Organ-like based on micro-fluidic chip as well as preparation method and application of organ-like

Abstract

The invention provides an organoid based on a micro-fluidic chip as well as a preparation method and application of the organoid. The organoid based on the micro-fluidic chip comprises the micro-fluidic chip and organoid microspheres, the micro-fluidic chip comprises an inner cavity, the inner cavity comprises capture card embedding structures which are alternately arranged in a quincunx shape, and each capture card embedding structure comprises a capture card embedding micro-column and a liquid guide channel; the organ-like microspheres are hydrogel microspheres loaded with cells. Through a 3D biological printing technology, batch organoid microspheres uniform in structure and stable in function can be rapidly prepared, and the situation that the experiment progress is slow due to the difference between models in a drug screening experiment is improved; the organ-like model with the characteristics simulates the flowing of human blood and better reproduces a human microenvironment; the capture rate of organoid microspheres is increased, the space utilization rate in the chip is improved, the usage amount of reagents and drugs in a drug screening experiment is reduced, and the scientific research cost is reduced; the problem that the micro-fluidic chip type organs are difficult to visualize and traceable can be solved.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to an organoid based on a microfluidic chip and a preparation method and application thereof. Background Art

[0002] Species differences between animals and humans have become the main obstacle limiting the use of animal models in drug development and screening. The main disadvantage of 2D (Two-dimensional) cell lines is that they lack hierarchical structure, dimensionality, cell diversity, and cell-cell or cell-matrix interactions, which makes it impossible for cell lines to accurately simulate the cell functions present in tissues. However, the emergence of organoid models has gradually broken this situation. Organoids, as the name suggests, refer to an in vitro organ model established to simulate human organs. These models have more complex and diverse cell types and more stable genomes, and can better reflect the interactions between cells and between cells and the microenvironment. This makes organoid models an important tool that is closer to human physiological conditions and can more accurately predict drug responses and toxicity, bringing new hope and opportunities to the field of drug research and development.

[0003] Currently, research on organoids is showing a spurt of growth, and various organoid models have been established like mushrooms after a spring rain. However, the large number of models does not mean that drug screening will embark on an easy road. The important limiting factor is that there are many uncontrollable factors in the model construction process, making it difficult to ensure that the organoid models constructed in different batches or even in the same batch are highly stable and consistent. The inconsistency between research and development and application is an important reason hindering the vigorous development of organoids. Therefore, in order to solve this problem, it is crucial to build a reproducible organoid model with stable structure and function for large-scale experiments.

[0004] At the same time, a single organoid model cannot perfectly replicate the complex and diverse physiological conditions in the human body, including the lack of a fluid environment and an integrated microenvironment formed by multi-organ tissues.

[0005] It usually takes more time to build a multifunctional system, which also applies to the design of organoid models. In this process, it is crucial to observe and judge the status of organoid microspheres in a timely manner. This is a key step to ensure the successful construction of organoid models and save manpower and material resources. Therefore, while improving the function of organoid models, taking into account convenient observation and tracking analysis capabilities is one of the important criteria for evaluating the pros and cons of microfluidic chip structure design. At this stage, the construction of most microfluidic chip models cannot be separated from the filling of various collagens. The addition of collagen is to simulate the function of the extracellular matrix in a real physiological environment, but the opaque nature of collagen has also brought many inconveniences to subsequent observations. How to deal with the "relationship between collagen and microfluidic chips" and realize more and more complete functions is an issue that needs to be deeply considered in the process of microfluidic chip design. Summary of the invention

[0006] The purpose of this application is to provide an organoid based on a microfluidic chip to improve the stability and consistency of the organoid model so that the organoid model can better reproduce the human microenvironment.

[0007] The first aspect of the present application provides an organoid based on a microfluidic chip, which includes a microfluidic chip and organoid microspheres. The microfluidic chip includes an inner chamber, the inner chamber includes a capture card structure arranged alternately in a plum blossom shape, the capture card structure includes capture card microcolumns and liquid diversion channels; the organoid microspheres are hydrogel microspheres loaded with cells.

[0008] In one embodiment of the present application, the cells are selected from at least one of human lung epithelial cells, human umbilical vein endothelial cells, and human lung fibroblasts; wherein the diameter of the organoid microspheres is h μm, and h satisfies 220≤h≤300.

[0009] In one embodiment of the present application, the material of the hydrogel microspheres is selected from at least one of sodium alginate, gelatin, collagen, and polyvinyl alcohol.

[0010] In one embodiment of the present application, the material of the microfluidic chip is selected from at least one of polydimethylsiloxane, polymethyl methacrylate, polyimide, and glass.

[0011] In one embodiment of the present application, the length of the inner chamber is 16 mm to 24 mm, and the width of the inner chamber is 11 mm to 15 mm.

[0012] In one embodiment of the present application, the capture embedded card structure satisfies at least one of the following characteristics: (1) the capture embedded card structure includes two half-moon shaped capture embedded card micro-columns, and the inner diameter of the half-moon is 260 μm to 280 μm; (2) the length of the capture embedded card structure is 1.5h to 1.8h, the width is 1.5h to 1.8h, and the height is 1.5h to 1.8h; (3) the interval between adjacent columns of the capture embedded card structure is 1.4h to 1.6h, and the interval between adjacent rows is 1.8h to 2h; (4) the shortest distance between the capture embedded card structure and the liquid inlet is 8h to 9h; (5) the shortest distance between the capture embedded card structure and the liquid outlet is 8h to 9h; (6) the liquid diversion channel is arranged between the two capture embedded card micro-columns of the capture embedded card structure; (7) the width of the liquid diversion channel is 0.5h to 0.8h.

[0013] In one embodiment of the present application, the method for preparing organoid microspheres includes: S1. spraying solution A into droplets into solution B through piezoelectric printing to form hydrogel microspheres; S2. treating the hydrogel microspheres step by step with type I rat tail collagen solution and dopamine hydrochloride solution to obtain functionalized hydrogel microspheres; S3. mixing different types of cells and functionalized hydrogel microspheres to obtain organoid microspheres.

[0014] In one embodiment of the present application, in step S1, the parameters of piezoelectric printing are: the concentration of solution A is 0.8wt% to 1.2wt%, the concentration of solution B is 0.8wt% to 1.2wt%, the print head height is 38mm to 42mm, the print spacing is 0.2mm to 0.3mm, the air pressure is 0.06Mpa to 0.08MPa, the opening time of the electric gate is 225ms to 275ms, and the closing time of the electric gate is 275ms to 325ms.

[0015] In one embodiment of the present application, the solute of solution A is selected from at least one of sodium alginate, gelatin, collagen, and polyvinyl alcohol; the solute of solution B is selected from at least one of calcium chloride, glutaraldehyde, and boric acid.

[0016] In one embodiment of the present application, in step S2, the concentration of the type I rat tail collagen solution is 8 mg / ml to 10 mg / ml, and the concentration of the dopamine hydrochloride solution is 0.15% to 0.25%.

[0017] In one embodiment of the present application, in step S3, the cells are selected from at least one of human lung epithelial cells, human umbilical vein endothelial cells, and human lung fibroblasts, and the total cell planting density is 0.6×10 4 / mm 2 to 1.0×10 4 / mm 2 .

[0018] The second aspect of the present application provides a method for preparing an organoid based on a microfluidic chip as described in the first aspect of the present application, comprising: (1) after adaptive culture of the organoid microspheres, gently blowing them 2-3 times with a pipette; (2) transferring the organoid microspheres to a microfluidic chip, and perfusing the organoid based on the microfluidic chip through an external circulation system.

[0019] Among them, the adaptive culture time is 8h to 16h, the number of organoid microspheres transferred to the microfluidic chip is 150 to 160, and the circulation system includes a driving pump, a controller, a liquid reservoir, a three-way valve and a pipeline.

[0020] The third aspect of the present application provides an application of the microfluidic chip-based organoid described in the first aspect of the present application or the microfluidic chip-based organoid prepared by the preparation method described in the second aspect in the treatment of acute lung injury with traditional Chinese medicine monomers.

[0021] Beneficial effects of this application:

[0022] The present application provides an organoid based on a microfluidic chip, which includes a microfluidic chip and organoid microspheres, the microfluidic chip includes an inner chamber, the inner chamber includes a capture card structure arranged alternately in a plum blossom shape, the capture card structure includes a capture card microcolumn and a liquid diversion channel; the organoid microspheres are hydrogel microspheres loaded with cells. Through 3D bioprinting technology, it is possible to quickly prepare a batch of organoid microspheres with uniform structure and stable function, and improve the slow progress of experiments caused by differences between models in drug screening experiments; the organoid model with the above characteristics simulates the flow of human blood and better reproduces the human microenvironment; increases the capture rate of organoid microspheres, improves the utilization rate of space in the chip, reduces the amount of reagents and drugs used in drug screening experiments, and reduces scientific research costs; it can overcome the problem that microfluidic chip organoids are difficult to visualize and trace.

[0023] Of course, implementing any product or method of the present application does not necessarily require achieving all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0025] Figure 1 This is a schematic diagram of the structure of the organoid based on the microfluidic chip prepared in Example 1 of the present application;

[0026] Figure 2 for Figure 1 Sectional view at AA in the middle;

[0027] Figure 3 This is a schematic diagram of the structure of the organoid based on the microfluidic chip prepared in Example 1 of the present application;

[0028] Figure 4 This is a schematic diagram of the structure of the organoid microspheres prepared in Example 1 of the present application;

[0029] Figure 5 The hydrogel microspheres prepared in Example 1 of the present application and the particle size distribution diagram;

[0030] Figure 6 These are light microscopic images of the microfluidic chip-based organoids prepared in Example 1 of the present application taken at continuous perfusion for 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h;

[0031] Figure 7 This is a light microscopic image of the microfluidic chip-based organoid prepared in Example 1 of the present application taken after continuous perfusion for 48 hours;

[0032] Figure 8 (a) A three-dimensional image of the organoid based on the microfluidic chip prepared in Example 1 of the present application after continuous perfusion for 48 hours under a confocal microscope; (b) DAPI staining results of the organoid microspheres;

[0033] Fig. 9 This is a result diagram of the detection of the inflammatory factor IL-1β content when the microfluidic chip-based organoid prepared in Example 1 of the present application is used as an organoid model;

[0034] Fig.10 This is a result diagram of the detection of the inflammatory factor IL-6 content when the microfluidic chip-based organoid prepared in Example 1 of the present application is used as an organoid model;

[0035] Fig.11 This is a result diagram of the detection of the inflammatory factor IL-1β content when the organoid microspheres prepared in Comparative Example 1 of the present application are used as an organoid model;

[0036] Fig.12 This is a graph showing the results of detecting the content of the inflammatory factor IL-6 when the organoid microspheres prepared in Comparative Example 1 of the present application are used as an organoid model.

[0037] Reference numerals: organoid based on microfluidic chip 1; microfluidic chip 10; organoid microsphere 11; immobilization chip 101; glass slide 102; liquid inlet 1011; liquid outlet 1012; inner chamber 1013; capture embedded card structure 1014; capture embedded card microcolumn 1015; capture embedded card microcolumn 1016;

[0038] The length of the capture card structure L1; the width of the liquid guide channel L2; the interval between adjacent columns L3; the width of the capture card structure w1; the interval between adjacent rows of the capture card structure w2; the shortest distance between the capture card structure and the liquid inlet w3; the shortest distance between the capture card structure and the liquid outlet w4;

[0039] Diameter of organoid microsphere h; height of capture card structure h1; hydrogel microsphere 1101; type I rat tail collagen and dopamine hydrochloride layer 1102; cell layer 1103.

[0040] In the drawings, the drawings are not necessarily drawn to scale. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the present application belong to the scope of protection of the present application.

[0042] The present application provides an organoid based on a microfluidic chip, which includes a microfluidic chip and organoid microspheres, the microfluidic chip includes an inner chamber, the inner chamber includes a capture card structure arranged alternately in a plum blossom shape, the capture card structure includes a capture card microcolumn and a liquid diversion channel; the organoid microspheres are hydrogel microspheres loaded with cells. Through 3D bioprinting technology, it is possible to quickly prepare a batch of organoid microspheres with uniform structure and stable function, and improve the slow progress of experiments caused by differences between models in drug screening experiments; the organoid model with the above characteristics simulates the flow of human blood and better reproduces the human microenvironment; increases the capture rate of organoid microspheres, improves the utilization rate of space in the chip, reduces the amount of reagents and drugs used in drug screening experiments, and reduces scientific research costs; it can overcome the problem that microfluidic chip organoids are difficult to visualize and trace.

[0043] In one embodiment of the present application, the cells are selected from at least one of human lung epithelial cells, human umbilical vein endothelial cells, and human lung fibroblasts; wherein the diameter of the organoid microsphere is hμm, and h satisfies 220≤h≤300. For example, h can be 220, 230, 240, 250, 260, 270, 280, 290, 300, or a range consisting of any two of these values. In the present application, the organoid microspheres can be used as a universal base material for constructing organoid models, and a variety of organoid models can be formed by changing the cell types used. The present application better simulates the real physiological environment of the human body by regulating the cell types selected from the above range and regulating the diameter of the organoid microspheres within the above range.

[0044] In one embodiment of the present application, the material of the hydrogel microspheres is selected from at least one of sodium alginate, gelatin, collagen, and polyvinyl alcohol.

[0045] In one embodiment of the present application, the material of the microfluidic chip is selected from at least one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyimide (PI), and glass. The microfluidic chip prepared by selecting the above materials is helpful to observe the contents in the microfluidic chip visually or through a microscope, improving the situation that the chip organoids are difficult to visualize and trace; at the same time, it is easy to shape and can be molded with high resolution.

[0046] In the present application, the above materials can be conventional substances available on the market, and there is no particular restriction on their sources, as long as the purpose of the present application can be achieved.

[0047] In one embodiment of the present application, the length of the inner chamber is 16mm to 24mm, and the width of the inner chamber is 11mm to 15mm. For example, the length of the inner chamber can be 16mm, 17mm, 18mm, 19mm, 20mm, 21mm, 22mm, 23mm, 24mm, or a range consisting of any two of these values; the width of the inner chamber can be 11mm, 12mm, 13mm, 14mm, 15mm, or a range consisting of any two of these values. By regulating the length and width of the inner chamber within the above range, the space of the inner chamber can be reasonably regulated, and the distribution state of the organoid microspheres therein can be regulated, thereby improving the utilization rate of the space in the microfluidic chip and making it better reproduce the human microenvironment.

[0048] In one embodiment of the present application, Figure 1 , Figure 2 and Figure 3 As shown, the capture embedded card structure 1014 satisfies at least one of the following characteristics: (1) the capture embedded card structure 1014 includes two half-moon-shaped capture embedded card micro-pillars, and the inner diameter of the half-moon is 260 μm to 280 μm; (2) the length L1 of the capture embedded card structure 1014 is 1.5h to 1.8h, the width w1 is 1.5h to 1.8h, and the height h1 is 1.5h to 1.8h; (3) the interval L3 between adjacent columns of the capture embedded card structure 1014 is 1. 4h to 1.6h, and the interval w2 between adjacent rows is 1.8h to 2h; (4) the shortest distance w3 between the capture embedded card structure 1014 and the liquid inlet 1011 is 8h to 9h; (5) the shortest distance w4 between the capture embedded card structure 1014 and the liquid outlet 1012 is 8h to 9h; (6) the liquid diversion channel is arranged between the two capture embedded card microcolumns of the capture embedded card structure 1014; (7) the width L2 of the liquid diversion channel is 0.5h to 0.8h.

[0049] In some embodiments, the height of the capture card structure is the height of the inner chamber.

[0050] In some embodiments, the thickness of the solidified chip is greater than the height of the inner chamber, which is beneficial for regulating the distribution state of the organoid microspheres therein.

[0051] In one embodiment of the present application, Figure 2 As shown, the capture card structure 1014 includes two half-moon-shaped capture card micro-pillars, and the inner diameter of the half-moon is 260 μm to 280 μm. For example, the inner diameter can be 260 μm, 265 μm, 270 μm, 275 μm, 280 μm, or a range consisting of any two values ​​therein. Therefore, by regulating the inner diameter of the half-moon-shaped capture card micro-pillar within the above range, the ability of the capture card structure to capture a single hydrogel microsphere can be improved, and the risk of clogging in the microfluidic chip can be reduced.

[0052] In one embodiment of the present application, Figure 2 As shown, the length L1 of the capture embedded card structure is 1.5h to 1.8h, the width is 1.5h to 1.8h, and the height is 1.5h to 1.8h. For example, the length of the capture embedded card structure can be 1.5h, 1.6h, 1.7h, 1.8h, or a range consisting of any two of the values; the width can be 1.5h, 1.6h, 1.7h, 1.8h, or a range consisting of any two of the values; the height can be 1.5h, 1.6h, 1.7h, 1.8h, or a range consisting of any two of the values. By regulating the length, width, and height of the capture embedded card structure within the above range, the ability to capture hydrogel microspheres can be improved while avoiding the situation where a single capture embedded card structure captures multiple organoid microspheres at the same time, thereby improving the experimental observation effect and accuracy.

[0053] In one embodiment of the present application, Figure 2 As shown, the adjacent column spacing L3 of the capture card structure 1014 is 1.4h to 1.6h; the adjacent row spacing w2 is 1.8h to 2h. For example, the adjacent column spacing of the capture card structure can be 1.4h, 1.45h, 1.5h, 1.55h, 1.6h or a range consisting of any two of these values; the adjacent row spacing can be 1.8h, 1.85h, 1.9h, 1.95h, 2h or a range consisting of any two of these values. By regulating the adjacent column spacing and adjacent row spacing of the capture card structure within the above range, it is beneficial for the organoid microspheres to shuttle between the side-by-side structures with the liquid without obstacles, and the probability of the organoid microspheres being successfully captured following the flow of the liquid is increased, the flow of human blood is simulated, and the human microenvironment is better reproduced.

[0054] In one embodiment of the present application, Figure 2As shown, the shortest distance w3 between the capture card structure 1014 and the liquid inlet 1011 is 8h to 9h. For example, the shortest distance between the capture card structure and the liquid inlet can be 8h, 8.2h, 8.4h, 8.6h, 8.8h, 9h, or a range consisting of any two of these values. By regulating the shortest distance between the capture card structure and the liquid inlet within the above range, the space utilization rate of the chamber of the microfluidic chip is improved, and at the same time, it is beneficial for the organoid microspheres to shuttle unimpeded when entering the inner chamber with the liquid, simulating the flow of human blood and better reproducing the human microenvironment.

[0055] In one embodiment of the present application, Figure 2 As shown, the shortest distance w4 between the capture card structure 1014 and the liquid outlet 1012 is 8h to 9h. For example, the shortest distance between the capture card structure and the liquid outlet can be 8h, 8.2h, 8.4h, 8.6h, 8.8h, 9h, or a range consisting of any two of these values. By regulating the shortest distance between the capture card structure and the liquid outlet within the above range, the space of the inner chamber can be utilized to the maximum extent, the number of organoid microspheres in the inner chamber can be increased, and at the same time, the organoid microspheres can be shuttled unimpeded in the microfluidic chip, reducing the risk of blockage in the chip, reducing the amount of reagents and drugs used in drug screening experiments, and reducing the cost of scientific research.

[0056] In one embodiment of the present application, Figure 2 As shown, the liquid diversion channel is arranged between the two capture embedded micro-columns of the capture embedded card structure 1014; the width L2 of the liquid diversion channel is 0.5h to 0.8h. For example, the width of the liquid diversion channel can be 0.5h, 0.6h, 0.7h, 0.8h or a range consisting of any two values ​​therein. By regulating the width of the liquid diversion channel within the above range, while ensuring that the cell feeding fluid can flow smoothly, the efficiency of the organoid microspheres being captured and embedded is increased, the function of the organoid model based on the microfluidic chip is improved, and the human microenvironment is better reproduced.

[0057] In the present application, the organoid based on the microfluidic chip includes a microfluidic chip and an organoid microsphere, the microfluidic chip includes a curing chip and a glass slide, one surface of the curing chip is provided with a groove, a capture card structure is provided in the groove, and the capture card structure includes two capture card microcolumns. The surface of the curing material provided with the groove is directly contacted with the glass slide to form an inner chamber. The surface of the curing chip away from the glass slide is provided with a liquid inlet and a liquid outlet, and both the liquid inlet and the liquid outlet are communicated with the inner chamber.

[0058] Specifically, Figure 1 and Figure 2As shown, the organoid 1 based on the microfluidic chip includes a microfluidic chip 10 and an organoid microsphere 11. The microfluidic chip 10 includes a curing chip 101 and a glass slide 102. A groove is provided on one surface of the curing chip 101, and a capture card structure 1014 is provided in the groove. The capture card structure 1014 includes capture card microcolumns 1015 and 1016. The surface of the curing material provided with the groove directly contacts the glass slide 102 to form an inner chamber 1013. The surface of the curing chip 101 away from the glass slide 102 is provided with a liquid inlet 1011 and a liquid outlet 1012, and both the liquid inlet 1011 and the liquid outlet 1012 are communicated with the inner chamber 1013.

[0059] In one embodiment of the present application, the method for preparing organoid microspheres includes: S1. spraying solution A into droplets into solution B through piezoelectric printing to form hydrogel microspheres; S2. treating the hydrogel microspheres step by step with type I rat tail collagen solution and dopamine hydrochloride solution to obtain functionalized hydrogel microspheres; S3. mixing different types of cells and functionalized hydrogel microspheres to obtain organoid microspheres.

[0060] In some embodiments, a 3D bioprinter is used to spray solution A into uniform droplets into solution B, and the two rapidly undergo a cross-linking reaction at room temperature, thereby forming wet hydrogel microspheres with good biocompatibility.

[0061] In some embodiments, the solutes of solution A and solution B are dissolved in ultrapure water, shaken sufficiently to completely dissolve, and then sterile filtered. The present application has no particular restrictions on the shaking method, as long as the purpose of the present application can be achieved. For example, the shaking can be shaken by a shaking table shaker, or by magnetic stirring.

[0062] The present application has no particular restrictions on the method of aseptic filtration, as long as the purpose of the present application can be achieved. For example, it can be filtered using a 0.22 μm sterile filter. In one embodiment of the present application, in step S1, the parameters of piezoelectric printing are: the concentration of solution A is 0.8wt% to 1.2wt%, the concentration of solution B is 0.8wt% to 1.2wt%, the print head height is 38mm to 42mm, the print spacing is 0.2mm to 0.3mm, the air pressure is 0.06Mpa to 0.08MPa, the opening time of the electric gate is 225ms to 275ms, and the closing time of the electric gate is 275ms to 325ms. For example, the concentration of solution A can be 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt% or a range consisting of any two of the values ​​therein; the concentration of solution B can be 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt% or a range consisting of any two of the values ​​therein; the print head height can be 38mm, 39mm, 40mm, 41mm, 42mm or a range consisting of any two of the values ​​therein; the print spacing can be 0.2mm, 0.22mm, 0.24mm, 0.26mm, 0.28mm, 0.3mm or a range consisting of any two of the values ​​therein. The air pressure can be 0.06Mpa, 0.07Mpa, 0.08MPa or a range consisting of any two of them; the opening time of the electric gate can be 225ms, 230ms, 235ms, 240ms, 245ms, 250ms, 255ms, 260ms, 265ms, 275ms or a range consisting of any two of them; the closing time of the electric gate can be 275ms, 280ms, 285ms, 290ms, 295ms, 300ms, 305ms, 310ms, 315ms, 320ms, 325ms or a range consisting of any two of them. By regulating the concentrations of solution A and solution B within the scope of this application, the printed hydrogel microspheres are of a fixed shape and will not have a tailing phenomenon, and are not easy to cause clogging of the print nozzle; regulating the air pressure within the scope of this application improves the printing balling rate of the hydrogel microspheres. Therefore, the present application can obtain hydrogel microspheres with regular shapes and appropriate sizes by adjusting the printing parameters of piezoelectric printing within the scope of the present application.

[0063] In one embodiment of the present application, the solute of solution A is selected from at least one of sodium alginate, gelatin, collagen, and polyvinyl alcohol, and the solute of solution B is selected from at least one of calcium chloride, glutaraldehyde, and boric acid.

[0064] In some embodiments, the diameter of the print head is 0.1 mm to 0.15 mm. For example, the diameter can be 0.1 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, 0.15 mm, or a range consisting of any two of the values. By selecting a print head with the above diameter range, it is helpful to form hydrogel microspheres of appropriate size.

[0065] In one embodiment of the present application, in step S2, the concentration of the type I rat tail collagen solution is 8 mg / ml to 10 mg / ml, and the concentration of the dopamine hydrochloride solution is 0.15% to 0.25%. For example, the concentration of the type I rat tail collagen solution can be 8 mg / ml, 8.5 mg / ml, 9 mg / ml, 9.5 mg / ml, 10 mg / ml, or a range consisting of any two of these values; the concentration of the dopamine hydrochloride solution can be 0.15%, 0.17%, 0.19%, 0.2%, 0.22%, 0.23%, 0.25%, or a range consisting of any two of these values.

[0066] In some embodiments, the mass ratio of the hydrogel microspheres to type I rat tail collagen is 1:(0.3-0.5). For example, the mass ratio can be 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5, or a range consisting of any two of these values. By regulating the mass ratio of the hydrogel microspheres to type I rat tail collagen within the scope of this application, it is ensured that type I rat tail collagen is uniformly attached to the surface of the hydrogel microspheres, simulating the function of the extracellular matrix in a real physiological environment, while avoiding the addition of collagen to the microfluidic chip, and facilitating the observation of the state of the organoid microspheres in the microfluidic chip.

[0067] In some embodiments, the process of treating the hydrogel microspheres with the type I rat tail collagen solution is as follows: after the type I rat tail collagen solution and the hydrogel microspheres are mixed, the two are fully contacted by vortexing.

[0068] In some embodiments, the volume ratio of the hydrogel microspheres treated with type I rat tail collagen to dopamine hydrochloride is (120-130): 1. For example, the volume ratio can be 120: 1, 122: 1, 124: 1, 126: 1, 128: 1, 130: 1, or a range consisting of any two of these values. By regulating the volume ratio within the scope of this application, it is ensured that type I rat tail collagen is uniformly attached to the surface of the hydrogel microspheres, simulating the function of the extracellular matrix in a real physiological environment, while avoiding the addition of collagen in the microfluidic chip, and facilitating the observation of the state of the organoid microspheres in the microfluidic chip.

[0069] In some embodiments, the process of treating the hydrogel microspheres treated with type I rat tail collagen with a dopamine hydrochloride solution is as follows: mixing the dopamine hydrochloride solution with the hydrogel microspheres treated with type I rat tail collagen, and then rotating the mixture on a suspension instrument for 1 to 2 hours to allow the two to fully contact each other.

[0070] In some embodiments, based on the mass of the organoid microspheres, the sum of the mass percentages of type I rat tail collagen and dopamine hydrochloride is 30% to 35%. For example, the sum of the mass percentages of type I rat tail collagen and dopamine hydrochloride can be 30%, 31%, 32%, 33%, 34%, 35%, or a range consisting of any two of these values.

[0071] In one embodiment of the present application, in step S3, the cells are selected from at least one of human lung epithelial cells, human umbilical vein endothelial cells, and human lung fibroblasts, and the total cell planting density is 0.6×10 4 / mm 2 to 1.0×10 4 / mm 2 When the cells are selected from human lung epithelial cells, human umbilical vein endothelial cells, and human lung fibroblasts, the planting density of the cells is the total cell planting density. For example, the total cell planting density can be 0.6×10 4 / mm 2 , 0.7×10 4 / mm 2 , 0.8×10 4 / mm 2 , 0.9×10 4 / mm 2 , 1.0×10 4 / mm 2 Or a range consisting of any two of the values. In the present application, the organoid microspheres can be used as a universal base material for building organoid models, and various organoid models can be formed by changing the cell types used. The present application selects the above cells and regulates the cell planting density within the above range, which can simulate the real physiological environment of the human body.

[0072] In some embodiments, there is no particular limitation on the order of implanting cells in the organoid microspheres, as long as the purpose of the present application can be achieved. For example, the implantation order may be to first implant human lung epithelial cells, then to implant human lung fibroblasts and human umbilical vein endothelial cells simultaneously; or, the implantation order may be to first implant human lung epithelial cells, then to implant human lung fibroblasts, and then to implant human umbilical vein endothelial cells; or, the implantation order may be to first implant human lung epithelial cells, then to implant human umbilical vein endothelial cells, and then to implant human lung fibroblasts.

[0073] In some embodiments, such as Figure 4As shown, the organoid microsphere 11 includes a hydrogel microsphere 1101 , a type I rat tail collagen and dopamine hydrochloride layer 1102 , and a loaded cell layer 1103 .

[0074] In some embodiments, the cell passage number used in the present application is within ten passages.

[0075] The second aspect of the present application provides a method for preparing an organoid based on a microfluidic chip as described in the first aspect of the present application, comprising: (1) after adaptive culture of the organoid microspheres, gently blowing them 2-3 times with a pipette; (2) transferring the organoid microspheres to a microfluidic chip, and perfusing the organoid based on the microfluidic chip through an external circulation system.

[0076] In some embodiments, after the organoid microspheres are adaptively cultured, they are gently blown 2-3 times using a pipette, wherein the adaptive culture time is 8h to 16h, for example, the culture time is 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h or a range consisting of any two of these values. By regulating the adaptive culture time within the above range, the cells and microspheres are more firmly attached, which can simulate the real physiological environment of the human body.

[0077] In some embodiments, the number of organoid microspheres transferred to the microfluidic chip is 150 to 160, for example, the number can be 150, 152, 154, 156, 158, 160 or a range consisting of any two values ​​therein.

[0078] In some embodiments, perfusion of microfluidic chip-based organoids is performed through an external circulation system, which includes a drive pump, a controller, a reservoir, a three-way valve and a pipeline, and the reservoir can be a 1.5 ml plastic centrifuge tube.

[0079] In some embodiments, the circulation system and the microfluidic chip need to be sterilized before use, and the sterilization step includes at least two of alcohol sterilization, ultraviolet light or high-pressure steam sterilization.

[0080] In some embodiments, before the microfluidic chip-based organoid is connected to an external circulation system, 0.8 mL to 1.2 mL of complete culture medium is pre-added to the reservoir. For example, the pre-added amount of complete culture medium can be 0.8 mL, 0.9 mL, 1 mL, 1.1 mL, 1.2 mL, or a range consisting of any two of these values. The controller operates the drive pump to perfuse the culture medium to reduce the risk of bubbles in the pipeline when the microfluidic chip-based organoid is subsequently connected, thereby reducing the risk of affecting cell growth in the organoid microspheres.

[0081] In some embodiments, the complete culture medium is composed of basal culture medium, fetal bovine serum and double antibody, wherein the proportion of each component is: 10% fetal bovine serum, 1% double antibody and 89% basal culture medium.

[0082] In some embodiments, the perfusion rate is 5 μL / min to 20 μL / min, and the perfusion time is 8 h to 16 h. For example, the perfusion rate can be 5 μL / min, 7 μL / min, 9 μL / min, 10 μL / min, 12 μL / min, 14 μL / min, 16 μL / min, 18 μL / min, 20 μL / min, or a range consisting of any two of them; the perfusion time can be 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, or a range consisting of any two of them.

[0083] In the present application, the solute in solution A, the solute in solution B, type I rat tail collagen, dopamine hydrochloride, glass slide, basal culture medium, fetal bovine serum, and bispecific antibodies are all conventional substances available on the market. There is no particular restriction on their sources as long as the purpose of the present application can be achieved.

[0084] This application has no special restrictions on the 3D bioprinter used, the drive pump, controller, liquid reservoir, three-way valve and pipeline in the circulation system, as long as the purpose of this application can be achieved.

[0085] The third aspect of the present application provides an application of the microfluidic chip-based organoid described in the first aspect of the present application or the microfluidic chip-based organoid prepared by the preparation method described in the second aspect in the treatment of acute lung injury with traditional Chinese medicine monomers.

[0086] Example

[0087] The following examples and comparative examples are given to more specifically describe the embodiments of the present application. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.

[0088] Test methods and equipment:

[0089] Morphology characterization and particle size statistics:

[0090] The printed hydrogel microspheres were placed under an inverted microscope fluorescence optical imaging analysis system (Olympus IX73), and the morphology of the hydrogel microspheres after the cross-linking reaction was observed and photographed;

[0091] The printed hydrogel microspheres were observed and photographed under an optical microscope, and the photos were imported into the image J software to count the particle size of the hydrogel microspheres. The obtained data were then imported into the Origin software to count the particle size distribution and draw a statistical diagram. Figure 5 .

[0092] After continuous perfusion for 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, the organoids based on the microfluidic chip were transferred from the perfusion system and placed under an optical microscope to observe their growth status and take photos to record them. Figure 6 .

[0093] The microfluidic chip-based organoids that had been perfused for 48 h were transferred from the perfusion system and placed under an inverted microscope fluorescence optical imaging analysis system (Olympus IX73) to observe their growth status and take photos to record them. Figure 7 .

[0094] DAPI staining:

[0095] The organoids based on the microfluidic chip that had been continuously perfused for 48 hours were removed from the perfusion system and slowly passed PBS through the injection port for washing. The washing was completed 3 times, 3 minutes each time. After the washing was completed, 150 μL of DAPI dye (Maokang Bio, MX4208) was passed through the light-proof and incubated for 20 minutes. After repeating the above washing operation, a 3D image was taken using the bright field of a confocal microscope to obtain Figure 8 (a); laser photography was performed with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Figure 8 (b).

[0096] Inflammatory factor detection:

[0097] (1) Organoid microspheres prepared from the same batch were transferred into a single well of a 96-well plate and a microfluidic chip, respectively, to form a single organoid model (75 organoid microspheres per well) and an organoid model based on a microfluidic chip (each sample contained 150 organoid microspheres). Three different groups (control group, model group, and drug-treated group) were set up in each of the two models, and each group had 4 parallel samples.

[0098] (2) Control group: The samples were treated with dimethyl sulfoxide (DMSO) solution (dissolved in culture medium, DMEM high glucose complete culture medium: MEM complete culture medium = 3:1) at a concentration of 0.5%.

[0099] The volume of DMSO solution added to a single well of a 96-well plate was 300 μL, and the time was 48 h without replacement;

[0100] For organoids based on microfluidic chips, the volume of DMSO solution is 150 μL, the solution is replaced every 24 h, the total liquid volume is 300 μL, and the time is 48 h.

[0101] (3) Model group: lipopolysaccharide (LPS, sigma, C0056) solution was used (samples were modeled separately, LPS concentration was 50 μg / mL, dissolved in culture medium (DMEM high glucose complete medium: MEM complete culture = 3:1)).

[0102] The volume of modeling liquid added to a single well in a 96-well plate was 300 μL, and the modeling time was 48 h, without replacing the culture medium in the middle;

[0103] For organoids based on microfluidic chips, the LPS concentration used was 50 μg / mL, the liquid volume was 150 μL, the modeling time was 48 h, the culture medium was replaced every 24 h, the total liquid volume was 300 μL, and the modeling took 48 h.

[0104] (4) Drug administration group: The samples were modeled and administered, and the modeling steps were the same as (3). At the same time, the therapeutic drug (liquiritin) was added together with LPS. The concentration of LPS was 50 μg / mL, and the concentration of the therapeutic drug was 200 μmol / L. They were dissolved in culture medium (DMEM high-glucose complete medium: MEM complete culture = 3:1) and administered after 48 hours of modeling.

[0105] The supernatants of the three groups of samples from the two models were collected respectively, and the supernatants were centrifuged at 1000g, 4°C, 15min, and the centrifugal speed was 8500rpm to remove cell debris. The samples were collected for Elisa detection. The detection steps were as follows:

[0106] The interleukin 1β (IL-1β) kit (MULTI SCIENCES, EK101B-96) and interleukin-6 (IL-6) kit (Signalway Antibody, EK1217) were used to collect samples from the control group, model group, and drug-treated group of the two models for Elisa detection. The specific experimental steps refer to the instructions of the kit. The content of inflammatory factors in different groups was made into Fig. 9 , Fig.10 , Figure 11 to Figure 12 .

[0107] Example 1

[0108] <Preparation of organoid microspheres>

[0109] (1) In the preparation stage before printing: use ultrapure water to dissolve sodium alginate powder (S100127, Aladdin) and calcium chloride particles (C110769, Aladdin), and vortex them thoroughly to make them completely dissolved. The concentration of sodium alginate solution is 1wt%, and the concentration of calcium chloride solution is 1wt%. Then use a 0.22μm sterile filter to filter and sterilize them separately. The final prepared solution can be stored at room temperature for use.

[0110] A 3D bioprinter (3DD-G05, regenHU, Switzerland) was used to spray the sodium alginate solution into a uniform droplet into the calcium chloride solution. The two rapidly cross-linked at room temperature to form wet hydrogel microspheres with good biocompatibility. During the printing process, a print head (900005897, regenHU, Switzerland) was used, the electric valve opening time was 250ms, the electric valve closing time was 300ms, the print head height was 40mm, the print spacing was 0.25mm, and the air pressure was 0.07MPa. Figure 5 The hydrogel microspheres shown have regular shapes.

[0111] (2) mixing the hydrogel microspheres obtained in step (1) with type I rat tail collagen in a mass ratio of 1:0.4, wherein the concentration of type I rat tail collagen solution (Corning, 354249) is 8 mg / ml, and vortexing the mixture thoroughly to make the two closely contact, and then placing the hydrogel microsphere-type I rat tail collagen suspension at 4° C. for 7 days to allow them to fully contact;

[0112] Use a pipette to remove the supernatant (without hydrogel microspheres) in the suspension, and add 10 mL of 0.2% dopamine hydrochloride solution (prepared with Tris buffer at pH 8.5, sterilized by filtration with a 0.22 μm filter membrane) per 2.5 mL of hydrogel microspheres treated with type I rat tail collagen. Place the hydrogel microsphere suspension treated with type I rat tail collagen and dopamine hydrochloride on a suspension instrument and rotate for 1.5 hours to complete the dopamine hydrochloride treatment process, then wash the microspheres with Tris buffer, remove the washing solution after 5 minutes, and obtain functionalized hydrogel microspheres, which are stored at 4°C in a conventional serum-free culture medium for use.

[0113] (3) Cell planting: In this example, Beas-2b human lung epithelial cells, MRC-5 human lung fibroblasts and HUVEC human umbilical vein endothelial cells (all from ATCC cell bank) were used. The three cells were mixed with functionalized hydrogel microspheres in sequence. The mixing order was as follows: Beas-2b cells were first mixed and co-cultured with functionalized hydrogel microspheres alone for 2 hours, and then HUVEC and MRC-5 cells were added and continued to be mixed and cultured for 2 hours. The mixing process took place in a 1.5mL Ep tube. Each Ep tube contained about 75 microspheres. The ratio of the three cells added was Beas-2b:MRC-5:HUVEC=1:2:1, the total number of cells was 150,000, and the total amount of culture medium in the tube was 700 μL (DMEM high glucose complete medium:MEM complete culture=3:1, containing 10% fetal bovine serum and 1% double antibody). The cell planting density was 0.94×10 4 / mm 2 , and obtain organoid microspheres.

[0114] After completing the mixing process, let the EP tube stand for 10 minutes, remove excess culture medium in the tube, leaving 300 μL of culture medium containing organoid microspheres, and use a Pasteur pipette to transfer the solution in the tube and the organoid microspheres to a U-shaped 96-well plate. Each EP tube corresponds to one well of the 96-well plate.

[0115] Among them, based on the mass of the organoid microspheres, the sum of the mass percentages of type I rat tail collagen and dopamine hydrochloride is 31.36%.

[0116] <Preparation of Microfluidic Chip>

[0117] (1) PDMS curing: Prepare a PDMS kit (Sigma, 184, 761028), which contains Part A (PDMS base reagent) and Part B (curing agent). Accurately weigh the two parts according to the ratio of Part A: Part B = 10:1. Mix the two weighed reagents and stir vigorously until a large number of white bubbles appear in the solution. After stopping stirring, place the PDMS mixed liquid in a container and evacuate the vacuum to remove the bubbles in the liquid until the solution is transparent. This process takes 1 hour;

[0118] After the PDMS mixture is vacuumed, the chip mold (the chip mold is prepared by photolithography process, with a diameter of 4 inches, the mold is wrapped with tin foil, and only the structural surface is reserved to prevent subsequent liquid from overflowing) is placed on a weighing balance, the PDMS mixture is poured into the chip mold, 25g of PDMS mixture is added, and it is placed on a heating plate, keeping the mold horizontal, setting the temperature for 80h and the time for 3h, and removing the cured PDMS from the chip mold to obtain a cured PDMS cured chip with a thickness of 3mm.

[0119] (2) Preparation of microfluidic chip: Use a cutter to cut off the excess part of the PDMS cured chip, leaving only the chip structure area required for subsequent bonding. Use a puncher to punch holes in the cut PDMS cured chip, leaving liquid inlets and outlets;

[0120] Then use a plasma cleaner to clean the surface of the PDMS cured chip to ensure that it is clean and has strong bonding ability, and put the glass slide into the cleaner together. The parameters of the plasma cleaner are set to 760V and the time is 2min. After cleaning, the PDMS chip and the glass slide are bonded one by one to obtain a microfluidic chip.

[0121] The microfluidic chip includes an inner chamber, which includes capture card structures arranged alternately in a plum blossom shape. The thickness of the solidified chip is 3mm, the size of the inner chamber is 20mm×13mm, and the height is 400μm; the capture card structure includes capture card microcolumns and liquid diversion channels. In the capture card structure, the inner diameter of the half-moon structure is 270μm, the length of the capture card structure is 400μm, the width is 400μm, the height is 400μm, the interval between adjacent columns is 400μm, and the interval between adjacent rows is 485.5μm. The shortest distance between the capture card structure and the liquid inlet is 2.2mm, and the shortest distance from the liquid outlet is 2.2mm; the width of the liquid diversion channel is 180μm.

[0122] <Preparation of organoids based on microfluidic chips>

[0123] The organoid microspheres were placed in a U-shaped 96-well plate and cultured for 12 hours. Then, they were gently blown three times with a pipette (too much blowing can easily cause cells to fall off the microspheres) to make them in a single dispersed state in the well plate. The organoid microspheres in every two wells of the 96-well plate were transferred to a microfluidic chip (containing 150 organoid microspheres). After the transfer was completed, they were cultured for 12 hours to obtain organoids based on microfluidic chips.

[0124] Then connect the external circulation system, which consists of a drive pump, controller, reservoir (1.5mL EP tube), three-way valve, and pipeline. Before the entire system is operated, the materials related to the circulation system are disinfected with alcohol three times and sterilized by ultraviolet for 4 hours; before use, the 1.5mL EP tube, pipeline and microfluidic chip need to be sterilized by high-pressure steam in addition to the above treatment. Before connecting the microfluidic chip-based organoid to the external circulation system, add 1mL of complete culture medium to the 1.5mL EP medium tube, and run the drive pump through the controller to perfuse the culture medium to ensure that there are no bubbles in the pipeline when the microfluidic chip-based organoid is subsequently connected, thereby reducing the impact on the growth of cells in the organoid microspheres.

[0125] After completing the pre-perfusion, the organoids based on the microfluidic chip were connected to the external circulation system in the inlet and outlet directions, and perfusion culture was started. The perfusion rate was set to 10 μL / min. After 12 hours of perfusion, the organoid model based on the microfluidic chip was modeled and administered.

[0126] Comparative Example 1

[0127] Organoid microspheres were prepared according to <Preparation of Organoid Microspheres> in Example 1, and organoid microspheres with regular shape and a diameter of 262 μm were cultured in U-shaped 96-well plates. After 12 h of adaptive culture, they were used as a separate organoid model for inflammatory factor detection.

[0128] The preparation parameters and performance parameters of the examples and comparative examples in this application are shown in Table 1.

[0129] Table 1

[0130]

[0131] Through Example 1 and Comparative Example 1, Figures 5 to 12 It can be seen that when the microfluidic chip-based organoids prepared in the embodiments of the present application are used in the treatment of acute lung injury with traditional Chinese medicine monomers, they can better replicate the complex and diverse physiological conditions in the human body when facing the occurrence and treatment of inflammation, which demonstrates that the microfluidic chip-based organoid model of the present application can better reproduce the human microenvironment. At the same time, the preparation method of the organoid microspheres of the present application can improve the stability and consistency of the prepared organoid microspheres.

[0132] Figure 5 It can be seen that the hydrogel microspheres prepared in the examples of the present application have regular shapes and uniform sizes. Figure 6 and Figure 7 It can be seen that the microfluidic chip prepared in the embodiment of the present application has an excellent fixation effect on the organoid microspheres, which can make the cell feeding fluid flow smoothly, and each capture card structure corresponds to a single organoid microsphere. The organoids based on the microfluidic chip can better reproduce the human microenvironment.

[0133] Figure 8 It can be seen that the microfluidic chip prepared in the embodiment of the present application will not affect the physiological state of the organoid microspheres, which shows that the microfluidic chip structure can non-destructively fix the organoid microspheres, while also meeting the needs of visualization and traceable observation of the organoid microspheres in the microfluidic chip.

[0134] Figures 9 to 12It can be seen that when the microfluidic chip-based organoids prepared in the embodiments of the present application are used in the treatment of acute lung injury with traditional Chinese medicine monomers, they can better replicate the complex and diverse physiological conditions in the human body when facing the occurrence and treatment of inflammation, which shows that the microfluidic chip-based organoids prepared in the embodiments of the present application can better reproduce the human microenvironment.

[0135] It should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0136] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. An organoid based on a microfluidic chip, comprising a microfluidic chip and organoid microspheres, wherein the microfluidic chip comprises an inner chamber, wherein the inner chamber comprises capture card structures alternately arranged in a plum blossom shape, wherein the capture card structures comprise capture card microcolumns and liquid diversion channels; and the organoid microspheres are hydrogel microspheres loaded with cells.

2. The microfluidic chip-based organoid according to claim 1, wherein: The cells are selected from at least one of human lung epithelial cells, human umbilical vein endothelial cells, and human lung fibroblasts; Wherein, the diameter of the organoid microsphere is h μm, and h satisfies 220≤h≤300.

3. The microfluidic chip-based organoid according to claim 1, wherein: The material of the hydrogel microspheres is selected from at least one of sodium alginate, gelatin, collagen and polyvinyl alcohol.

4. The microfluidic chip-based organoid according to claim 1, wherein: The material of the microfluidic chip is selected from at least one of polydimethylsiloxane, polymethyl methacrylate, polyimide and glass.

5. The microfluidic chip-based organoid according to claim 2, wherein: The length of the inner chamber is 16 mm to 24 mm, and the width of the inner chamber is 11 mm to 15 mm.

6. The microfluidic chip-based organoid according to claim 2, wherein: The capture card structure satisfies at least one of the following characteristics: (1) The capture embedded card structure includes two half-moon-shaped capture embedded card micro-pillars, and the inner diameter of the half-moon is 260 μm to 280 μm; (2) The capture card structure has a length of 1.5h to 1.8h, a width of 1.5h to 1.8h, and a height of 1.5h to 1.8h; (3) The interval between adjacent columns of the capture card structure is 1.4h to 1.6h, and the interval between adjacent rows is 1.8h to 2h; (4) The shortest distance between the capture card structure and the liquid inlet is 8h to 9h; (5) The shortest distance between the capture card structure and the liquid outlet is 8h to 9h; (6) The liquid guide channel is disposed between two capture embedded card micro-columns of the capture embedded card structure; (7) The width of the liquid guiding channel is 0.5h to 0.8h.

7. The microfluidic chip-based organoid according to claim 1, wherein: The method for preparing the organoid microspheres comprises: S1. Solution A is formed into droplets and ejected into solution B by piezoelectric printing to form hydrogel microspheres; S2. treating the hydrogel microspheres in steps using a type I rat tail collagen solution and a dopamine hydrochloride solution to obtain functionalized hydrogel microspheres; S3. Mix different types of cells and functionalized hydrogel microspheres to obtain organoid microspheres.

8. The microfluidic chip-based organoid according to claim 7, wherein: In step S1, the piezoelectric printing parameters are: the concentration of solution A is 0.8wt% to 1.2wt%, the concentration of solution B is 0.8wt% to 1.2wt%, the print head height is 38mm to 42mm, the print spacing is 0.2mm to 0.3mm, the air pressure is 0.06Mpa to 0.08MPa, the opening time of the electric gate is 225ms to 275ms, and the closing time of the electric gate is 275ms to 325ms; The solute of the solution A is selected from at least one of sodium alginate, gelatin, collagen, and polyvinyl alcohol; the solute of the solution B is selected from at least one of calcium chloride, glutaraldehyde, and boric acid; In step S2, the concentration of the type I rat tail collagen solution is 8 mg / ml to 10 mg / ml, and the concentration of the dopamine hydrochloride solution is 0.15% to 0.25%; In step S3, the cells are selected from human lung epithelial cells, human umbilical vein endothelial cells, and human lung fibroblasts, and the total cell planting density is 0.6×10 4 / mm 2 to 1.0×10 4 / mm 2 .

9. A method for preparing an organoid based on a microfluidic chip according to any one of claims 1 to 8, comprising: (1) After the organoid microspheres have undergone adaptive culture, they are gently blown 2-3 times using a pipette; (2) transferring the organoid microspheres into the microfluidic chip, and performing perfusion of the organoids based on the microfluidic chip through an external circulation system; The adaptive culture time is 8 hours to 16 hours, the number of organoid microspheres transferred to the microfluidic chip is 150 to 160, and the circulation system includes a driving pump, a controller, a liquid reservoir, a three-way valve and a pipeline.

10. Use of the microfluidic chip-based organoid according to any one of claims 1 to 8 or the microfluidic chip-based organoid prepared by the preparation method according to claim 9 in the treatment of acute lung injury with traditional Chinese medicine monomers.

Images