CN118256348A

CN118256348A - Organoid co-culture chip and preparation method and application thereof

Info

Publication number: CN118256348A
Application number: CN202211642915.3A
Authority: CN
Inventors: 瓮喆; 唐冲; 穆伟华; 王洪琦; 吴俊龙; 赵政
Original assignee: Huada Qinglan Biotechnology Wuxi Co ltd
Current assignee: Huada Qinglan Biotechnology Wuxi Co ltd
Filing date: 2022-12-20
Publication date: 2024-06-28

Abstract

The invention relates to an organoid co-culture chip, which consists of a first fluid channel layer, a cell culture layer, a porous structure layer and a second fluid channel layer from top to bottom, wherein the first fluid channel layer is provided with a first fluid inlet, a first culture area and a first fluid outlet, the cell culture layer is provided with a first micropore for accommodating organoid cells, the porous structure layer is provided with a second micropore through which organoid cells cannot pass, and the second fluid channel layer is provided with a second fluid inlet, a second culture area and a second fluid outlet. The organoid co-culture chip can be used for simulating the co-culture of various organoid cells from various tissue sources.

Description

Organoid co-culture chip and preparation method and application thereof

Technical Field

The invention relates to the technical field of cell culture, in particular to an organoid co-culture chip and a preparation method and application thereof.

Background

For newly developed drugs, links such as cell tests, animal experiments, human body tests and the like are generally required. However, with the ethical concerns of animals in various countries, many animal experiments are gradually moving out of the approval allowable range of new drug tests. Therefore, the preparation of the cell organoids of animal experiment substitutes in vitro is used for simulating the metabolic process of the drugs in vivo, and the dynamic and pharmacodynamic evaluation is better, so that the urgent need of the current biopharmaceutical industry is met.

However, in the existing cell organoid culture method, two types of cells are usually inoculated on adjacent surfaces, specific cell tissue types are simulated through two-dimensional adherence culture, or a three-dimensional cell sphere-based simulated organoid is not adopted, no matter what cell culture method is adopted, a complex three-dimensional tissue environment in a body cannot be well simulated, and three-dimensional cell sphere-based simulated organoid co-culture cannot be well realized.

Therefore, there is a need in the art for a new organoid co-culture chip, and methods of making and using the same to address the above-mentioned problems.

Disclosure of Invention

The invention relates to an organoid co-culture chip, a preparation method and application thereof, and aims to solve the problem that the existing organoid co-culture effect is not ideal.

An aspect of the present invention provides an organoid co-culture chip, characterized in that the chip comprises a first fluid channel layer, a cell culture layer, a porous structure layer and a second fluid channel layer from top to bottom; wherein,

The first fluid channel layer has a first culture area on a lower surface thereof and a first fluid inlet and a first fluid outlet communicating with the first culture area, respectively;

The cell culture layer has a plurality of first microwells, the first microwells being through-holes and capable of accommodating cultured organoid cells;

The porous structure layer has a plurality of second micropores, the second micropores being through-holes having a size smaller than the cultured organoid cells such that the cultured organoid cells cannot pass through the second micropores;

the second fluid channel layer has a second culture area on an upper surface thereof and a second fluid inlet and a second fluid outlet in communication with the second culture area, respectively.

In some embodiments, the inner sidewall surface of the first microporous structure has a hydrophobic modification.

In some embodiments, the upper surface of the porous structure layer has an adhesion protein modification.

In some embodiments, the first microwells have a maximum cross-sectional dimension of 100 to 800 microns.

In some embodiments, the second microwells have a maximum cross-sectional dimension of 5 to 20 microns.

In some embodiments, the first fluid channel layer, the second fluid channel layer, the cell culture layer, and the porous structure layer are made of dimethylsilane, polymethyl methacrylate, or glass.

In some embodiments, the first fluid channel layer, the second fluid channel layer, the cell culture layer, and the porous structure layer are encapsulated by plasma bonding and/or adhesive material bonding between the layers.

In another aspect, the present invention provides a method for preparing any one of the organoid co-culture chips described above, comprising the steps of:

i) Preparing a first fluid channel layer, a second fluid channel layer, a cell culture layer and a porous structure layer respectively;

performing hydrophobic modification on the inner side wall of the first microporous structure of the cell culture layer to form a hydrophobic layer for promoting 3D cell generation;

Performing adhesion modification on the upper surface of the porous structure layer to form an adhesion layer for promoting cell adhesion;

packaging the modified cell culture layer and the porous structure layer by adopting a mode of bonding by plasma and/or bonding materials to obtain an intermediate layer;

ii) packaging the first fluid channel layer, the second fluid channel layer, the cell culture layer and the porous structure layer by adopting a mode of plasma bonding and/or bonding of bonding materials, so as to obtain the chip.

In some embodiments, step i) further comprises hydrophobically modifying the inner sidewall surface of the first microwell of the cell culture layer.

In some embodiments, step i) further comprises performing an adhesion protein modification to the upper surface of the porous structure layer.

Another aspect of the invention provides a method of organoid co-culture comprising the steps of:

S1: inoculating a first organoid cell into a first culture area of a first fluid channel layer of any of the organoid co-culture chips, allowing the first organoid cell to fall into a first microwell of the cell culture layer, and adhering to the upper surface of the porous structure layer at the bottom of the first microwell for growth;

S2: and inoculating the second organoid cells into the first culture area of the first fluid channel layer of the organoid co-culture chip, and enabling the second organoid cells to fall into the first micropores of the cell culture layer to grow.

In some embodiments, the organoid co-culture method further comprises step S3: injecting a first medium suitable for culturing the first organoid into the second culture zone and injecting a second medium suitable for culturing the second organoid into the first culture zone.

In some embodiments, step S1 comprises: after the first organoid cells fall into the first micropores of the cell culture layer, standing for a second preset time to enable the first organoid cells to adhere to the upper surface of the porous structure layer for growth; preferably, the second preset time is at least 4 hours.

In some embodiments, step S1 further comprises: after inoculating the first organoid cells into the first culture zone of the first fluid channel layer, allowing the first organoid cells to stand for a first preset time to fall into the first microwells of the cell culture layer, and then rinsing the excess first organoid cells; preferably, the first preset time is at least 30 minutes.

In some embodiments, step S2 comprises: standing for a fourth preset time after the second organoid cells fall into the first microwells of the cell culture layer to allow the second organoid cells to grow; preferably, the fourth preset time is at least 4 hours.

In some embodiments, step S2 further comprises: after inoculating the second organoid cells into the first culture area of the first fluid channel layer, allowing the second organoid cells to stand for a third preset time to fall into the first microwells of the cell culture layer, and then rinsing the excess second organoid cells; preferably, the third preset time is at least 30 minutes.

In some embodiments, the first organoid cell is a cell capable of growing adherently on the upper surface of the porous structure layer or capable of growing as a 3D cell sphere in the first microwell.

In some embodiments, the first organoid cell is an epithelial cell, an endothelial cell, or a fibroblast.

In some embodiments, the second organoid cell is a cell capable of growing into a 3D cell sphere in the first microwell, preferably a parenchymal cell, a stem cell, or a tumor cell.

In another aspect, the invention provides the use of any of the organoid co-culture chips described above in organoid co-culture.

In addition, the organoid co-culture chip has simple structure and low preparation cost, can be used for simulating the co-culture of multiple organoid cells with multiple tissue sources, reproduces the fine structure of complex tissues, can observe the change condition of at least two organoid cells in the chip in real time, simplifies the related experimental process, and is beneficial to large-scale production.

Drawings

The invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic diagram of the structure of layers of an organoid co-culture chip according to an embodiment of the invention.

FIG. 2 is a schematic cross-sectional view of an organoid co-culture chip according to an embodiment of the invention.

Reference numerals:

110: a first fluid channel layer; 111: a first fluid inlet; 112: a first culture region; 113: a first fluid outlet;

120: a cell culture layer; 121: a first microwell;

130: a porous structural layer; 131: a second microwell;

140: a second fluid channel layer; 141: a second fluid inlet; 142: a second culture region; 143: and a second fluid outlet.

Detailed Description

Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are merely for explaining the technical principles of the present invention, and are not intended to limit the scope of the present invention. For example, although described in one embodiment in connection with myocardial fibroblasts and neural stem cells, the teachings of the present invention are not so limited, and the chip may obviously be applied to co-culturing other cells, such as gastric mucosal cells and adenoid cystic carcinoma cells, or 3D cell spheres, without departing from the spirit and scope of the present invention.

In the present invention, unless explicitly specified and limited otherwise, the terms "connected," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; may be a mechanical connection or may be a communication between the interiors of two members. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art in a specific case.

In the present invention, the terms "bottom," "top," "left," "right," "upper," "lower," "center," "inner," "outer," and the like refer to an orientation or positional relationship based on that shown in the drawings, or that is commonly put in place when the device or element is used, merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be configured and operated in a specific orientation, and therefore should not be construed as limiting the present invention. In some embodiments, the orientation or positional relationship indicated by the terms "bottom," "top," "upper," "lower," are based on the orientation shown in fig. 2.

Furthermore, the terms "first," "second," "third," "fourth," "fifth," and the like are used merely for distinguishing between descriptions and not for indicating or implying a relative importance.

In the present invention, the terms "and/or", describing the association relationship of the associated objects, means that there may be three relationships, for example, a and/or B, may mean: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the present invention, the term "dimension" generally refers to the length, diameter, or width of the geometric shape or geometrically shaped element being described. The term "cross-sectional dimension" generally refers to a measure of the longest distance or maximum distance between two points on the edge of a cross-section of a space, object or element having a circular or non-circular cross-section. It should be understood that the "cross-sectional dimension" of a circular cross-section is the "diameter" of the circular cross-section. For circular cross-sections, the terms "cross-sectional dimension" and "diameter" may be used interchangeably.

The organoid co-culture chip of the invention may be used to co-culture a variety of organoids, for example, two or more organoids. The plurality of organoids may include a plurality of 3D cytoballs, or include 3D cytoballs and 2D cultured cells (i.e., adherent cultured cells). In some embodiments, the organoid co-culture chip of the invention can be used to culture two different 3D cell spheres. In some embodiments, the organoid co-culture chip of the invention can be used to culture an adherent culture cell and a 3D cell pellet. In the present invention, the terms "chip" and "organoid co-culture chip" are used interchangeably.

The term "organoid" as used herein refers to a cell structure that is artificially cultured, formed from the same kind of cells, and has a structure and function similar to that of an organ or tissue. Cells that make up the organoid are able to gradually differentiate, mimicking the structure and/or function of a tissue or organ. Organoids are typically three-dimensional (3D) cell aggregates, but may also be organoids formed from cells that are cultured in an adherent culture (i.e., 2D culture), such as cells that are cultured in an adherent culture on a porous surface, which cells can form a dense cellular structure, as well as mimic a tissue or organ.

In the present invention, "cell mass", "cell pellet", "3D cell pellet", "three-dimensional cell pellet" are used interchangeably.

The term "seeded cells" as used herein refers to cells that are injected into the organoid co-culture chip of the invention to form organoids. The seeded cells may be single cells, which are contained in a cell suspension, and may form 3D cell pellets or adherent cultured cells (i.e., 2D organoids) in the organoid co-culture chip of the invention. The seeding cells may also be 3D cell pellets, which may be injected as 3D cell pellets into the organoid co-culture chip of the invention and continue to grow as 3D cell pellets.

The invention provides an organoid co-culture chip, a preparation method and application thereof, wherein a cell culture layer is arranged between a first fluid channel layer and a second fluid channel layer to form a co-culture system, which can well simulate a complex three-dimensional tissue environment in a body and realize co-culture of cells of at least two organoids.

Organoid co-culture chip

As shown in fig. 1 and 2, the chip of the present invention includes a first fluid channel layer 110, a cell culture layer 120, a porous structure layer 130, and a second fluid channel layer 140, which are four-layered structures. The first fluid channel layer 110, the cell culture layer 120, the porous structure layer 130, and the second fluid channel layer 140 are sequentially disposed from top to bottom (i.e., in the top-to-bottom direction of fig. 2). To clearly show the structure of the first fluid channel layer 110, the cell culture layer 120, the porous structure layer 130 and the second fluid channel layer 140, the first fluid channel layer 110, the cell culture layer 120, the porous structure layer 130 and the second fluid channel layer 140 are sequentially shown in a staggered manner in fig. 1 so as to avoid obscuring a part or all of the structure of any one of the layers.

The first fluid channel layer 110, the second fluid channel layer 140, the cell culture layer 120 and the porous structure layer 130 are made of biocompatible materials, preferably transparent materials such as Polydimethylsilane (PDMS), polymethyl methacrylate (PMMA) or glass. The materials selected for the first fluid channel layer 110, the second fluid channel layer 140, the cell culture layer 120 and the porous structure layer 130 may be the same or different, and the present invention is not limited in any way.

The first fluid channel layer 110 and the cell culture layer 120, the cell culture layer 120 and the porous structure layer 130, and the porous structure layer 130 and the second fluid channel layer 140 can be encapsulated by adopting a plasma bonding mode; or can also be packaged by adopting a bonding mode of bonding materials; or may be packaged in a combination of plasma bonding and adhesive material bonding.

Preferably, the plasma may be selected from oxygen, nitrogen, helium, neon, argon, krypton, xenon and/or radon plasmas, which may be selected as the case may be by a person skilled in the art.

Preferably, the adhesive material is a glue, such as polydimethylsilane, ultraviolet curable optical glue (NOA 73), nitrile rubber, polyisobutylene, polyacrylate, and other tacky materials.

As shown in fig. 1 and 2, the first fluid channel layer 110 is provided with a first fluid channel comprising a first fluid inlet 111, a first culture zone 112 and a first fluid outlet 113 (wherein the first fluid inlet 111 and the first fluid outlet 113 are not shown in fig. 2). The first fluid inlet 111 and the first fluid outlet 113 communicate with the first culture zone 112. The first fluid inlet 111 and the first fluid outlet 113 are opened at the surface of the first fluid passage layer 110, and may be on the upper surface or the side. The first culture region 112 is located on the lower surface of the first fluid channel layer 110 without penetrating the upper surface thereof, the first culture region 112 is opposite to the upper surface of the cell culture layer 120, a first culture space is formed therebetween, and inoculated cells can be injected into the first culture space. In some embodiments, the first culture region 112 is a recessed region on the lower surface of the first fluid channel layer 110.

The cell culture layer 120 is disposed below the first fluid channel layer 110. As shown in fig. 1 and 2, the cell culture layer 120 is provided with one or more first microwells 121 for culturing cells, the first microwells 121 being for accommodating cultured organoid cells. The material of the cell culture layer is preferably hydrophobic, such as Polydimethylsiloxane (PDMS), so that the inner side walls of the first microwells are hydrophobic surfaces suitable for promoting 3D cell sphere growth. In some embodiments, the first microwell may also have a hydrophobic modification on its inner sidewall surface to facilitate 3D cell sphere growth. Hydrophobic modifications are well known to those skilled in the art and may be made, for example, with a hydrophobic reagent, which may be Pluronic-F127, polyethylene glycol-poly (L-lysine) (PEG-PLL), lipidure, or the like.

The first plurality of microwells may be distributed in an array. Preferably, the first plurality of micro-holes 121 are distributed in a rectangular shape, for example, in six columns and sixteen rows, or in eight columns and fifteen rows. Of course, the plurality of first micro holes 121 may be distributed in other shapes such as a circle, a ring, and a quincunx.

Further, the hole pitch (i.e., the distance between adjacent hole centers) between two adjacent first micro holes 121 is 400 to 2000 micrometers, and still further, the hole pitch is 600 to 1800 micrometers, for example, 800 micrometers, 1000 micrometers, 1200 micrometers, 1400 micrometers, 1600 micrometers, and the like.

The first micro-pores are sized so that the seeded cells can be received and grown therein. The cross-section (i.e., radial cross-section) of the first microwells 121 may be any shape, including, but not limited to, circular, oval, triangular, square, or other polygonal shape. Preferably, the maximum cross-sectional dimension of the first micro-holes 121 is 100-800 micrometers, and further, the maximum cross-sectional dimension of the first micro-holes 121 is 200-700 micrometers, for example, 300 micrometers, 400 micrometers, 500 micrometers, 600 micrometers, etc.

The maximum cross-sectional dimensions of all first microwells 121 may be the same to fit the same organoid cell sphere having the same size. Of course, the maximum cross-sectional dimensions of the different first microwells 121 may also be different to accommodate different kinds of cells or cell spheres having different sizes.

Preferably, the depth of the first micro-holes 121 is 100-500 micrometers, and further, the depth of the first micro-holes 121 is 200-400 micrometers, for example, 250 micrometers, 300 micrometers, 350 micrometers, etc.

Preferably, the first micro-holes 121 are through-holes and penetrate through the cell culture layer 120 in the height direction of the cell culture layer 120 (i.e., the bottom-to-top direction of fig. 2) such that the first micro-holes 121 communicate with the upper surface of the porous structure layer 130.

In the present invention, the number and the laying area of the first microwells are not particularly limited as long as the cell culture layer can be made strong enough to support the layer structure. In some embodiments, the first microwells may be located within a specific area in the middle of the cell culture layer.

The porous structure layer 130 is disposed under the cell culture layer 120. As shown in fig. 1 and 2, the porous structure layer 130 is provided with a plurality of second micropores 131. The second micro-holes 131 are through-holes penetrating the upper and lower surfaces of the porous structure layer such that the second fluid passage layer 140 and the cell culture layer 120 communicate with each other, so that the substances in the space above the porous structure layer 130 and the substances in the space below can be mass-exchanged through the second micro-holes 131. The second microwells 131 have a size smaller than that of the seeded cells such that the seeded cells and the formed organoid cells cannot pass through the second microwells. In some embodiments, the upper surface of the porous structure layer 130 may be coated with a substance that promotes cell adhesion. Such cell adhesion promoting substances are well known to those skilled in the art and may be, for example, proteins or Matrigel, such as fibrin, collagen, matrigel, etc.

The porous structure layer 130 may be closely attached to the lower surface of the cell culture layer 120; alternatively, the porous structure layer 130 may have a certain gap with the lower surface of the cell culture layer 120. In some embodiments, the gap is configured to not allow 3D cell spheres in a first microwell to move through the gap into other first microwells.

The plurality of second microwells may be distributed in an array. Preferably, the plurality of second micro-holes 131 are distributed in a rectangular shape, for example, in sixteen columns and twenty rows, or in ten columns and twenty rows. Of course, the plurality of second micro holes 131 may be distributed in other shapes such as a circle, a ring, and a quincunx.

In some embodiments, the second laying range of the plurality of second micro holes 131 may be made larger, smaller, or equal to the first laying range of the plurality of first micro holes 121.

Further, the hole pitch (i.e., the distance between adjacent hole centers) between two second micro holes 131 adjacently is 50 to 600 micrometers, and still further, the hole pitch is 100 to 500 micrometers, for example, 150 micrometers, 200 micrometers, 300 micrometers, 400 micrometers, etc.

The cross-section (i.e., radial cross-section) of the second microwells 131 may be any shape, including, but not limited to, circular, oval, triangular, square, or other polygonal shape. Preferably, the second micro-holes 131 have a maximum cross-sectional dimension of 5-20 microns, and further, the second micro-holes 131 have a maximum cross-sectional dimension of 7-18 microns, e.g., 9 microns, 11 microns, 13 microns, 15 microns, etc. The maximum cross-sectional dimensions of all second microwells may or may not be identical.

Preferably, the depth of the second micro-holes 131 is 100-500 micrometers, and further, the depth of the second micro-holes 131 is 200-400 micrometers, for example, 250 micrometers, 300 micrometers, 350 micrometers, etc.

In the present invention, the number and the laying area of the second micropores are not particularly limited as long as the porous structure layer can be made strong enough to support the layer structure. In some embodiments, the second micropores may be located within a specific area in the middle of the porous structure layer, or may extend throughout the porous structure layer.

In some embodiments, the first microwells are disposed in a first region on the cell culture layer, the second microwells are disposed in a second region on the porous structure layer, and the vertical projection of the second region completely encloses the vertical projection of the first region, i.e., the vertical projection of the first region is contained within the vertical projection of the second region.

The second fluid channel layer 140 is disposed below the porous structure layer 130. As shown in fig. 1 and 2, the second fluid channel layer 140 is provided with a second fluid channel comprising a second fluid inlet 141, a second culture zone 142 and a second fluid outlet 143 (wherein the second fluid inlet 141 and the second fluid outlet 143 are not shown in fig. 2). The second fluid inlet 141 and the second fluid outlet 143 are in communication with the second culture zone 142. The second fluid inlet 141 and the second fluid outlet 143 are opened at the surface of the second fluid passage layer 140, and may be at the lower surface or the side. The second culturing region 142 is located on the upper surface of the second fluid channel layer 140 without penetrating the lower surface thereof, and the second culturing region 142 is opposite to the lower surface of the porous structure layer 130, with a second culturing space formed therebetween. In some embodiments, the second culture region 142 is a recessed region on the upper surface of the second fluid channel layer 140.

In some embodiments, a plurality of organoid co-culture chips of the invention may be juxtaposed on a single large multi-channel chip, the plurality of organoid co-culture chips being arranged adjacent to one another, each organoid co-culture chip having an independent fluid channel, cell culture layer and porous structural layer, the plurality of organoid co-culture chips not being in communication with one another for independent co-culture of groups of organoids, respectively. The multi-channel chip may be formed by packaging a first fluid channel layer including a plurality of sets of first fluid channels, a cell culture layer, a porous structure layer, and a second fluid channel layer including a plurality of sets of second fluid channels, wherein boundaries of each organoid co-culture chip are correspondingly defined on the first fluid channel layer, the cell culture layer, the porous structure layer, and the second fluid channel layer including the plurality of sets of second fluid channels, respectively, and the boundaries of each organoid co-culture chip are packaged at the time of packaging such that the plurality of organoid co-culture chips are not in communication with each other, and each organoid co-culture chip has an independent first fluid channel, cell culture layer, porous structure layer, and second fluid channel. The number of organoid co-culture chips present on a multi-channel chip may be at least 2, at least 5, at least 10, e.g. 10-20.

Preparation method of organoid co-culture chip

The preparation method of the organoid co-culture chip can comprise the following steps:

ii) packaging the four-layer structure to form the chip.

Wherein the first fluid channel layer, the second fluid channel layer, the cell culture layer and the porous structure layer are made of biocompatible materials, preferably transparent materials, such as polymeric materials, e.g. materials such as polydimethylsilane, polymethyl methacrylate or glass.

In some embodiments, step i) may comprise: and respectively preparing a first fluid channel layer, a second fluid channel layer, a cell culture layer and a mould of the porous structure layer by photoetching, 3D printing or nanoimprint and other methods, then pouring a polymer material into the mould, curing and forming, and removing the mould to obtain each layer of structure made of the polymer material.

For the first and second fluid channel layers, the respective layer structures having the first and second fluid channels may be prepared first, and then the respective fluid inlets and fluid outlets may be prepared by punching.

In some embodiments, step i) may further comprise hydrophobically modifying the inner sidewall surface of the first microwell of the cell culture layer, for example, using a hydrophobic agent. Reagents for hydrophobic modification are well known to those skilled in the art and may be, for example, pluronic-F127, polyethylene glycol-poly (L-lysine) (PEG-PLL) or Lipidure, etc.

In some embodiments, step i) may further comprise coating the upper surface of the porous structure layer with a substance that promotes cell adhesion. Substances which promote cell adhesion are well known to those skilled in the art and may be, for example, proteins or Matrigel, such as fibrin, collagen, matrigel, etc.

In some embodiments, step ii) comprises: and packaging the four-layer structure layer by adopting a mode of plasma bonding and/or bonding materials.

In some embodiments, the plasma may be selected from oxygen, nitrogen, helium, neon, argon, krypton, xenon, and/or radon plasmas.

In some embodiments, the adhesive material is a glue, such as polydimethylsilane, ultraviolet curable optical glue (NOA 73), nitrile rubber, polyisobutylene, polyacrylate, and other tacky materials.

Organoid co-culture method

The method for carrying out the co-culture of various organoids by using the chip comprises the following steps:

s1: inoculating first organoid cells into a first culture area of a first fluid channel layer, allowing the first organoid cells to fall into first micropores of the cell culture layer, and adhering the bottom of the first micropores to the upper surface of the porous structure layer for growth;

S2: the second organoid cells are seeded into the first culture region of the first fluid channel layer such that the second organoid cells fall into the first microwells of the cell culture layer and grow.

In some embodiments, step S1 comprises: and standing for a second preset time after the first organoid cells fall into the first micropores of the cell culture layer, so that the first organoid cells adhere to the upper surface of the porous structure layer for growth.

The second predetermined time is a time sufficient for the first organoid cells to adhere to the upper surface of the porous structure layer for growth. The length of the second preset time may be set and adjusted by one skilled in the art according to the kind of the first organoid cell, the culture condition, and/or the purpose of the culture, etc. In some embodiments, the second preset time is at least 4 hours, such as may be at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, or at least 12 hours, such as 12-24 hours.

In some embodiments, step S1 further comprises: after seeding the first organoid cells into the first culture zone of the first fluid channel layer, allowing the first organoid cells to stand for a first preset time to drop into the first microwells of the cell culture layer, and then rinsing the excess first organoid cells.

The second preset time is a time sufficient to drop at least a portion of the first organoid cells into the first microwells of the cell culture layer. The first preset time can be flexibly adjusted and set according to the actual effect by those skilled in the art. In some embodiments, the first preset time is at least 20 minutes, at least 30 minutes, or at least 40 minutes.

In some embodiments, excess first organoid cells may be rinsed in step S1 with culture solution, buffer solution, or water.

The first organoid may be a 2D adherent culture organoid or a 3D cytosphere. The first organoid cells may be seeded in the form of a cell suspension or 3D cell pellet. When the first organoid cell is seeded in the form of a cell suspension, it can be grown adherent to the upper surface of the porous structure layer or as a 3D cell sphere in the first microwell adhering to the upper surface of the porous structure layer, depending on the particular cell type. When the first organoid cells are seeded in the form of 3D cell pellets, the first organoid cells can adhere to the upper surface of the porous cell layer and continue to grow as 3D cell pellets.

In some embodiments, the first is seeded in the form of 3D cell spheres, which may be liver parenchymal cell 3D cell spheres or glial cell 3D cell spheres, or the like.

In some preferred embodiments, the first organoid cells are seeded in the form of a cell suspension and the first organoid cells tend to grow as 2D organoids, such as epithelial cells, endothelial cells, fibroblasts, etc., on the upper surface of the porous structural layer. In some embodiments, the first organoid cell may be a kidney epithelial cell, a vascular endothelial cell, or a cardiac fibroblast cell, or the like.

In some embodiments, step S2 comprises: after the second organoid cells fall into the first microwells of the cell culture layer, allowing the second organoid cells to stand for a fourth predetermined time to allow the second organoid cells to grow.

The fourth preset time is a time sufficient to grow the second organoid cells. The length of the fourth preset time may be set and adjusted by a person skilled in the art according to the kind of the second organoid cell, the culture condition, and/or the purpose of the culture, etc. In some embodiments, the second preset time is at least 4 hours, such as may be at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 10 hours, or at least 12 hours, such as 12-24 hours.

In some embodiments, step S2 further comprises: after seeding the second organoid cells into the first culture zone of the first fluid channel layer, allowing the second organoid cells to stand for a third predetermined time to drop into the first microwells of the cell culture layer, and then rinsing the excess second organoid cells.

The third preset time is a time sufficient for at least a portion of the second organoid cells to fall into the first microwells of the cell culture layer. The third preset time can be flexibly adjusted and set according to the actual effect by those skilled in the art. In some embodiments, the third preset time is at least 20 minutes, at least 30 minutes, or at least 40 minutes.

In some embodiments, excess second organoid cells may be rinsed in step S2 with culture solution, buffer solution or water.

Wherein the second organoid is preferably a 3D cytosphere. The second organoid may be inoculated directly in the form of a cell suspension or a 3D cell pellet. When the second organoid cell is seeded in the form of a cell suspension, it can grow as a 3D cell sphere in the first microwell. The second organoid cell grows over the first organoid cell. In some embodiments, the second organoid cell can be grown adherent to the first organoid cell. In a preferred embodiment, the second organoid is seeded in the form of a 3D cytosphere. In some embodiments, the second organoid cell may be a 3D cell sphere of a parenchymal cell, e.g., a 3D cell sphere of a stem cell, tumor cell, such as a 3D cell sphere of a neural stem cell, glial cell, liver cancer cell, kidney cancer cell.

The first organoid cell and the second organoid cell are preferably cells that will have interactions in vivo. In some embodiments, the first organoid cell is a vascular endothelial cell and the second organoid cell is a neural stem cell 3D cytosphere. In other embodiments, the first organoid cell is a vascular endothelial cell and the second organoid cell is a glioma cell 3D cell pellet. In other embodiments, the first organoid cell is a cardiac fibroblast and the second organoid cell is a neural stem cell 3D cytosphere. In other embodiments, the first organoid cell is a vascular endothelial cell and the second organoid cell is a hepatic parenchymal cell 3D cytosphere. In other embodiments, the first organoid cell is a kidney epithelial cell and the second organoid cell is a kidney cancer cell 3D glomeruli.

In some embodiments, the chip of the invention is sterilized prior to use in organoid co-culture. The sterilization may be ultraviolet light sterilization and/or irradiation sterilization.

In some embodiments, the chip of the invention is washed prior to organoid co-cultivation using the chip. The washing may be performed using a buffer, a medium, or water.

In some embodiments, the culturing of the first organoid and the second organoid in step S3 is perfusion culturing. The perfusion culture of the first organoid and the perfusion culture of the second organoid may be performed separately using appropriate flow rates to simulate the microenvironment of the different organoids.

In some embodiments, the culturing of the first organoid and the second organoid in step S3 lasts for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or long term culturing.

In co-cultivation of the first organoid and the second organoid, the first organoid may be in nutrient exchange with the first medium in the second cultivation area through the second micropores comprised by the porous structure layer, the second organoid may be in nutrient exchange with the second medium in the first cultivation area, and the first organoid and the second organoid may be in nutrient exchange through interactions between cells. Through co-culture of two kinds of organs, the upper and lower two-layer fluid channels are utilized to exchange different tissue microenvironment nutrient substances, so that a co-culture system of the two kinds of organs can be formed.

According to the organoid co-culture chip, the cell culture layer 120 is arranged between the first fluid channel layer 110 and the second fluid channel layer 140, the porous structure layer 130 is arranged at the bottom of the cell culture layer 120, at least two organoid cells can be sequentially inoculated in the cell culture layer 120, the first organoid cell inoculated firstly can exchange nutrients through culture mediums in the porous structure layer 130 and the second fluid channel layer 140, the second organoid cell inoculated later can exchange nutrients with the culture mediums in the first fluid channel layer 110, and the culture mediums and the nutrients infused into the first fluid channel layer 110 and the second fluid channel layer 140 can be respectively and accurately regulated, so that interaction of the first organoid cell and the second organoid cell can be realized, further, the first organoid cell and the second organoid cell can exchange nutrients through the interaction, a complex three-dimensional tissue environment in a body can be well simulated, and co-culture of at least two organoid cells is realized. In addition, the organoid co-culture chip has simple structure and low preparation cost, can be used for simulating the co-culture of various organoids with various tissue sources, reproduces the fine structure of complex tissues, can observe the change condition of at least two organoid cells in the chip in real time, simplifies the related experimental process, and is beneficial to large-scale production.

Example 1 preparation of organoid Co-culture chip

The prepared organoid co-culture chip consists of a first fluid channel layer, a cell culture layer, a porous structure layer and a second fluid channel layer from top to bottom, wherein the first fluid channel layer is provided with a first fluid inlet 111, a first culture area 112 and a first fluid outlet 113, the cell culture layer is provided with a first micropore 121 for containing organoid cells, the porous structure layer is provided with a second micropore 131 through which organoid cells cannot pass, and the second fluid channel layer is provided with a second fluid inlet 141, a second culture area 142 and a second fluid outlet 143.

Taking Polydimethylsiloxane (PDMS) material as an example, designing a four-layer structure through a computer, preparing a die with the four-layer structure through methods such as photoetching, nanoimprint and the like, then carrying out rotary die processing on the die with each layer of structure by using the PDMS material, punching by using a puncher at a corresponding position, and preparing a fluid inlet and a fluid outlet. The vertical inner side surface of the first micropore of the cell culture layer is modified by Pluronic-F127 hydrophobic reagent, the upper surface of the porous structure layer is modified by fibroblast to facilitate cell adhesion, and then a four-layer structure is sealed layer by using a plasma cleaner or PDMS glue.

Example 2 application of organoid Co-culture chip

Before cell culture, sterilization is first performed. In this example, ultraviolet rays were used for sterilization for 1 hour, and irradiation sterilization was performed for 1 hour.

The chip was washed 4-5 times with PBS and then two cells were seeded one after the other in the first culture zone. This example exemplifies co-culture of mouse cardiac fibroblasts-mouse neural stem cells.

Firstly, inoculating the mouse myocardial fibroblasts into a first culture area through a first fluid inlet, settling the cells at the bottom of a first micropore of a cell culture layer, standing and culturing for 12-24 hours, and promoting the myocardial mouse fibroblasts to gather at the bottom of the first micropore and the upper surface of a porous structure layer to form a uniformly distributed mouse myocardial fibroblasts layer.

And secondly, after the mouse myocardial fibroblast layer is formed, inoculating the 3D cell spheres of the mouse neural stem cells formed in advance in vitro into a first culture area through a first fluid inlet, settling the cells in first micropores of the cell culture layer, and standing for culturing for 12-24 hours to promote the generation of the 3D cell spheres of the mouse neural stem cells.

Injecting a special culture medium for the mouse myocardial fibroblasts into the second culture area, injecting a special culture medium for the mouse neural stem cells into the first culture area, and culturing the 3D cell spheres of the mouse myocardial fibroblasts layer-mouse neural stem cells for a long time. After 3 months, functional evaluation was performed, mainly comprising: two tissue sizes, cell activity, myocardial cell beating frequency, neural cell marker expression, etc.

The embodiments of the present invention are not limited to the examples described above, and those skilled in the art can make various changes and modifications in form and detail without departing from the spirit and scope of the present invention, which are considered to fall within the scope of the present invention.

Claims

1. The organoid co-culture chip is characterized by comprising a first fluid channel layer, a cell culture layer, a porous structure layer and a second fluid channel layer from top to bottom; wherein,

2. The organoid co-culture chip of claim 1, wherein an inner sidewall surface of the first microporous structure has a hydrophobic modification.

3. The organoid co-culture chip of claim 1 or 2, wherein the upper surface of the porous structural layer has an adhesion protein modification.

4. The organoid co-culture chip of any of claims 1-3, wherein the first microwell has a maximum cross-sectional dimension of 100-800 microns.

5. The organoid co-culture chip of any of claims 1-4, wherein the second microwells have a maximum cross-sectional dimension of 5-20 microns.

6. The organoid co-culture chip of any one of claims 1 to 5, wherein the first fluid channel layer, the second fluid channel layer, the cell culture layer, and the porous structure layer are made of dimethylsilane, polymethyl methacrylate, or glass.

7. An organoid co-culture chip according to any one of claims 1 to 5, wherein the first fluid channel layer, the second fluid channel layer, the cell culture layer and the porous structure layer are encapsulated by means of plasma bonding and/or adhesive material bonding between the layers.

8. A method of preparing the organoid co-culture chip of any of claims 1 to 7, comprising the steps of:

9. The method of claim 8, wherein step i) further comprises hydrophobically modifying the inner sidewall surface of the first microwell of the cell culture layer.

10. The production method according to claim 8 or 9, wherein step i) further comprises performing an adhesive protein modification to the upper surface of the porous structure layer.

11. A method of organoid co-culture comprising the steps of:

S1: inoculating a first organoid cell into a first culture region of a first fluid channel layer of the organoid co-culture chip of any of claims 1-7, allowing the first organoid cell to fall into a first microwell of the cell culture layer and adhere to the upper surface of the porous structure layer at the bottom of the first microwell for growth;

12. The organoid co-culture method according to claim 11, further comprising step S3: injecting a first medium suitable for culturing the first organoid into the second culture zone and injecting a second medium suitable for culturing the second organoid into the first culture zone.

13. A method of organoid co-culture according to claim 11 or 12, wherein step S1 comprises: after the first organoid cells fall into the first micropores of the cell culture layer, standing for a second preset time to enable the first organoid cells to adhere to the upper surface of the porous structure layer for growth; preferably, the second preset time is at least 4 hours.

14. A method of organoid co-culture according to any one of claims 11 to 13, wherein step S1 further comprises: after inoculating the first organoid cells into the first culture zone of the first fluid channel layer, allowing the first organoid cells to stand for a first preset time to fall into the first microwells of the cell culture layer, and then rinsing the excess first organoid cells; preferably, the first preset time is at least 30 minutes.

15. A method of organoid co-culture according to any one of claims 11 to 14, wherein step S2 comprises: standing for a fourth preset time after the second organoid cells fall into the first microwells of the cell culture layer to allow the second organoid cells to grow; preferably, the fourth preset time is at least 4 hours.

16. A method of organoid co-culture according to any one of claims 11 to 15, wherein step S2 further comprises: after inoculating the second organoid cells into the first culture area of the first fluid channel layer, allowing the second organoid cells to stand for a third preset time to fall into the first microwells of the cell culture layer, and then rinsing the excess second organoid cells; preferably, the third preset time is at least 30 minutes.

17. The method of any one of claims 11-16, wherein the first organoid cell is a cell capable of growing adherently to the upper surface of the porous structure layer or capable of growing as a 3D cell sphere in the first microwell.

18. The method of claim 17, wherein the first organoid cell is an epithelial cell, an endothelial cell, or a fibroblast.

19. The method according to any one of claims 11-18, wherein the second organoid cell is a cell, preferably a parenchymal cell, a stem cell or a tumor cell, capable of growing as a 3D cell sphere in the first microwell.

20. Use of the organoid co-culture chip of any of claims 1 to 7 in organoid co-culture.