Modularized multi-organ co-culture chip and preparation method thereof

Abstract

The invention provides a modular multi-organ co-culture chip and a preparation method thereof.The chip is of a double-layer structure composed of two sets of single-layer chips, PDMS serves as a main body material of the single-layer chips, the two sets of single-layer PDMS chips are bonded into a whole after being subjected to plasma treatment, and the complete double-layer structure chip is formed; four culture units are arranged in each group of single-layer chips, each culture unit comprises three sections of culture channels, and each section of culture channel is sequentially and respectively composed of a short culture medium channel, a gel channel and a long culture medium channel which are adjacent to each other; a plurality of square micro-columns which are arranged at equal intervals and are used for locally separating the gel from the culture medium are arranged among the adjacent short culture medium channels, gel channels and long culture medium channels on each section of culture channel, and the four culture units are connected through the same long culture medium channel; the chip not only can realize the functions of co-culture and mutual influence of multiple organs, but also is simple in structure and easy to operate.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a modular multi-organ co-culture chip and a preparation method thereof. Background Art

[0002] Organ-on-a-chip technology has been a revolutionary breakthrough in biomedical engineering in recent years. Combining microfluidics with biomaterials, it provides a highly controllable experimental platform that closely resembles the in vivo environment for drug screening, toxicology studies, and the construction of disease models. The basic structure of an organ-on-a-chip typically consists of components such as microfluidic channels, cell culture chambers, and sensors. By precisely controlling fluid flow, applying mechanical forces, and transmitting biochemical signals, it can simulate some or all of the physiological functions of human organs in vitro.

[0003] Early organ-on-a-chip research focused primarily on the simulation of single organs. For example, the lung-on-a-chip, by recreating the alveolar-capillary barrier, has delved into gas exchange mechanisms and inflammatory responses; the liver-on-a-chip, by mimicking the liver lobule structure, has systematically studied drug pharmacokinetics and toxicity mechanisms; the intestine-on-a-chip, by mimicking the intestinal epithelial barrier, has revealed the molecular mechanisms of nutrient absorption and drug delivery; and the kidney-on-a-chip, by recreating the glomerular filtration barrier, has provided an important tool for studying kidney disease mechanisms and assessing drug toxicity. These single-organ-on-a-chips have demonstrated significant advantages in drug screening and toxicology studies, but their application in systemic studies remains limited due to the lack of simulation of interactions between multiple organ systems. Although some studies have attempted to combine two or more chips to more realistically simulate the interactions of complex physiological systems in the human body, these approaches require the design of complex connection systems and precise fluid control mechanisms to ensure the most accurate simulation of complex physiological processes in the human body. Due to the complexity of the systems and the difficulty of operation, such studies are often time-consuming and require a high level of expertise and skills from researchers. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a modular multi-organ co-culture chip.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the above modular multi-organ co-culture chip.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] A modular multi-organ co-culture chip has a double-layer structure consisting of two groups of single-layer chips. Each group of single-layer chips contains four culture units, and each culture unit contains three culture channels. Each culture channel is composed of a short culture medium channel, a gel channel and a long culture medium channel in sequence. There are several equidistantly arranged square microcolumns between the adjacent short culture medium channels, gel channels and long culture medium channels on each culture channel for locally separating the gel and culture medium. The four culture units are connected by the same long culture medium channel.

[0008] In the modular multi-organ co-culture chip, the short culture medium channels are the first culture medium channel 2, the second culture medium channel 4, the third culture medium channel 5, and the fourth culture medium channel 6. The intermediate gel channels are the first gel channel 7, the second gel channel 8, the third gel channel 9, and the fourth gel channel 10. After the gel flows through the gel channels, the presence of the square micropillars prevents it from flowing outward due to surface tension and other factors, thus achieving the purpose of separating the gel from the culture medium.

[0009] Preferably, in the above-mentioned modular multi-organ co-culture chip, the single-layer chip uses PDMS as the main material. PDMS has good biocompatibility and air permeability, which can meet the requirements of long-term cell culture and simulation of the physiological dynamic environment in the body. In addition, PDMS also has good optical transparency and is suitable for live cell imaging and micromanipulation; the two groups of single-layer PDMS chips 12 are bonded into a whole after plasma treatment to form a complete double-layer structure chip.

[0010] Preferably, in the above-mentioned modular multi-organ co-culture chip, the ratio of PDMS prepolymer to curing agent used in the production of the single-layer chip is 10:1, and the chip is placed in an oven and heated at 80°C for 2 hours, then taken out and demolded, and the demolded chip is ultrasonically cleaned using anhydrous ethanol and ultrapure water as the cleaning liquids. After cleaning in each of the two cleaning liquids for 5 minutes, the chip is placed in an oven and heated at 120°C for 5 minutes to ensure that the residual moisture in the chip is completely evaporated to avoid affecting the subsequent surface treatment process.

[0011] Preferably, in the modular multi-organ co-culture chip, the short culture channel has a length of L1, a width of W1, and a depth of D1. The two ends of the main body of the short culture channel are respectively an inlet and a liquid outlet, the hole radius of the inlet and the liquid outlet is R1, and the angle between the inlet and the liquid outlet and the main body of the short culture channel is θ; the long culture channel has a length of L2, a width of W2, and a depth of D2, and is used to connect four culture units. The two ends of the long culture channel are respectively a long culture channel inlet 21 and a long culture channel outlet 22. The culture medium flows into the channel through the long culture channel inlet 21. After passing through the first culture unit, the metabolites of the cells cultured in the unit continue to flow through the second culture unit, and then flow through the third culture unit together with the metabolites of the first two culture units, and so on, flow through the fourth culture unit until it flows out from the liquid outlet 22 of the long culture medium channel. The length of the gel channel is L3, the width is W3, and the depth is D3. The depths of different culture medium channels are the same, that is, D1=D2, and the widths of different channels are the same, that is, W1=W2=W3. The side length of the square microcolumn between the culture medium channel and the gel channel is L3, the height is D3, and the equidistant interval between the microcolumns is S.

[0012] Preferably, in the modular multi-organ co-culture chip, the length of the short culture channel is L1 = 13 mm, 14 mm, 15 mm, 18 mm, 20 mm or 23 mm, preferably 18 mm, the width is W1 = 0.3 mm, 0.5 mm, 1 mm, 1.5 mm or 2 mm, preferably 1 mm, the depth is D1 = 1 mm, 2 mm, 3 mm, 5 mm or 7 mm, preferably 1 mm, and the hole radius of the liquid inlet and outlet is R1 = 0.2 mm, 0 .4mm, 0.8mm, 1.0mm or 1.3mm, preferably, 0.8mm, the angle between the liquid inlet and the liquid outlet and the short culture medium channel body is θ = 105°, 115°, 125°, 135° or 145°, preferably, 135°; the length of the long culture medium channel is L2 = 70mm, 75mm, 80mm, 85mm, 90mm or 100mm, preferably, 75mm, and the width is W2 = 0.3mm, 0.5mm, 1mm, 1.5m m or 2mm, preferably, 1mm, the depth is D2 = 1mm, 2mm, 3mm, 5mm or 7mm, preferably, 1mm; the length of the gel channel is L3 = 18mm, 19mm, 20mm, 23mm, 25mm or 28mm, preferably, 18mm, the width is W3 = 0.3mm, 0.5mm, 1mm, 1.5mm or 2mm, preferably, 1mm, the depth is D3 = 300μm, 700μm, 1000μm, 1500μm m or 2000 μm, preferably, 300 μm; the side length of the square microcolumn is L3 = 50 μm, 100 μm, 200 μm, 300 μm or 500 μm, preferably, 200 μm, the height is D3 = 300 μm, 700 μm, 1000 μm, 1500 μm or 2000 μm, preferably, 300 μm, and the equidistant spacing between the microcolumns is S = 50 μm, 100 μm, 200 μm, 300 μm or 500 μm, preferably, 200 μm.

[0013] Preferably, in the above-mentioned modular multi-organ co-culture chip, the length of the single-layer chip is 70 mm, 75 mm, 80 mm, 85 mm, 90 mm or 100 mm, preferably 70 mm, the width is 20 mm, 25 mm, 30 mm, 35 mm, 40 mm or 50 mm, preferably 20 mm, and the height is 3 mm, 4 mm, 5 mm, 8 mm or 10 mm, preferably 3 mm.

[0014] In the preparation method of the above-mentioned modular multi-organ co-culture chip, the single-layer chip uses PDMS as the main material. PDMS has good biocompatibility and breathability, which can meet the requirements of long-term cell culture and simulation of the physiological dynamic environment in the body. In addition, PDMS also has good optical transparency and is suitable for live cell imaging and micromanipulation; two sets of single-layer PDMS chips are bonded into a whole after plasma treatment to form a complete double-layer structure chip.

[0015] Preferably, in the preparation method of the above-mentioned modular multi-organ co-culture chip, the ratio of PDMS prepolymer to curing agent used in the production of the single-layer chip is 10:1, the chip is placed in an oven for heating, and then taken out and demolded. The mold is obtained by 3D printing technology. The demolded chip is ultrasonically cleaned, and the cleaning liquids are anhydrous ethanol and ultrapure water respectively. The chip is cleaned in the two cleaning liquids successively, and the chip is placed in an oven and heated until the residual water in the chip is completely evaporated.

[0016] Preferably, in the preparation method of the above-mentioned modular multi-organ co-culture chip, the ratio of PDMS prepolymer to curing agent used in the production of the single-layer chip is 10:1, and the chip is placed in an oven and heated at 80°C for 2 hours, then taken out and demolded. The mold is obtained by 3D printing technology. The demolded chip is ultrasonically cleaned, and the cleaning liquids are anhydrous ethanol and ultrapure water, respectively. After cleaning in each of the two cleaning liquids for 5 minutes, the chip is placed in an oven and heated at 120°C for 5 minutes to ensure that the residual water in the chip is completely evaporated to avoid affecting the subsequent surface treatment process.

[0017] Beneficial effects:

[0018] The above-mentioned modular multi-organ co-culture chip contains four culture units, and the four culture units are connected by a long culture medium channel. It can not only simulate the tissue structure of multiple organs and their respective mechanical microenvironments, but also explore the interactions between organs, realize the functions of multi-organ co-culture and mutual influence, and has a simple structure and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the structure of the modular multi-organ co-culture chip of the present invention;

[0020] Figure 2 This is a layered diagram of the modular multi-organ co-culture chip of the present invention;

[0021] Figure 3 This is a top view of the modular multi-organ co-culture chip of the present invention.

[0022] In the figure: 1-modular multi-organ co-culture chip, 2-first culture medium channel, 3-long culture medium channel, 4-second culture medium channel, 5-third culture medium channel, 6-fourth culture medium channel, 7-first gel channel, 8-second gel channel, 9-third gel channel, 10-fourth gel channel, 11-square microcolumn, 12-single-layer chip, 13-first culture medium channel inlet, 14-first culture medium channel outlet, 15-second culture medium channel inlet, 16-second culture medium channel outlet, 17-first Three culture medium channel inlets, 18-third culture medium channel outlet, 19-fourth culture medium channel inlet, 20-fourth culture medium channel outlet, 21-long culture medium channel inlet, 22-long culture medium channel outlet, 23-first gel channel inlet, 24-first gel channel outlet, 25-second gel channel inlet, 26-second gel channel outlet, 27-third gel channel inlet, 28-third gel channel outlet, 29-fourth gel channel inlet, 30-fourth gel channel outlet. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0024] Example 1

[0025] like Figure 1-3 As shown, a modular multi-organ co-culture chip 1 has a double-layer structure consisting of two groups of single-layer chips 12. Each group of single-layer chips contains four culture units, and each culture unit contains three culture channels. Each culture channel is composed of a short culture medium channel, a gel channel and a long culture medium channel 3 adjacent to each other. There are several square microcolumns 11 with equal intervals of 200 μm (or 50 μm, 100 μm, 200 μm, 300 μm or 500 μm) between the adjacent short culture medium channels, gel channels and long culture medium channels on each culture channel for locally separating the gel and the culture medium. The four culture units are connected by the same long culture medium channel.

[0026] The short culture medium channels are respectively the first culture medium channel 2, the second culture medium channel 4, the third culture medium channel 5 and the fourth culture medium channel 6, and the middle gel channels are respectively the first gel channel 7, the second gel channel 8, the third gel channel 9 and the fourth gel channel 10.

[0027] The corresponding liquid inlets on each channel: the first culture medium channel liquid inlet 13, the second culture medium channel liquid inlet 15, the third culture medium channel liquid inlet 17, the fourth culture medium channel liquid inlet 19, the long culture medium channel liquid inlet 21, the first gel channel liquid inlet 23, the second gel channel liquid inlet 25, the third gel channel liquid inlet 27, and the fourth gel channel liquid inlet 29 are located on the same side; the corresponding liquid outlets: the first culture medium channel liquid outlet 14, the second culture medium channel liquid outlet 16, the third culture medium channel liquid outlet 18, the fourth culture medium channel liquid outlet 20, the long culture medium channel liquid outlet 22, the first gel channel liquid outlet 24, the second gel channel liquid outlet 26, the third gel channel liquid outlet 28, and the fourth gel channel liquid outlet 30 are located on the other side.

[0028] The preparation method of the modular multi-organ co-culture chip is as follows:

[0029] (1) Draw the chip mold diagram in SOLIDWORKS (design the first culture medium channel 2, long culture medium channel 3, second culture medium channel 4, third culture medium channel 5, fourth culture medium channel 6, first gel channel 7, second gel channel 8, third gel channel 9, fourth gel channel 10, and square microcolumns 11);

[0030] (2) Use 3D printing to process the mold drawing into a physical object;

[0031] (3) demolding the chip mold using trimethylchlorosilane under vacuum;

[0032] (4) Use Dow Corning 184PDMS prepolymer, where the ratio of glue A to glue B is 10:1, and stir the two until fine bubbles appear;

[0033] (5) Place the mixed PDMS in a vacuum pump and use negative pressure to expel the bubbles in the mixture. This process takes about 20 minutes.

[0034] (6) Use a syringe to slowly cover the chip mold with a release agent on the surface with bubble-free PDMS. When it is about 1 mm thick (the thickness and other dimensions can be adjusted according to actual needs), place the mold in a vacuum pump again to expel the bubbles generated by the PDMS on the mold surface during this process.

[0035] (7) Repeat step (6) 2-3 times;

[0036] (8) Place the mold covered with PDMS in an oven and heat at 80°C for 2 hours;

[0037] (9) Remove the chip from the mold and perform ultrasonic cleaning on it. The cleaning solutions are anhydrous ethanol and ultrapure water. After rinsing for 5 minutes in each of the two cleaning solutions, the chip is placed in an oven and heated at 120°C for 5 minutes to ensure that the remaining water in the chip is completely evaporated.

[0038] (10) Using a plasma cleaning agent to perform oxygen plasma treatment on the PDMS chip (Plasma), the treated single-layer chip 12 is placed under a microscope for alignment and bonding;

[0039] (11) Place the bonded double-layer chip in an oven and heat at 120°C for 2 hours;

[0040] (12) Place the chip in a high-pressure steam sterilizer for sterilization and it is ready for use.

[0041] The length of the short culture medium channel is L1, the width is W1, and the depth is D1. The two ends of the main body of the short culture medium channel are respectively a liquid inlet and a liquid outlet. The hole radius of the liquid inlet and the liquid outlet is R1, and the angle between the liquid inlet and the liquid outlet and the main body of the short culture medium channel is θ; the length of the long culture medium channel is L2, the width is W2, and the depth is D2. It is used to connect four culture units. The two ends of the long culture medium channel are respectively a long culture medium channel liquid inlet 21 and a long culture medium channel liquid outlet 22. The culture medium flows into the channel through the long culture medium channel liquid inlet 21 and passes through the first culture unit. Then, the metabolic products of the cells cultured in this unit continue to flow through the second culture unit, and then the metabolic products of the first two culture units flow through the third culture unit, and so on, flow through the fourth culture unit, until it flows out from the liquid outlet 22 of the long culture medium channel. The length of the gel channel is L3, the width is W3, and the depth is D3. The depths of different culture medium channels are the same, that is, D1=D2, and the widths of different channels are the same, that is, W1=W2=W3. The side length of the square microcolumn between the culture medium channel and the gel channel is L3, the height is D3, and the equidistant interval between the microcolumns is S.

[0042] The length of the short culture medium channel is L1=18mm (or 13mm, 14mm, 15mm, 20mm or 23mm), the width is W1=1mm (or 0.3mm, 0.5mm, 1.5mm or 2mm), the depth is D1=1mm (or 2mm, 3mm, 5mm or 7mm), the hole radius of the liquid inlet and the liquid outlet is R1=0.8mm (or 0.2mm, 0.4mm, 1.0mm or 1.3mm), and the angle between the liquid inlet and the liquid outlet and the main body of the short culture medium channel is θ=135° (or 105°, 115°, 125° or 145°); the length of the long culture medium channel is L2=75mm (or 70mm, 80mm, 85mm, 90mm or 100mm), the width is W2=1mm (or 0.3mm, 0.5mm, 1.5 mm or 2mm), the depth is D2 = 1mm (can also be 2mm, 3mm, 5mm or 7mm); the length of the gel channel is L3 = 18mm (can also be 19mm, 20mm, 23mm, 25mm or 28mm), the width is W3 = 1mm (can also be 0.3mm, 0.5mm, 1.5mm or 2mm), the depth is D3 = 300μm (can also be 700μm, 1000μm , 1500μm or 2000μm); the side length of the square microcolumn is L3=200μm (it can also be 50μm, 100μm, 300μm or 500μm), the height is D3=300μm (it can also be 700μm, 1000μm, 1500μm or 2000μm), and the equidistant spacing between the microcolumns is S=200μm (it can also be 50μm, 100μm, 300μm or 500μm).

[0043] The length of the single-layer chip is 70 mm (or 75 mm, 80 mm, 85 mm, 90 mm or 100 mm), the width is 20 mm (or 25 mm, 30 mm, 35 mm, 40 mm or 50 mm), and the height is 3 mm (or 4 mm, 5 mm, 8 mm or 10 mm).

[0044] The modular multi-organ co-culture chip can culture two or more organs in the same chip. During application:

[0045] (1) Sterilize the modular multi-organ co-culture chip with high-pressure steam;

[0046] (2) Prepare a 10% GelMa hydrogel solution, protect from light, and sterilize using a 0.22 μm sterile syringe filter;

[0047] (3) Resuspending cells a of a simulated organ or physiological structure A in the prepared GelMa hydrogel solution, using a syringe pump to introduce the hydrogel solution containing cells a into the gel channel (7) at a flow rate of 20 μL / min, and then irradiating with a 405 nm light source for 15 to 30 seconds to cause gelation;

[0048] (4) The same as the operation in step (3), repeat the above operation for cells b, c, and d of the simulated organs or physiological structures B, C, and D respectively;

[0049] (5) Injecting culture medium into the corresponding liquid inlets of the first culture medium channel 2, the second culture medium channel 4, the third culture medium channel 5, and the fourth culture medium channel 6, respectively, and placing the chip in a 37°C incubator for 5 minutes, then removing the chip and removing the culture medium;

[0050] (6) Injecting culture medium containing nutrients and growth factors required for culturing cells a, b, c, and d into the corresponding liquid inlets of the first culture medium channel 2, the second culture medium channel 4, the third culture medium channel 5, and the fourth culture medium channel 6, respectively. For example, injecting DMEM culture medium supplemented with L-glutamine, HEPES buffer, and double antibodies into the culture medium channel next to the hepatocyte gel channel, and circulating the culture medium in each channel at an appropriate speed.

[0051] (7) Adding ordinary culture medium to the long culture medium channel 3, and similarly, allowing the culture medium to circulate in the channel at a flow rate of 10 μL / min;

[0052] (8) Adding drugs, such as cisplatin and vancomycin, to the long culture medium channel 3 can simulate the effects of different doses of drugs on multiple organs on the chip by adjusting the flow rate or drug concentration, or simulate the time-dependent effects of drugs, such as observing the absorption, metabolism, and excretion processes of drugs in various organs as the culture time changes after the drug is injected.

[0053] In the above-mentioned application process, after the cells are planted in the chip, the inlet and outlet of each channel are connected to the silicone hose on the peristaltic pump by a capillary, and the microstructure design of each channel is combined with the peristaltic pump. The flow rate of the long culture medium channel is controlled by a single pump, and the state of multi-organ collaboration is reproduced in vitro by structural design (such as channel connection, micro-column separation), fluid control (peristaltic pump, channel size) and metabolic pathway simulation (channel drug addition). The dynamic balance between multiple organs is achieved, and the gel and culture medium are separated by micro-columns to ensure the stability and directionality of fluid flow. This design can still accurately simulate physiological flow rates and reduce human operation errors while simplifying the control system. In addition, by channel drug addition and overall circulation mode (such as three-way valve design in Example 2 below), the effect of drugs on other organs after metabolism by specific organs (such as liver) can be studied in a directed manner. At the same time, multiple culture units are connected in series through long culture medium channels to simulate the time-dependent effect of drugs in vivo, filling the gap in systemic drug evaluation.

[0054] Example 2

[0055] During the application of the modular multi-organ co-culture chip described in Example 1:

[0056] (1) The only difference from Example 1 is the location of drug addition and the culture fluid circulation method in this embodiment: in Example 1, the location of drug addition is the liquid inlet 21 of the long culture medium channel, while in this embodiment, the location of drug addition is the liquid inlet 13 of the first culture medium channel; in Example 1, the culture fluid circulation method is a separate circulation, and each short culture medium channel is connected to an external liquid storage bottle, and the liquid circulating in the channel is always the liquid in the corresponding liquid storage bottle, that is, each culture unit is relatively independent, and only the effect of the drug on a single organ is studied, while the circulation method in this embodiment is a whole circulation, and except for the first culture medium channel 2, the liquid in the other three short culture medium channels will not return to the corresponding liquid storage bottle, but will be collected to the liquid inlet 21 of the long culture medium channel through a number of three-way valves, and then enter the collection device through the channel;

[0057] (2) The same as the first five steps in Example 1, culturing the organ or tissue structure to be simulated in the experiment in the modular multi-organ co-culture chip;

[0058] (3) Injecting a culture medium containing nutrients and growth factors required for culturing cells into the first culture medium channel 2. In addition, adding drugs at the concentration required for the experiment to the culture medium allows the drugs to pass through the first gel channel 7 and diffuse into the long culture medium channel 3 together with the cell metabolites in the channel: the GeIMa hydrogel solution has a porous structure after solidification. The porosity of the 10% GelMa hydrogel solution used is about 0.6. The culture medium can enter the gel through these pores. As the culture medium slowly flows in the first culture medium channel 2, a portion of the culture medium can gradually pass through the gel channel and enter the long culture medium channel 3.

[0059] (4) The culture medium containing the nutrients and growth factors required for culturing cells is injected into the second culture medium channel 4, the third culture medium channel 5 and the fourth culture medium channel 6 respectively. Three three-way valves are used to transport the drug metabolites of the cultured different organs or tissue structures together with the culture medium back to the long culture medium channel 3, wherein the two ends of the first three-way valve are used to connect the pipes connected to the second culture medium channel outlet 16 and the fourth culture medium channel outlet 20, and the third port of the valve is connected to one end of the second three-way valve. The other two ends of the second three-way valve are connected to the third culture medium channel outlet 18 and one end of the third three-way valve respectively, and the other two interfaces of the third three-way valve are connected to the liquid storage bottle and the long culture medium channel 3 respectively. The long culture medium channel outlet 22 is connected to the liquid storage bottle through a silicone hose to realize the circulation of the liquid.

[0060] (5) The drug addition and liquid circulation model used can simulate the effects of the drug's metabolites on other organs in the body after it is metabolized by a specific organ (such as the liver), and also reflects the effects of these metabolites on the original metabolic organ itself.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. The improvements and modifications made by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. A modular multi-organ co-culture chip, characterized by: It has a double-layer structure consisting of two groups of single-layer chips. Each group of single-layer chips contains four culture units, and each culture unit contains three culture channels. Each culture channel is composed of a short culture medium channel, a gel channel and a long culture medium channel in sequence. There are several equidistantly arranged square microcolumns between the adjacent short culture medium channels, gel channels and long culture medium channels on each culture channel for locally separating the gel and the culture medium. The four culture units are connected by the same long culture medium channel.

2. The modular multi-organ co-culture chip according to claim 1, characterized in that: The length of the short culture medium channel is L1, the width is W1, and the depth is D1. The two ends of the main body of the short culture medium channel are respectively the liquid inlet and the liquid outlet. The hole radius of the liquid inlet and the liquid outlet is R1, and the angle between the liquid inlet and the liquid outlet and the main body of the short culture medium channel is θ; the length of the long culture medium channel is L2, the width is W2, and the depth is D2, which is used to connect four culture units. The length of the gel channel is L3, the width is W3, and the depth is D3. The depths of different culture medium channels are the same, that is, D1=D2, and the widths of different channels are the same, that is, W1=W2=W3. The side length of the square microcolumn between the culture medium channel and the gel channel is L3, the height is D3, and the equidistant spacing between the microcolumns is S.

3. The modular multi-organ co-culture chip according to claim 2, characterized in that: The length of the short culture medium channel is L1 = 13 mm, 14 mm, 15 mm, 18 mm, 20 mm or 23 mm, the width is W1 = 0.3 mm, 0.5 mm, 1 mm, 1.5 mm or 2 mm, the depth is D1 = 1 mm, 2 mm, 3 mm, 5 mm or 7 mm, the hole radius of the liquid inlet and the liquid outlet is R1 = 0.2 mm, 0.4 mm, 0.8 mm, 1.0 mm or 1.3 mm, and the angle between the liquid inlet and the liquid outlet and the short culture medium channel body is θ = 105°, 115°, 125°, 135° or 145°; the length of the long culture medium channel is L2 = 70 mm, 75 mm, 80 mm, 85 mm, 90 mm or 100 mm, the width is W2 = 0.3 mm, 0.5 mm, 1 mm, 1.5 mm or 2 mm, the depth is D1 = 1 mm, 2 mm, 3 mm, 5 mm or 7 mm, the hole radius of the liquid inlet and the liquid outlet is R1 = 0.2 mm, 0.4 mm, 0.8 mm, 1.0 mm or 1.3 mm, and the angle between the liquid inlet and the liquid outlet and the short culture medium channel body is θ = 105°, 115°, 125°, 135° or 145°. 5mm or 2mm, and a depth of D2 = 1mm, 2mm, 3mm, 5mm or 7mm; the length of the gel channel is L3 = 18mm, 19mm, 20mm, 23mm, 25mm or 28mm, the width is W3 = 0.3mm, 0.5mm, 1mm, 1.5mm or 2mm, and the depth is D3 = 300μm, 700μm, 1000μm, 1500μm or 2000μm; the side length of the square microcolumn is L3 = 50μm, 100μm, 200μm, 300μm or 500μm, the height is D3 = 300μm, 700μm, 1000μm, 1500μm or 2000μm, and the equidistant spacing between the microcolumns is S = 50μm, 100μm, 200μm, 300μm or 500μm.

4. The modular multi-organ co-culture chip according to claim 3, characterized in that: The length of the short culture medium channel is L1=18 mm, the width is W1=1 mm, the depth is D1=1 mm, and the hole radius of the liquid inlet and outlet is R i =0.8mm, the angle between the liquid inlet and outlet and the main body of the short culture medium channel is θ=135°; the length of the long culture medium channel is L2=75mm, the width is W2=1mm, and the depth is D2=1mm; the length of the gel channel is L3=18mm, the width is W3=1mm, and the depth is D3=300μm; the side length of the square microcolumn is L3=200μm, the height is D3=300μm, and the equidistant spacing between the microcolumns is S=200μm.

5. The modular multi-organ co-culture chip according to claim 2, characterized in that: The single-layer chip has a length of 70 mm, 75 mm, 80 mm, 85 mm, 90 mm or 100 mm, a width of 20 mm, 25 mm, 30 mm, 35 mm, 40 mm or 50 mm, and a height of 3 mm, 4 mm, 5 mm, 8 mm or 10 mm.

6. The modular multi-organ co-culture chip according to claim 5, characterized in that: The single-layer chip has a length of 70 mm, a width of 20 mm, and a height of 3 mm.

7. The method for preparing the modular multi-organ co-culture chip according to any one of claims 1 to 6, characterized in that: The single-layer chip uses PDMS as the main material. Two sets of single-layer PDMS chips are bonded into a whole after plasma treatment to form a complete double-layer structure chip.

8. The method for preparing a modular multi-organ co-culture chip according to claim 7, characterized in that: The single-layer chip is produced using a 10:1 ratio of PDMS prepolymer to curing agent. The chip is placed in an oven for heating and then removed from the mold. The mold is obtained by 3D printing technology. The demolded chip is ultrasonically cleaned using anhydrous ethanol and ultrapure water as cleaning fluids. The chip is cleaned in both cleaning fluids in succession and then placed in an oven and heated until any residual moisture in the chip is completely evaporated.

Images