Cell trap array for standardization and controllable regeneration of organoid, preparation method of cell trap array and organoid
By preparing the first matrix with a growth array and performing pre-curing treatment of extracellular matrix, the problem of difficult control of intercellular communication network, mechanical force action and ECM signal distribution during organoid regeneration is solved, and the standardization and controllability of the organoid regeneration process is achieved.
Patent Information
- Application Number
- CN202411987278.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-05-09
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to simultaneously control the intercellular communication network, mechanical force action and spatial distribution of ECM signals during self-organization of organoids, resulting in phenotypic heterogeneity during organoid regeneration, affecting subsequent research and application.
By providing a first matrix with a growth array, cells are added to the growth module and cultured to obtain a multicellular spheroid. Then, extracellular matrix is added to the module and pre-cured to form an extracellular matrix-multicellular spheroid inclusion, and finally cured to prepare a cell well array for organoid regeneration.
The standardization, controllability, high-throughput and customization of the organoid regeneration process is achieved, and the problems of organoid regeneration consistency and uncustomization in traditional methods are solved, which enhances the repeatability of the regeneration process.
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Figure CN119955711A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological tissue engineering, and in particular to a cell trap array for organoid regeneration 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 method for preparing a cell trap array for standardized and controllable regeneration of organoids, wherein the prepared cell trap array is conducive to achieving standardized and controllable regeneration of organoids.
[0006] The technical problem to be solved by the present invention is also to provide a cell trap array for organoid regeneration, which is conducive to achieving high-throughput and customizable organoid culture in a uniform, stable, controllable and customizable manner.
[0007] In order to solve the above technical problems, the present invention provides a method for preparing a cell trap array for standardized and controllable regeneration of organoids, comprising the following steps:
[0008] Providing a first substrate having a growth array, wherein the growth array includes a plurality of growth modules capable of constraining cell movement;
[0009] adding cells to the growth module, wherein the cells are cultured to obtain multicellular spheroids;
[0010] Adding an extracellular matrix to the growth module containing the multicellular spheroid to cover the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix;
[0011] Pre-curing the extracellular matrix so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body;
[0012] separating the first matrix and the extracellular matrix-multicellular spheroid inclusions;
[0013] The extracellular matrix-multicellular spheroid inclusions are solidified to obtain a cell trap array for organoid regeneration.
[0014] In some embodiments, the growth module comprises one of microwells, microcolumns, and microbeads.
[0015] In some embodiments, providing a first substrate having a growth array includes:
[0016] preparing a first matrix;
[0017] Prepare a mold, wherein the mold is provided with a protrusion array, wherein the protrusion array includes a plurality of protrusions;
[0018] The protrusion array of the mold is pressed onto the first substrate, and the mold is separated after standing to obtain a first substrate with a micropore growth array.
[0019] In some embodiments, the growth array is a growth array with a preset structure customized according to the needs of the target organoid;
[0020] The preset structure includes the size of a single growth module, the shape of a single growth module, the distance between growth modules, the connection method between growth modules, the shape of the connection channel between growth modules, the size of the connection channel between growth modules, and the spatial arrangement of growth modules.
[0021] In some embodiments, the first matrix comprises a porous material, and the first matrix does not undergo a cross-linking reaction with the extracellular matrix.
[0022] In some embodiments, the first matrix comprises agarose gel.
[0023] In some embodiments, adding cells to the growth module comprises:
[0024] Customize preset regimes based on the needs of target organoids;
[0025] Cells are added to the growth module according to the preset schedule.
[0026] In some embodiments, the preset regime includes the type of cells added to a single growth module and the number of cells added to a single growth module.
[0027] In some embodiments, the cells include one or more of somatic cells, adult stem cells, and pluripotent stem cells.
[0028] In some embodiments, the cells are cultured to obtain multicellular spheroids, comprising:
[0029] The cells self-assemble within the growth modules to obtain multicellular spheroids.
[0030] In some embodiments, the extracellular matrix comprises a hydrogel;
[0031] The hydrogel includes collagen, Matrigel, fibrinogen and prothrombin.
[0032] In some embodiments, the extracellular matrix is prepared by the following method:
[0033] Add Tris HCl and NaCl solution to fibrinogen to obtain solution A;
[0034] Add Ca-containing 2+ Liquid, containing Mg 2+ Liquid B is then mixed with prothrombin and collagen to obtain liquid B;
[0035] The extracellular matrix was obtained by mixing solution A and solution B.
[0036] In some embodiments, the extracellular matrix is subjected to a pre-curing treatment, comprising:
[0037] The extracellular matrix includes hydrogel, and the extracellular matrix is dehydrated to allow the extracellular matrix to wrap the multicellular spheroid and form an extracellular matrix-multicellular spheroid inclusion body, while the extracellular matrix-multicellular spheroid inclusion body has a physical strength sufficient to be separated from the first matrix.
[0038] In some embodiments, the dehydrating the extracellular matrix comprises:
[0039] Applying negative pressure on one side of the extracellular matrix to absorb water from the extracellular matrix to achieve dehydration of the extracellular matrix; and / or
[0040] The first matrix is provided with pores. Negative pressure is applied to one side of the first matrix to absorb water from the extracellular matrix through the pores of the first matrix, thereby achieving dehydration of the extracellular matrix.
[0041] In some embodiments, the solidification treatment of the extracellular matrix-multicellular spheroid inclusions comprises:
[0042] The extracellular matrix includes hydrogel, and a cross-linking agent is added to the extracellular matrix-multicellular spheroid inclusion body to achieve solidification treatment.
[0043] In some embodiments, the solidification treatment of the extracellular matrix-multicellular spheroid inclusions further comprises:
[0044] During the solidification process, the organoid chip is added to the extracellular matrix-multicellular spheroid inclusion body.
[0045] In order to solve the above problems, 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.
[0046] And, an organoid, wherein the organoid is cultured by the cell well array for organoid regeneration.
[0047] The implementation of the present invention has the following beneficial effects:
[0048] The present invention proposes a method for preparing a cell trap array for standardized and controllable regeneration of organoids. The prepared cell trap array is conducive to the controllable and standardized regeneration of organoids, and is conducive to high-throughput pathological analysis. The growth module shape, spacing, connectivity, extracellular matrix and the types and numbers of cells contained therein of the cell trap array jointly determine the organoid regeneration process, and 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 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, and provides a basis for subsequent organoid scientific research and clinical diagnosis and treatment applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0049] Figure 1 A flow chart of a method for preparing a cell trap array for standardized and controllable regeneration of organoids provided in Example 1;
[0050] Figure 2 The organoids are derived from human airway basal cells and cultured using the cell well array for organoid regeneration prepared in Example 1;
[0051] Figure 3 This is a schematic diagram of the structure of the dehydration module in Example 1;
[0052] Figure 4 is a cross-sectional view of the dehydration module in Example 1;
[0053] Figure 5 It is a schematic diagram of the structure of the automated manufacturing system of the cell well array with human-machine interaction in Example 1;
[0054] Figure 6 The organoids are derived from mixed human airway basal cells / alveolar type II cells cultured using the cell well array for organoid regeneration prepared in Example 2;
[0055] Figure 7 The organoids are derived from human colon cancer cells and cultured using the cell well array for organoid regeneration prepared in Example 3;
[0056] Figure 8 The small intestinal organoids are derived from induced pluripotent stem cells (iPSCs) and cultured using the cell well array for organoid regeneration prepared in Example 4. DETAILED DESCRIPTION
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] To solve the above problems, the present invention provides a method for preparing a cell trap array for standardized and controllable regeneration of organoids, comprising the following steps:
[0065] (1) providing a first substrate having a growth array, wherein the growth array includes a plurality of growth modules capable of constraining cell movement;
[0066] In some embodiments, the growth module includes one of micropores, microcolumns, and microbeads. Micropores refer to holes with diameters usually between a few microns and hundreds of microns, which can promote the exchange of substances and the growth of cells. Microporous structures are commonly used on the surface of biomaterials and can provide space for cell attachment and expansion, especially in tissue engineering, which helps to build a 3D cell culture environment. Micropillars refer to tiny protrusions with a columnar structure on the surface, which are usually used to mimic the cell growth environment in an organism. The microcolumn structure can provide mechanical stimulation or guide the directional growth of cells. By designing different microcolumn sizes and spacings, the arrangement, morphology, and migration of cells can be regulated. Microbeads are usually spherical particles with diameters between a few microns and hundreds of microns, which are commonly used in drug delivery, cell culture, or separation technology. In cell culture, microbeads can be used as scaffolds to help cells attach and promote their growth.
[0067] Preferably, providing a first substrate provided with a growth array comprises:
[0068] preparing a first matrix;
[0069] Prepare a mold, wherein the mold is provided with a protrusion array, wherein the protrusion array includes a plurality of protrusions;
[0070] The protrusion array of the mold is pressed onto the first substrate, and the mold is separated after standing to obtain a first substrate with a micropore growth array.
[0071] In some embodiments, the growth array is a growth array with a preset structure customized according to the needs of the target organoid; the growth array is a growth array with a preset structure customized according to the needs of the target organoid; the preset structure includes the size of a single growth module, the shape of a single growth module, the distance between growth modules, the connection method between growth modules, the shape of the connection channel between growth modules, the size of the connection channel between growth modules, and the spatial arrangement of growth modules.
[0072] Preferably, the size of the single growth module includes the hole depth, opening size, and holding volume of the single growth module; the shape of the single growth module includes a cone-like, a cylinder-like, a cuboid, and a hemisphere; the distance between the growth modules is the distance between adjacent growth modules, which can be equidistant or unequal, and the distance between the growth modules varies according to the needs of the target organoid. The connection between the growth modules can be that all adjacent growth modules are independent of each other, or that all adjacent growth modules are connected, or that some adjacent growth modules are independent of each other and some adjacent growth modules are connected, and the connection between the growth modules varies according to the needs of the target organoid. The spatial arrangement of the growth modules is the spatial arrangement pattern formed by the combination of all growth modules, and the spatial arrangement of the growth modules varies according to the needs of the target organoid.
[0073] In some embodiments, the size, shape, spacing, and spatial distribution of the growth array on the mold are designed according to experimental needs, and a three-dimensional stereogram of the mold is drawn using a 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. In addition, it is not limited to the growth array, and other shapes that can provide physical boundaries and constrain cell movement can also be used.
[0074] It should be noted that the preset structure includes the size of a single growth module, the shape of a single growth module, the distance between growth modules, the connection between growth modules, and the spatial arrangement of growth modules. The structure of the above growth modules determines the number, type, source and interaction ability of the starting cells and the physical boundaries of organoid growth. A growth 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 extracellular matrix 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.
[0075] In addition, the embodiments of providing the first matrix with a growth 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 growth array by customizing the growth 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.
[0076] Furthermore, the first matrix is an important carrier for culturing multicellular spheroids. In some embodiments, the first matrix includes a porous material, and the first matrix does not undergo a cross-linking reaction with the extracellular matrix.
[0077] Preferably, the first matrix includes agarose gel. Due to its inert chemical properties, agarose gel cannot form a covalent connection with all current biohydrogels, and biohydrogels are more commonly used extracellular matrices. In the subsequent preparation method, biohydrogels used to make organoids, including collagen, Matrigel, fibrinogen-thrombin, etc., can form an entangled state by dehydration, and can provide a gel with physical strength beyond cross-linking in a short time. The presence of pores in agarose gel allows it to be dehydrated without direct contact with the biohydrogel.
[0078] In other embodiments, a first matrix body is provided, pores are set on the first matrix body, and the first matrix and the extracellular matrix do not undergo cross-linking reaction. In these embodiments, the first matrix is not required to be made of porous material, but the pores are artificially increased, so that the extracellular matrix (mainly hydrogel) can be dehydrated without direct contact with it.
[0079] (2) adding cells to the growth module, and culturing the cells to obtain multicellular spheroids;
[0080] In some embodiments, adding cells to the growth module comprises:
[0081] Customize preset regimes based on the needs of target organoids;
[0082] Cells are added to the growth module according to the preset schedule.
[0083] In some embodiments, the cells are cultured to obtain multicellular spheroids, comprising:
[0084] The cells self-assemble within the growth modules to obtain multicellular spheroids.
[0085] Preferably, the preset system includes the type of cells added to a single growth module, the number of cells added to a single growth module; 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.
[0086] 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.
[0087] (3) adding an extracellular matrix to the growth module containing the multicellular spheroid and covering the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix;
[0088] After the cells self-assemble in the growth module, an extracellular matrix is added to the growth module. The extracellular matrix is a preset extracellular matrix customized according to the needs of the target organoid. This approach can freely control the composition of the extracellular matrix (ECM), and the components of the extracellular matrix around each organoid can be standardized and controllable.
[0089] In some embodiments, the extracellular matrix comprises a hydrogel; the hydrogel comprises collagen, Matrigel, fibrinogen, and prothrombin.
[0090] Preferably, the first matrix includes agarose gel, and hydrogel is added to the multicellular spheroid, and then the hydrogel is dehydrated to form a temporary steady state of entanglement rather than cross-linking. Since the agarose gel cannot be cross-linked with the hydrogel, the agarose gel can be easily peeled off from the hydrogel, and the self-assembled multicellular spheroid will remain in the hydrogel. Subsequently, the self-assembled multicellular spheroid will form an organoid through morphogenesis. The cell signals related to each cell in the organoid are constrained by cell-to-cell interactions in a specific spatiotemporal pattern and directly affect the subsequent morphogenesis process, so this technology is named cell trap array.
[0091] More preferably, the extracellular matrix is prepared by the following method: adding Tris HCl and NaCl solution to fibrinogen to obtain solution A;
[0092] Add Ca-containing 2+ Liquid, containing Mg 2+ Liquid B is then mixed with prothrombin and collagen to obtain liquid B;
[0093] The extracellular matrix was obtained by mixing solution A and solution B.
[0094] The extracellular matrix forms an entangled state by dehydration, and the method of temporarily increasing the physical strength of the hydrogel can freely determine the composition of the extracellular matrix (ECM) and ensure that the ECM around each organoid is uniform.
[0095] (4) pre-curing the extracellular matrix so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body;
[0096] In some embodiments, the extracellular matrix is subjected to a pre-curing treatment, comprising:
[0097] The extracellular matrix includes hydrogel, and the extracellular matrix is dehydrated to allow the extracellular matrix to wrap the multicellular spheroid and form an extracellular matrix-multicellular spheroid inclusion body, while the extracellular matrix-multicellular spheroid inclusion body has a physical strength sufficient to be separated from the first matrix.
[0098] It should be noted that the biological hydrogels used to make organoids, including collagen, Matrigel matrix glue, fibrinogen-thrombin, etc., can form an entangled state through dehydration, which can provide physical strength beyond cross-linking in a short time.
[0099] Furthermore, in some embodiments, the dehydration treatment of the extracellular matrix comprises:
[0100] Applying negative pressure on one side of the extracellular matrix to absorb water from the extracellular matrix to achieve dehydration of the extracellular matrix; and / or
[0101] The first matrix is provided with pores. Negative pressure is applied to one side of the first matrix to absorb water from the extracellular matrix through the pores of the first matrix, thereby achieving dehydration of the extracellular matrix.
[0102] In some embodiments, the first matrix includes agarose gel. Due to its inert chemical properties, agarose gel cannot form a covalent bond with the hydrogel, and the pores of agarose gel allow it to dehydrate the hydrogel without directly contacting the hydrogel.
[0103] In other embodiments, a first matrix body is provided, pores are set on the first matrix body, and the first matrix and the extracellular matrix do not undergo cross-linking reaction. In these embodiments, the first matrix is not required to be made of porous material, but the pores are artificially increased, so that the hydrogel can be dehydrated later without direct contact with it.
[0104] In the embodiment of pre-curing by dehydration, the method of forming an entangled state by dehydration and temporarily increasing the physical strength of the hydrogel can freely determine the composition of the extracellular matrix (ECM) and ensure that the ECM around each organoid is uniform.
[0105] (5) separating the first matrix and the extracellular matrix-multicellular spheroid inclusions;
[0106] In some embodiments, the first matrix cannot cross-link with the extracellular matrix. Therefore, the first matrix can be easily peeled off from the hydrogel, and the self-assembled multicellular spheroids will remain in the hydrogel. The self-assembled multicellular spheroids will then form organoids through morphogenesis. The cell signals associated with each cell in the organoid are constrained in a specific spatiotemporal pattern by cell-to-cell interactions and directly affect the subsequent morphogenesis process. Therefore, this technology is named cell trap array.
[0107] (6) The extracellular matrix-multicellular spheroid inclusions are solidified to obtain a cell trap array for organoid regeneration.
[0108] In some embodiments, the solidification treatment of the extracellular matrix-multicellular spheroid inclusions comprises:
[0109] The extracellular matrix includes hydrogel, and a cross-linking agent is added to the extracellular matrix-multicellular spheroid inclusion body to achieve solidification treatment.
[0110] The solidification treatment of the extracellular matrix-multicellular spheroid inclusions further comprises:
[0111] During the solidification process, the organoid chip is added to the extracellular matrix-multicellular spheroid inclusion body.
[0112] 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.
[0113] In some embodiments, the present invention is prepared by a human-machine interactive cell trap array automated manufacturing system.
[0114] like Figure 5 As shown, the human-machine interactive cell trap array automated manufacturing system includes a first matrix, 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 3-4The first matrix 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 growth module in the cell inoculation area to obtain the growth module 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 growth module position image fed back by the industrial camera to implant cells into the growth module corresponding to the growth module position image. The dehydration module 2 plays the role of supporting and fixing the first matrix 1. One side of the dehydration module 2 is used to fix the first matrix 1, and the other side is provided with a through hole for connecting with the dehydration equipment to realize the dehydration of the first matrix 1, and indirectly realize the dehydration treatment of the extracellular matrix.
[0115] Specifically, a first matrix 1 is provided with a growth array, wherein the growth array includes a plurality of growth modules capable of constraining cell movement;
[0116] Fixing the first matrix 1 in the dehydration module 2;
[0117] Adding cells into the growth module by means of a pipette of the robot arm 3, and culturing the cells to obtain multicellular spheroids;
[0118] Adding an extracellular matrix to the growth module containing the multicellular spheroid to cover the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix;
[0119] Dehydrating the extracellular matrix using a dehydration module 2 to complete pre-curing, so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body;
[0120] separating the first matrix and the extracellular matrix-multicellular spheroid inclusions;
[0121] A cross-linking agent is added to the extracellular matrix-multicellular spheroid inclusion body to achieve a solidification treatment, thereby obtaining a cell trap array for organoid regeneration.
[0122] 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.
[0123] And, an organoid, wherein the organoid is cultured by the cell well array for organoid regeneration.
[0124] 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.
[0125] The present invention is further described below with specific embodiments:
[0126] Example 1
[0127] This embodiment provides a method for preparing a cell trap array for standardized and controllable regeneration of organoids, comprising the following steps:
[0128] Providing a first substrate having a microporous growth array, wherein the microporous growth array comprises a plurality of microporous (growth modules), and the first substrate comprises agarose gel;
[0129] Cells are added to the microwells, the cells including human adult stem cells, tumor cells, induced stem cells (iPSCs), embryonic stem cells, pluripotent stem cells derived from different germ layers differentiated from the above cells, and multi-source cells obtained by mixing these cells. The cells are cultured to obtain multicellular spheroids; specifically, the first matrix is placed 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.
[0130] Adding an extracellular matrix into the microwell containing the multicellular spheroid to cover the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix and comprises a hydrogel;
[0131] The extracellular matrix is pre-cured so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body, and the pre-curing treatment includes dehydrating the extracellular matrix so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body, and the extracellular matrix-multicellular spheroid inclusion body has a physical strength sufficient to be separated from the first matrix. Specifically, the method comprises: Figures 3-4 The dehydration module shown performs dehydration processing.
[0132] separating the first matrix and the extracellular matrix-multicellular spheroid inclusions;
[0133] The extracellular matrix-multicellular spheroid inclusions are solidified, and the solidification treatment includes adding a cross-linking agent to the extracellular matrix-multicellular spheroid inclusions to achieve solidification treatment to obtain a cell trap array for organoid regeneration.
[0134] Figure 1 A flow chart of a method for preparing a cell trap array for standardized and controllable regeneration of organoids provided in Example 1, wherein Part A represents adding cells to a first matrix provided with a microporous growth array; Part B represents culturing cells to obtain multicellular spheroids, adding extracellular matrix to the micropores containing the multicellular spheroids and covering the multicellular spheroids; Part C represents separating the ECM from the microarray by demolding, leaving the cell spheroids in the ECM, and finally obtaining a cell trap array for organoid regeneration.
[0135] Figure 2 The organoids are derived from human airway basal cells and cultured using the cell well array for organoid regeneration prepared in Example 1.
[0136] Example 2
[0137] This embodiment provides a method for preparing a cell trap array for standardized and controllable regeneration of organoids, comprising the following steps:
[0138] A first matrix provided with a microporous growth array is provided, wherein the microporous growth array includes a plurality of micropores capable of constraining cell movement, and a method for preparing the first matrix provided with the microporous growth array includes: preparing the first matrix; preparing a mold, wherein the mold is provided with a protrusion array, wherein the protrusion array includes a plurality of protrusions; pressing the protrusion array of the mold onto the first matrix, separating the mold after standing, and obtaining the first matrix provided with the microporous growth array, wherein the first matrix includes agarose gel;
[0139] Adding cells into the microwells, the cells comprising mixed human airway basal cells / alveolar type II cells, and the cells are cultured to obtain multicellular spheroids;
[0140] Adding an extracellular matrix into the microwell containing the multicellular spheroid to cover the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix and comprises a hydrogel;
[0141] The extracellular matrix is pre-cured so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body. The pre-curing treatment includes dehydrating the extracellular matrix so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body, and at the same time, the extracellular matrix-multicellular spheroid inclusion body has a physical strength sufficient to be separated from the first matrix.
[0142] separating the first matrix and the extracellular matrix-multicellular spheroid inclusions;
[0143] The extracellular matrix-multicellular spheroid inclusions are solidified, and the solidification treatment includes adding a cross-linking agent to the extracellular matrix-multicellular spheroid inclusions to achieve solidification treatment to obtain a cell trap array for organoid regeneration.
[0144] Figure 6 The organoids are derived from mixed human airway basal cells / alveolar type II cells and cultured using the cell well array for organoid regeneration prepared in Example 2.
[0145] Example 3
[0146] This embodiment provides a method for preparing a cell trap array for standardized and controllable regeneration of organoids, comprising the following steps:
[0147] Providing a first matrix with a growth array, the growth array comprising a plurality of growth modules; the first matrix comprising agarose gel;
[0148] Adding cells to the growth module, wherein the cells include human colon cancer cells, and the cells are cultured to obtain multicellular spheroids;
[0149] Adding an extracellular matrix to the growth module containing the multicellular spheroid and covering the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix and comprises a hydrogel;
[0150] The extracellular matrix is pre-cured so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body. The pre-curing treatment includes dehydrating the extracellular matrix so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body, and at the same time, the extracellular matrix-multicellular spheroid inclusion body has a physical strength sufficient to be separated from the first matrix.
[0151] separating the first matrix and the extracellular matrix-multicellular spheroid inclusions;
[0152] The extracellular matrix-multicellular spheroid inclusions are solidified, and the solidification treatment includes adding a cross-linking agent to the extracellular matrix-multicellular spheroid inclusions to achieve solidification treatment to obtain a cell trap array for organoid regeneration.
[0153] Figure 7 The organoids are derived from human colon cancer cells and cultured using the cell well array for organoid regeneration prepared in Example 3.
[0154] Example 4
[0155] This embodiment provides a method for preparing a cell trap array for standardized and controllable regeneration of organoids, comprising the following steps:
[0156] Providing a first matrix with a growth array, the growth array comprising a plurality of growth modules; the first matrix comprising agarose gel;
[0157] Adding cells to the growth module, wherein the cells include induced pluripotent stem cells (iPSCs) and Hindgut endoderm cells differentiated therefrom, and culturing the cells to obtain multicellular spheroids;
[0158] Adding an extracellular matrix to the growth module containing the multicellular spheroid and covering the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix, and the extracellular matrix includes a hydrogel, wherein Tris HCl and NaCl solution are added to fibrinogen to obtain solution A; adding Ca-containing solution to Matrigel 2+ Liquid, containing Mg 2+ Liquid A is then mixed with prothrombin and collagen to obtain liquid B; liquid A and liquid B are mixed to obtain extracellular matrix.
[0159] The extracellular matrix is pre-cured so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body. The pre-curing treatment includes dehydrating the extracellular matrix so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body, and at the same time, the extracellular matrix-multicellular spheroid inclusion body has a physical strength sufficient to be separated from the first matrix.
[0160] separating the first matrix and the extracellular matrix-multicellular spheroid inclusions;
[0161] The extracellular matrix-multicellular spheroid inclusions are solidified, and the solidification treatment includes adding a cross-linking agent to the extracellular matrix-multicellular spheroid inclusions to achieve solidification treatment to obtain a cell trap array for organoid regeneration.
[0162] Figure 8 The small intestinal organoids are derived from induced pluripotent stem cells (iPSCs) and cultured using the cell well array for organoid regeneration prepared in Example 4.
[0163] The present invention proposes a method for preparing a cell trap array for standardized and controllable regeneration of organoids, which can customize the mode and intensity of cell-cell interactions and cell-microenvironment interactions 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.
[0164] 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.
[0165] 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 method for preparing a cell trap array for standardized and controllable regeneration of organoids, characterized in that: The following steps are involved: Providing a first substrate having a growth array, wherein the growth array includes a plurality of growth modules capable of constraining cell movement; adding cells to the growth module, wherein the cells are cultured to obtain multicellular spheroids; Adding an extracellular matrix to the growth module containing the multicellular spheroid to cover the multicellular spheroid, wherein the extracellular matrix does not chemically react with the first matrix; Pre-curing the extracellular matrix so that the extracellular matrix wraps around the multicellular spheroid to form an extracellular matrix-multicellular spheroid inclusion body; separating the first matrix and the extracellular matrix-multicellular spheroid inclusions; The extracellular matrix-multicellular spheroid inclusions are solidified to obtain a cell trap array for organoid regeneration.
2. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The growth module includes one of micropores, microcolumns and microbeads.
3. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The method of providing a first substrate having a growth array comprises: preparing a first matrix; 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 first substrate, and the mold is separated after standing to obtain a first substrate with a micropore growth array.
4. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The growth array is a growth array with a preset structure customized according to the needs of the target organoid; The preset structure includes the size of a single growth module, the shape of a single growth module, the distance between growth modules, the connection method between growth modules, the shape of the connection channel between growth modules, the size of the connection channel between growth modules, and the spatial arrangement of growth modules.
5. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The first matrix includes a porous material, and no cross-linking reaction occurs between the first matrix and the extracellular matrix.
6. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 5, characterized in that: The first matrix includes agarose gel.
7. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: Adding cells to the growth module comprises: Customize preset regimes based on the needs of target organoids; Cells are added to the growth module according to the preset schedule.
8. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 7, characterized in that: The preset system includes the type of cells added to a single growth module and the number of cells added to a single growth module.
9. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The cells include one or more of somatic cells, adult stem cells, and pluripotent stem cells.
10. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The cells are cultured to obtain a multicellular spheroid, comprising: The cells self-assemble within the growth modules to obtain multicellular spheroids.
11. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The extracellular matrix includes a hydrogel; The hydrogel includes collagen, Matrigel, fibrinogen and prothrombin.
12. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The extracellular matrix is prepared by the following method: Add Tris HCl and NaCl solution to fibrinogen to obtain solution A; Add Ca-containing 2+ Liquid, containing Mg 2+ Liquid B is then mixed with prothrombin and collagen to obtain liquid B; The extracellular matrix was obtained by mixing solution A and solution B.
13. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The extracellular matrix is subjected to a pre-curing treatment, comprising: The extracellular matrix includes hydrogel, and the extracellular matrix is dehydrated to allow the extracellular matrix to wrap the multicellular spheroid and form an extracellular matrix-multicellular spheroid inclusion body, while the extracellular matrix-multicellular spheroid inclusion body has a physical strength sufficient to be separated from the first matrix.
14. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 13, characterized in that: The dehydration treatment of the extracellular matrix comprises: Applying negative pressure on one side of the extracellular matrix to absorb water from the extracellular matrix to achieve dehydration of the extracellular matrix; and / or The first matrix is provided with pores. Negative pressure is applied to one side of the first matrix to absorb water from the extracellular matrix through the pores of the first matrix, thereby achieving dehydration of the extracellular matrix.
15. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The solidification treatment of the extracellular matrix-multicellular spheroid inclusions comprises: The extracellular matrix includes hydrogel, and a cross-linking agent is added to the extracellular matrix-multicellular spheroid inclusion body to achieve solidification treatment.
16. The method for preparing a cell trap array for standardized and controllable regeneration of organoids according to claim 1, characterized in that: The solidification treatment of the extracellular matrix-multicellular spheroid inclusions further comprises: During the solidification process, the organoid chip is added to the extracellular matrix-multicellular spheroid inclusion body.
17. A cell trap array for standardized and controllable regeneration of organoids, characterized in that: The cell trap array is prepared by the method for preparing a cell trap array for standardized and controllable regeneration of organoids as described in any one of claims 1 to 16.
18. An organoid, characterized in that The organoid is obtained by culturing the cell well array for standardized and controllable regeneration of organoids as described in claim 17.