Matrigel for preparing cell trap array for regeneration of organoid, cell trap array, preparation method of cell trap array and organoid

Abstract

The invention relates to the technical field of biological tissue engineering, and discloses matrigel for preparing a cell trap array for organoid regeneration, the matrigel comprises a solution A and a solution B, the mass ratio of the solution A to the solution B is (0.9-1.1): 1, the solution A comprises fibrinogen, trihydroxymethyl aminomethane hydrochloride and NaCl, and the solution B comprises matrigel, Ca < 2 + >, Mg < 2 + >, thrombin and collagen. The matrigel for preparing the cell trap array for organoid regeneration, provided by the invention, is beneficial to realizing standardization and controllability of organoid regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of biological tissue engineering, and in particular to a matrix gel for preparing a cell trap array for organoid regeneration, a cell trap array and a preparation method thereof, and an organoid. Background Art

[0002] Organoids are three-dimensional in vitro cultures formed by self-organization of adult stem cells or pluripotent stem cells. Since the emergence of organoid technology, a variety of renewable or non-renewable tissues and organs, including intestinal epithelium, liver, skin, heart, brain, etc., have been successfully produced under laboratory conditions. Organoids retain the characteristics of the structure, function and genetic background of the source organs, which makes them have extremely broad application prospects in the fields of personalized medicine, drug development, organ transplantation, and cutting-edge biomedical research.

[0003] Traditional organoid culture systems are based on manual operations to construct a three-dimensional culture microenvironment of matrix gel, drive stem cell proliferation and differentiation by manipulating biochemical signals such as cytokines, and form organoids under the action of self-organization mechanisms driven by intercellular communication. There are three main factors that control these self-organization processes: (1) the nature of the intercellular communication network, which corresponds to the type, number and spatial distribution of the starting cells; (2) the spatial distribution of mechanical forces and ECM (extracellular matrix) signals, which corresponds to the composition and degree of cross-linking of the ECM around the organoid; and (3) the geometric shape of the organoid, which corresponds to the physical boundary around the organoid.

[0004] In the traditional dome culture method, there is no mechanism to control the above three factors at the same time, which leads to differences in the starting conditions of different organoids in the dome. These differences will lead to uncontrollable phenotypic heterogeneity between the regenerated organoids, which will bring difficulties to subsequent research and application. Although some tissue engineering technologies, such as microarrays and organ chips, have solved the problem of controllability of the organoid regeneration process to a certain extent, there is currently no technology that can control the three factors of self-organization at the same time. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a matrix gel for preparing a cell trap array for organoid regeneration, which is conducive to achieving standardization and controllability of organoid regeneration.

[0006] The technical problem to be solved by the present invention is also to provide a cell trap array for organoid regeneration and a preparation method thereof, which is conducive to achieving standardization and controllability of organoid regeneration.

[0007] In order to solve the above technical problems, a matrix gel for preparing a cell trap array for organoid regeneration comprises liquid A and liquid B, wherein the mass ratio of the liquid A to the liquid B is (0.9-1.1):1, the liquid A comprises fibrinogen, tris(hydroxymethyl)aminomethane hydrochloride) and NaCl, and the liquid B comprises matrix gel, Ca 2+ Mg 2+ , thrombin, collagen.

[0008] In some embodiments, in the solution A, the concentration of the fibrinogen is 95 mg / ml to 105 mg / ml, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride is 45 mM to 55 mM, and the concentration of NaCl is 145 mM to 155 mM.

[0009] In some embodiments, in the B solution, Ca 2+ and Mg 2+ The concentration of is 0.5mM~1.5mM, the concentration of thrombin is 1.5μg / ml~2.5μg / ml, and the concentration of collagen is 0.5mg / ml~1.5mg / ml.

[0010] In some embodiments, the collagen is type I collagen.

[0011] In order to solve the above technical problems, the present invention provides a method for preparing a cell trap array for organoid regeneration, comprising the following steps:

[0012] Providing an agarose gel layer having a microwell array, wherein the microwell array comprises a plurality of microwells;

[0013] adding cells into the microwells, and culturing the cells to obtain multicellular spheroids;

[0014] Adding the A solution and the B solution of the matrix gel for preparing the cell trap array for organoid regeneration according to any one of claims 1 to 4 into the microwell containing the multicellular spheroid to cover the multicellular spheroid;

[0015] Dehydrating the matrix gel so that the matrix gel wraps the multicellular spheroid to form a matrix gel-multicellular spheroid inclusion body;

[0016] separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body;

[0017] The matrix gel-multicellular spheroid inclusions are solidified to obtain a cell trap array for organoid regeneration.

[0018] In some embodiments, the A solution and the B solution are added in a mass ratio of 1:(0.95-1.05).

[0019] In some embodiments, the matrix gel-multicellular spheroid inclusions are solidified, comprising:

[0020] Covering the matrix gel-multicellular spheroid inclusions with a C solution of matrix gel for preparing a cell trap array for organoid regeneration, recovering the C solution after culturing for a first preset time, and then covering the matrix gel-multicellular spheroid inclusions with A solution and B solution until the matrix gel-multicellular spheroid inclusions are completely covered, and then culturing for a second preset time;

[0021] The C solution includes thrombin, Ca 2+ and Mg 2+ , where the concentration of thrombin is 95μg / ml~105μg / ml, Ca 2+ and Mg 2+ The concentration is 0.5mM~1.5mM.

[0022] In some embodiments, providing an agarose gel layer having a microwell array comprises:

[0023] preparing an agarose gel layer;

[0024] Prepare a mold, wherein the mold is provided with a protrusion array, wherein the protrusion array includes a plurality of protrusions;

[0025] The protrusion array of the mold is pressed onto the agarose gel layer, and the mold is separated after standing to obtain an agarose gel layer with a micropore array.

[0026] In some embodiments, the microwell array is a microwell array with a preset structure customized according to the needs of the target organoid;

[0027] The preset structure includes the size of a single micropore, the shape of a single micropore, the distance between micropores, the connection method between micropores, the shape of the connecting channel between micropores, the size of the connecting channel between micropores, and the spatial arrangement of micropores.

[0028] In some embodiments, the dehydration treatment of the matrix glue comprises:

[0029] Applying negative pressure on one side of the matrix gel to absorb water from the matrix gel to achieve dehydration of the matrix gel; and / or

[0030] Negative pressure is applied to one side of the agarose gel layer to absorb water from the matrix gel through the pores of the agarose gel, thereby achieving dehydration of the matrix gel.

[0031] In order to solve the above technical problems, the present invention also provides a cell trap array for organoid regeneration, and the cell trap array for organoid regeneration is prepared by the preparation method of the cell trap array for organoid regeneration.

[0032] The implementation of the present invention has the following beneficial effects:

[0033] The matrix gel provided by the present invention is used to prepare a cell trap array for organoid regeneration, wherein the matrix gel is wrapped around the organoid cells, and the matrix gel can form a temporary steady state of entanglement rather than cross-linking by dehydration, temporarily increasing the physical strength of the matrix gel to achieve a cell trap array of a specific shape. The matrix gel can be used to customize the components of the extracellular matrix used in the organoid culture process, and can ensure that the extracellular matrix around each organoid is uniform.

[0034] The present invention proposes a method for preparing a cell trap array for organoid regeneration, which can customize the mode and strength of cell-cell interaction and cell-microenvironment interaction to constrain the spatial distribution of cell signal activation in organoids. Compared with traditional organoid regeneration methods, it solves the consistency and non-customizability problems of existing arrayed organoid regeneration. It enhances the consistency and repeatability of the organoid regeneration process, providing a basis for subsequent organoid scientific research and clinical diagnosis and treatment applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a physical picture of adding solution A and solution B of matrix gel into the microwell containing the multicellular spheroid and covering the multicellular spheroid in Example 1;

[0036] Figure 2 This is a physical picture of the matrix gel-multicellular spheroid inclusion body formed after the matrix gel is dehydrated in Example 1;

[0037] Figure 3 This is a schematic diagram of the structure of the dehydration module in Example 1;

[0038] Figure 4 is a cross-sectional view of the dehydration module in Example 1;

[0039] Figure 5 It is a schematic diagram of the structure of the automated manufacturing system for cell well arrays with human-machine interaction in Example 1;

[0040] Figure 6 These are morphological observation pictures of airway epithelial organoids cultured using the cell well array for preparing organoid regeneration prepared in Example 1 on the 3rd, 5th and 7th days. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0042] In the description of the present invention, it is necessary to understand that the terms "length", "width", "up", "down", "left", "right", "horizontal", "top", "bottom", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0043] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0044] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or mutual communication; it can be a direct connection, or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0045] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes that the first feature is directly below and obliquely below the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0046] The disclosure below provides many different embodiments or examples to realize different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed. In addition, the examples of various specific processes and materials provided by the present invention, but those of ordinary skill in the art can be aware of the application of other processes and / or the use of other materials.

[0047] In the present invention, "preferred" and "better" are only used to describe implementation methods or embodiments with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features. In the present invention, when involving numerical ranges, unless otherwise specified, the two endpoints of the numerical range are included.

[0048] In order to solve the above problems, the present invention provides a matrix gel for preparing a cell trap array for organoid regeneration, comprising liquid A and liquid B, wherein the mass ratio of the liquid A to the liquid B is (0.9-1.1):1, the liquid A comprises fibrinogen, tris(hydroxymethyl)aminomethane hydrochloride) and NaCl, and the liquid B comprises matrix gel, Ca 2+ Mg 2+ , thrombin, collagen.

[0049] The matrix gel provided by the present invention is used to prepare a cell trap array for organoid regeneration, wherein the matrix gel is wrapped around the organoid cells, and the matrix gel can form a temporary steady state of entanglement rather than cross-linking by dehydration, temporarily increasing the physical strength of the matrix gel to achieve a cell trap array of a specific shape. The matrix gel can be used to customize the components of the extracellular matrix used in the organoid culture process, and can ensure that the extracellular matrix around each organoid is uniform.

[0050] Specifically, in organoid culture, matrix gel plays a vital role, providing cells with a three-dimensional growth environment, simulating the extracellular matrix (ECM) in the body, thereby supporting cell adhesion, growth, differentiation and function. In the present invention, liquid A and liquid B are the main materials for forming cell traps and are also the main materials for making organoid culture.

[0051] In the A solution, fibrinogen is a protein with coagulation function synthesized by the liver and is a precursor of fibrin. Tris HCl, also known as Tris HCl, is a commonly used biochemical reagent, mainly used to adjust the pH value of the solution and as a buffer. In some embodiments, in the A solution, the concentration of the fibrinogen is 95mg / ml~105mg / ml, the concentration of the tris HCl is 45mM~55mM, and the concentration of NaCl is 145mM~155mM. Preferably, in the A solution, the concentration of the fibrinogen is 97mg / ml~103mg / ml, the concentration of the tris HCl is 48mM~52mM, and the concentration of NaCl is 146mM~154mM.

[0052] It should be noted that the fibrinogen concentration is moderately excessive so that it can form a better entangled state during the gel dehydration process, increase the strength and prevent damage during demolding. Tris(hydroxymethyl)aminomethane hydrochloride) ensures sufficient buffering to keep the pH stable when mixed with other gel components such as collagen. NaCl provides the appropriate solubility of the protein. However, when the fibrinogen concentration is too low, demolding may fail, and when it is too high, the overly hard extracellular matrix will affect the subsequent growth of organoids.

[0053] In the B solution, matrix glue, also known as Matrigel, is a matrix glue widely used in cell biology and tissue engineering research. It is derived from the basement membrane components of mouse sarcoma cells (EHS sarcoma) and is mainly composed of laminin, type IV collagen, nestin and other growth factors and extracellular matrix proteins. These components together constitute a complex three-dimensional network structure, similar to the microenvironment of cell growth in the body. Thrombin is a glycoprotein, a substance before thrombin activation, and a very important coagulation factor in the body. It participates in the endogenous coagulation pathway and the exogenous coagulation pathway of the body; type I collagen is a helical structure protein composed of three polypeptide chains. These peptide chains are tightly bound together by hydrogen bonds to form a stable triple helix structure. It is a natural protein widely present in human skin, bones, tendons and other tissues. It has high tension and strength and can provide stable structural support for tissues. Ca 2+ Mg 2+ The source of the raw material may be a calcium chloride solution or a magnesium chloride solution, without limitation.

[0054] In some embodiments, in the B solution, Ca 2+ and Mg 2+ The concentration of the B solution is 0.5mM to 1.5mM; the concentration of the thrombin is 1.5μg / ml to 2.5μg / ml, and the concentration of the collagen is 0.5mg / ml to 1.5mg / ml.2+ and Mg 2+ The concentration of calcium and magnesium ions is 0.7mM to 1.2mM; the concentration of thrombin is 1.8μg / ml to 2.4μg / ml, and the concentration of collagen is 0.6mg / ml to 1.1mg / ml. It should be noted that within the range of the above embodiment, the concentration of calcium and magnesium ions is the concentration required for the maximum activity of thrombin. Too little will lead to activation disorders, and too much will affect cell activity. Excessive collagen concentration will change the curing mode of the entire composite hydrogel, resulting in its inability to solidify, and too little will weaken the gel strength.

[0055] Preferably, the liquid A and the liquid B are mixed in a mass ratio of 1:(0.95-1.05). More preferably, the liquid A and the liquid B are mixed in a mass ratio of 1:1. The equal ratio combination of liquid A and liquid B can better prevent uneven gel curing, which may cause breakage during subsequent demolding.

[0056] Thrombin turns into thrombin after activation, and thrombin has the ability to activate fibrinogen. When thrombin acts on fibrinogen, it causes a series of biochemical reactions, ultimately generating fibrin monomers. These monomers are further polymerized into water-insoluble fibrin polymers under the catalysis of soluble fibrinogen polymers. These polymers interweave in the solution to form a three-dimensional network structure, namely biohydrogel. This hydrogel has good biocompatibility and degradability, and can provide a good growth environment for cells.

[0057] The present invention provides a method for preparing a cell trap array for organoid regeneration, comprising the following steps:

[0058] (1) providing an agarose gel layer having a microwell array, wherein the microwell array comprises a plurality of microwells;

[0059] In some embodiments, providing an agarose gel layer having a microwell array comprises:

[0060] preparing an agarose gel layer;

[0061] Prepare a mold, wherein the mold is provided with a protrusion array, wherein the protrusion array includes a plurality of protrusions;

[0062] The protrusion array of the mold is pressed onto the agarose gel layer, and the mold is separated after standing to obtain an agarose gel layer with a micropore array.

[0063] Preferably, the micropore array is a micropore array with a preset structure customized according to the needs of the target organoid; the preset structure includes the size of a single micropore, the shape of a single micropore, the distance between micropores, the connection method between micropores, the shape of the connecting channels between micropores, the size of the connecting channels between micropores, and the spatial arrangement of micropores.

[0064] In some embodiments, the size of the single micropore includes the pore depth, opening size, and holding volume of the single micropore; the shape of the single micropore includes a cone-like, a cylinder-like, a cuboid, and a hemisphere; the distance between the micropores is the distance between adjacent micropores, which can be equidistant or unequal, and the distance between the micropores varies according to the needs of the target organoid. The connection between the micropores can be that all adjacent micropores are independent of each other, or that all adjacent micropores are connected, or that some adjacent micropores are independent of each other and some adjacent micropores are connected, and the connection between the micropores varies according to the needs of the target organoid. The spatial arrangement of the micropores is a spatial arrangement pattern formed by the combination of all micropores, and the spatial arrangement of the micropores varies according to the needs of the target organoid.

[0065] In some embodiments, the size, shape, spacing, and spatial distribution of the micropore array on the mold are designed according to experimental needs, and a three-dimensional stereogram of the mold is drawn using three-dimensional drawing software; the material, precision, and other parameters of the 3D printer are set, and the mold is printed using a 3D printer.

[0066] It should be noted that the preset structure includes the size of a single micropore, the shape of a single micropore, the distance between micropores, the connection method between micropores, and the spatial arrangement of micropores. The structure of the above micropores determines the number, type, source and interaction ability of the starting cells and the physical boundaries of organoid growth. The micropore array with a preset structure customized according to the needs of the target organoid can ensure that when the organoid regenerates, the state, number, type, spatial distribution of the starting cells and the composition of the matrix gel around each organoid can be standardized and controllable, thereby ensuring that the batch effect of the regeneration process is extremely low and has strong consistency.

[0067] In addition, the embodiments of providing the agarose gel with a micropore array are not limited to the above examples, and can also be directly prepared by 3D printing. The present invention can customize the size, spacing, shape, and information exchange channels between different holes of the micropore array by customizing the micropore array with a preset structure according to the needs of the target organoid. The process of cell self-organization is controlled by controlling the number, type, and cell spacing of the starting cells.

[0068] Furthermore, the agarose gel is an important carrier for culturing multicellular spheroids. Due to its inert chemical properties, the agarose gel cannot form a covalent connection with the matrix gel provided by the present invention. In the subsequent preparation method, the matrix gel provided by the present invention, including collagen, Matrigel, fibrinogen-thrombin, etc., can form an entangled state by dehydration, and can provide a gel with a physical strength beyond that after cross-linking in a short time. The pores of the agarose gel allow it to be dehydrated without direct contact with the matrix gel.

[0069] (2) adding cells into the microwells, and culturing the cells to obtain multicellular spheroids;

[0070] In some embodiments, adding cells to the microwells comprises:

[0071] Customize preset regimes based on the needs of target organoids;

[0072] Cells are added to the microwells according to the preset protocol.

[0073] In some embodiments, the cells are cultured to obtain multicellular spheroids, comprising:

[0074] The cells self-assemble within the microwells to obtain multicellular spheroids.

[0075] Preferably, the preset system includes the type of cells added to a single microwell and the number of cells added to a single microwell; the cells include one or more of somatic cells, adult stem cells, and pluripotent stem cells. The process of cell self-organization is controlled by controlling the number, type, and cell spacing of the starting cells.

[0076] Multicellular spheroids and organoids are the main representative models in 3D cell culture. Multicellular spheroids usually refer to comprehensive and compact cell aggregates composed of a single type of cells (epithelial cells, mesenchymal cells, endothelial cells, etc.). They may be formed by cell lines, single cells, or crypts in biopsy tissues. Different types of cells can also be mixed to form randomly distributed or stratified spheroids based on cell types.

[0077] (3) adding the matrix gel solution A and solution B into the microwells containing the multicellular spheroids to cover the multicellular spheroids;

[0078] After the cells self-assemble in the micropores, liquid A and liquid B are added to the micropores. Preferably, the liquid A and the liquid B are mixed in a mass ratio of 1: (0.95-1.05). More preferably, the liquid A and the liquid B are mixed in a mass ratio of 1: 1. Thrombin is converted into thrombin after activation, and thrombin has the ability to activate fibrinogen. When thrombin acts on fibrinogen, it causes a series of biochemical reactions, eventually generating fibrin monomers. These monomers are further polymerized into water-insoluble fibrin polymers under the catalysis of soluble fibrinogen polymers. These polymers are intertwined in the solution to form a three-dimensional network structure, namely a biohydrogel. This hydrogel has good biocompatibility and degradability, and can provide a good growth environment for cells.

[0079] (4) dehydrating the matrix gel so that the matrix gel wraps around the multicellular spheroid to form a matrix gel-multicellular spheroid inclusion body;

[0080] In some embodiments, the dehydration treatment of the matrix glue comprises:

[0081] Applying negative pressure on one side of the matrix gel to absorb water from the matrix gel to achieve dehydration of the matrix gel; and / or

[0082] Negative pressure is applied to one side of the agarose gel layer to absorb water from the matrix gel through the pores of the agarose gel, thereby achieving dehydration of the matrix gel.

[0083] Due to its inert chemical properties, agarose gel cannot form a covalent bond with the matrix gel, and the presence of pores in agarose gel allows it to be dehydrated without direct contact with the matrix gel. In the embodiment of pre-curing by dehydration, the matrix gel is formed into a temporary steady state of entanglement rather than cross-linking. Since agarose gel cannot be cross-linked with the matrix gel, it can be easily peeled off from the matrix gel, and the self-assembled multicellular spheroids will remain in the matrix gel. Subsequently, the self-assembled multicellular spheroids will form organoids through morphogenesis. The cell signals related to each cell in the organoid are constrained to a specific spatiotemporal pattern by cell-to-cell interactions and directly affect the subsequent morphogenesis process. Therefore, this technology is named cell trap array. In addition, the method of forming an entangled state by dehydration and temporarily increasing the physical strength of the hydrogel can freely determine the components of the ECM and ensure that the ECM around each organoid is uniform.

[0084] Preferably, the dehydration treatment of the matrix glue comprises:

[0085] 1) Place the side of the agarose gel layer without the microporous array on the filter paper of the negative pressure suction device, and continue to absorb water from the agarose gel through the filter paper. At the same time, continue to add liquid A and liquid B on top. After evenly dropping, start the negative pressure suction device.

[0086] 2) When liquid A and liquid B fill the entire array holes, stop the negative pressure suction device and cover the agarose gel layer with liquid A and liquid B again.

[0087] 3) Place the side of the agarose gel layer with the microwell array on the center side of the Confocal dish glass, cover it with the lid and place it in a cell culture incubator to solidify for 10 to 15 minutes.

[0088] (5) separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body;

[0089] In some embodiments, agarose gel cannot cross-link with matrigel, so agarose gel can be easily peeled off from matrigel, and the self-assembled multicellular spheroids will remain in the matrigel.

[0090] (6) The matrix gel-multicellular spheroid inclusions are solidified to obtain a cell trap array for organoid regeneration.

[0091] In some embodiments, the solidification treatment of the matrix gel-multicellular spheroid inclusions comprises:

[0092] The matrix gel-multicellular spheroid inclusions are covered with the matrix gel C solution, and the C solution is recovered after culturing for a first preset time, and then the matrix gel-multicellular spheroid inclusions are covered with A solution and B solution until the matrix gel-multicellular spheroid inclusions are completely covered, and then cultured for a second preset time.

[0093] In some embodiments, the C solution includes thrombin, Ca 2+ Mg 2+ , the concentration of thrombin is 95μg / ml~105μg / ml, the Ca 2+ and Mg 2+ The concentration of thrombin is 0.5mM to 1.5mM. Preferably, the concentration of thrombin is 97μg / ml to 102μg / ml, and the Ca 2+ and Mg 2+ The concentration is 0.6mM~1.2mM.

[0094] It should be noted that after the initial curing, the hydrogel is not in a cross-linked curing form, but in an entangled state, and an additional cross-linking curing step is required, otherwise the gel will quickly loosen and degrade during subsequent organoid culture. Here, liquid C plays a cross-linking role. Alternatively, other cross-linking agents can be added, such as a 0.1%-1% (w / v) mixed solution of genipin and PBS.

[0095] Preferably, the first preset time is 15 minutes to 20 minutes; the second preset time is 40 minutes to 60 minutes.

[0096] The solidification treatment of the matrix gel-multicellular spheroid inclusions further comprises:

[0097] During the solidification process, the organoid chip is added to the matrix gel-multicellular spheroid inclusions.

[0098] The present invention facilitates the customization of complex organoid chips, the designs of which include multiple tissue sources, microenvironments, blood vessels or neural connections, etc., and solves the current problem of poor tissue morphology reproduction in organ chips.

[0099] In some embodiments, the present invention is prepared by a human-machine interactive cell well array automated manufacturing system.

[0100] like Figure 5 As shown, the human-machine interactive cell trap array automated manufacturing system includes an agarose gel layer, a dehydration module 2, a robotic arm 3, a workbench 4, an industrial camera and a control module. The workbench 4 is provided with an array production area and a cell seeding area. The cell seeding area includes Figure 3-4 The agarose gel layer 1 and the dehydration module 2 shown are connected to the dehydration equipment. The industrial camera is arranged above the cell inoculation area, and is used to perform visual imaging of the micropore array in the cell inoculation area to obtain the micropore array position image. The control module is electrically connected to the mechanical arm 3 and the industrial camera. The driving end of the mechanical arm 3 is provided with a pipette. The control module controls the mechanical arm 3 to drive the pipette according to the micropore array position image fed back by the industrial camera to implant cells into the micropore array corresponding to the micropore array position image. The dehydration module 2 plays the role of supporting and fixing the agarose gel layer 1. One side of the dehydration module 2 is used to fix the agarose gel layer 1, and the other side is provided with a through hole for connecting with the dehydration equipment to realize the dehydration of the agarose gel layer 1, and indirectly realize the dehydration treatment of the matrix glue.

[0101] Specifically, an agarose gel layer 1 having a micropore array is provided, wherein the micropore array comprises a plurality of micropores;

[0102] Fixing the agarose gel layer 1 in the dehydration module 2;

[0103] Add cells into the microwell array by using a pipette of the robot arm 3, and culture the cells to obtain multicellular spheroids;

[0104] Adding solution A and solution B of matrix gel to the microwell array containing the multicellular spheroids to cover the multicellular spheroids;

[0105] Dehydrating the matrix glue using a dehydration module 2 to complete pre-curing, so that the matrix glue wraps the multicellular spheroid to form a matrix glue-multicellular spheroid inclusion body;

[0106] separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body;

[0107] A cross-linking agent is added to the matrix gel-multicellular spheroid inclusions to achieve solidification treatment, thereby obtaining a cell trap array for organoid regeneration.

[0108] Correspondingly, the present invention also provides a cell trap array for organoid regeneration, wherein the cell trap array is prepared by the preparation method of the cell trap array for standardized and controllable regeneration of organoids.

[0109] Correspondingly, the present invention also provides a cell trap array for organoid regeneration, wherein the cell trap array is prepared by the preparation method of the cell trap array for organoid regeneration.

[0110] In summary, the present invention proposes a cell trap array for organoid regeneration and a preparation method thereof, which can realize the customization of the mode and intensity of cell-cell interaction and cell-microenvironment interaction to constrain the spatial distribution of cell signal activation in organoids. Compared with the traditional organoid regeneration method, it solves the consistency and non-customizability problems of existing arrayed organoid regeneration. It enhances the consistency and repeatability of the organoid regeneration process, and provides a basis for subsequent organoid scientific research and clinical diagnosis and treatment applications.

[0111] The present invention is further described below with specific embodiments:

[0112] Example 1

[0113] This embodiment provides a matrix gel for preparing a cell trap array for organoid regeneration, comprising liquid A and liquid B, wherein the mass ratio of the liquid A to the liquid B is 1:1, the liquid A comprises fibrinogen, tris hydroxymethylaminomethane hydrochloride (Tris HCl), and NaCl, and the liquid B comprises Matrigel, Ca 2+ Mg 2+ , thrombin, collagen.

[0114] In the solution A, the concentration of fibrinogen is 100 mg / ml, the concentration of Tris HCl is 50 mM, and the concentration of NaCl is 150 mM;

[0115] In the B solution, Ca 2+ and Mg 2+ The concentration of is 1 mM, the concentration of thrombin is 2 μg / ml, the collagen is type I collagen, and the concentration of collagen is 1 mg / ml.

[0116] The specific products of each reagent are:

[0117] High concentration low growth factor phenol red-free Matrigel, ABW, Cat#082723;

[0118] bovine fibrinogen, Solarbio, Cat#F8051;

[0119] Thrombin, Solarbio, Cat#T8021;

[0120] 1.5 M Tris-HCl buffer (pH 8.8), Solarbio, T1010;

[0121] Collgen Type I, CORNING, 354249;

[0122] The matrix gel provided in Example 1 was used to prepare a cell trap array for organoid regeneration. The preparation method of the cell trap array for organoid regeneration is as follows:

[0123] Providing an agarose gel layer having a microwell array, wherein the microwell array comprises a plurality of microwells;

[0124] Add cells to the microwells, and culture the cells to obtain multicellular spheroids; specifically, place the first matrix in Figures 3-4 The dehydration module is then placed in the dehydration module containing the first matrix. Figure 5 In the human-machine interactive cell well array automated manufacturing system shown, a robotic arm is used to add cells to the microwells.

[0125] Adding solution A and solution B of matrix gel to the microwells containing the multicellular spheroids to cover the multicellular spheroids;

[0126] The matrix gel is dehydrated, that is, negative pressure is applied to one side of the agarose gel layer to absorb water from the matrix gel through the pores of the agarose gel, thereby dehydrating the matrix gel so that the matrix gel wraps the multicellular spheroid to form a matrix gel-multicellular spheroid inclusion body; specifically, the following method is used: Figures 3-4 The dehydration module shown performs dehydration processing.

[0127] separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body;

[0128] The matrix gel-multicellular spheroid inclusions are solidified to obtain a cell trap array for organoid regeneration. Figure 1 This is a physical picture of adding solution A and solution B of matrix gel into the microwell containing the multicellular spheroid and covering the multicellular spheroid in Example 1;

[0129] Figure 2 This is a physical picture of the matrix gel-multicellular spheroid inclusion body formed after the matrix gel is dehydrated in Example 1;

[0130] Figure 6 These are morphological observation pictures of airway epithelial organoids cultured using the cell well array for preparing organoid regeneration prepared in Example 1 on the 3rd, 5th and 7th days.

[0131] Example 2

[0132] This embodiment provides a matrix gel for preparing a cell trap array for organoid regeneration, comprising liquid A and liquid B, wherein the mass ratio of the liquid A to the liquid B is 1:1, the liquid A comprises fibrinogen, tris hydroxymethylaminomethane hydrochloride (Tris HCl), and NaCl, and the liquid B comprises Matrigel, Ca 2+ Mg 2+ , thrombin, collagen.

[0133] In the solution A, the concentration of fibrinogen is 103 mg / ml, the concentration of Tris HCl is 52 mM, and the concentration of NaCl is 153 mM;

[0134] In the B solution, Ca 2+ and Mg 2+ The concentration of is 0.9 mM, the concentration of thrombin is 2.1 μg / ml, the collagen is type I collagen, and the concentration of collagen is 1.1 mg / ml.

[0135] The specific products of each reagent are:

[0136] High concentration low growth factor phenol red-free Matrigel, ABW, Cat#082723;

[0137] bovine fibrinogen, Solarbio, Cat#F8051;

[0138] Thrombin, Solarbio, Cat#T8021;

[0139] 1.5 M Tris-HCl buffer (pH 8.8), Solarbio, T1010;

[0140] Collgen Type I, CORNING, 354249;

[0141] The matrix gel provided in Example 2 was used to prepare a cell trap array for organoid regeneration. The preparation method of the cell trap array for organoid regeneration is as follows:

[0142] Providing an agarose gel layer having a microwell array, wherein the microwell array comprises a plurality of microwells;

[0143] adding cells into the microwells, and culturing the cells to obtain multicellular spheroids;

[0144] Adding solution A and solution B of matrix gel to the microwells containing the multicellular spheroids to cover the multicellular spheroids;

[0145] Dehydrating the matrix gel, i.e., applying negative pressure on one side of the agarose gel layer to absorb water from the matrix gel through the pores of the agarose gel, thereby dehydrating the matrix gel, so that the matrix gel wraps the multicellular spheroid to form a matrix gel-multicellular spheroid inclusion body;

[0146] separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body;

[0147] The matrix gel-multicellular spheroid inclusions are solidified, specifically comprising: covering the matrix gel-multicellular spheroid inclusions with matrix gel C solution, recovering the C solution after 10 minutes of culture, and then covering the matrix gel-multicellular spheroid inclusions with A solution and B solution until the matrix gel-multicellular spheroid inclusions are completely covered, and then culturing for 40 minutes to obtain a cell trap array for organoid regeneration. The C solution includes thrombin, Ca 2+ and Mg 2+ , where the concentration of thrombin is 105 μg / ml, Ca 2+ and Mg 2+ The concentration is 1.2 mM.

[0148] Example 3

[0149] This embodiment provides a matrix gel for preparing a cell trap array for organoid regeneration, comprising liquid A and liquid B, wherein the mass ratio of the liquid A to the liquid B is 1:1, the liquid A comprises fibrinogen, tris hydroxymethylaminomethane hydrochloride (Tris HCl), and NaCl, and the liquid B comprises Matrigel, Ca 2+ Mg 2+ , thrombin, collagen.

[0150] In the solution A, the concentration of fibrinogen is 97 mg / ml, the concentration of Tris HCl is 51 mM, and the concentration of NaCl is 154 mM;

[0151] In the B solution, Ca 2+ and Mg 2+ The concentration of is 1.2 mM, the concentration of thrombin is 1.8 μg / ml, the collagen is type I collagen, and the concentration of collagen is 1.3 mg / ml.

[0152] The specific products of each reagent are:

[0153] High concentration low growth factor phenol red-free Matrigel, ABW, Cat#082723;

[0154] bovine fibrinogen, Solarbio, Cat#F8051;

[0155] Thrombin, Solarbio, Cat#T8021;

[0156] PBS buffer, Boster Biotechnology, Cat# PYG0021;

[0157] 1.5 M Tris-HCl buffer (pH 8.8), Solarbio, T1010;

[0158] Collgen Type I, CORNING, 354249;

[0159] The matrix gel provided in Example 3 was used to prepare a cell trap array for organoid regeneration. The preparation method of the cell trap array for organoid regeneration is as follows:

[0160] Providing an agarose gel layer having a microwell array, wherein the microwell array comprises a plurality of microwells;

[0161] adding cells into the microwells, and culturing the cells to obtain multicellular spheroids;

[0162] Adding solution A and solution B of matrix gel to the microwells containing the multicellular spheroids to cover the multicellular spheroids;

[0163] Dehydrating the matrix gel, i.e., applying negative pressure on one side of the agarose gel layer to absorb water from the matrix gel through the pores of the agarose gel, thereby dehydrating the matrix gel, so that the matrix gel wraps the multicellular spheroid to form a matrix gel-multicellular spheroid inclusion body;

[0164] separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body;

[0165] The matrix gel-multicellular spheroid inclusions are solidified, specifically comprising: adding 0.5% (w / v) genipin and PBS buffer mixture to the matrix gel-multicellular spheroid inclusions to obtain a cell trap array for organoid regeneration.

[0166] Example 4

[0167] This embodiment provides a matrix gel for preparing a cell trap array for organoid regeneration, comprising liquid A and liquid B, wherein the mass ratio of the liquid A to the liquid B is 1:1, the liquid A comprises fibrinogen, tris hydroxymethylaminomethane hydrochloride (Tris HCl), and NaCl, and the liquid B comprises Matrigel, Ca 2+ Mg 2+ , thrombin, collagen.

[0168] In the solution A, the concentration of fibrinogen is 100 mg / ml, the concentration of Tris HCl is 50 mM, and the concentration of NaCl is 150 mM;

[0169] In the B solution, Ca 2+ and Mg 2+ The concentration of is 1 mM, the concentration of thrombin is 2 μg / ml, the collagen is type I collagen, and the concentration of collagen is 1 mg / ml.

[0170] The specific products of each reagent are:

[0171] High concentration low growth factor phenol red-free Matrigel, ABW, Cat#082723;

[0172] bovine fibrinogen, Solarbio, Cat#F8051;

[0173] Thrombin, Solarbio, Cat#T8021;

[0174] 1.5 M Tris-HCl buffer (pH 8.8), Solarbio, T1010;

[0175] Collgen Type I, CORNING, 354249;

[0176] The matrix gel provided in Example 4 was used to prepare a cell trap array for organoid regeneration. The preparation method of the cell trap array for organoid regeneration is as follows:

[0177] (1) providing an agarose gel layer having a microwell array, wherein the microwell array comprises a plurality of microwells;

[0178] (2) adding cells into the microwells, and culturing the cells to obtain multicellular spheroids;

[0179] (3) adding solution A and solution B of matrix gel to the microwells containing the multicellular spheroids to cover the multicellular spheroids;

[0180] (4) dehydrating the matrix gel, i.e., applying negative pressure on one side of the agarose gel layer to absorb water from the matrix gel through the pores of the agarose gel, thereby dehydrating the matrix gel so that the matrix gel wraps the multicellular spheroid to form a matrix gel-multicellular spheroid inclusion body, specifically comprising:

[0181] 1) Place the side of the agarose gel layer without the microporous array on the filter paper of the negative pressure suction device, and continue to absorb water from the agarose gel through the filter paper. At the same time, continue to add liquid A and liquid B on top. After evenly dropping, start the negative pressure suction device.

[0182] 2) When liquid A and liquid B fill the entire array holes, stop the negative pressure suction device and cover the agarose gel layer with liquid A and liquid B again.

[0183] 3) Place the side of the agarose gel layer with the microwell array on the center side of the Confocal dish glass, cover it with the lid and place it in a cell culture incubator to solidify for 10 to 15 minutes.

[0184] (5) separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body;

[0185] (6) solidifying the matrix gel-multicellular spheroid inclusions, specifically comprising: covering the matrix gel-multicellular spheroid inclusions with matrix gel C solution, recovering the C solution after culturing for 10 minutes, and then covering the matrix gel-multicellular spheroid inclusions with A solution and B solution until the matrix gel-multicellular spheroid inclusions are completely covered, and culturing for another 40 minutes to obtain a cell trap array for organoid regeneration. The C solution includes thrombin, Ca 2+ and Mg 2+ , where the concentration of thrombin is 105 μg / ml, Ca 2+ and Mg 2+ The concentration is 1.2 mM.

[0186] The matrix gel provided by the present invention is used to prepare a cell trap array for organoid regeneration, wherein the matrix gel is wrapped around the organoid cells, and the matrix gel can form a temporary steady state of entanglement rather than cross-linking by dehydration, and temporarily increase the physical strength of the matrix gel to achieve a cell trap array of a specific shape. The matrix gel can be used to customize the components of the matrix gel used in the organoid culture process, and it can be ensured that the matrix gel around each organoid is uniform. The cell trap array for organoid regeneration can realize the customization of the mode and intensity of cell-cell interaction, and the cell-microenvironment interaction to constrain the spatial distribution of cell signal activation in the organoid. Compared with the traditional organoid regeneration method, the consistency and non-customization problems of the existing arrayed organoid regeneration are solved. The consistency and repeatability of the organoid regeneration process are enhanced, providing a basis for subsequent organoid scientific research and clinical diagnosis and treatment applications.

[0187] In the description of this specification, the description with reference to the terms "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0188] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A matrix gel for preparing a cell trap array for organoid regeneration, characterized in that: The invention comprises liquid A and liquid B, wherein the mass ratio of the liquid A to the liquid B is (0.9-1.1):1, the liquid A comprises fibrinogen, tris(hydroxymethyl)aminomethane hydrochloride) and NaCl, and the liquid B comprises matrix glue, Ca 2+ Mg 2+ , thrombin, collagen.

2. The matrix gel for preparing a cell trap array for organoid regeneration according to claim 1, characterized in that: In the liquid A, the concentration of the fibrinogen is 95 mg / ml to 105 mg / ml, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride is 45 mM to 55 mM, and the concentration of NaCl is 145 mM to 155 mM.

3. The matrix gel for preparing a cell trap array for organoid regeneration according to claim 1, characterized in that: In the B solution, Ca 2+ and Mg 2+ The concentration of is 0.5mM~1.5mM, the concentration of thrombin is 1.5μg / ml~2.5μg / ml, and the concentration of collagen is 0.5mg / ml~1.5mg / ml.

4. The matrix gel for preparing a cell trap array for organoid regeneration according to claim 1, characterized in that: The collagen is type I collagen.

5. A method for preparing a cell trap array for organoid regeneration, characterized in that: The following steps are involved: Providing an agarose gel layer having a microwell array, wherein the microwell array comprises a plurality of microwells; adding cells into the microwells, and culturing the cells to obtain multicellular spheroids; Adding the A solution and the B solution of the matrix gel for preparing the cell trap array for organoid regeneration according to any one of claims 1 to 4 into the microwell containing the multicellular spheroid to cover the multicellular spheroid; Dehydrating the matrix gel so that the matrix gel wraps the multicellular spheroid to form a matrix gel-multicellular spheroid inclusion body; separating the agarose gel layer and the matrix gel-multicellular spheroid inclusion body; The matrix gel-multicellular spheroid inclusions are solidified to obtain a cell trap array for organoid regeneration.

6. The method for preparing a cell trap array for organoid regeneration according to claim 5, characterized in that: The A liquid and the B liquid are added in a mass ratio of 1:(0.95-1.05).

7. The method for preparing a cell trap array for organoid regeneration according to claim 5, characterized in that: The matrix gel-multicellular spheroid inclusions are solidified, comprising: Covering the matrix gel-multicellular spheroid inclusions with a C solution of matrix gel for preparing a cell trap array for organoid regeneration, recovering the C solution after culturing for a first preset time, and then covering the matrix gel-multicellular spheroid inclusions with A solution and B solution until the matrix gel-multicellular spheroid inclusions are completely covered, and then culturing for a second preset time; The C solution includes thrombin, Ca 2+ and Mg 2+ , where the concentration of thrombin is 95μg / ml~105μg / ml, Ca 2+ and Mg 2+ The concentration is 0.5mM~1.5mM.

8. The method for preparing a cell trap array for organoid regeneration according to claim 5, characterized in that: The method provides an agarose gel layer provided with a micropore array, comprising: preparing an agarose gel layer; Prepare a mold, wherein the mold is provided with a protrusion array, wherein the protrusion array includes a plurality of protrusions; The protrusion array of the mold is pressed onto the agarose gel layer, and the mold is separated after standing to obtain an agarose gel layer with a micropore array.

9. The method for preparing a cell trap array for organoid regeneration according to claim 5, characterized in that: The microwell array is a microwell array with a preset structure customized according to the needs of the target organoid; The preset structure includes the size of a single micropore, the shape of a single micropore, the distance between micropores, the connection method between micropores, the shape of the connecting channel between micropores, the size of the connecting channel between micropores, and the spatial arrangement of micropores.

10. The method for preparing a cell trap array for organoid regeneration according to claim 5, characterized in that: The step of dehydrating the matrix glue comprises: Applying negative pressure on one side of the matrix gel to absorb water from the matrix gel to achieve dehydration of the matrix gel; and / or Negative pressure is applied to one side of the agarose gel layer to absorb water from the matrix gel through the pores of the agarose gel, thereby achieving dehydration of the matrix gel.

11. A cell trap array for organoid regeneration, characterized in that: The cell trap array for organoid regeneration is prepared by the preparation method of the cell trap array for organoid regeneration according to any one of claims 5 to 10.

12. An organoid, characterized in that The organoid is cultured by the cell well array for organoid regeneration as claimed in claim 11.

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