gas mixing channel 12 and is mixed into gases with different oxygen concentrations. The gases then flow through different gas covering channels 13 and are discharged from the gas outlet 131 .

[0117] The cross-sectional view of the chip of the branch culture and observation channel 125 area of ​​the present invention is shown in FIG. Figure 5As shown in FIG. C, the gas diffuses from the gas-covered channel 13 to the branch culture and observation channel 125 through the 50-μm PDMS membrane in the direction of the arrow in the figure, and the cell 4 (taking yeast as an example) is trapped in the trap structure 1253.

[0118] In the present invention, the size of each structure and the number of injection ports can be adjusted according to experimental requirements.

[0119] This embodiment also provides a method for manufacturing the above-mentioned microfluidic chip, and the preparation process diagram is shown in FIG. Figure 5 As shown in A.

[0120] The preparation method mainly includes the following steps:

[0121] Step S1: preparing molds for the culture layer 2 chip and the gas and cell sampling layer 1 chip respectively.

[0122] Step S2: coating the prepared material on the mold and performing a curing process.

[0123] Step S3 : aligning the sample inlet connection area 142 with the sample inlet through hole 1251 and the liquid outlet connection area 144 with the sample outlet through hole 1252 .

[0124] Step S4: After drilling holes at the gas inlet 11 , the gas outlet 131 , the culture medium inlet 21 , the injection hole 146 on the injection channel 141 and the liquid outlet 145 on the liquid outlet channel 143 , the chip is subjected to surface plasma treatment and packaged with the glass slide 3 .

[0125] The specific steps are as follows:

[0126] (1) Preparation of mold. Use L-Edit to draw and print it on a plastic film or optical mask as an exposure mask (structures of the same height in the chip are located on the same plastic film or optical mask) for subsequent experiments. A total of two silicon wafer molds are required, one for making the gas and cell sampling layer 1 chip, and one for making the culture layer 2 chip. The preparation process of the two molds is basically the same, except that the masks used are different. Use SU8 photoresist, pre-spin at a speed of 500 rpm for 10 seconds when spreading the glue, and then set different speeds for 30 seconds according to different height requirements. After baking on a 95-degree hot plate for 2-30 minutes (the pre-baking time depends on the height), put it into the exposure machine for exposure. The exposure time needs to be determined by referring to the help document of the exposure machine and the actual light intensity. Then bake on a 95-degree hot plate for 2-15 minutes (the post-baking time depends on the height), and the mold can be obtained by development.

[0127] (2) Chip injection molding and curing treatment. The gas and cell sampling layer 1 chip and the culture layer 2 chip are both obtained by PDMS injection molding. When making the gas and cell sampling layer 1 chip, the ratio of A glue (monomer) to B glue (cross-linking agent) is 6:1. The two are mixed evenly, and after vacuuming to remove bubbles, injection molding is performed. The thickness is more than 3mm. When making the culture layer 2 chip, A glue (monomer) and B glue (cross-linking agent) are mixed at a ratio of 14:1. After vacuuming to remove bubbles, PDMS is spin-coated on the mold through a glue roller (2100rpm, 30s). A PDMS film with a thickness of about 50 microns can be obtained. This thickness allows the 70-micron-high sample inlet through-hole 1251 and the sample outlet through-hole 1252 to be exposed, while the 20-micron-high branch culture and observation channel 125 is covered (the height can be judged by microscopic observation to see whether the through-hole structure is exposed). To cure, place the two molds in a 70°C oven. Bake the gas and cell injection layer 1 chip for 40 minutes, and the culture layer 2 chip for 30 minutes. Remove the two chips, and the PDMS will have initially cured.

[0128] When preparing each chip layer, the ratio of glue A to glue B should be at least 5:1 to achieve crosslinking. Based on the preparation and curing time of the two chip layers, the ratio of glue A to glue B used in each layer can be adjusted to ensure that the two chip layers are completed in a similar time, thus saving overall preparation time.

[0129] (3) Chip bonding. Peel off the gas and cell sampling layer PDMS chip from the mold and trim it to a size that can cover the pattern of the culture layer 2 without affecting the surrounding patterns. Use a microscope to align the gas and cell sampling layer PDMS chip with the culture layer PDMS film chip on the mold. It is required that the gas covering channel 13 of the gas and cell sampling layer 1 completely covers the cell isolation structure 23 of the culture layer 2, and the sampling connection area 142 of the gas and cell sampling layer 1 covers the sampling through-hole 1251, and the liquid outlet connection area 144 covers the sampling through-hole 1252. Place it in a 70-degree oven and bake overnight. The PDMS will continue to solidify and the two layers of chips will be firmly bonded together. Peel off the two layers of chips from the mold and punch holes. Use a 0.5mm diameter puncher for the two gas inlets 11, the gas outlet 131, and the culture liquid inlet 21, and use a No. 18 puncher (0.9mm diameter) for the four sampling holes 146 and the four liquid outlet holes 145. After punching, the chip and the glass slide 3 are subjected to surface plasma treatment together, and the two are bonded together and placed in a 70-degree oven for baking overnight to complete the packaging.

[0130] The chip must be modified before loading cells. Specifically, it must be vacuumed for approximately 30 minutes to ensure smooth cell loading without creating bubbles in the branch culture and observation channel 125. It should be noted that after vacuuming, the internal pressure of the chip will gradually equilibrate with the external pressure, so the sample injection operation must be completed within 30 minutes after vacuuming.

[0131] The method for manufacturing a high-throughput microfluidic chip for cell culture that can generate an oxygen concentration gradient microenvironment provided by the present invention greatly simplifies the injection process through the layered connection structure of the injection through hole 1251, the sample outlet through hole 1252, the injection connection area 142, and the liquid outlet connection area 144, so that multiple cells can be directly loaded into the cell culture area under different oxygen environments through the injection area 14, thereby realizing high-throughput quantitative research on the response behavior of cells under different oxygen environments.

[0132] This embodiment also tests the gas concentration in the branch culture and observation channel 125 of the microfluidic chip prepared above to determine whether it is consistent with the preset concentration in each gas coverage channel 13. The details are as follows:

[0133] Detection was performed using the oxygen-sensitive fluorescent dye RTDP (tris(2,2′-bipyridyl)dichlororuthenium hexahydrate, Ru(BPY)3). 1 mg / ml RTDP was introduced into the culture layer from the culture medium inlet 21 at a flow rate of 250 μL / h, and the fluorescence intensity of each channel was measured. The fluorescence intensity of RTDP in the presence of oxygen, I, decreased compared to the intensity in the absence of oxygen, I0, following the Stern-Volmer equation: I0 / I = 1 + K q [O2], where K q is the quenching constant. According to the Stern-Volmer equation, the oxygen concentration is calculated by measuring the fluorescence intensity, that is, [O2] = (I0 / I-1) / K q , K q The calculation formula is K q =(I0 / I 20.9 -1) / [20.9%], of which I 20.9 is the fluorescence intensity value in air-saturated medium.

[0134] The gas environment was switched from pure air to pure nitrogen and then back to pure air. Finally, air and nitrogen were added to the two inlets respectively. The dynamic changes of RTDP fluorescence in the branch culture and observation channel 125 of 6 different cell isolation structures were measured (see the results). Figure 8 ) and the fluorescence intensity at different positions along the branch culture and observation channel 125 (results see Figure 9 The channel length of 0 μm corresponds to the downstream edge of the trap structure 1253. The results show that the oxygen concentration is stable in the culture area.

[0135] Finally, this embodiment also calculates the oxygen concentration [O2] generated in the branch culture and observation channels 125 in the six different cell isolation structures 23 (see the results). Figure 10), it was found that the experimental and theoretical (preset) values ​​were in good agreement, indicating that the chip of the present invention can achieve the expected oxygen concentration gradient.

[0136] Example 2

[0137] This example uses the chip prepared in Example 1 to culture and observe yeast. The method mainly includes the following steps:

[0138] Step S1: Four different yeast solutions are introduced into the chip through the four injection holes 146 of the injection area 14 of the gas and cell injection layer 1 respectively.

[0139] Step S2: different yeast solutions are allowed to pass through the channels formed by the corresponding injection connection areas 142 and the injection through holes 1251 in the culture layer 2 and enter the different branch culture and observation channels 125 of the culture layer 1 .

[0140] Step S3: block the four injection holes 146 and add culture medium through the culture medium inlet 21.

[0141] Step S4: nitrogen and air with equal pressures are introduced to observe different yeasts under different oxygen concentration environments.

[0142] The yeast used in this example was constructed from the BY4741 strain (MATa-his3Δ1-leu2Δ0-met15Δ0-ura3Δ0, i.e., the male (a-type) yeast BY4741 was used as the starting strain, and the first copy of the his3 gene, all copies of the leu2 gene, all copies of the met15 gene, and all copies of the ura3 gene were knocked out, making it unable to synthesize leucine, methionine, and uracil). GFP markers (specifically SKN7-gfp, MSN2-gfp, ADE12-gfp, and PYK1-gfp) were added after different target genes in the strain to obtain four different yeast strains.

[0143] Each yeast strain was inoculated into SD medium containing 2% glucose and cultured at 30°C overnight. Then, 20 μL of saturated culture was diluted into 2 ml of fresh growth medium and cultured at 30°C for 4-5 h to ensure that the yeast was in the logarithmic growth phase before cell loading.

[0144] The specific steps are as follows:

[0145] (1) Cell loading. In order to increase the probability of yeast entering the branch culture and observation channel 125, the yeast suspension is first centrifuged and concentrated (concentrated to about 10 8A 20-microliter yeast suspension is pipetted into the corresponding injection hole 146 using a pipette. Pressure is applied by a syringe, forcing the yeast into the injection area 14 under applied pressure. The yeast then flows through the channel formed by the injection connection area 142 and the injection through-hole 1251 into the culture layer chip, where it is trapped in the yeast culture area (trap structure 124). After loading, the four injection holes 146 are blocked.

[0146] (2) Prepare the culture medium. Use a 1 ml syringe to draw up the yeast culture medium. Gently tap the syringe tube to remove any bubbles. Connect the hose and connector and set aside.

[0147] (3) Prepare nitrogen and air. Compressed air is produced by an Elveflow PG113 pressure generator, and high-purity compressed nitrogen is produced by an AYAN-20L nitrogen generator. Both are regulated by an MBS-PR-200E1 pressure regulator before output. Connecting hoses and connectors are kept ready for use.

[0148] (4) Experimental setup. Insert the syringe connector into the culture medium inlet 21, mount the syringe on the Langer syringe pump, and set the flow rate to 250 μL / h. Insert the nitrogen and air connectors into the corresponding gas inlets 11, and set the pressure to 750 mbar.

[0149] (5) Results shooting and processing. Fluorescence scanning and shooting were performed using a Ti-E microscope. The experimental images obtained are as follows: Figure 6 、 Figure 7 shown. Figure 6 This is a partial physical image of the structure of a high-throughput microfluidic chip for yeast culture that can generate an oxygen concentration gradient microenvironment provided by an embodiment of the present invention. The enlarged area is a phase contrast image of the chamber that confines the yeast. Figure 7 Fluorescence images (time series) of different yeast cells loaded onto a high-throughput microfluidic chip for yeast culture that can generate oxygen concentration gradient microenvironments, as provided by an embodiment of the present invention, demonstrate the absence of contamination between different yeasts. The cellseg program can be used to identify cell boundaries using phase contrast images and track cells in time to obtain single-cell trajectories, enabling the simultaneous observation of the response behaviors of four yeast strains (including transcription factors and downstream proteins) under six different oxygen environments.

[0150] Example 3

[0151] This embodiment provides a microfluidic chip for culturing Escherichia coli, which has basically the same chip structure as that of Example 1, except that the height of the trap structure 1253 is changed to 1.2 microns, the narrowest channel is 3 microns, and the width of the opening on one side of the U-shaped structure facing the sample injection hole 1251 is 35 microns to facilitate the capture of Escherichia coli.

[0152] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A parallel cell culture microfluidic chip, characterized in that: It includes a gas and cell sampling layer, a culture layer and a glass slide connected in sequence from top to bottom; The gas and cell sampling layer includes multiple gas inlets, gas mixing channels, and multiple gas covering channels; the multiple gas inlets respectively introduce different gases, the gas mixing channels mix different gases in different proportions to obtain multiple mixed gases, each of the mixed gases correspondingly introduces into one of the multiple gas covering channels; the gas covering channels have gas outlets; The gas and cell sampling layer further comprises a sampling area, wherein the sampling area comprises a plurality of independent sampling modules and liquid outlet modules, wherein the sampling modules comprise a sampling channel and a sampling connection area that are interconnected, and the liquid outlet module comprises a liquid outlet channel and a liquid outlet connection area that are interconnected; The culture layer includes: a culture liquid inlet, a culture liquid main channel and a plurality of cell isolation structures connected to the culture liquid main channel, each of the cell isolation structures includes: a gas-liquid premixing channel and a plurality of branch culture and observation channels, and the gas-liquid premixing channel is connected to the plurality of branch culture and observation channels through a fence structure; Each of the branch culture and observation channels is a U-shaped structure, with a sample inlet through-hole and a sample outlet through-hole respectively provided at the end points of two sides of the U-shaped structure, the two sides of the U-shaped structure being connected by a plurality of trap structures, and the trap structure can capture at least one cell on a side facing one side of the U-shaped structure having the sample inlet through-hole; The gas covering channel covers the cell isolation structure, which can fully exchange the gas between the corresponding gas covering channels and the cell isolation structures above and below; the sampling connection area of ​​each sampling module is respectively connected to the sampling through hole of a branch culture and observation channel in each cell isolation structure to transport a type of cell to be cultured to the corresponding branch culture and observation channel, and the liquid outlet connection area of ​​the liquid outlet module is connected to the sampling through hole of the branch culture and observation channel.

2. The parallel cell culture microfluidic chip according to claim 1, characterized in that: The sampling area includes a plurality of independent liquid outlet modules, the number of which is consistent with the number of the sampling modules, and is used to discharge the liquid in the branch culture and observation channels connected to each of the sampling modules.

3. The parallel cell culture microfluidic chip according to claim 2, characterized in that: The trap structure is dumbbell-shaped; And / or, in each of the injection modules, each of the injection channels includes an injection hole; And / or, in each of the liquid outlet modules, each of the liquid outlet channels includes a liquid outlet hole.

4. The parallel cell culture microfluidic chip according to any one of claims 1 to 3, characterized in that: The sampling module comprises one sampling channel and a plurality of sampling connection areas connected in series through the sampling channel, and each of the sampling connection areas is simultaneously connected to the sampling through holes of a plurality of the branch culture and observation channels; And / or, the liquid outlet module includes one liquid outlet channel and multiple liquid outlet connection areas connected in series through the liquid outlet channel, and each liquid outlet connection area is simultaneously connected to the sample outlet holes of multiple branch culture and observation channels.

5. The parallel cell culture microfluidic chip according to any one of claims 1 to 3, characterized in that: The gas mixing channel includes two gas inlet channels and K gas distribution units, K is an integer greater than or equal to 1, each gas inlet channel is connected to a gas inlet, the number of the gas inlets is 2, each gas distribution unit includes a horizontal flow channel and multiple downstream flow channels, the gas is distributed through the horizontal flow channel and flows into the multiple downstream flow channels, and the downstream flow channels mix the two gases evenly; each gas inlet channel is connected to the horizontal flow channel of the first gas distribution unit, when K is 1, the multiple downstream flow channels of the first gas distribution unit are respectively connected to the multiple gas covering channels in a one-to-one correspondence; when K is greater than 1, the K gas distribution units are arranged in sequence along the gas flow direction, the multiple downstream flow channels of the first gas distribution unit are connected to the horizontal flow channel of the second gas distribution unit, the multiple downstream flow channels of the second gas distribution unit are connected to the horizontal flow channel of the third gas distribution unit, and so on, the multiple downstream flow channels of the last gas distribution unit are respectively connected to the multiple gas covering channels in a one-to-one correspondence.

6. The parallel cell culture microfluidic chip according to claim 4, characterized in that: The gas mixing channel includes two gas inlet channels and K gas distribution units, K is an integer greater than or equal to 1, each gas inlet channel is connected to a gas inlet, the number of the gas inlets is 2, each gas distribution unit includes a horizontal flow channel and multiple downstream flow channels, the gas is distributed through the horizontal flow channel and flows into the multiple downstream flow channels, and the downstream flow channels mix the two gases evenly; each gas inlet channel is connected to the horizontal flow channel of the first gas distribution unit, when K is 1, the multiple downstream flow channels of the first gas distribution unit are respectively connected to the multiple gas covering channels in a one-to-one correspondence; when K is greater than 1, the K gas distribution units are arranged in sequence along the gas flow direction, the multiple downstream flow channels of the first gas distribution unit are connected to the horizontal flow channel of the second gas distribution unit, the multiple downstream flow channels of the second gas distribution unit are connected to the horizontal flow channel of the third gas distribution unit, and so on, the multiple downstream flow channels of the last gas distribution unit are respectively connected to the multiple gas covering channels in a one-to-one correspondence.

7. The parallel cell culture microfluidic chip according to any one of claims 1 to 3 and 6, characterized in that: The material of the gas and cell sampling layer and the culture layer is polydimethylsiloxane; the sum of the wall thicknesses of the corresponding covering contact of the gas covering channel and the gas-liquid premixing channel is 20-80 microns; the sum of the wall thicknesses of the corresponding covering contact of the gas covering channel and the branch culture and observation channel is 20-80 microns.

8. The parallel cell culture microfluidic chip according to claim 4, characterized in that: The material of the gas and cell sampling layer and the culture layer is polydimethylsiloxane; the sum of the wall thicknesses of the corresponding covering contact of the gas covering channel and the gas-liquid premixing channel is 20-80 microns; the sum of the wall thicknesses of the corresponding covering contact of the gas covering channel and the branch culture and observation channel is 20-80 microns.

9. The parallel cell culture microfluidic chip according to claim 5, characterized in that: The material of the gas and cell sampling layer and the culture layer is polydimethylsiloxane; the sum of the wall thicknesses of the corresponding covering contact of the gas covering channel and the gas-liquid premixing channel is 20-80 microns; the sum of the wall thicknesses of the corresponding covering contact of the gas covering channel and the branch culture and observation channel is 20-80 microns.

10. The parallel cell culture microfluidic chip according to claim 7, characterized in that: The sum of the wall thicknesses of the gas-covered channel and the gas-liquid premixing channel in corresponding covering contact is 50 microns; the sum of the wall thicknesses of the gas-covered channel and the branch culture and observation channel in corresponding covering contact is 50 microns.

11. The parallel cell culture microfluidic chip according to claim 8 or 9, characterized in that: The sum of the wall thicknesses of the gas-covered channel and the gas-liquid premixing channel in corresponding covering contact is 50 microns; the sum of the wall thicknesses of the gas-covered channel and the branch culture and observation channel in corresponding covering contact is 50 microns.

12. A method for preparing the parallel cell culture microfluidic chip according to any one of claims 1 to 11, characterized in that: include: Step S1: preparing molds for the gas and cell injection layers and the culture layer respectively; Step S2: coating the materials for preparing the parallel cell culture microfluidic chip on the mold and curing them to obtain the gas and cell sampling layers and the culture layer; Step S3: Covering the gas and cell sampling layer on the culture layer so that the gas covering channel covers the cell isolation structure, and aligning the sampling connection area with the sampling through hole and the liquid outlet connection area with the sampling through hole, and then bonding them; Step S4: After drilling holes at the gas inlet, gas outlet, culture liquid inlet, the sample injection hole on the sample injection channel, and the liquid outlet hole on the liquid outlet channel, the glass slide is bonded under the culture layer.

13. A cell culture method, characterized in that: Cells are cultured using the parallel cell culture microfluidic chip according to any one of claims 1 to 11.

14. The method according to claim 13, characterized in that include: (1) vacuum treatment of the parallel cell culture microfluidic chip; (2) injecting the cell solution into the injection holes on the injection channel of the gas and cell injection layer, so that the cell solution enters each cell isolation structure of the culture layer through the injection holes; (3) Close the injection hole and add culture medium through the culture medium inlet; (4) After introducing different gases into each gas inlet, cell culture is carried out.

15. Use of the parallel cell culture microfluidic chip according to any one of claims 1 to 11 or the method according to claim 13 or 14 for simultaneously culturing different cells in different gas environments.

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