Construction method of organ chip

Abstract

The invention discloses a construction method of an organ chip. The construction method comprises the following steps: constructing a chip base structure comprising a first chamber, a first film, a second chamber, a second film and a third chamber which are stacked in sequence; two-dimensional adherent cells or three-dimensional cells are inoculated in at least one of the first chamber, the second chamber, and the third chamber. According to the construction method, a chip base structure with a structure of a first chamber, a first membrane, a second chamber, a second membrane and a third chamber which are sequentially stacked is constructed, more types of cells can be inoculated in the first chamber, the third chamber and the second chamber, and a more complex in-vivo cell growth environment can be simulated, or in other words, the cell growth environment can be simulated. The environment constructed by the embodiment is closer to the actual growth environment of in-vivo cells.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and more particularly to a method for constructing an organ-on-a-chip. Background Technology

[0002] Organ-on-a-chip technology originated from microfluidic chip technology. It is a technology that uses microfluidics to simulate the microenvironment in the body and to construct physiological and pathological models of human tissues and organs by culturing 2D and 3D cells in the microenvironment.

[0003] This technology is an important research tool in the biomedical field, and it is widely used in drug sensitivity, drug efficacy detection, pharmacokinetics research, and precision medicine. Compared with traditional preclinical cell-level evaluation and animal-level evaluation models, organ-on-a-chip technology can more accurately reproduce the internal environment of the human body, thus obtaining predictive results that are more relevant to clinical data.

[0004] Microfluidic channels are the basic element of organ-on-a-chip. The flow of culture medium within the channels simulates the circulatory environment of body fluids, allowing cells to achieve a growth state more similar to that in vivo. Different organ models have their own unique technical elements. For example, the blood-brain barrier model requires the organ-on-a-chip to have biophysical structures that simulate the barrier; the lung model requires the chip to have force stimulation that simulates the expansion and contraction of alveoli; the neural organoid model requires the chip to have the function of simulating electrophysiological signals; and the multicellular organ model requires the chip to have the function of co-culturing multiple cells. From the perspective of the complexity of the in vivo environment, the more cell types that an organ-on-a-chip can be compatible with, the higher the similarity between the organ-on-a-chip and the real organ.

[0005] However, the culture medium composition varies for different types of cells in vitro. To construct multi-cell organ-on-a-chip, it is necessary to divide the growth environment of different cells spatially on the one hand, and to ensure that different types of cells can exchange signals and factors on the other hand.

[0006] Existing technologies typically divide the flow channel into upper and lower layers by placing a semi-permeable membrane in the middle of the flow channel. Different types of cells are cultured on both sides of the semi-permeable membrane, and the upper and lower flow channels can provide corresponding culture environments for the cells in their respective channels. Cells on both sides of the semi-permeable membrane can exchange factors, signals, etc. through the pores of the semi-permeable membrane. This technology is generally used to construct two-dimensional co-culture models of vascular endothelial cells and simulated target organ cells. However, it is not suitable for constructing more independent environmental structures or three-dimensional growth spaces that more closely resemble the in vivo cell growth state. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing organ-on-a-chip, which can provide more independent growth environments and a three-dimensional growth space that is closer to the in vivo cell growth state.

[0008] To achieve the above-mentioned objective, one embodiment of the present invention provides a method for constructing an organ-on-a-chip. The method includes:

[0009] Construct a chip substrate consisting of a first chamber, a first membrane, a second chamber, a second membrane, and a third chamber stacked sequentially;

[0010] Two-dimensional adherent cells or three-dimensional cells are inoculated in at least one of the first, second, and third chambers.

[0011] Preferably, the step "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells" includes:

[0012] Two-dimensional adherent cells or three-dimensional cells of simulated organ types are inoculated in at least one of the first, second, and third chambers.

[0013] Preferably, the step "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells" further includes:

[0014] In two adjacent chambers of the first, second, and third chambers, one is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types, and the other is inoculated with two-dimensional adherent cells simulating epithelial tissue.

[0015] Preferably, the step "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells" includes:

[0016] Two-dimensional adherent cells or three-dimensional cells simulating organ types are inoculated in the second chamber, and two-dimensional adherent cells simulating epithelial tissue are either not inoculated in the first and third chambers, or are selectively inoculated in one or both; or,

[0017] Two-dimensional adherent cells or three-dimensional cells simulating organ types are inoculated in either the first or third chamber, or in both. In the second chamber, no cells are inoculated, or two-dimensional adherent cells simulating epithelial tissue are inoculated.

[0018] Preferably, two-dimensional adherent cells simulating epithelial tissue are first inoculated, followed by two-dimensional adherent cells or three-dimensional cells simulating organ types.

[0019] Alternatively, two-dimensional or three-dimensional adherent cells simulating organ types can be inoculated first, followed by two-dimensional adherent cells simulating epithelial tissue.

[0020] Preferably, the two-dimensional adherent cells simulating epithelial tissue are set as epithelial cells, or a combination of epithelial cells and immune cells;

[0021] When two-dimensional or three-dimensional adherent cells of simulated organ types are inoculated in the second chamber, and two-dimensional adherent cells of simulated epithelial tissue are inoculated in both the first and third chambers: the two-dimensional adherent cells of simulated epithelial tissue inoculated in the first chamber are the first cell line, and the two-dimensional adherent cells of simulated epithelial tissue inoculated in the third chamber are the second cell line.

[0022] The first cell line is a first epithelial cell or a combination of a first epithelial cell and a first immune cell;

[0023] The second cell line is a combination of second epithelial cells or second immune cells;

[0024] The first epithelial cell and the second epithelial cell may be the same or different, and the first immune cell and the second immune cell may be the same or different.

[0025] Preferably, when two-dimensional adherent cells or three-dimensional cells of simulated organ types are seeded in both the first and third chambers, and two-dimensional adherent cells of simulated epithelial tissue are not seeded in the second chamber, or are seeded in the second chamber:

[0026] The two-dimensional or three-dimensional adherent cells of the simulated organ type inoculated in the first chamber may be the same or different organ types as the two-dimensional or three-dimensional adherent cells of the simulated organ type inoculated in the third chamber.

[0027] Preferably, when two-dimensional adherent cells are seeded in the first chamber, the surface of the first membrane facing away from the second membrane is used as the support surface;

[0028] When seeding two-dimensional adherent cells in the third chamber, the surface of the second membrane facing away from the first membrane is used as the support surface;

[0029] When two-dimensional adherent cells are seeded in the second chamber, the surface of the first membrane facing the second membrane is used as the supporting surface, and / or the surface of the second membrane facing the first membrane is used as the supporting surface.

[0030] In the step “inoculating at least one of the first, second, and third chambers with two-dimensional or three-dimensional adherent cells”: the support surface is immersed in the corresponding two-dimensional adherent cell suspension to inoculate the corresponding two-dimensional adherent cells on the support surface.

[0031] Preferably, in the step "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells":

[0032] Two-dimensional adherent cell suspension is filled into the first, second, or third chamber to be inoculated in an upflow manner in the first direction, and then sealed. The chip substrate is positioned with the support surface facing upward and immersed in the corresponding two-dimensional adherent cell suspension so as to seed the two-dimensional adherent cells on the support surface.

[0033] The first direction is parallel to the first membrane.

[0034] Preferably, the first chamber and the third chamber are interconnected by a passage, and the second chamber is independent of the first chamber and the third chamber;

[0035] The chip substrate also includes a first inlet, a first outlet, a second inlet, and a second outlet;

[0036] The first inlet and the first end of the channel are respectively connected to the opposite ends of the first chamber in the first direction, and the first outlet and the second end of the channel are respectively connected to the opposite ends of the third chamber in the first direction;

[0037] In the first direction, the first inlet and the first outlet are located at the same end of the chip substrate;

[0038] The second inlet and the second outlet are respectively connected to the two opposite ends of the second chamber in the first direction.

[0039] Preferably, the first chamber, the second chamber, and the third chamber are independent of each other;

[0040] The chip substrate also includes a first inlet, a first outlet, a second inlet, a second outlet, a third inlet, and a third outlet;

[0041] The first inlet and the first outlet are respectively connected to the two opposite ends of the first chamber in the first direction;

[0042] The third inlet and the third outlet are respectively connected to the opposite ends of the third chamber in the first direction;

[0043] The second inlet and the second outlet are respectively connected to the two opposite ends of the second chamber in the first direction.

[0044] Preferably, the step of "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells" is performed before:

[0045] Surface modification is performed on any one or more or all of the following surfaces: the surface of the first membrane facing away from the second membrane, the surface of the second membrane facing away from the first membrane, the surface of the first membrane facing the second membrane, and the surface of the second membrane facing the first membrane.

[0046] Preferably, this occurs after the step "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells":

[0047] When any one of the first, second, and third chambers is inoculated with three-dimensional cells, it is sealed, and a continuously flowing culture medium of the appropriate phase is introduced into the adjacent chambers.

[0048] When the first, second, and third chambers are all uninoculated with three-dimensional cells, a continuously flowing culture medium of the appropriate type is introduced into each of the three chambers.

[0049] Preferably, in the step "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells":

[0050] Two-dimensional adherent cells or three-dimensional cells simulating lung organ types were inoculated in the second chamber;

[0051] After step “inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells”, a suitable culture medium and air are alternately and periodically introduced into the first, second, and / or third chambers, and the air pressure is adjusted to periodically fluctuate so that the first and / or second membranes undergo stretching and deformation.

[0052] Compared with the prior art, the beneficial effects of the present invention are as follows: by constructing a chip substrate with a structure of a first chamber, a first membrane, a second chamber, a second membrane, and a third chamber stacked sequentially, more types of cells can be seeded in the first chamber, the third chamber, and the second chamber to simulate more types of cell growth microenvironments and construct a more complex co-culture model; at the same time, the first chamber, the second chamber, and the third chamber can provide a more comprehensive and balanced culture medium or mechanical stimulation for the three-dimensional growth space. In short, this embodiment can simulate a more complex in vivo cell growth environment, or in other words, the environment constructed in this embodiment is closer to the actual in vivo cell growth environment. Attached Figure Description

[0053] Figure 1 This is a flowchart of the construction method of the present invention;

[0054] Figure 2a This is an exploded view from a first perspective of the chip substrate constructed in the construction method of the present invention;

[0055] Figure 2b yes Figure 2a Another exploded view of the chip substrate;

[0056] Figure 2c yes Figure 2a Exploded cross-sectional view of the chip substrate;

[0057] Figure 3a This is an exploded view from a first perspective of the second embodiment of the chip substrate constructed in the construction method of the present invention;

[0058] Figure 3b yes Figure 3a Another exploded view of the chip substrate;

[0059] Figure 3c yes Figure 3aExploded cross-sectional view of the middle chip structure. Detailed implementation manners

[0060] The present invention will be described in detail below in conjunction with the specific implementation manners shown in the drawings. However, these implementation manners do not limit the present invention, and any structural, method, or functional transformation made by those of ordinary skill in the art based on these implementation manners is included within the protection scope of the present invention.

[0061] Unless otherwise specifically stated, the technical terms or scientific terms used in the present invention should have the ordinary meaning understood by those of ordinary skill in the field to which the present invention pertains. The "including" or "comprising" used in the present invention neither limits the mentioned shapes, numbers, steps, actions, operations, components, elements, and / or their groups, nor excludes the occurrence or addition of one or more other different shapes, numbers, steps, actions, operations, components, elements, and / or their groups. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity and order of the indicated technical features.

[0062] In addition, for the numbers "S200", "S400", "S402", "S404", etc. of each step in the present application, they are only used for the convenience of description and cannot be understood as indicating or implying relative importance or implicitly indicating the order of the indicated steps. For example, step S402 and step S404 are not limited by the numerical sizes of the numbers "S402" and "S404" to mean that step S402 is prior to step S404 in implementation. In fact, except as clearly stated later, step S402 can be implemented prior to step S404, synchronously with step S404, or later than step S404.

[0063] Refer Figure 1 , in the first implementation manner of the present invention, a method for constructing an organ chip is provided.

[0064] The construction method can be used to prepare a biochip of an organ or an organoid, which are collectively referred to as organ chips in the present invention.

[0065] Specifically, the construction method includes the following steps:

[0066] S200, chip structure construction;

[0067] S400, seeding.

[0068] Specifically, in step S200, the construction includes stacking a chip structure of a first chamber, a first membrane, a second chamber, a second membrane, and a third chamber in sequence.

[0069] Combined with the attached Figures 2a to 2cThe diagram shows the specific structure of the chip substrate 100 constructed in step S200 of the first embodiment.

[0070] The chip substrate 100 includes a first chamber 10, a first membrane 40, a second chamber 20, a second membrane 50, and a third chamber 30 stacked sequentially.

[0071] The first chamber 10, the second chamber 20, and the third chamber 30 can be used to seed cells, specifically two-dimensional adherent cells or three-dimensional cells, to construct different types of organ-on-a-chip.

[0072] Furthermore, the first chamber 10, the second chamber 20, and the third chamber 30 can be used to circulate substances required to simulate the microenvironment within the body; these substances can be liquids or gases.

[0073] For example, the first chamber 10, the second chamber 20, and the third chamber 30 are used to circulate culture medium to simulate the body fluid circulation environment or to simulate force stimulation, or the first chamber 10, the second chamber 20, and the third chamber 30 are used to circulate gas to simulate force stimulation, but are not limited thereto.

[0074] From the perspective of physical structure, the chip substrate 100 includes a first partition 101, a second partition 102 and a third partition 103 stacked in sequence.

[0075] The first chamber 10 includes a recessed structure 1011 at the first partition 101 and / or a recessed structure at the second partition 102.

[0076] In other words, the first chamber 10 may be defined solely by a recessed structure 1011 formed by the recessed surface of the first partition 101 facing the second partition 102; or solely by a recessed structure formed by the recessed surface S1 of the second partition 102 facing the first partition 101; or by a combination of the recessed structure 1011 formed by the recessed surface of the first partition 101 facing the second partition 102 and the recessed structure formed by the recessed surface S1 of the second partition 102 facing the first partition 101.

[0077] Similarly, the third chamber 30 includes a recessed structure at the second partition 102 and / or a recessed structure 1031 at the third partition 103.

[0078] In other words, the third chamber 30 may be defined solely by a recessed structure formed by the recessed surface S2 of the second partition 102 facing the third partition 103; or solely by a recessed structure 1031 formed by the recessed surface of the third partition 103 facing the second partition 102; or by a combination of the recessed structure formed by the recessed surface S2 of the second partition 102 facing the third partition 103 and the recessed structure 1031 formed by the recessed surface of the third partition 103 facing the second partition 102.

[0079] The second chamber 20 includes a passage S located in the second partition 102.

[0080] In other words, the second chamber 20 is completely located in the second partition 102, and the second chamber 20 is defined by forming a channel S in the second partition 102.

[0081] Of course, in alternative embodiments, the second chamber 20 may also extend from the second partition 102 to the first partition 101, and / or from the second partition 102 to the third partition 103.

[0082] Specifically, in the chip substrate 100 shown in the attached figure, the first membrane 40, the surface S1 of the second partition 102 facing the first partition 101, and the first recessed structure 1011 surround to form a first chamber 10, the first membrane 40, the channel S, and the second membrane 50 surround to form a second chamber 20, and the surface S2 of the second membrane 50 and the second partition 102 facing the third partition 103, and the third recessed structure 1031 surround to form a third chamber 30.

[0083] The first membrane 40 forms part of the boundary between the second chamber 20 and the first chamber 10, that is, the first membrane 40 is arranged at the boundary between the second chamber 20 and the first chamber 10.

[0084] Understandably, the first membrane 40 may be located in the first partition 101, or between the first partition 101 and the second partition 102, or in the second partition 102.

[0085] The second membrane 50 serves as part of the boundary between the second chamber 20 and the third chamber 30; that is, the second membrane 50 is arranged at the boundary between the second chamber 20 and the third chamber 30.

[0086] Similarly, it is understood that the second membrane 50 may be located in the third partition 103, or between the second partition 102 and the third partition 103, or in the second partition 102.

[0087] Here, both the first membrane 40 and the second membrane 50 can be used for cell inoculation. At this time, the cells are immersed in the flowing material in the first chamber 10, the second chamber 20 and the third chamber 30, which can simulate the growth of cells in the microenvironment.

[0088] Both the first membrane 40 and the second membrane 50 are semi-permeable membranes and can also be used for mass exchange.

[0089] "Substance exchange" refers to the exchange of factors, signals, etc. between cells on both sides of a semipermeable membrane, which can simulate the exchange of signals and factors between different types of cells.

[0090] The semipermeable membrane can be a flat membrane or a three-dimensional structure membrane, to be suitable for seeding different types of cells.

[0091] When the semipermeable membrane is a flat membrane, the flat membrane can have various different pore sizes to adapt to different application scenarios.

[0092] For example, flat membranes with pore sizes of 0.1 nm to 1 nm can be used to retain small molecules, such as sodium ions, glucose, lactic acid, and ammonia ions, which facilitates the exchange of small molecule nutrients and metabolic waste in the system.

[0093] Flat membranes with pore sizes of 1 nm to 100 nm can be used to retain macromolecules such as proteins, polysaccharides, and DNA, for the retention and collection of system metabolites.

[0094] Flat membranes with pore sizes of 5 nm to 1 μm can trap particulate matter, latex, micelles, etc., for drug or factor delivery.

[0095] Flat membranes with pore sizes of 100nm to 10μm can retain some biological tissues, such as viruses, bacteria, mycoplasma, cells, and cell exosomes, and are used for the retention of biological tissues.

[0096] Flat membranes with pore sizes of 10μm to 1mm can trap biological tissue aggregates, such as organoids, cell clusters, embryos, isolated tissues and organs, and are used for the trapping of biological aggregates.

[0097] When the semipermeable membrane is a three-dimensional structure membrane, the bottom of the three-dimensional structure membrane has multiple arrayed recesses, and the minimum size of the inscribed circle of the recess is in the range of 5μm to 5cm.

[0098] For example, when the minimum size of the inscribed circle is in the range of 5 μm to 10 μm, three-dimensional structured membranes are used for single-cell culture.

[0099] When the minimum size of the inscribed circle is in the range of 10μm to 1000μm, three-dimensional structured membranes are used to form and culture single cell clusters, etc.

[0100] When the minimum size of the inscribed circle is in the range of 100 μm to 5 cm, the three-dimensional structure membrane is used to form and culture individual organoids.

[0101] The specific materials and structures of the semipermeable membranes described above can be implemented using feasible technologies disclosed in the art, and will not be elaborated further.

[0102] Next, continue to participate Figure 1 In the construction method of the present invention, the S400 inoculation step includes:

[0103] Two-dimensional adherent cells or three-dimensional cells are seeded in at least one of the first chamber 10, the second chamber 20 and the third chamber 30.

[0104] Two-dimensional adherent cells refer to cells that spread out and grow on a supporting surface, and the cell population in this state is roughly arranged in layers.

[0105] Three-dimensional cells refer to cells supported by scaffolds made of different materials with three-dimensional structures. Cells in this state migrate and grow in the three-dimensional spatial structure of the scaffold in the form of single cells or clusters of cells.

[0106] Specifically, scaffolds suitable for three-dimensional cells come in various forms, such as traditional nanofiber scaffolds and porous scaffolds, as well as emerging matrix scaffolds formed from collagen hydrogels. These are all conventional choices for scaffolds in the art. In the following text, matrix scaffolds are used as an example to describe in detail the three-dimensional cell seeding in the chip substrate 100. However, it is understood that other conventional scaffolds besides matrix scaffolds are also suitable for three-dimensional cell seeding in the chip substrate 100 of this application.

[0107] Furthermore, the "inoculation of at least one two-dimensional adherent cell or three-dimensional cell in the first chamber 10, the second chamber 20, and the third chamber 30" includes various implementation methods, which can be summarized into the following three categories:

[0108] The first category involves seeding two-dimensional adherent cells or three-dimensional cells in any one of the first chamber 10, the second chamber 20, and the third chamber 30, while leaving the other two unseeded.

[0109] The second type involves seeding two-dimensional adherent cells or three-dimensional cells in any two of the first chamber 10, the second chamber 20, and the third chamber 30, while leaving the remaining chamber unseeded.

[0110] The third type involves seeding two-dimensional adherent cells or three-dimensional cells in chamber 10, chamber 20, and chamber 30.

[0111] The first type of implementation described above can be further implemented in various ways. For example, in one implementation, the first chamber 10 is inoculated with two-dimensional adherent cells or three-dimensional cells simulating organ types; from the perspective of different chambers, in one variation, the second chamber 10 is inoculated with two-dimensional adherent cells or three-dimensional cells simulating organ types; from the perspective of different cell types, in yet another variation, the first chamber 10 is inoculated with two-dimensional adherent cells or three-dimensional cells simulating epithelial tissue; and so on. That is, depending on the different chambers inoculated and the different cell types, the first type of implementation can have a variety of different implementations.

[0112] Similarly, the second type of implementation described above can be further implemented in various ways. For example, in one implementation, the first chamber 10 is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types, and the second chamber 20 is inoculated with two-dimensional or three-dimensional adherent cells simulating epithelial tissue. From the perspective of different chambers, in one variation, the third chamber 10 is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types, and the second chamber 20 is inoculated with two-dimensional or three-dimensional adherent cells simulating epithelial tissue. From the perspective of different cell types, in yet another variation, the first chamber 10 is inoculated with two-dimensional or three-dimensional adherent cells simulating epithelial tissue, and the second chamber 20 is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types. And so on. That is, depending on the different chambers inoculated and the different cell types, the second type of implementation can have a variety of different implementations.

[0113] Similarly, the second type of implementation described above can be further implemented in various ways. For example, in one implementation, the first chamber 10 is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types, the second chamber 20 is inoculated with two-dimensional or three-dimensional adherent cells simulating epithelial tissue, and the third chamber 10 is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types. From the perspective of different cell types, in another variation, the first chamber 10 is inoculated with two-dimensional or three-dimensional adherent cells simulating epithelial tissue, the second chamber 20 is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types, and the third chamber 10 is inoculated with two-dimensional or three-dimensional adherent cells simulating epithelial tissue.

[0114] The various implementation methods described above can be further implemented in multiple ways depending on the type of organ being simulated, such as simulating the lungs, brain, or liver.

[0115] In summary, by constructing a chip substrate with a structure of sequentially stacked first chamber, first membrane, second chamber, second membrane, and third chamber, more types of cells can be seeded in the first, third, and second chambers to simulate more types of cell growth microenvironments and construct more complex co-culture models. At the same time, the first, second, and third chambers can provide a more comprehensive and balanced culture medium or mechanical stimulation for the three-dimensional growth space. In short, this embodiment can simulate a more complex in vivo cell growth environment, or in other words, the environment constructed in this embodiment is closer to the actual in vivo cell growth environment.

[0116] As mentioned above, both the first membrane 40 and the second membrane 50 can be used for cell seeding.

[0117] For example, in step S400: when two-dimensional adherent cells are seeded in the first chamber 10, the surface of the first membrane 40 facing away from the second membrane 50 is used as the supporting surface, and the seeded two-dimensional adherent cells adhere to the surface of the first membrane 40 facing away from the second membrane 50; when two-dimensional adherent cells are seeded in the third chamber 30, the surface of the second membrane 50 facing away from the first membrane 40 is used as the supporting surface, and the seeded two-dimensional adherent cells adhere to the surface of the second membrane 50 facing away from the first membrane 40; when two-dimensional adherent cells are seeded in the second chamber 20, the surface of the first membrane 40 facing the second membrane 50 is used as the supporting surface, and the seeded two-dimensional adherent cells adhere to the surface of the first membrane 40 facing the second membrane 50; and / or, the surface of the second membrane 50 facing the first membrane 40 is used as the supporting surface, and the seeded two-dimensional adherent cells adhere to the surface of the second membrane 50 facing the first membrane 40.

[0118] Furthermore, in one embodiment of the present invention, before step S400, the construction method further includes:

[0119] S300, surface modification of the first film 40 and the second film 50.

[0120] In other words, before cell seeding in the first chamber 10, the second chamber 20 and the third chamber 30, the surface of the first membrane 40 and the second membrane 50 are modified in advance to increase the cell seeding probability in step S400 and to increase the success rate of cell culture in the constructed organ-on-a-chip.

[0121] Specifically, step S300 may involve surface modification of any of the support surfaces, including surface modification of one or a combination of the following four surfaces: the surface of the first membrane 40 facing away from the second membrane 50, the surface of the second membrane 50 facing away from the first membrane 40, the surface of the first membrane 40 facing the second membrane 50, and the surface of the second membrane 50 facing the first membrane 40.

[0122] Preferably, surface modification can be performed on at least one of the surfaces of the first membrane 40 facing away from the second membrane 50 and the surfaces of the first membrane 40 facing the second membrane 50, and on at least one of the surfaces of the second membrane 50 facing away from the first membrane 40 and the surfaces of the second membrane 50 facing the first membrane 40.

[0123] More preferably, the surfaces of the first membrane 40 facing away from the second membrane 50, the second membrane 50 facing away from the first membrane 40, the first membrane 40 facing the second membrane 50, and the second membrane 50 facing the first membrane 40 are all surface-modified.

[0124] In one embodiment, in step S300, a surface modification liquid is used to modify the surface of the first film 40 and the second film 50.

[0125] Preferably, the surface modification solution is a protein-containing surface modification solution.

[0126] More preferably, the surface modification liquid is a surface modification liquid containing collagen.

[0127] In one embodiment, the surface modification solution is prepared by mixing a 1.25 mg / mL collagen solution with phosphate-buffered saline (PBS) at a volume ratio of 0.05:1 to 1:0.05 to obtain the surface modification solution.

[0128] For example, 1 volume of collagen solution can be mixed with 19 volumes of PBS to obtain a surface modification solution.

[0129] In one specific embodiment, step S300 includes:

[0130] S302, fill at least one of the first chamber 10 and the third chamber 30 with the surface modification liquid, and / or fill the second chamber 20 and maintain it for a first duration;

[0131] S304, drain the surface modification solution from the chip substrate 100, and then air dry it in a cell-friendly temperature environment.

[0132] In step S302, the first duration is preferably set to 45 minutes or more.

[0133] Preferably, in step S304, the chip substrate 100 can be air-dried in an incubator with a suitable temperature environment for cells.

[0134] The suitable temperature environment for the cells is, for example, 36°C to 38°C, or 37°C to 38°C, or 37°C.

[0135] Next, in step S400, when at least one of the first chamber 10, the second chamber 20 and the third chamber 30 is inoculated with three-dimensional cells, the chamber inoculated with three-dimensional cells is named a three-dimensional inoculation chamber, and the inoculation method of three-dimensional cells is described in detail.

[0136] It is understandable that when the first chamber 10 is inoculated with three-dimensional cells, the "three-dimensional inoculation chamber" described below can refer to the first chamber 10; similarly, when the second chamber 20 and the third chamber 30 are inoculated with three-dimensional cells, the "three-dimensional inoculation chamber" described below can refer to the second chamber 20 and the third chamber 30.

[0137] In one embodiment, three-dimensional cells are inoculated using any one of the first chamber 10, the second chamber 20, and the third chamber 30 as a three-dimensional inoculation chamber, specifically including:

[0138] The inoculation solution formed by cells and scaffolds is filled into the three-dimensional inoculation chamber.

[0139] As mentioned earlier, the scaffolds used here come in various forms, such as traditional nanofiber scaffolds and porous scaffolds, as well as emerging matrix scaffolds formed from collagen hydrogels, which are all conventional choices for scaffolds in this field.

[0140] Next, using a matrix scaffold as an example, we will further elaborate on the seeding methods for three-dimensional cells.

[0141] In one embodiment, the step of "filling the three-dimensional inoculation chamber with the inoculation solution formed by the cells and scaffold" includes:

[0142] Organoids were digested using trypsin, and cell suspensions were collected.

[0143] The inoculation solution was prepared using a matrix scaffold formed from collagen hydrogel and the cell suspension.

[0144] The inoculation solution is then filled into the three-dimensional inoculation chamber.

[0145] The cells in the cell suspension exist in the form of single cells, or in the form of aggregated cells, or in the form of a combination of single cells and aggregated cells.

[0146] Specifically, in the step "digesting organoids with trypsin and collecting cell suspension", after digesting the organoids, single cells or cell clusters with a size not exceeding 20-100 μm are screened to prepare cell suspension.

[0147] For example, after organoids are digested, they are passed through a sieve with a pore size of 20–100 μm, thereby producing a cell suspension from single cells and aggregated cells that can pass through the sieve.

[0148] The preferred cell density in the cell suspension is 10. 5 ~10 6 per mL.

[0149] In one embodiment, in the step of "preparing the inoculation solution using a matrix scaffold formed of collagen hydrogel and the cell suspension", the matrix scaffold and the cell suspension are mixed in a volume ratio of not less than 2:3 to prepare the inoculation solution.

[0150] As mentioned earlier, the three-dimensional inoculation chamber can specifically be any one of the first chamber 10, the second chamber 20, and the third chamber 30. Here, we take the inoculation of three-dimensional cells in the second chamber 20 as an example, that is, the three-dimensional inoculation chamber is the second chamber 20. Accordingly, combined with Figure 2a and Figure 2b The specific operation of step "filling the three-dimensional inoculation chamber with the inoculation solution" in the chip substrate 100 can be as follows:

[0151] The inoculation solution is injected into the second chamber 20 through the second inlet H21 until the second chamber 20 is full.

[0152] Understandably, during the process of injecting the inoculum into the second chamber 20, the gas inside the second chamber 20 is discharged through the second outlet H22.

[0153] Preferably, in one embodiment, after the inoculation solution fills the three-dimensional inoculation chamber, the matrix adhesive is cured, and then the three-dimensional inoculation chamber is sealed.

[0154] Specifically, taking the three-dimensional inoculation room as the second chamber 20 as an example, the second chamber 20 can be sealed by blocking the second inlet H21 and the second outlet H22.

[0155] Alternatively, the chip substrate 100 can be placed in a cell-appropriate temperature environment for 15–30 minutes to allow the matrix gel to solidify.

[0156] For example, the chip substrate 100 is placed in a cell culture incubator with a suitable cell temperature environment and treated for 15–30 minutes.

[0157] For example, the suitable temperature environment for the cells is 36℃~38℃, or 37℃~38℃, or 37℃.

[0158] Optionally, the organoids may be any one of intestinal organoids, intestinal cancer organoids, liver organoids, liver cancer organoids, lung organoids, lung cancer organoids, stomach organoids, stomach cancer organoids, pancreatic organoids, pancreatic cancer organoids, brain organoids, and heart organoids.

[0159] More preferably, in the step of "filling the three-dimensional inoculation chamber with the inoculation solution formed by the cells and scaffold", the inoculation solution is filled into the three-dimensional inoculation chamber after the liquids in the first chamber 10, the second chamber 20 and the third chamber 30 have been emptied in advance.

[0160] In other words, before injecting the inoculation solution into the three-dimensional inoculation chamber, it is necessary to ensure that there is no liquid in the first chamber 10, the second chamber 20, and the third chamber 30.

[0161] For example, if the first membrane 40 and the second membrane 50 need to be surface-modified before inoculation, then the surface modification liquid that may still remain in the first chamber 10, the second chamber 20 and the third chamber 30 need to be drained before injecting the inoculation solution into the three-dimensional inoculation chamber.

[0162] For example, if two-dimensional adherent cells are pre-inoculated in at least one of the first chamber 10, the second chamber 20, and the third chamber 30 before inoculation in the three-dimensional inoculation chamber, then the cell suspension of two-dimensional adherent cells that may remain or have seeped into the first chamber 10, the second chamber 20, and the third chamber 30 needs to be drained before injecting the inoculation solution into the three-dimensional inoculation chamber.

[0163] Next, in step S400, when at least one of the first chamber 10, the second chamber 20 and the third chamber 30 is inoculated with two-dimensional adherent cells, the chamber inoculated with two-dimensional adherent cells is named the two-dimensional inoculation chamber, and the specific inoculation method of two-dimensional adherent cells is described in detail.

[0164] It is understandable that when two-dimensional adherent cells are inoculated in the first chamber 10, the term "two-dimensional inoculation chamber" as used below can refer to the first chamber 10; similarly, when two-dimensional adherent cells are inoculated in the second chamber 20 and the third chamber 30, the term "two-dimensional inoculation chamber" as used below can refer to the second chamber 20 and the third chamber 30.

[0165] In one embodiment, two-dimensional adherent cells are inoculated using any one of the first chamber 10, the second chamber 20, and the third chamber 30 as a two-dimensional inoculation chamber, specifically including:

[0166] The support surface is immersed in the corresponding two-dimensional adherent cell suspension to seed the corresponding two-dimensional adherent cells on the support surface.

[0167] For example, when seeding two-dimensional adherent cells into the first chamber 10, the surface of the first membrane 40 facing away from the second membrane 50 is used as a support surface, and the surface of the first membrane 40 facing away from the second membrane 50 is immersed in the corresponding two-dimensional adherent cell suspension, so as to seed the corresponding two-dimensional adherent cells on the surface of the first membrane 40 facing away from the second membrane 50.

[0168] Similarly, when the surface of the second membrane 50 facing away from the first membrane 40 is used as the support surface, or the surface of the first membrane 40 facing the second membrane 50 is used as the support surface, or the surface of the second membrane 50 facing the first membrane 40 is used as the support surface, these support surfaces are also immersed in the corresponding two-dimensional adherent cell suspension to facilitate inoculation.

[0169] In a preferred embodiment, in step S400, whenever two-dimensional adherent cell seeding is performed:

[0170] The two-dimensional adherent cell suspension is injected into the two-dimensional inoculation chamber in an upflow manner in the first direction;

[0171] Then, the two-dimensional inoculation chamber is sealed, and the chip substrate 100 is positioned with the support surface facing upward and immersed in the corresponding two-dimensional adherent cell suspension to inoculate the two-dimensional adherent cells on the support surface; the first direction is parallel to the first membrane 10.

[0172] In this way, injecting the two-dimensional adherent cell suspension in an upflow manner can facilitate the smooth discharge of gas in the two-dimensional inoculation chamber, avoid the generation of air bubbles, stabilize the flow state of the suspension, and at the same time, the two-dimensional adherent cell suspension submerges the support surface to be inoculated, with the support surface facing upwards, so that the cells in the two-dimensional adherent cell suspension can settle and adhere to the support surface under their own gravity, thereby completing efficient inoculation.

[0173] In this application, the "upflow mode" refers to a flow mode in which fluid enters from the bottom of the two-dimensional inoculation chamber and the liquid level gradually rises until it reaches the outlet, based on the orientation relationship between the fluid inlet of the two-dimensional inoculation chamber at the bottom of the two-dimensional inoculation chamber and the fluid outlet at the top of the two-dimensional inoculation chamber.

[0174] Combination Figures 2a to 2c The chip substrate 100 has a first chamber 10 and a third chamber 30 connected to each other, and a second chamber 20 independent of the first chamber 10 and the third chamber 30.

[0175] Here, "the first chamber 10 and the third chamber 30 are interconnected" means that the first chamber 10 and the third chamber 30 are structurally interconnected, and the fluid required to simulate the microenvironment in vivo can flow directly between the first chamber 10 and the third chamber 30.

[0176] The phrase "the second chamber 20 is independent of the first chamber 10 and the third chamber 30" means that the second chamber 20 is structurally isolated from the first chamber 10 and the third chamber 30, and the fluid flowing in the first chamber 10 and the third chamber 30 cannot directly flow into the second chamber 20.

[0177] It should be noted that although the first membrane 40 is used for material exchange between the second chamber 20 and the first chamber 10, this "material exchange" is not the same as the aforementioned "circulation". This still falls under the category of the second chamber 20 being structurally isolated from the first chamber 10.

[0178] Similarly, although the second membrane 50 is used for the exchange of matter between the second chamber 20 and the third chamber 30, this "exchange of matter" is not the same as the aforementioned "circulation". It still falls under the category of the second chamber 20 being structurally isolated from the third chamber 30.

[0179] More specifically, the chip substrate 100 also includes a second inlet H21 and a second outlet H22.

[0180] The second inlet H21 and the second outlet H22 are connected to the second chamber 20, and are respectively connected to the two opposite ends of the second chamber 20 in the first direction.

[0181] For example, with Figure 2aIn terms of orientation, the second inlet H21 is connected to the left end of the second chamber 20 shown in the diagram, and the second outlet H22 is connected to the right end of the second chamber 20 shown in the diagram.

[0182] More specifically, participants Figure 2c The chip substrate 100 includes two extension pipes C, one of which connects to the second inlet H21 and the second chamber 20, and the other extension pipe C connects to the second outlet H22 and the second chamber 20.

[0183] The extension pipe C includes a through hole C1 penetrating the first partition 101 and an opening C2 formed on the second partition 102. Of course, in variant embodiments, the arrangement of the extension pipe C is not limited to this, as long as the second inlet H21 and the second chamber 20, and the second outlet H22 and the second chamber 20 are respectively connected.

[0184] In one embodiment, when two-dimensional adherent cells are seeded in the second chamber 20, the two-dimensional adherent cells can be seeded using the surface of the first membrane 40 facing the second membrane 50 as the supporting surface, and when the two-dimensional adherent cells are seeded using the surface of the second membrane 50 facing the first membrane 40 as the supporting surface, specifically:

[0185] First, position the chip substrate 100 such that the second inlet H21 is at the bottom of the second chamber 20 and the second outlet H22 is at the top of the second chamber 20 (for example, the chip substrate 100 is placed vertically). Then, inject a two-dimensional wall-adhering suspension into the second chamber 20 from the second inlet H21 in an upflow manner until the liquid surface reaches the second outlet H22.

[0186] Then, the second chamber 20 is sealed (e.g., the second inlet H21 and the second outlet H22 are blocked), and the chip substrate 100 is positioned so that the surface of the first membrane 40 faces the second membrane 50 with the surface horizontally upward (to...). Figure 2a (View angle horizontally inverted), at this time the surface of the first membrane 40 facing the second membrane 50 is immersed in the two-dimensional adherent cell suspension and maintained for a period of time, for example, about 24h to 72h, so as to seed the two-dimensional adherent cells on the surface of the first membrane 40 facing the second membrane 50.

[0187] Then the two-dimensional adherent cell suspension in the second chamber 20 was drained;

[0188] Next, position the chip substrate 100 such that the second inlet H21 is at the bottom of the second chamber 20 and the second outlet H22 is at the top of the second chamber 20 (for example, the chip substrate 100 is placed vertically). Inject two-dimensional wall-adhering suspension into the second chamber 20 from the second inlet H21 in an upflow manner until the liquid surface reaches the second outlet H22.

[0189] Then, the second chamber 20 is sealed (e.g., the second inlet H21 and the second outlet H22 are blocked), and the chip substrate 100 is positioned so that the surface of the second membrane 50 facing the first membrane 40 is horizontally upward (so that...). Figure 2a With the viewing angle horizontal and upright, the surface of the second membrane 50 facing the first membrane 40 is immersed in a two-dimensional adherent cell suspension for a period of time, for example, about 24 hours to 72 hours, so as to seed the two-dimensional adherent cells on the surface of the second membrane 50 facing the first membrane 40.

[0190] Of course, the two-dimensional adherent cells seeded on the surface of the first membrane 40 facing the second membrane 50 and the surface of the second membrane 50 facing the first membrane 40 can be the same or different.

[0191] Alternatively, in another variation, when two-dimensional adherent cells are seeded in the second chamber 20, they may be seeded only on one of the surfaces of the first membrane 40 facing the second membrane 50 and the second membrane 50 facing the first membrane 40, while the other surface remains unseeded.

[0192] In one specific embodiment, the chip substrate 100 further includes a first outlet H11 (or a third outlet H31), a first inlet H12 (or a third inlet H32), and a channel 60.

[0193] The passageway 60 connects the first chamber 10 and the third chamber 30, and the passageway 60 is separated from the second chamber 20.

[0194] Here, the “separation between channel 60 and second chamber 20” means that channel 60 and second chamber 20 are not structurally connected to each other, and the substance flowing through channel 60 will not enter second chamber 20 through channel 60.

[0195] "Channel 60 connects the first chamber 10 and the third chamber 30" means that the first chamber 10 and the third chamber 30 can be interconnected structurally through channel 60.

[0196] In the attached drawings, the channel 60 is located outside the second chamber 20; the channel 60 passes through the surface S1 of the second partition 102 facing the first partition 101 and the surface S2 of the second partition 102 facing the third partition 103, and connects the recessed structure 1011 and the recessed structure 1031 to connect the first chamber 10 and the third chamber 30.

[0197] The first inlet H12 is connected to the first chamber 10, and the first end of the first inlet H12 and the first end of the channel 60 are respectively connected to the opposite ends of the first chamber 10 in the first direction.

[0198] For example, with Figure 2a In terms of orientation, the first inlet H12 is connected to the left end of the first chamber 10 shown in the figure (e.g., the left end region A2 of the first chamber 10), and the first end of the channel 60 is connected to the right end of the first chamber 10 shown in the figure (e.g., the rightmost end of the right end region A1 of the first chamber 10).

[0199] The first exit H11 connects to the third chamber 30, and the second ends of the first exit H11 and the passage 60 are respectively connected to the opposite ends of the third chamber 30 in the first direction.

[0200] For example, with Figure 2a In terms of orientation, the first exit H11 connects to the left end of the third chamber 30 shown in the diagram (e.g., the left end region A4 of the third chamber 30), and the second end of the passage 60 connects to the right end of the third chamber 30 shown in the diagram (e.g., the rightmost end of the right end region A3 of the third chamber 30).

[0201] Thus, when fluid (such as the surface modification solution, two-dimensional adherent cell suspension, culture medium, or inoculation solution) is injected from the first inlet H12, the fluid flows along the direction of the first inlet H12, the first chamber 10, the channel 60, the third chamber 30, and the first outlet H11; the first end of the channel 60 constitutes the fluid outlet of the first chamber 10, and the second end of the channel 60 constitutes the fluid inlet of the third chamber 30.

[0202] In one embodiment, when two-dimensional adherent cells are seeded in the first chamber 10 with the surface of the first membrane 40 facing away from the second membrane 50 (or in the third chamber 30 with the surface of the second membrane 50 facing away from the first membrane 30) as the supporting surface:

[0203] First, position the chip substrate 100 so that the first inlet H12 is at the bottom of the first chamber 10 and the first end of the channel 60 is at the top of the first chamber 10 (e.g., the chip substrate 100 is placed vertically). Inject a two-dimensional adherent cell suspension into the first chamber 10 through the first inlet H12 until the liquid level reaches the first end of the channel 60. Then, position the chip substrate 100 so that the second end of the channel 60 is at the bottom of the third chamber 30 and the first outlet H11 is at the top of the third chamber 30 (e.g., the chip substrate 100 is rotated 180° from its vertical position and placed upside down). Continue to inject the cell suspension to be inoculated into the first chamber 10 through the first inlet H12. The cell suspension to be inoculated is injected into the third chamber 30 from the second end of the channel 60 until the liquid level reaches the first outlet H11.

[0204] Then, the first chamber 10 and the third chamber 30 are sealed (e.g., the first inlet H12 and the first outlet H11 are blocked), and the chip substrate 100 is positioned so that the surface of the first membrane 40 facing away from the second membrane 50 is horizontally upward. At this time, the surface of the first membrane 40 facing away from the second membrane 50 is immersed in a two-dimensional adherent cell suspension and maintained for a period of time to seed the two-dimensional adherent cells on the surface of the first membrane 40 facing away from the second membrane 50 (or, the chip substrate 100 is positioned so that the surface of the second membrane 50 facing away from the first membrane 30 is horizontally upward, and the surface of the second membrane 50 facing away from the first membrane 30 is immersed in a two-dimensional adherent cell suspension and maintained for a period of time, for example, about 24 hours to 72 hours, to seed the two-dimensional adherent cells on the surface of the second membrane 50 facing away from the first membrane 30).

[0205] Further, in step S400, the two-dimensional adherent cells inoculated in the two-dimensional inoculation chamber can specifically be two-dimensional adherent cells simulating organ types. Accordingly, step S400 includes:

[0206] Configure a two-dimensional adherent cell suspension to simulate organ types.

[0207] More specifically, step S400 includes: digesting the organoids with trypsin, and then adjusting the cell density, for example, to a cell density of 102. 5 ~10 8 The cells were collected at a density of 1 cell / mL, and a two-dimensional adherent cell suspension was obtained.

[0208] Preferably, while inoculating two-dimensional adherent cells of simulated organ types in a two-dimensional inoculation chamber, pericytes or human fibroblasts can also be further inoculated.

[0209] Correspondingly, the preparation method for suspensions of pericytes or human fibroblasts is the same as that for two-dimensional adherent cell suspensions simulating organ types.

[0210] In one embodiment, in step S400, the two-dimensional adherent cells inoculated in the two-dimensional inoculation chamber may specifically be two-dimensional adherent cells simulating epithelial tissue. Accordingly, step S400 includes:

[0211] Prepare a two-dimensional adherent cell suspension to simulate epithelial tissue.

[0212] More specifically, the two-dimensional adherent cells that simulate epithelial tissue can be set as epithelial cells, or a combination of epithelial cells and immune cells.

[0213] Accordingly, step S400 specifically includes:

[0214] Prepare a two-dimensional adherent cell suspension of epithelial cells; specifically, for example, digest the epithelial tissue with trypsin and then adjust the cell density, for example, to a cell density of 102. 5 ~10 8 The number of cells / mL was collected, and finally a two-dimensional adherent cell suspension of epithelial cells was obtained.

[0215] And / or, prepare a two-dimensional adherent cell suspension of immune cells; specifically, for example, differentiate immune cells in a multi-well plate for a predetermined time period, then digest them with trypsin, and then adjust the cell density, for example, to a cell density of 2.5 × 10⁻⁶. 5 ~10×10 5 The cells were collected at a density of 1 cell / mL, and finally a two-dimensional adherent cell suspension of immune cells was obtained.

[0216] The perforated plate can be a six-hole plate, or other perforated plates known in the art can be selected according to actual needs.

[0217] Preferably, in the step of "preparing a two-dimensional adherent cell suspension of immune cells", 5-500 ng / mL of phorbol 12-myristate 13-acetate (PMA), 20-2000 IU / mL of recombinant interferon-γ (IFN-γ), or 10 -6 ~10 -5 1,25-dihydroxyvitamin D3 (VD3) at mol / L was used as a differentiating agent.

[0218] The preset time period is preferably more than 40 hours, and more preferably around 48 hours.

[0219] The two-dimensional adherent cells of the simulated organ type are one or more combinations of cells or organoids derived from normal tissues or diseases such as the intestine, liver, lung, stomach, pancreas, brain, and heart, or cells or organoids derived from stem cells such as IPSC and MSC.

[0220] In other words, the two-dimensional adherent cells that simulate organ types can be one or a combination of several of the following: intestinal cells, intestinal cancer cells, hepatocytes, liver cancer cells, lung cells, lung cancer cells, gastric cells, gastric cancer cells, pancreatic cells, pancreatic cancer cells, brain cells, and heart cells.

[0221] The colorectal cancer cells can be any one or both of human clonal colon adenocarcinoma cells (Caco-2 cells) and human colorectal cancer cells (HTB-37 cells).

[0222] The hepatocytes are preferably human hepatic stellate cells (LX-2 cells).

[0223] The liver cancer cells are preferably human liver cancer tissue cells (HepG2 cells).

[0224] The lung cells are preferably human alveolar epithelial cells (HPAEpiC).

[0225] The brain cells are preferably astrocytes.

[0226] The gastric cells are preferably human gastric parietal cells.

[0227] The gastric cancer cells are preferably human gastric cancer cells (MGC-803 cells).

[0228] The pancreatic cancer cells are preferably human pancreatic cancer cells (PANC-1 cells).

[0229] The heart cells are preferably cardiomyocytes.

[0230] Of course, the above are just some examples of organ cells and cells. The present invention is not limited to these, and other applicable organ cells and cells known in the art can also be used.

[0231] The epithelial cells mentioned can specifically be vascular endothelial cells, human hepatobiliary epithelial cells (e.g., HIBEC), human pancreatic duct epithelial cells, human bronchial epithelial cells (e.g., 16HBE14o-), gastric mucosal epithelial cells, intestinal mucosal epithelial cells, or neuroepithelial cells. The selection can be based on the needs of the two-dimensional inoculation chamber for inoculating two-dimensional adherent cells simulating epithelial tissue, that is, based on the type of organ being simulated by the organ-on-a-chip to be constructed.

[0232] The vascular endothelial cells mentioned are human umbilical vein endothelial cells (HUVECs).

[0233] The immune cells mentioned can specifically be monocytes / macrophages, granulocytes, dendritic cells, mast cells, or human lymphocytes (e.g., U-937).

[0234] Furthermore, in one embodiment, in step S400, at least one of the first chamber 10, the second chamber 20, and the third chamber 30 is inoculated with two-dimensional adherent cells or three-dimensional cells of a simulated organ type.

[0235] Specifically, for example, in one embodiment, two-dimensional adherent cells or three-dimensional cells of simulated organ type can be inoculated in the second chamber 20, while no inoculation is performed in the first chamber 10 and the third chamber 30, or two-dimensional adherent cells or three-dimensional cells of simulated organ type can be inoculated in one of them, or two-dimensional adherent cells or three-dimensional cells of simulated epithelial tissue can be inoculated in one of them, or both can be inoculated with two-dimensional adherent cells or three-dimensional cells of simulated epithelial tissue.

[0236] In another variation, two-dimensional adherent cells or three-dimensional cells of simulated organ type may be inoculated in the first chamber 10, while neither the first chamber 10 nor the second chamber 20 may be inoculated, or one of the two chambers may be inoculated with two-dimensional adherent cells or three-dimensional cells of simulated organ type, or one of the two chambers may be inoculated with two-dimensional adherent cells or three-dimensional cells of simulated epithelial tissue, or both may be inoculated with two-dimensional adherent cells or three-dimensional cells of simulated epithelial tissue.

[0237] Furthermore, in one embodiment, in step S400, in two adjacent chambers of the first chamber 10, the second chamber 20, and the third chamber 30, one is inoculated with two-dimensional adherent cells or three-dimensional cells simulating organ types, and the other is inoculated with two-dimensional adherent cells simulating epithelial tissue.

[0238] Specifically, for example, in one embodiment, two-dimensional adherent cells or three-dimensional cells simulating epithelial tissue can be inoculated in the first chamber 10, two-dimensional adherent cells or three-dimensional cells simulating organ types can be inoculated in the second chamber 20, and no cells can be inoculated in the third chamber 30, or two-dimensional adherent cells or three-dimensional cells simulating organ types can be inoculated, or two-dimensional adherent cells or three-dimensional cells simulating epithelial tissue can be inoculated.

[0239] Furthermore, when two-dimensional adherent cells or three-dimensional cells simulating organ types are seeded in both the first chamber 10 and the third chamber 30, and the second chamber 20 is not seeded, or is seeded with two-dimensional adherent cells simulating epithelial tissue: the two-dimensional adherent cells or three-dimensional cells simulating organ types seeded in the first chamber 10 are of the same or different organ types as those seeded in the third chamber 20. Thus, co-culture of multiple different organ types can be achieved.

[0240] Additionally, preferably, when at least one of the first chamber 10, the second chamber 20, and the third chamber 30 is inoculated with two-dimensional adherent cells or three-dimensional cells of a simulated organ type, and at least one of the third chambers is inoculated with two-dimensional adherent cells or three-dimensional cells of a simulated epithelial tissue:

[0241] Two-dimensional adherent cells simulating epithelial tissue can be inoculated first, followed by two-dimensional or three-dimensional adherent cells simulating organ types.

[0242] Alternatively, two-dimensional or three-dimensional adherent cells simulating organ types can be inoculated first, followed by two-dimensional adherent cells simulating epithelial tissue.

[0243] Furthermore, in one embodiment, the construction method further includes:

[0244] S600, cell culture.

[0245] Specifically, when any one of the first chamber 10, the second chamber 20, and the third chamber 30 is seeded with three-dimensional cells, it is sealed and a continuously flowing culture medium of the appropriate phase is introduced into the adjacent other chambers.

[0246] When the first chamber 10, the second chamber 20, and the third chamber 30 are all uninoculated with three-dimensional cells, a continuously flowing culture medium of the appropriate phase is introduced into each of the three chambers.

[0247] In other words, in step S600, the three-dimensional inoculation chamber is sealed; while the two-dimensional inoculation chamber or the uninoculated chamber is vented with culture medium.

[0248] This allows for the simulation of the in vivo microenvironment, thereby enabling the culture of inoculated three-dimensional cells and / or two-dimensional adherent cells.

[0249] Specifically, a suitable culture medium is used in step S600 according to the cell type inoculated in step S400.

[0250] The culture medium can specifically be a special culture medium.

[0251] For example, when a two-dimensional inoculation chamber is inoculated with two-dimensional adherent cells simulating epithelial tissue, in step S600, a special culture medium adapted to the two-dimensional adherent cells can be introduced into the two-dimensional inoculation chamber; further, for example, if the two-dimensional adherent cells are any one of vascular endothelial cells, human hepatobiliary epithelial cells, human pancreatic duct epithelial cells, human bronchial epithelial cells, gastric mucosal epithelial cells, intestinal mucosal epithelial cells, neuroepithelial cells, or any combination of any one with immune cells, then the appropriate special culture medium is any one of the following: vascular endothelial cell special culture medium, human hepatobiliary epithelial cell special culture medium, human pancreatic duct epithelial cell special culture medium, human bronchial epithelial cell special culture medium, gastric mucosal epithelial cell special culture medium, intestinal mucosal epithelial cell special culture medium, or neuroepithelial cell special culture medium.

[0252] For example, when a two-dimensional inoculation chamber is inoculated with two-dimensional adherent cells that simulate organ types, in step S600, a special culture medium adapted to the two-dimensional adherent cells can be introduced into the two-dimensional inoculation chamber; further, for example, if the two-dimensional adherent cells are human clonal colon adenocarcinoma cells (Caco-2 cells), human colorectal cancer cells (HTB-37 cells), human hepatic stellate cells (LX-2 cells), human liver cancer tissue cells (HepG2 cells), human alveolar epithelial cells (HPAEpiC), astrocytes, human gastric parietal cells, human gastric cancer cells (MGC-803 cells), or human pancreatic cancer cells (PANC-1 cells). For any type of cardiomyocyte, the appropriate culture medium can be any one of the following: human clonal colon adenocarcinoma cell (Caco-2 cells), human colorectal cancer cell (HTB-37 cells), human hepatic stellate cell (LX-2 cells), human liver cancer tissue cell (HepG2 cells), human alveolar epithelial cell (HPAEpiC) culture medium, astrocyte culture medium, human gastric parietal cell culture medium, human gastric cancer cell (MGC-803 cells), human pancreatic cancer cell (PANC-1 cells), or cardiomyocyte culture medium.

[0253] The specific components of these specialized culture media are common knowledge in the field and will not be elaborated upon.

[0254] Alternatively, the culture medium may specifically be a mixed culture medium.

[0255] In one embodiment, the mixed culture medium is adapted to two different types of epithelial cells, for example, it is a mixture of any two of the following: vascular endothelial cell culture medium, human hepatobiliary duct epithelial cell culture medium, human pancreatic duct epithelial cell culture medium, human bronchial epithelial cell culture medium, gastric mucosal epithelial cell culture medium, intestinal mucosal epithelial cell culture medium, and neuroepithelial cell culture medium.

[0256] In another embodiment, the mixed culture medium is adapted to two-dimensional adherent cells simulating epithelial tissue and two-dimensional adherent cells simulating organ types. For example, it is prepared by mixing any one or more of the following: vascular endothelial cell culture medium, human hepatobiliary duct epithelial cell culture medium, human pancreatic duct epithelial cell culture medium, human bronchial epithelial cell culture medium, gastric mucosal epithelial cell culture medium, intestinal mucosal epithelial cell culture medium, and neuroepithelial cell culture medium with organoid culture medium at a volume ratio of 0.1:1 to 1:0.1, and adding supplements and cell growth factors.

[0257] Preferably, the supplement is insulin-transferrin-selenium.

[0258] Preferably, the amount of the supplement added is 0.1% to 10% of the volume of the mixed culture medium.

[0259] Preferably, the cell growth factor is Culture Boost cell growth factor.

[0260] Preferably, the amount of cell growth factor added is 0.2% to 20% of the volume of the mixed culture medium.

[0261] Furthermore, in one embodiment, step S600 further includes:

[0262] Connect the chip substrate 100, one or more liquid storage chambers, and one or more peristaltic pumps via hoses and connectors.

[0263] Specifically, with Figure 2a and Figure 2b Taking the chip substrate 100 shown as an example, when the second chamber 20 constitutes a three-dimensional inoculation chamber and the first chamber 10 and the third chamber 30 constitute a two-dimensional inoculation chamber, a liquid storage chamber and a peristaltic pump can be used. The outlet of the liquid storage chamber is connected to the first inlet H12 after passing through the peristaltic pump, and the first outlet H11 is connected to the inlet of the liquid storage chamber. Thus, when the peristaltic pump is started, the culture medium in the liquid storage chamber enters the first chamber 10 through the first inlet H12, then enters the third chamber 30, and finally flows out through the first outlet H11 and returns to the liquid storage chamber.

[0264] Of course, two storage chambers and one peristaltic pump can also be used, with the outlet of storage chamber A connected to the first inlet H12 after passing through the peristaltic pump, and the first outlet H11 connected to the inlet of storage chamber B. Thus, when the peristaltic pump is started, the culture medium in storage chamber A enters the first chamber 10 through the first inlet H12, then enters the third chamber 30, and finally flows out through the first outlet H11 and returns to storage chamber B.

[0265] Again Figure 2a and Figure 2b Taking the chip substrate 100 shown as an example, when the second chamber 20 constitutes a two-dimensional inoculation chamber and the first chamber 10 and the third chamber 30 constitute a three-dimensional inoculation chamber, a storage chamber and a peristaltic pump can be used. The outlet of the storage chamber is connected to the second inlet H21 after passing through the peristaltic pump, and the second outlet H22 is connected to the inlet of the storage chamber. Thus, when the peristaltic pump is started, the culture medium in the storage chamber enters the second chamber 20 through the second inlet H21 and finally flows out through the second outlet H22 and returns to the storage chamber. Of course, two storage chambers and one peristaltic pump can also be used, and assembled into a flow path of storage chamber A, peristaltic pump, second inlet H21, second chamber 20, second outlet H22, and storage chamber B.

[0266] Again Figure 2a and Figure 2b Taking the chip substrate 100 shown as an example, when the first chamber 10, the second chamber 20 and the third chamber 30 all constitute a two-dimensional inoculation chamber, they can also be assembled into a flow path one consisting of a liquid storage chamber A, a peristaltic pump A, a second inlet H21, a second chamber 20, a second outlet H22, and a liquid storage chamber B (or a storage chamber A), and a flow path one consisting of a liquid storage chamber C, a peristaltic pump B, a first inlet H12, a first chamber 10, a third chamber 30, a first outlet H11, and a liquid storage chamber D (or a storage chamber C).

[0267] In another embodiment, multiple chip substrates 100 can be arranged in series or in parallel between the outlet and inlet of a liquid storage chamber, or in series or in parallel between two liquid storage chambers (e.g., between a first liquid storage chamber containing fresh culture medium and a second liquid storage chamber for recovering culture medium), thereby enabling co-culture of multiple organ types.

[0268] Furthermore, in step S600, the flow rate of the culture medium can be controlled to achieve various different culture modes. For example, in one embodiment, the flow rate of the culture medium is constant, maintained at 1 μL / h-200 μL / h, and can be further cyclically flowed, for example, the culture medium is introduced for 2 hours, and then the culture medium is stopped for 2 hours, and this is carried out in a cycle; in another embodiment, the flow rate of the culture medium is controlled to vary within 1 μL / h-200 μL / h, for example, the culture medium is introduced at 20 μL / h for 2 hours, and then the culture medium is introduced at 120 μL / h for 2 hours, and this is carried out in a cycle.

[0269] Furthermore, in one embodiment of the present invention, when two-dimensional adherent cells or three-dimensional cells simulating lung organ types are inoculated in the second chamber 20, step S600 includes:

[0270] The appropriate culture medium and air are alternately and periodically introduced into the first chamber 10, the second chamber 20 and / or the third chamber 30.

[0271] That is to say, step S600 includes several cycles; and in each cycle:

[0272] Culture medium is introduced into the first chamber 10, the second chamber 20 and / or the third chamber 30 without air, and maintained for a period of time, such as 2 hours, to simulate the internal microenvironment;

[0273] Afterwards, stop the flow of culture medium and introduce air into the first chamber 10, the second chamber 20 and / or the third chamber 30, and maintain this for a period of time, such as 2 hours, to simulate the stimulation of force.

[0274] In addition, when air is introduced into the first chamber 10, the second chamber 20 and / or the third chamber 30, the air pressure is adjusted to periodically fluctuate so that at least one of the first membrane 40 or the second membrane 50 undergoes expansion and contraction deformation.

[0275] For example, when inoculating three-dimensional cells simulating lung organs into the second chamber 20, a suitable culture medium and air can be alternately and periodically introduced into any one or both of the first chamber 10 and the third chamber 30; when inoculating two-dimensional adherent cells simulating lung organs into the second chamber 20, a suitable culture medium and air can be alternately and periodically introduced into any one or both of the first chamber 10, the second chamber 20 and the third chamber 30.

[0276] In one specific embodiment, in step S400, two-dimensional adherent cells or three-dimensional cells simulating organ types are seeded in the second chamber 20, and two-dimensional adherent cells simulating epithelial tissue are seeded in both the first chamber 10 and the third chamber 30.

[0277] Among them, the two-dimensional adherent cells of simulated epithelial tissue in chamber 10 are the first cell line, and the two-dimensional adherent cells of simulated epithelial tissue in chamber 30 are the second cell line.

[0278] Preferably, the first cell line is a first epithelial cell or is inoculated with a first epithelial cell and a first immune cell.

[0279] In other words, first epithelial cells are seeded on the surface of the first membrane 40 opposite to the second membrane 50 to form an epithelial layer, thereby simulating the epithelial microenvironment in the first chamber 10; or, first epithelial cells and first immune cells are seeded on the surface of the first membrane 40 opposite to the second membrane 50 to form an immune-functional epithelial layer, thereby simulating an immune-functional epithelial microenvironment in the first chamber 10.

[0280] Similarly, the second cell line is a second epithelial cell or is inoculated with a second epithelial cell and a second immune cell.

[0281] The first cell line and the second cell line can be set to be the same or different.

[0282] Specifically, the first epithelial cell and the second epithelial cell may be the same or different, and the first immune cell and the second immune cell may be the same or different.

[0283] For example, in one embodiment, the first cell line is a first epithelial cell and the second cell line is a second epithelial cell, wherein the first epithelial cell and the second epithelial cell may be the same or different.

[0284] Alternatively, in another embodiment, the first cell line is a first epithelial cell, and the second cell line is a second epithelial cell and a second immune cell, wherein the first epithelial cell and the second epithelial cell may be the same or different.

[0285] Alternatively, in another embodiment, the first cell line is a first epithelial cell and a first immune cell, and the second cell line is a second epithelial cell, wherein the first epithelial cell and the second epithelial cell may be the same or different.

[0286] Alternatively, in another embodiment, the first cell line is a first epithelial cell and a first immune cell, and the second cell line is a second epithelial cell and a second immune cell. The first epithelial cell and the second epithelial cell may be the same or different, and the first immune cell and the second immune cell may be the same or different.

[0287] When the first cell line consists of first epithelial cells and first immune cells, the first epithelial cells are first inoculated on the surface of the first membrane 40 opposite to the second membrane 50, and then the first immune cells are inoculated.

[0288] Similarly, when the second cell line is a second epithelial cell and a second immune cell, the second epithelial cell is first inoculated on the surface of the second membrane 50 opposite to the first membrane 40, and then the second immune cell is inoculated.

[0289] The construction method of the first embodiment of the present invention has been described above. Below, the present invention also provides a second embodiment of the construction method.

[0290] The difference between the second embodiment and the first embodiment described above is that: the channel 60 is eliminated, the first chamber 10 and the third chamber 30 are independent of each other (and are no longer interconnected as in the first embodiment); and the liquid injection / flow method in steps S400 and S600 related thereto.

[0291] The following is a detailed explanation of these differences.

[0292] First, see Figures 3a to 3c The diagram shows the structure of the chip substrate 100a constructed in step S200 of the construction method of the second embodiment of the present invention. For components / structures with the same names as those in the first embodiment, they are marked with an additional "a" or "'" in the drawings of the second embodiment.

[0293] In the first embodiment, the first chamber 10 and the third chamber 30 are interconnected; in contrast, in the second embodiment, the first chamber 10a and the third chamber 30a are independent of each other.

[0294] Here, "the first chamber 10a and the third chamber 30a are independent of each other" means that the first chamber 10a is structurally separated from the third chamber 30a. Thus, in this embodiment, the first chamber 10a, the second chamber 20a and the third chamber 30a can each independently circulate the required substances without interfering with each other.

[0295] Specifically, in the first embodiment, the first chamber 10 and the third chamber 30 are interconnected through a passage 60; in contrast, the passage 60 is omitted in the second embodiment.

[0296] Furthermore, in the first embodiment, the first chamber 10 and the third chamber 30 introduce fluid (such as cell suspension to be inoculated, culture medium, surface modification solution, etc.) through a common first inlet H12 (or third inlet H32); in contrast, in the second embodiment, the chip substrate 100a includes a first outlet H11', a first inlet H12', a third outlet H31' and a third inlet H32'.

[0297] Specifically, in this embodiment, the first chamber 10a includes a first region A1' that overlaps with the second chamber 20a and two second regions A2' that protrude from the second chamber 20a. The two second regions A2' are located at opposite ends of the first chamber 10a in a first direction.

[0298] The first outlet H11' and the first inlet H12' are connected to the first chamber 10a, specifically to the opposite ends of the first chamber 10a in the first direction, such as the second region A2'.

[0299] Similarly, the third chamber 30a includes a third region A3' that overlaps with the second chamber 20a and two fourth regions A4' that protrude from the second chamber 20a, the two fourth regions A4' being located at opposite ends of the third chamber 30a in the first direction.

[0300] The third outlet H31' and the third inlet H32' connect to the third chamber 30a, specifically to the opposite ends of the third chamber 30a in the first direction, such as the fourth area A4'.

[0301] Here, the third outlet H31' and the third inlet H32' both penetrate the first partition 101a. The second partition 102a has a second through hole P2 and a third through hole P3 that are independent of the second chamber 20a. The third outlet H31' is connected to the fourth region A4' of the third chamber 30a through the second through hole P2, and the third inlet H32' is connected to the fourth region A4' of the third chamber 30a through the third through hole P3.

[0302] Corresponding to the structure of the chip substrate 100a, in this second embodiment, when the support surface (i.e. the surface of the first membrane 40a facing away from the second membrane 50a) in the first chamber 10a is surface modified, or when two-dimensional adherent cells are seeded in the first chamber 10a, or when three-dimensional cells are seeded in the first chamber 10a, or when culture medium is introduced into the first chamber 10a, the relevant fluids, such as surface modification solution, or two-dimensional adherent cell suspension, or seeding solution, or culture medium, enter the first chamber 10a from the first inlet H12' and can then leave the first chamber 10a through the first outlet H11' without entering the third chamber 30a.

[0303] Similarly, when the support surface in the third chamber 30a (i.e. the surface of the second membrane 50a facing away from the first membrane 40a) is modified, or when the third chamber 30a is seeded with two-dimensional adherent cells, or when the third chamber 30a is seeded with three-dimensional cells, or when culture medium is introduced into the third chamber 30a, the relevant fluids, such as surface modification solution, or two-dimensional adherent cell suspension, or seeding solution, or culture medium, enter the third chamber 30a from the third inlet H32' and can then leave the third chamber 30a through the third outlet H31' without flowing through the first chamber 10a.

[0304] In particular, when two-dimensional adherent cells are seeded in the first chamber 10a or the third chamber 30a, the two-dimensional adherent cell suspension is filled into the corresponding first chamber 10a or the third chamber 30a in an upflow manner; then the corresponding first chamber 10a or the third chamber 30a is closed, and the chip substrate 100a is positioned with the support surface facing upwards for cell seeding.

[0305] Specifically, when filling the corresponding first chamber 10a with a two-dimensional adherent cell suspension in an upflow manner, the operation can be as follows: the chip substrate 100a is positioned such that the first inlet H12' is at the bottom of the first chamber 10a and the first outlet H11' is at the top of the first chamber 10a (for example, the chip substrate 100a is placed vertically), and the two-dimensional adherent cell suspension is injected into the first chamber 10a from the first inlet H12' until the liquid level reaches the first outlet H11'.

[0306] When filling the corresponding third chamber 30a with a two-dimensional adherent cell suspension in an upflow manner, the specific operation can be as follows: Position the chip substrate 100a such that the third inlet H32' is at the bottom of the third chamber 30a and the third outlet H31' is at the top of the third chamber 30a (for example, the chip substrate 100a is placed vertically), and inject the two-dimensional adherent cell suspension into the third chamber 30a from the third inlet H32' until the liquid level reaches the third outlet H31'.

[0307] Corresponding to the appendix Figure 3a In the chip substrate 100a, when the second chamber 20a constitutes a three-dimensional inoculation chamber and the first chamber 10a and the third chamber 30a constitute a two-dimensional inoculation chamber, the specific operation of step S600 can be as follows:

[0308] The first universal culture medium is placed in the first storage container, and the outlet of the first storage container is connected to the first inlet H12' through the first inlet pipe. The first universal culture medium can be pumped into the first chamber 10a through the first inlet H12', and the first outlet H11' is connected to the first outlet pipe to discharge the first universal culture medium from the first chamber 10a; that is, a path is formed of the first storage container, the first inlet H12', the first chamber 10a, and the first outlet H11'.

[0309] Furthermore, the second universal culture medium is placed in the second storage container, and the outlet of the second storage container is connected to the third inlet H32' through the second inlet pipe. The second universal culture medium can be pumped into the third chamber 30a through the third inlet H32', and the third outlet H31' is connected to the third outlet pipe to discharge the second universal culture medium from the third chamber 30a; that is, a path three is formed, consisting of the second storage container, the third inlet H32', the third chamber 30a, and the third outlet H31'.

[0310] Of course, the first outlet pipe can be connected to the first storage container, and the second outlet pipe can be connected to the second storage container to realize the recycling of the culture medium; or, the first outlet pipe and the second outlet pipe can be connected to a separate or shared collection container to recycle the culture medium.

[0311] In one alternative implementation, the second liquid storage container may be omitted, and the first outlet H11' may be connected to the third inlet H32', while the third outlet H31' may be connected to the first liquid storage container or other separate collection container through the third liquid outlet pipeline.

[0312] When the first chamber 10a, the second chamber 20a, and the third chamber 30a all constitute a two-dimensional inoculation chamber, in addition to the above-mentioned path one and path three, path two can also be assembled into a third liquid storage container, a second inlet H21', a second chamber 20a, a second outlet H22', and a third liquid storage container (or collection container).

[0313] In another embodiment, multiple chip substrates 100a can be arranged in series or in parallel between the outlet and inlet of a liquid storage chamber, or in series or in parallel between two liquid storage chambers (e.g., between a first liquid storage chamber containing fresh culture medium and a second liquid storage chamber for recovering culture medium), thereby enabling co-culture of multiple organ types.

[0314] Furthermore, based on the chip substrate 100a of this embodiment, the flow rate control of the culture medium is the same as that of the first embodiment described above, which can be constant flow rate, periodic flow, or flow rate variation.

[0315] In addition, in this second embodiment, when two-dimensional adherent cells or three-dimensional cells simulating lung organ types are seeded in the second chamber 20, step S600 further includes:

[0316] A suitable culture medium and air are periodically introduced into one or both of the first chamber 10a and the third chamber 30a.

[0317] Specifically, for example, it could be: periodically introducing a compatible culture medium (e.g., the first universal culture medium) and air into the first chamber 10a alternately, and continuously introducing a compatible culture medium (e.g., the second universal culture medium) into the third chamber 30a.

[0318] Alternatively, a suitable culture medium (e.g., the first universal culture medium) may be continuously introduced into the first chamber 10a, while a suitable culture medium (e.g., the second universal culture medium) and air may be alternately and periodically introduced into the third chamber 30a.

[0319] Alternatively, it can be done by alternately and periodically introducing a suitable culture medium (e.g., the first universal culture medium) and air into the first chamber 10a, and alternately and periodically introducing a suitable culture medium (e.g., the second universal culture medium) and air into the third chamber 30a.

[0320] Here, the phrase "alternatingly and periodically introducing a suitable culture medium (e.g., the first universal culture medium) and air into the first chamber 10a" refers to the following process in each of several cycles: introducing culture medium into the first chamber 10a without introducing air, maintaining this for a period of time, for example, 2 hours, to simulate the internal microenvironment; then stopping the introduction of culture medium into the first chamber 10a and introducing air, maintaining this for a period of time, for example, 2 hours, to simulate the stimulation of force.

[0321] The phrase "alternatingly and periodically introducing a suitable culture medium (e.g., the second universal culture medium) and air into the third chamber 30a" refers to the following process in each of several cycles: introducing culture medium into the third chamber 30a without introducing air, maintaining this for a period of time, for example, 2 hours, to simulate the internal microenvironment; then stopping the introduction of culture medium into the third chamber 30a and introducing air, maintaining this for a period of time, for example, 2 hours, to simulate the stimulation of force.

[0322] In addition, when air is introduced into either the first chamber 10a or the third chamber 30a, the air pressure is adjusted to periodically fluctuate so that the first membrane 40a or the second membrane 50a undergoes expansion and contraction deformation.

[0323] The differences between the second embodiment and the first embodiment have been described above. Apart from these, other technologies of the second embodiment (such as those described in the first embodiment but not mentioned in the above description of the second embodiment) are the same as those of the first embodiment and can be understood by referring to the first embodiment. They will not be repeated here.

[0324] The following are some specific application examples using the described construction method. These examples can be implemented using... Figure 2a The chip substrate 100 shown can be used to implement this, or it can be implemented using... Figure 3a The chip substrate 100 shown is implemented accordingly.

[0325] Inoculation of first and second cell lines

[0326] Specifically, in these application examples, the techniques of any of the embodiments described in the first and second embodiments above can be used to seed the first cell line B (specifically including the first epithelial cells B1 and the first immune cells B2) onto the surface of the first membrane 40 (or 40a) opposite to the second membrane 50 (or 50a), and the second cell line C (specifically including the second epithelial cells C1 and the second immune cells C2) onto the surface of the second membrane 50 (or 50a) opposite to the first membrane 40 (or 40a), as shown in Table 1.

[0327] Table 1 (where " / " indicates that no corresponding cell seeding was performed)

[0328]

[0329]

[0330] Inoculation of three-dimensional cells simulating organ types

[0331] Specifically, the techniques described in any of the first and second embodiments above can be used to inoculate three-dimensional cells of simulated organ types into the second chamber 20 (or 20a), and the organ type D from which the cells originate is shown in Table 2.

[0332] Table 2

[0333] D Example 1F Heart organoids Example 2F liver organoids Example 3F Organoids from liver cancer Example 4F gastric organoids Example 5F Gastric cancer organoids Example 6F Pancreatic organoids Example 7F Pancreatic cancer organoids Example 8F Lung organoids Example 9F Lung cancer organoids Example 10F Intestinal organoids Example 11F Colorectal cancer organoids Example 12F Brain organoids

[0334] As described above, a series of specific application examples employing the first and second embodiments of the construction method are provided herein, including:

[0335] The first group consists of application examples formed by combinations of each of Examples 1F to 12F and each of Examples 1A to 4A; wherein, for example, the application example formed by the combination of Examples 1F and 2A refers to an application example in which HUVEC and U-937 are seeded on the surface of the first membrane 40 (or 40a) opposite to the second membrane 50 (or 50a), HUVEC is seeded on the surface of the second membrane 50 (or 50a) opposite to the first membrane 40 (or 40a), and three-dimensional cells simulating the heart are seeded in the second chamber 20 (or 20a).

[0336] The second group consists of combinations of Examples 2F and 3F with each of Examples 5A to 9A, forming application examples; among them, the organ-on-a-chip constructed by this group of application examples is a liver organ-on-a-chip or a liver cancer organ-on-a-chip.

[0337] The third group consists of combinations of Examples 4F and 5F with each of Examples 10A to 14A, forming application examples; among them, the organ-on-a-chip constructed by this group of application examples is a gastric organ-on-a-chip or a gastric cancer organ-on-a-chip.

[0338] The fourth group consists of combinations of Examples 6F and 7F with each of Examples 15A to 19A, forming application examples; among them, the organ-on-a-chip constructed in this group of application examples is a pancreatic organ-on-a-chip or a pancreatic cancer organ-on-a-chip.

[0339] The fifth group consists of combinations of Examples 8F and 9F with each of Examples 20A to 24A, forming application examples; among them, the organ-on-a-chip constructed by this group of application examples is a lung organ-on-a-chip or a lung cancer organ-on-a-chip.

[0340] The sixth group consists of combinations of Examples 10F and 11F with each of Examples 25A to 29A, forming application examples; among them, the organ-on-a-chip constructed by this group of application examples is an intestinal organ-on-a-chip or an intestinal cancer organ-on-a-chip.

[0341] The sixth group consists of combinations of each of Examples 12F and Examples 30A to 34A, forming application examples; among them, the organ-on-a-chip constructed by this group of application examples is a brain organoid chip.

[0342] Seeding of two-dimensional adherent cells simulating organ types

[0343] Specifically, the techniques described in any of the first and second embodiments above can be used to seed two-dimensional adherent cells of simulated organ types into the second chamber 20 (or 20a), including two-dimensional adherent cells D1 seeded on the surface of the first membrane 40 (or 40a) facing the second membrane 50 (or 50a) and two-dimensional adherent cells D2 seeded on the surface of the second membrane 50 (or 50a) facing the first membrane 40 (or 40a), as shown in Table 3.

[0344] Table 3

[0345]

[0346]

[0347] As described above, a series of specific application examples employing the first and second embodiments of the construction method are provided herein, including:

[0348] The first group consists of application examples formed by combinations of each of Examples 1G to 29G and each of Examples 1A to 4A; among which, for example, the application example formed by the combination of Examples 2G and 2A refers to the application example of seeding HUVEC and U-937 on the surface of the first membrane 40 (or 40a) away from the second membrane 50 (or 50a), seeding HUVEC on the surface of the second membrane 50 (or 50a) away from the first membrane 40 (or 40a), seeding human fibroblasts on the surface of the first membrane 40 (or 40a) facing the second membrane 50 (or 50a), and seeding cardiomyocytes on the surface of the second membrane 50 (or 50a) facing the first membrane 40 (or 40a).

[0349] The second group consists of combinations of each of Examples 5G to 9G and each of Examples 5A to 9A, forming application examples; among which, the organ-on-a-chip constructed by this group of application examples is a liver organ-on-a-chip or a liver cancer organ-on-a-chip.

[0350] The third group consists of combinations of each of Examples 10G to 14G and each of Examples 10A to 14A, forming application examples; among which, the organ-on-a-chip constructed by this group of application examples is a gastric organ-on-a-chip or a gastric cancer organ-on-a-chip.

[0351] The fourth group consists of combinations of each of Examples 15G to 19G and each of Examples 15A to 19A, forming application examples; among which, the organ-on-a-chip constructed by this group of application examples is a pancreatic organ-on-a-chip or a pancreatic cancer organ-on-a-chip.

[0352] The fifth group consists of combinations of each of Examples 20G to 21G and each of Examples 20A to 24A, forming application examples; among which, the organ-on-a-chip constructed by this group of application examples is a lung organ-on-a-chip or a lung cancer organ-on-a-chip.

[0353] The sixth group consists of combinations of each of Examples 22G to 25G and each of Examples 25A to 29A, forming application examples; wherein the organ-on-a-chip constructed by this group of application examples is an intestinal organ-on-a-chip or an intestinal cancer organ-on-a-chip.

[0354] The sixth group consists of combinations of each of Examples 26G to 29G and each of Examples 30A to 34A, forming application examples; among them, the organ-on-a-chip constructed by this group of application examples is a brain organoid chip.

[0355] In summary, the beneficial effects of the present invention are as follows: the construction method constructs a chip substrate with a structure of a first chamber, a first membrane, a second chamber, a second membrane, and a third chamber stacked sequentially. More types of cells can be seeded in the first, third, and second chambers to simulate more types of cell growth microenvironments and construct a more complex co-culture model. At the same time, the first, second, and third chambers can provide a more comprehensive and balanced culture medium or mechanical stimulation for the three-dimensional growth space. In short, this embodiment can simulate a more complex in vivo cell growth environment, or in other words, the environment constructed in this embodiment is closer to the actual in vivo cell growth environment.

[0356] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0357] The detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing an organ-on-a-chip, characterized in that, The construction method includes: Construct a chip substrate consisting of a first chamber, a first membrane, a second chamber, a second membrane, and a third chamber stacked sequentially; Two-dimensional adherent cells or three-dimensional cells are inoculated in at least one of the first, second, and third chambers.

2. The method for constructing an organ-on-a-chip according to claim 1, characterized in that, The step "seeding two-dimensional adherent cells or three-dimensional cells in at least one of the first, second, and third chambers" includes: In two adjacent chambers of the first, second, and third chambers, one is inoculated with two-dimensional or three-dimensional adherent cells simulating organ types, and the other is inoculated with two-dimensional adherent cells simulating epithelial tissue.

3. The method for constructing an organ-on-a-chip according to claim 1 or 2, characterized in that, The step "seeding two-dimensional adherent cells or three-dimensional cells in at least one of the first, second, and third chambers" includes: Two-dimensional adherent cells or three-dimensional cells simulating organ types are inoculated in the second chamber, and two-dimensional adherent cells simulating epithelial tissue are either not inoculated in the first and third chambers, or are selectively inoculated in one or both; or, Two-dimensional adherent cells or three-dimensional cells simulating organ types are inoculated in either the first or third chamber, or in both. In the second chamber, no cells are inoculated, or two-dimensional adherent cells simulating epithelial tissue are inoculated.

4. The method for constructing an organ-on-a-chip according to claim 3, characterized in that, First, inoculate with two-dimensional adherent cells that simulate epithelial tissue, then inoculate with two-dimensional adherent cells or three-dimensional cells that simulate organ types; Alternatively, two-dimensional or three-dimensional adherent cells simulating organ types can be inoculated first, followed by two-dimensional adherent cells simulating epithelial tissue.

5. The method for constructing an organ-on-a-chip according to claim 3, characterized in that, Two-dimensional adherent cells simulating epithelial tissue are set as epithelial cells, or a combination of epithelial cells and immune cells; When two-dimensional or three-dimensional adherent cells of simulated organ types are inoculated in the second chamber, and two-dimensional adherent cells of simulated epithelial tissue are inoculated in both the first and third chambers: the two-dimensional adherent cells of simulated epithelial tissue inoculated in the first chamber are the first cell line, and the two-dimensional adherent cells of simulated epithelial tissue inoculated in the third chamber are the second cell line. The first cell line is a first epithelial cell or a combination of a first epithelial cell and a first immune cell; The second cell line is a combination of second epithelial cells or second immune cells; The first epithelial cell and the second epithelial cell may be the same or different, and the first immune cell and the second immune cell may be the same or different.

6. The method for constructing an organ-on-a-chip according to claim 3, characterized in that, When two-dimensional adherent cells or three-dimensional cells of simulated organ types are inoculated in both the first and third chambers, and two-dimensional adherent cells of simulated epithelial tissue are not inoculated in the second chamber, or are inoculated in the second chamber: The two-dimensional or three-dimensional adherent cells of the simulated organ type inoculated in the first chamber may be the same or different organ types as the two-dimensional or three-dimensional adherent cells of the simulated organ type inoculated in the third chamber.

7. The method for constructing an organ-on-a-chip according to claim 1, characterized in that, When two-dimensional adherent cells are seeded in the first chamber, the surface of the first membrane facing away from the second membrane is used as the supporting surface. When seeding two-dimensional adherent cells in the third chamber, the surface of the second membrane facing away from the first membrane is used as the support surface; When two-dimensional adherent cells are seeded in the second chamber, the surface of the first membrane facing the second membrane is used as the supporting surface, and / or the surface of the second membrane facing the first membrane is used as the supporting surface. In the step "Seedling two-dimensional adherent cells or three-dimensional cells in at least one of the first, second, and third chambers": Two-dimensional adherent cell suspension is filled into the first, second, or third chamber to be inoculated in an upflow manner in the first direction, and then sealed. The chip substrate is positioned with the support surface facing upward and immersed in the corresponding two-dimensional adherent cell suspension so as to seed the two-dimensional adherent cells on the support surface. The first direction is parallel to the first membrane.

8. The method for constructing an organ-on-a-chip according to claim 1, characterized in that, The first chamber and the third chamber are connected to each other via a passage, and the second chamber is independent of the first chamber and the third chamber; The chip substrate also includes a first inlet, a first outlet, a second inlet, and a second outlet; The first inlet and the first end of the channel are respectively connected to the opposite ends of the first chamber in the first direction, and the first outlet and the second end of the channel are respectively connected to the opposite ends of the third chamber in the first direction; In the first direction, the first inlet and the first outlet are located at the same end of the chip substrate; The second inlet and the second outlet are respectively connected to the two opposite ends of the second chamber in the first direction.

9. The method for constructing an organ-on-a-chip according to claim 1, characterized in that, The first chamber, the second chamber, and the third chamber are independent of each other; The chip substrate also includes a first inlet, a first outlet, a second inlet, a second outlet, a third inlet, and a third outlet; The first inlet and the first outlet are respectively connected to the two opposite ends of the first chamber in the first direction; The third inlet and the third outlet are respectively connected to the opposite ends of the third chamber in the first direction; The second inlet and the second outlet are respectively connected to the two opposite ends of the second chamber in the first direction.

10. The method for constructing an organ-on-a-chip according to claim 1, characterized in that, In the step "Seedling two-dimensional adherent cells or three-dimensional cells in at least one of the first, second, and third chambers": Two-dimensional adherent cells or three-dimensional cells simulating lung organ types were inoculated in the second chamber; After step "inoculating at least one of the first, second, and third chambers with two-dimensional adherent cells or three-dimensional cells", a suitable culture medium and air are alternately and periodically introduced into the first, second, and / or third chambers, and the air pressure is adjusted to periodically fluctuate so that the first and / or second membranes undergo stretching and deformation.

Images