Micro-fluidic chip for interaction of intercellular secretory factors and application of micro-fluidic chip

Abstract

The invention belongs to the technical field of micro-fluidic chips, and particularly relates to an intercellular secretion factor interaction micro-fluidic chip and application thereof. The microfluidic chip comprises an intermediate culture layer, a first culture layer and a second culture layer, wherein the first culture layer and the second culture layer are detachably connected with the intermediate culture layer respectively; culture chambers are arranged in the middle culture layer, the first culture layer and the second culture layer, and a plurality of micro-channels communicated with the culture chambers are uniformly arranged on the periphery of each culture chamber; the culture chambers in the middle culture layer, the first culture layer and the second culture layer are mutually communicated through the micro-channels; independent perfusion channels and liquid outlet channels are arranged in the middle culture layer, the first culture layer and the second culture layer, and each independent perfusion channel and each independent liquid outlet channel are connected with an independent liquid inlet and an independent liquid outlet which are formed in each culture layer. The micro-fluidic chip provided by the invention is simple in structure, can realize separation of multiple cells in time and space at the same time, and meets the in-situ observation requirements.

Description

Technical Field

[0001] The invention belongs to the technical field of microfluidic chips, and in particular relates to a microfluidic chip for cell-cell secretory factor interaction and application thereof. Background Art

[0002] The complexity of the cell models commonly used for modeling ranges from two-dimensional (2D) cell culture to simple three-dimensional (3D) cell culture. At present, the three-dimensional culture method has been upgraded to 3D microsphere and organoid culture. As a special existence of in vitro culture, organoids have all the functions of organs in the body and can fully meet the needs of establishing a bionic model in vitro for research.

[0003] In current cutting-edge research, the important physiological state of the human body is not only composed of cells, but also the influence of blood perfusion. Microfluidics technology is characterized by a microchannel network. Through micromachining technology, microchannels, micropumps, microvalves, microreservoirs, microelectrodes, microdetection elements and other functional components are integrated on chip materials like integrated circuits. With the support of microfluidics, a high degree of imitation of the perfusion of blood in the body has also been achieved. Therefore, combining microfluidics with three-dimensional cultured organoids to establish a bionic in vitro model can serve as the basis for the establishment of various disease models.

[0004] At present, there are also multi-cell co-culture chips, which are tumor organoids containing a certain number of immune cells mixed with matrix gel and injected into the middle chamber of the 3D microfluidic culture device. After incubation at 37°C for 30 minutes, the matrix gel solidifies, and the culture medium or drugs are injected into the chambers on both sides, and cultured in a cell culture incubator at 37°C and 5% CO2. However, it is cultured after the organoids are mixed with other cells. Mixed culture cannot distinguish the two cells for separate detection. Even if the detection is performed, it will be difficult to distinguish between cells and organoids, and the observation effect is not good, which may eventually lead to inaccurate test results. There are also similar methods that combine multiple chambers on the same plane, connect them in series through microfluidic channels to complete the co-culture of different cells and organoids, and detect the changes of different factors therein. However, the above two types of classic microfluidic chips may have the same problem. First, if there is enough space for cell and organoid culture on the chip, the chip may be designed into a different shape, or the overall volume may be increased, which will not be able to adapt to the fixing frames of various microscopes, and in situ observation and detection are often impossible. If the shape is designed to fit the morphology observed under a microscope, it may not be possible to ensure the number of organoids in the chamber and sufficient growth space. Secondly, although this type of chip is very good at separating cells, since multiple cells exist on the same chip, it is impossible to add or remove a single organoid or cell for testing in the middle of the experiment. When a cell needs to be tested, it is difficult to take out the cells or organoids in another channel or chamber to complete another experiment.

[0005] An optimized in vitro research model, in addition to mimicking the effects of blood flow, is not rigorous if it simply relies on the function of parenchymal cells. The human body is a large interactive whole, and parenchymal cells are also affected by non-parenchymal cells. Therefore, it is necessary to provide a multi-cell co-culture system that can meet the conditions for interaction between cells and present the pathological and physiological state of tissues and organs in vitro in a more rigorous and scientific manner. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a microfluidic chip for cell-cell secretory factor interaction and its application, which has solved the problem in the prior art that in multi-cell co-culture, inability to meet in situ observation and inability to present the pathophysiology of tissues and organs in vitro.

[0007] To achieve the above object, the present invention adopts the following technical solution.

[0008] The present invention provides a microfluidic chip for cell-cell secretory factor interaction, comprising: an intermediate culture layer and a first culture layer and a second culture layer detachably connected to the top surface and the bottom surface of the intermediate culture layer respectively;

[0009] The intermediate culture layer, the first culture layer, and the second culture layer are each provided with a culture chamber, and a plurality of microchannels connected to the culture chamber are evenly arranged around each culture chamber; the microchannels interconnect the culture chambers in the intermediate culture layer, the first culture layer, and the second culture layer, and form a closed loop between each culture chamber; the intermediate culture layer, the first culture layer, and the second culture layer are each provided with a separate perfusion channel and a liquid outlet channel, and each of the separate perfusion channel and the liquid outlet channel is connected to a separate liquid inlet and a separate liquid outlet arranged on the intermediate culture layer, the first culture layer, and the second culture layer;

[0010] The height of the culture chamber is 1 mm to 1.5 mm; the diameter of the microchannel is 0.5 mm to 0.7 mm.

[0011] In the present invention, by continuously perfusing the culture fluid into the perfusion channel of each culture layer, the perfusion state of human blood can be simulated. The provision of multiple culture layers can realize the co-culture of multiple cells. Under the action of the microchannel, the interaction of cytokines or metabolites between different cells can also be realized, laying a foundation for the study of various diseases. In the present invention, the microchannel is annular to minimize the spatial position, and the culture medium can be evenly interconnected in each direction. If one of the microchannels is accidentally blocked, the microchannels on both sides can replace its function and complete the interconnection. Various cytokines and drugs can communicate and interact through the microchannels evenly arranged around. And the microfluidics provided in the present invention can simultaneously achieve separation in time and space when co-culturing multiple cells, and meet the needs of in situ observation.

[0012] In the present invention, the height of the culture chamber is related to the type of cells to be cultured. In some embodiments, the height of the culture chamber is 1 mm or 1.5 mm. When the height of the culture chamber is 1.5 mm, the height of the culture chamber is relatively high, and the matrix gel can be filled in the culture chamber, which is suitable for culturing any organoids and can avoid clogging of the microchannels; when the height of the culture chamber is 1 mm, it can be used to culture adherent cells that do not require high depth.

[0013] In some embodiments, the diameter of the microchannel is 0.5 mm to 0.7 mm; for example, 0.5 mm, 0.6 mm or 0.7 mm. If the diameter of the microchannel is less than 0.5 mm, the diameter of the microchannel is too thin, which may easily cause blockage. If the diameter of the microchannel is greater than 0.7 mm, the diameter of the microchannel is too thick, which may cause cells in different culture layers to exchange positions.

[0014] In some embodiments, the culture chamber comprises: a first culture chamber, an intermediate culture chamber, and a second culture chamber; the microchannel comprises a first microchannel, an intermediate microchannel, and a second microchannel;

[0015] The first microchannel is disposed in the first culture layer and extends downward in the first culture layer, and its channel opening is located on the bottom surface of the first culture layer; the second microchannel is disposed in the second culture layer and extends upward in the second culture layer, and its channel opening is located on the top surface of the second culture layer;

[0016] The intermediate microchannel is arranged to penetrate the intermediate culture layer, the channel opening located on the top surface of the intermediate culture layer is connected to the channel opening of the first microchannel; the channel opening located on the bottom surface of the intermediate culture layer is connected to the channel opening of the second microchannel;

[0017] The number of the first microchannels, the middle microchannels and the second microchannels is the same, and the number is 8 to 12. For example, the number of the first microchannels, the middle microchannels and the second microchannels is 8, 9, 10, 11 or 12 respectively.

[0018] In the present invention, the number of microchannels mainly affects the liquid exchange between different culture layers. When the number of microchannels is less than 8, insufficient liquid exchange may occur. When the number of microchannels is greater than 12, the chip cost is too high and it will affect the setting of the perfusion channel and the liquid outlet channel.

[0019] In a specific embodiment of the present invention, the number of the first microchannels, the middle microchannels and the second microchannels is 12.

[0020] In some embodiments, the number of the intermediate culture layers is 0 to 3; for example, the number of the intermediate culture layers is 0, 1, 2 or 3.

[0021] When the number of the intermediate culture layers is 0, the channel opening of the first microchannel is connected to the channel opening of the second microchannel.

[0022] In some embodiments, the liquid inlet and the liquid outlet are distributed on both sides of the first culture chamber, the middle culture chamber and the second culture chamber.

[0023] In some embodiments, the liquid inlet includes a first liquid inlet, a middle liquid inlet, and a second liquid inlet; the liquid outlet includes a first liquid outlet, a middle liquid outlet, and a second liquid outlet;

[0024] The first liquid inlet, the middle liquid inlet and the second liquid inlet are located on the top surface or the side surface of the first culture layer, the middle culture layer and the second culture layer; the first liquid outlet, the middle liquid outlet and the second liquid outlet are located on the top surface or the side surface of the first culture layer, the middle culture layer and the second culture layer.

[0025] In some embodiments, the first liquid inlet, the middle liquid inlet and the second liquid inlet are located on the sides of the first culture layer, the middle culture layer and the second culture layer; the first liquid outlet, the middle liquid outlet and the second liquid outlet are located on the sides of the first culture layer, the middle culture layer and the second culture layer.

[0026] In some embodiments, the first liquid inlet, the middle liquid inlet and the second liquid inlet are located on the top surfaces of the first culture layer, the middle culture layer and the second culture layer; the first liquid outlet, the middle liquid outlet and the second liquid outlet are located on the top surfaces of the first culture layer, the middle culture layer and the second culture layer.

[0027] In some embodiments, the first liquid inlet, the middle liquid inlet and the second liquid inlet are located on the top surfaces of the first culture layer, the middle culture layer and the second culture layer; the first liquid outlet, the middle liquid outlet and the second liquid outlet are located on the sides of the first culture layer, the middle culture layer and the second culture layer.

[0028] In some embodiments, when the first liquid inlet, the middle liquid inlet and the second liquid inlet are located on the top surfaces of the first culture layer, the middle culture layer and the second culture layer, a plurality of through holes are provided on the first culture layer and the middle culture layer, which are respectively connected with the middle liquid inlet and the second liquid inlet.

[0029] In some embodiments, the culture chambers are all cylindrical, cube-shaped, or cuboid-shaped;

[0030] When the first culture chamber, the middle culture chamber and the lower cell culture chamber are cylindrical, the bottom diameter of the cylinder is 6 mm to 8 mm; for example, 6 mm, 7 mm or 8 mm;

[0031] When the first culture chamber, the middle culture chamber and the lower cell culture chamber are cubes, the side length of the cube is 6 mm to 8 mm; for example, 6 mm, 7 mm or 8 mm;

[0032] When the first culture chamber, the middle culture chamber and the lower cell culture chamber are rectangular parallelepipeds, the length of the cube is 6mm to 10mm, and the width is 6mm to 8mm; for example, the length is 6mm, 7mm, 8mm, 9mm or 10mm, and the width is 6mm, 7mm or 8mm;.

[0033] In a specific embodiment of the present invention, the first culture chamber is a cylinder with a bottom diameter of 8 mm.

[0034] In a specific embodiment of the present invention, the second culture chamber is a cylinder with a bottom diameter of 6 mm.

[0035] In a specific embodiment of the present invention, the second culture chamber is filled with matrix gel. The three-dimensional wrapping state of the matrix gel is used to adapt to the culture of any organoid. In the present invention, when the bottom surface of the culture chamber is circular, the area of ​​the bottom surface is larger, which is suitable for the culture of adherent cells and can meet the treatment of most cells.

[0036] In some embodiments, the first culture layer, the middle culture layer and the second culture layer are rectangular parallelepipeds; the length of the rectangular parallelepiped is 50 mm to 60 mm, the width is 20 mm to 25 mm, and the height is 2.5 mm to 3 mm.

[0037] In some embodiments, it further comprises a fixed mold for combining the first culture layer, the middle culture layer and the second culture layer;

[0038] The fixed mold includes: a base in a rectangular shape, with four screws detachably connected to the base and perpendicular to the base; when the microfluidic chip is located on the base, the microfluidic chip is located between the four screws, and the screws are detachably connected to one side of the long side of the microfluidic chip.

[0039] The present invention also provides application of the microfluidic chip for cell-cell secretory factor interaction in multi-cell co-culture.

[0040] In some embodiments, the microfluidic chip for cell-cell secretory factor interaction is used to co-culture at least two of immune cells, organoid endothelial cells, fibroblasts, or organoids from different tissues.

[0041] In some specific embodiments, the microfluidic chip for cell-cell secretory factor interaction described in the present invention is used in the co-culture of immune cells THP-1 and liver organoids.

[0042] In some embodiments, in an ultra-clean workbench, the liquid inlet of the microfluidic chip is connected to a syringe through a hose via a Luer connector and a flat needle, the liquid outlet is connected to a waste liquid bottle through a hose, and a bacterial air filter is attached; the digested hepatobiliary organoids are resuspended in matrix gel, and then manually injected into the second culture chamber at a speed of 10 μL / second using a syringe, and then cell differentiation culture medium is pumped in to differentiate and culture the digested hepatobiliary organoids. After the differentiation is completed, THP-1 cells are injected into the first culture chamber using a syringe, and the perfusion of the first culture layer is stopped after the injection, and the perfusion of the second culture layer is maintained, waiting for the THP-1 cells to adhere to the wall, and then THP-1 cell culture medium is injected from the first liquid inlet to co-culture the hepatobiliary organoids and THP-1 cells.

[0043] The disinfected microfluidic chip for cell-cell secretion factor interaction and the connecting hose, flat needle, Luer connector, waste bottle and 0.22 μm bacterial air filter were placed in the clean bench for

[0044] Based on the technical solution of the present invention, compared with the prior art, the present invention has the following beneficial effects:

[0045] The microfluidic chip provided by the present invention has a simple structure and is easy to operate when in use. It can co-culture up to 5 different cells at the same time, and the culture layer can be disassembled or different cells can be added at any time during the culture process. Different experimental requirements can be met for different cells at the same time. At different times, a certain layer of cells (organoids) can be taken out and tested separately without affecting the normal perfusion culture of cells (organoids) in the remaining layers of the chip. Therefore, the microfluidic chip provided by the present invention can achieve separation in time and space at the same time, and meets the needs of in situ observation. The number and type of cells to be cultured can be selected according to the actual needs, and the number of intermediate culture layers and the size of the culture chamber can be selected, which is convenient for scientific researchers to use. It is a special and practical co-culture chip.

[0046] The microfluidic chip provided by the present invention can be detachably connected between each culture layer, which can facilitate the detection of cells in different culture layers without cell contamination. At the same time, by adding an intermediate layer of the chip, the purpose of increasing the types of co-cultured cells (organoids) according to experimental requirements can be achieved. The microfluidic chip provided by the present invention has an overall chip size and shape that can fit the fixed frame of the microscope, and the thickness of the single layer can allow the confocal microscope to penetrate, and each layer can be taken out separately for processing and observation. It solves the problem of different chip shapes, excessive chip thickness, and the inability of the microscope to penetrate and focus in the prior art.

[0047] The microfluidic chip provided by the present invention is detachable and convenient for subsequent chip cleaning, ensuring that the chip can be reused multiple times, with a wide range of applications, and greatly increasing the number of times the chip is used. In addition, in the present invention, the diameter of the microchannel is only 0.5 mm, so when the cells are pushed, the cell suspension will be more easily injected into the disc with a large space, rather than into the upper culture chamber, so the role of intercellular factors is studied, providing more reliable research data for constructing an in vitro model to study pathological and physiological progress.

[0048] The microfluidic chip provided in the present invention can simulate the perfusion state of human blood by continuously perfusing culture fluid into the perfusion channel of each culture layer. The multiple culture layers are provided to realize the co-culture of multiple cells. Under the action of the microchannel, the interaction of cytokines or metabolites between different cells in each culture chamber can be ensured, providing more reliable research data for constructing an in vitro model to study pathological and physiological progress.

[0049] The present invention uses immune cells THP-1 and liver organoids as research objects, and simulates the pathophysiological state of the liver on an in vitro chip. Microchannels are used to ensure that the factors secreted by cells in different culture layers can communicate with each other, and that immune cells and parenchymal cells can influence each other. The many inflammatory factors secreted by immune cells under pathological conditions can produce an effect similar to the in vivo inflammatory state on parenchymal cells through the liquid interaction microchannels in the culture chambers of different culture layers. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a cross-sectional view of the microfluidic chip for cell-cell secretory factor interaction provided in Example 1 of the present invention.

[0051] Figure 2 This is a top view of the first culture layer of the microfluidic chip for cell-cell secretion factor interaction provided in Example 1 of the present invention.

[0052] Figure 3 This is a top view of the second culture layer of the microfluidic chip for cell-cell secretion factor interaction provided in Example 1 of the present invention.

[0053] Figure 4 This is a schematic diagram of the microfluidic chip for cell-cell secretion factor interaction provided in Example 1 of the present invention being fixed in a fixed mold.

[0054] Figure 5 This is a cross-sectional view of the microfluidic chip for cell-cell secretory factor interaction provided in Example 2 of the present invention.

[0055] Figure 6 This is a top view of the microfluidic chip for cell-cell secretory factor interaction provided in Example 2 of the present invention, wherein the solid line portion in the figure represents the structure of the first culture layer, and the dotted line portion represents the structure of the second culture layer.

[0056] Figure 7 This is a top view of the first culture layer of the microfluidic chip for cell-cell secretion factor interaction provided in Example 2 of the present invention.

[0057] Figure 8 This is a top view of the second culture layer of the microfluidic chip for cell-cell secretion factor interaction provided in Example 2 of the present invention.

[0058] Fig. 9 Schematic diagram of culturing immune cells THP-1 and liver organoids using Example 2 of the present invention.

[0059] Fig.10 The scale bar is 50μm, and the morphology and status of organoids at different differentiation stages after induction with human hepatobiliary differentiation medium correspond to different culture days.

[0060] Fig.11 These are the HE staining results of undifferentiated bile duct organoids and differentiated mature liver organoids with a scale of 50 μm. The two pictures on the right correspond to the enlarged images of the boxed parts in the two pictures on the left, and the scales of the two pictures on the left are 100 μm.

[0061] Fig.12 The figure shows the result of immunofluorescence staining of undifferentiated bile duct organoids cultured on a chip; SOX9 and EPCAM are stem cell markers, CK19 is a bile duct marker, ZO-1 is a tight junction protein, and DAPI is a nuclear fluorescent dye.

[0062] Fig.13 This is a three-dimensional spherical structure diagram of liver organoids.

[0063] Fig.14 This is the result of screening the culture medium ratio under co-culture conditions; among them, Fig.14 A in the figure represents the statistical results of the differences in cell activity of organoids under different ratios of culture medium in different time gradients; Fig.14 B in the figure shows the statistical results of the difference in cell culture activity of THP-1 in different proportions of culture medium at different time gradients.

[0064] Fig.15 Bright field images of THP-1 organoid co-culture and induction.

[0065] Fig.16 This is the result of the organoid co-culture live-death experiment; Fig.16 A in the figure indicates living cells fluorescently labeled by AM reaction; Fig.16 B in the figure indicates dead cells fluorescently labeled by PI reaction; Fig.16 C in the figure represents the overlay of two fluorescence images.

[0066] Reference numerals:

[0067] 1-middle culture layer, 2-first culture layer, 3-second culture layer, 4-first culture chamber, 5-middle culture chamber, 6-second culture chamber, 7-first microchannel, 8-middle microchannel, 9-second microchannel, 10-first liquid inlet, 11-middle liquid inlet, 12-second liquid inlet, 13-first liquid outlet, 14-middle liquid outlet, 15-second liquid outlet, 16-through hole, 17-base, 18-screw, 19-magnet. DETAILED DESCRIPTION

[0068] The experimental methods in the following examples of the present invention without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0069] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0070] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0071] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0072] The following describes the invention in conjunction with specific embodiments.

[0073] Example 1: A microfluidic chip for cell-cell secretory factor interaction

[0074] like Figure 1 to Figure 3 As shown, a microfluidic chip for cell-cell secretion factor interaction (the material is PDMS and PC board formed by photolithography technology), comprising: an intermediate culture layer 1 and a first culture layer 2 and a second culture layer 3 detachably connected to the top surface and the bottom surface of the intermediate culture layer 1 respectively;

[0075] The middle culture layer 1, the first culture layer 2 and the second culture layer 3 are all provided with culture chambers, and a plurality of microchannels connected to the culture chambers are evenly arranged around each culture chamber; the microchannels interconnect the culture chambers in the middle culture layer 1, the first culture layer 2 and the second culture layer 3, and form a closed loop between each culture chamber; the middle culture layer 1, the first culture layer 2 and the second culture layer 3 are provided with separate perfusion channels and liquid outlet channels, and each separate perfusion channel and liquid outlet channel is connected to a separate liquid inlet and a separate liquid outlet arranged on the middle culture layer 1, the first culture layer 2 and the second culture layer 3;

[0076] The culture chambers are all cylindrical, cubic, or cuboid; the height of the culture chambers is 1 mm to 1.5 mm; and the diameter of the microchannels is 0.5 to 0.8 mm.

[0077] In the present invention, multiple culture layers are provided, and each culture layer is provided with a separate liquid inlet, perfusion channel, liquid outlet channel and liquid outlet, so that cells in multiple culture layers can be cultured simultaneously. At the same time, since microchannels are provided around the culture chambers of each culture layer, the microchannels can connect the culture chambers of each culture layer, realize the circulation of liquids and the interaction of cytokines between different culture layers, realize the co-culture of multiple cells, and lay a foundation for the research of various diseases.

[0078] Specifically, a magnet 19 is disposed on one side corresponding to the long side of the first culture layer 2 , the middle culture layer 1 and the second culture layer 3 , thereby realizing a detachable connection between the first culture layer 2 , the middle culture layer 1 and the second culture layer 3 .

[0079] Specifically, in this embodiment, the height of the first culture chamber 4 is 1 mm; the height of the intermediate culture chamber 5 is 1.5 mm; the height of the second culture chamber 6 is 1 mm or 1.5 mm, and the appropriate height of the culture chamber 5 can be selected according to the cells to be cultured; the first culture chamber 4, the intermediate culture chamber 5 and the second culture layer 3 are cylinders, and the bottom diameter of the cylinder is 6 mm to 8 mm.

[0080] Specifically, in this embodiment, the bottom diameter of the first culture chamber 4 is 8 mm, the bottom diameter of the second culture chamber 6 is 6 mm, and the diameter of the middle culture chamber 5 is 6 mm or 8 mm. The appropriate culture chamber size can be selected according to the cells to be cultured. In this embodiment, the first culture chamber 4 can meet the requirements of 1×10 ^5 Quantitative cell culture.

[0081] Further, the microchannel includes a first microchannel 7, an intermediate microchannel 8 and a second microchannel 9, and the number of the three microchannels is the same, which is 8 to 12; specifically, in this embodiment, the number of the first microchannel 7, the intermediate microchannel 8 and the second microchannel 9 is uniformly 12;

[0082] The first microchannel 7 is disposed in the first culture layer 2 and extends downward in the first culture layer 2, and its channel opening is located at the bottom surface of the first culture layer 2; the second microchannel 9 is disposed in the second culture layer 3 and extends upward in the second culture layer 3, and its channel opening is located at the top surface of the second culture layer 3;

[0083] The middle microchannel 8 is arranged through the middle culture layer 1 , and the channel opening located on the top surface of the middle culture layer 1 is connected to the channel opening of the first microchannel 7 ; the channel opening located on the bottom surface of the middle culture layer 1 is connected to the channel opening of the second microchannel 9 .

[0084] Furthermore, the number of the intermediate culture layers 1 is 0 to 5; specifically, in this embodiment, the number of the intermediate culture layers 1 is 1 (eg Figure 1Further, the number of the intermediate culture layers 1 can be adjusted according to the actual use requirements, such as Figure 4 In the example, the number of the intermediate culture layers 1 is two.

[0085] Furthermore, the liquid inlet and the liquid outlet are distributed on both sides of the first culture chamber 4 , the middle culture chamber 5 and the second culture chamber 6 .

[0086] Further, the liquid inlet includes a first liquid inlet 10, a middle liquid inlet 11 and a second liquid inlet 12; the liquid outlet includes a first liquid outlet 13, a middle liquid outlet 14 and a second liquid outlet 15;

[0087] The first liquid inlet 10, the middle liquid inlet 11 and the second liquid inlet 12 are located on the sides of the first culture layer 2, the middle culture layer 1 and the second culture layer 3; the first liquid outlet 13, the middle liquid outlet 14 and the second liquid outlet 15 are located on the sides of the first culture layer 2, the middle culture layer 1 and the second culture layer 3.

[0088] Furthermore, the first culture layer 2, the middle culture layer 1 and the second culture layer 3 are cuboids; the length of the cuboids is 50 mm to 60 mm, the width is 20 mm to 25 mm, and the height is 2.5 mm to 3 mm. Specifically, in this embodiment, each culture layer is 60 mm long, 25 mm wide, and the single layer is 2.5 mm high.

[0089] Furthermore, it also includes a fixed mold for combining the first culture layer 2, the middle culture layer 1 and the second culture layer 3;

[0090] The fixed mold includes: a base 17, which is in a rectangular shape, and four screws 18 are detachably connected to the base 17 and are perpendicular to the base 17; when the microfluidic chip is located on the base 17, the microfluidic chip is located between the four screws 18, and the screws 18 are detachably connected to one side of the long side of the microfluidic chip (such as Figure 4 The fixed mold can fix multiple culture layers together to avoid the situation where the microchannels of each layer cannot realize the interaction between liquid and cytokines due to the offset of the position of a culture layer.

[0091] Example 2: A microfluidic chip for cell-cell secretory factor interaction

[0092] like Figures 5 to 8 As shown, a microfluidic chip for cell-cell secretion factor interaction (the material is PDMS and PC board formed by photolithography technology), comprising: an intermediate culture layer 1 and a first culture layer 2 and a second culture layer 3 detachably connected to the top surface and the bottom surface of the intermediate culture layer 1 respectively;

[0093] The middle culture layer 1, the first culture layer 2 and the second culture layer 3 are all provided with culture chambers, and a plurality of microchannels connected to the culture chambers are evenly arranged around each culture chamber; the microchannels interconnect the culture chambers in the middle culture layer 1, the first culture layer 2 and the second culture layer 3, and form a closed loop between each culture chamber; the middle culture layer 1, the first culture layer 2 and the second culture layer 3 are provided with separate perfusion channels and liquid outlet channels, and each separate perfusion channel and liquid outlet channel is connected to a separate liquid inlet and a separate liquid outlet arranged on the middle culture layer 1, the first culture layer 2 and the second culture layer 3;

[0094] The culture chambers are all cylindrical, cubic, or cuboid; the height of the culture chambers is 1 mm to 1.5 mm; and the diameter of the microchannels is 0.5 to 0.8 mm.

[0095] In the present invention, multiple culture layers are provided, and each culture layer is provided with a separate liquid inlet, perfusion channel, liquid outlet channel and liquid outlet, so that cells in multiple culture layers can be cultured simultaneously. At the same time, since microchannels are provided around the culture chambers of each culture layer, the microchannels can connect the culture chambers of each culture layer, realize the circulation of liquids and the interaction of cytokines between different culture layers, realize the co-culture of multiple cells, and lay a foundation for the research of various diseases.

[0096] Specifically, a magnet 19 is disposed on one side corresponding to the wide side of the first culture layer 2 , the middle culture layer 1 and the second culture layer 3 , so as to realize a detachable connection between the first culture layer 2 , the middle culture layer 1 and the second culture layer 3 .

[0097] Specifically, in this embodiment, the height of the first culture chamber 4 is 1 mm; the height of the second culture chamber 6 is 1.5 mm.

[0098] Further, the microchannel includes a first microchannel 7, an intermediate microchannel 8 and a second microchannel 9, and the number of the three microchannels is the same, which is 8 to 12; specifically, in this embodiment, the number of the first microchannel 7, the intermediate microchannel 8 and the second microchannel 9 is uniformly 12;

[0099] The first microchannel 7 is disposed in the first culture layer 2 and extends downward in the first culture layer 2, and its channel opening is located at the bottom surface of the first culture layer 2; the second microchannel 9 is disposed in the second culture layer 3 and extends upward in the second culture layer 3, and its channel opening is located at the top surface of the second culture layer;

[0100] The middle microchannel 8 is arranged through the middle culture layer 1 , and the channel opening located on the top surface of the middle culture layer 1 is connected to the channel opening of the first microchannel 7 ; the channel opening located on the bottom surface of the middle culture layer 1 is connected to the channel opening of the second microchannel 9 .

[0101] Further, the number of the intermediate culture layers 1 is 0 to 5; specifically, in this embodiment, the number of the intermediate culture layers 1 is 0 (eg Figure 5 , Figure 6 As shown), therefore, the channel opening of the first microchannel 7 is connected to the channel opening of the second microchannel 9.

[0102] Furthermore, the liquid inlet and the liquid outlet are distributed on both sides of the first culture chamber 4 , the middle culture chamber 5 and the second culture chamber 6 .

[0103] Furthermore, the liquid inlet includes a first liquid inlet 10, a middle liquid inlet 11 and a second liquid inlet 12; the liquid outlet includes a first liquid outlet 13, a middle liquid outlet 14 and a second liquid outlet 15;

[0104] The first liquid inlet 10, the middle liquid inlet 11 and the second liquid inlet 12 are located on the top surfaces of the first culture layer 2, the middle culture layer 1 and the second culture layer 3; the first liquid outlet 13, the middle liquid outlet 14 and the second liquid outlet 15 are located on the top surfaces of the first culture layer 2, the middle culture layer 1 and the second culture layer 3. When the first liquid inlet 10, the middle liquid inlet 11 and the second liquid inlet 12 are located on the top surfaces of the first culture layer 2, the middle culture layer 1 and the second culture layer 3, a plurality of through holes 16 are provided on the first culture layer 2 and the middle culture layer 1, which are respectively connected with the middle liquid inlet 11 and the second liquid inlet 12.

[0105] The first culture chamber 4, the middle culture chamber 5 and the second culture layer 3 are cylindrical, and the bottom diameter of the cylinder is 6 mm to 8 mm.

[0106] Specifically, in this embodiment, the bottom diameter of the first culture chamber 4 is 8 mm, and the bottom diameter of the second culture chamber 6 is 6 mm.

[0107] Furthermore, the first culture layer 2, the middle culture layer 1 and the second culture layer 3 are rectangular parallelepipeds; the length of the rectangular parallelepiped is 50 mm to 60 mm, the width is 20 mm to 25 mm, and the height is 2.5 mm to 3 mm.

[0108] Specifically, in this embodiment, each culture layer is 60 mm long, 25 mm wide, and the single layer height is 2.5 mm.

[0109] Furthermore, it also includes a fixed mold for combining the first culture layer 2, the middle culture layer 1 and the second culture layer 3;

[0110] The fixed mold includes: a base 17, which is in a rectangular shape, and four screws 18 are detachably connected to the base 17 and are perpendicular to the base 17; when the microfluidic chip is located on the base 17, the microfluidic chip is located between the four screws 18, and the screws 18 are detachably connected to one side of the long side of the microfluidic chip.

[0111] The microfluidic chip for cell-cell secretory factor interaction provided in this embodiment and the sterilized connecting accessories (combined connecting hose, flat needle, Luer connector, waste liquid bottle and 0.22 μm bacterial air filter after high pressure at 121° C. for 20 minutes and drying in a drying oven for 12 hours) are placed in an ultraviolet disinfected clean bench and assembled and fixed to ensure that the chip and connecting accessories are sterile.

[0112] The liquid inlet of the microfluidic chip is connected to the syringe through a hose through a Luer connector and a flat needle. After the upper and lower layers of the chip are aligned, the first culture layer 2 and the second culture layer 3 are fixed with a fixed mold, and the liquid outlet is connected to the waste liquid bottle through a hose, and at the same time connected to the additional bacterial air filter through a flat needle to ensure the supply of oxygen and carbon dioxide in the chip system and isolate the bacterial environment. The overall liquid perfusion is pumped by a micro-injection pump to achieve dynamic perfusion in the chip and maintain a sterile culture state.

[0113] In this embodiment, if Fig. 9 As shown, immune cell THP-1 and liver organoids were used as research objects. The hepatobiliary organoids were cut into pieces with tissue scissors in a clean bench after UV disinfection in advance, washed 10-15 times with DMEM-F / 12 medium containing 1% BSA until the supernatant was clear and transparent, and the tissue digestion solution (manufacturer: Baizhen Biology, catalog number K601008) was selected to digest the tissue for 10 minutes until small cell clumps appeared. Centrifuge at 290G for 5 minutes, wash the digested cell pellet again with cold DMEM / F12, and wrap the cell clumps with matrix gel (manufacturer: Corning; catalog number 354234), and 50μL per well was planted in a 24-well plate.

[0114] After standing for half an hour to allow the gel to solidify, liver organoid expansion medium (manufacturer: Baizhen Biotechnology, product number K2008-HLD) was added. After 15 days of culture, the cells were passaged to obtain purified organoids.

[0115] The purified 1-2 generation human bile duct organoids were digested with organoid digestion solution (manufacturer: Baizhen Biotechnology, catalog number E238001) for two minutes until small cell clumps appeared, centrifuged at 290G for 5 minutes, washed with ice-cold DMEM / F12 digestion solution, centrifuged at 200G for 5 minutes, and 500 cell clumps / single chip were added to 300μL of matrix gel (manufacturer: Corning; catalog number 354234). The resuspended matrix gel was manually injected into the second culture chamber 6 on ice using a 1mL syringe at a speed of 10μL / second. It can be seen that the matrix gel is evenly spread on the bottom of the lower disc, forming a 3D structure with a certain depth. Wait for 30 minutes until the matrix gel is completely solidified, and the cell clumps can be evenly spread in the matrix gel of the bottom disc.

[0116] Through the liquid inlets of the first culture layer 2 and the second culture layer 3, a 5 mL syringe was used to connect a thin hose with an inner diameter of 1.5 mm and an outer diameter of 1.5 mm. The perfusion speed was adjusted to 0.8 μL / min through a microinjection pump (manufacturer: Corning; product number 354234), and human mature liver organoid expansion culture medium (manufacturer: Baizhen Bio, product number K2008-HLD) was pumped to culture the organoids in the chip for 5 days.

[0117] When the cell clumps were observed to be vacuolar and about 500 μm in size under a microscope, the pumping medium was replaced with human mature liver organoid differentiation medium (manufacturer: Baizhen Biotechnology, product number K2008-HLH) for the first stage of induction, and the medium was pumped at a rate of 1 μL / min for 5 days.

[0118] The culture medium was replaced with human mature liver organoid differentiation medium (manufacturer: Baizhen Biotechnology, product number K2008-HLH) for the first stage of induction. The medium was pumped at a rate of 1 μL / min for 10 days. The state of the organoids was observed under a microscope. The organoids had a dense structure, tighter connections between cells, and did not collapse after perfusion when wrapped with matrix gel, but still showed a vacuolar spherical morphology of about 500 μm in size.

[0119] When the second stage of induction of human mature liver organoids reaches the 7th day, THP-1 cell culture is added, and the THP-1 cells frozen in the -80°C refrigerator are quickly thawed in a water bath heated to 37°C in advance, and aspirated using a 1mL sterile pipette tip in an ultraviolet-sterilized clean bench in advance, and added into the culture medium at a ratio of 1:9, and centrifuged at 1200rpm for 3min to obtain a cell pellet.

[0120] Resuspend the cells in 300 μL mixed culture medium at a number of 1*10^5, add 100 ng / ml phorbol ester (PMA, dissolved in DMSO), and inject into the first culture chamber 4 in the first culture layer 2 through a 1 mL syringe through the channel. Stop the perfusion of the first culture layer 2, maintain the perfusion of the second culture layer 3, wait for 6 hours for THP-1 cells to adhere to the wall, and perfuse the upper layer with 100 ng / mL phorbol ester (PMA, dissolved in DMSO) THP-1 commercial culture medium (manufacturer: Pu Nuo Sai; item number CM-0233; RPMI-1640, NaHCO31.5g / L glucose 2.5g / L. Sodium Pyruvate 0.11g / L and 0.05mM β-mercaptoethanol, high-quality fetal bovine blood, 10%). After perfusion for 2 days, replace the mixed culture medium for the next step of pathological model induction. In this embodiment, the mixed culture medium used is THP-1 culture medium (manufacturer: Procell; catalog number CM-0233) and human mature liver organoid culture medium (manufacturer: Procell; catalog number CM-0233) mixed in different proportions.

[0121] like Fig.10 Shown are bright field images of the organoid induction culture process. It can be seen that the organoid morphology becomes denser from the first stage to the second stage as the induction time increases, and the organoids are evenly distributed in the matrix gel and have a full morphology.

[0122] like Fig.11 Shown is HE staining of organoid paraffin sections. Compared with undifferentiated single-layer vesicle-like organoids, the cells of mature organoids that have completed differentiation are morphologically similar to solid organoids, and the connections between cells are tighter.

[0123] like Fig.12 The image below shows an organoid immunofluorescence experiment, which shows the functional status of the organoid. The image was taken using a Leica confocal microscope. In the undifferentiated bile duct organoid, CK19, a marker of bile duct function, was clearly expressed, while Epcam and SOX9, markers of primary stem cell function, were also clearly expressed. The organoid had a good functional status and was an undifferentiated primary bile duct organoid with high stemness.

[0124] like Fig.13 As shown, the organoid morphology (hemisphere) under the confocal layer scanning superposition can show the single-layer vesicle-like three-dimensional structure of the organoid, which is morphologically similar to human hepatobiliary organoids.

[0125] like Fig.14 The figure shows the proliferation of organoids (HLO) and the survival rate of THP-1 under different ratios. As the time gradient increases, organoids and THP-1 cells in co-culture treated with different ratios of culture medium show different activities. CCK8 reagent is used for microplate reader quantification. After comprehensive consideration, a suitable culture medium ratio for both is selected for mixed culture perfusion to ensure the activity of the two cells in the chip.

[0126] Judging from the CCK8 cell proliferation test, the cell proliferation state in this model is best when the mixed culture medium ratio of THP-1 culture medium (manufacturer: Prosai; catalog number CM-0233) and human mature liver organoid culture medium (manufacturer: Prosai; catalog number CM-0233) is 1:2.

[0127] like Fig.15 Shown is a bright field image of organoids (HLO) and THP-1 cells co-cultured. Focusing on different levels, it can be seen that both the cells and organoids are in good condition and are located at different levels, and can be completely distinguished.

[0128] like Fig.16 The figure shows an organoid live-death experiment. After imaging with a Leica confocal microscope, green fluorescence represents AM binding to active cells, and red fluorescence represents PI binding to dead cells. It can be seen that the organoids after co-culture have good activity.

[0129] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0130] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A microfluidic chip for cell-cell secretory factor interaction, characterized in that: include: An intermediate culture layer (1), and a first culture layer (2) and a second culture layer (3) which are detachably connected to the top surface and the bottom surface of the intermediate culture layer (1), respectively; The intermediate culture layer (1), the first culture layer (2) and the second culture layer (3) are each provided with a culture chamber, and a plurality of microchannels connected to the culture chamber are evenly arranged around each culture chamber; the microchannels interconnect the culture chambers in the intermediate culture layer (1), the first culture layer (2) and the second culture layer (3), and form a closed loop between each culture chamber; the intermediate culture layer (1), the first culture layer (2) and the second culture layer (3) are provided with separate perfusion channels and liquid outlet channels, and each of the separate perfusion channels and liquid outlet channels is connected to a separate liquid inlet and a separate liquid outlet arranged on the intermediate culture layer (1), the first culture layer (2) and the second culture layer (3); The height of the culture chamber is 1 mm to 1.5 mm; the diameter of the microchannel is 0.5 mm to 0.7 mm.

2. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 1, characterized in that: The culture chamber comprises: a first culture chamber (4), an intermediate culture chamber (5) and a second culture chamber (6); the microchannel comprises a first microchannel (7), an intermediate microchannel (8) and a second microchannel (9); The first microchannel (7) is arranged in the first culture layer (2) and extends downward in the first culture layer (2), and its channel opening is located on the bottom surface of the first culture layer (2); the second microchannel (9) is arranged in the second culture layer (3) and extends upward in the second culture layer (3), and its channel opening is located on the top surface of the second culture layer (3); The intermediate microchannel (8) is arranged to penetrate the intermediate culture layer (1), the channel opening located on the top surface of the intermediate culture layer (1) is connected to the channel opening of the first microchannel (7); the channel opening located on the bottom surface of the intermediate culture layer (1) is connected to the channel opening of the second microchannel (9); The number of the first microchannels (7), the middle microchannels (8) and the second microchannels (9) is the same, and the number is 8 to 12.

3. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 2, characterized in that: The number of the intermediate culture layers (1) is 0 to 3; When the number of the intermediate culture layers (1) is 0, the channel opening of the first microchannel (7) is connected to the channel opening of the second microchannel (9).

4. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 1, characterized in that: The liquid inlet and the liquid outlet are distributed on both sides of the first culture chamber (4), the middle culture chamber (5) and the second culture chamber (6).

5. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 4, characterized in that: The liquid inlet comprises a first liquid inlet (10), an intermediate liquid inlet (11) and a second liquid inlet (12); the liquid outlet comprises a first liquid outlet (13), an intermediate liquid outlet (14) and a second liquid outlet (15); The first liquid inlet (10), the middle liquid inlet (11) and the second liquid inlet (12) are located on the top surface or the side surface of the first culture layer (2), the middle culture layer (1) and the second culture layer (3); the first liquid outlet (13), the middle liquid outlet (14) and the second liquid outlet (15) are located on the top surface or the side surface of the first culture layer (2), the middle culture layer (1) and the second culture layer (3).

6. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 5, characterized in that: When the first liquid inlet (10), the intermediate liquid inlet (11) and the second liquid inlet (12) are located on the top surfaces of the first culture layer (2), the intermediate culture layer (1) and the second culture layer (3), a plurality of through holes (16) are provided on the first culture layer (2) and the intermediate culture layer (1), respectively communicating with the intermediate liquid inlet (11) and the second liquid inlet (12).

7. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 1, characterized in that: The culture chambers are all cylindrical, cube-shaped, or cuboid-shaped; When the first culture chamber (4), the intermediate culture chamber (5) and the second culture layer (3) are cylindrical, the bottom diameter of the cylindrical body is 6 mm to 8 mm; When the first culture chamber (4), the intermediate culture chamber (5) and the second culture layer (3) are cubes, the side length of the cube is 6 mm to 8 mm; When the first culture chamber (4), the intermediate culture chamber (5) and the second culture layer (3) are rectangular parallelepipeds, the length of the cube is 6 mm to 10 mm, and the width is 6 mm to 8 mm.

8. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 1, characterized in that: The first culture layer (2), the middle culture layer (1) and the second culture layer (3) are rectangular parallelepipeds; the length of the rectangular parallelepiped is 50 mm to 60 mm, the width is 20 mm to 25 mm, and the height is 2.5 mm to 3 mm.

9. The microfluidic chip for cell-cell secretory factor interaction as claimed in claim 1, characterized in that: It also includes a fixed mold for combining the first culture layer (2), the middle culture layer (1) and the second culture layer (3); The fixed mold comprises: a base (17) in a rectangular shape, and four screws (18) are detachably connected to the base (17) and are perpendicular to the base (17); when the microfluidic chip is located on the base (17), the microfluidic chip is located between the four screws (18), and the screws (18) are detachably connected to one side of the long side of the microfluidic chip.

10. Use of the microfluidic chip for cell-cell secretory factor interaction as claimed in any one of claims 1 to 9 in multi-cell co-culture.

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