Intestinal Organ-on-a-Chip with Enhanced Barrier Function and Its Applications

Abstract

The present invention provides an intestinal organoid chip with enhanced barrier function and its application. The enhanced intestinal organoid chip includes at least one structural unit, and the structural unit includes: an upper sub-chip, a porous membrane, and a lower sub-chip; wherein, the upper sub-chip includes: a first flow channel, and the first flow channel is adapted to accommodate intestinal organoids and a first culture medium; the lower sub-chip includes: a second flow channel, and the second flow channel is adapted to accommodate endothelial cells and a second culture medium; the porous membrane is disposed above the second flow channel and covers at least a part of the bottom of the first flow channel; the projection of the first flow channel towards the lower sub-chip passes through the porous membrane and at least partially overlaps with the second flow channel. Among them, the width of the first flow channel is greater than the width of the second flow channel. The enhanced intestinal organoid chip of the present invention can be used to study the process of pathogen infection and pathogenic mechanism, and provides a research platform with enhanced barrier function for new therapies for intestinal-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biology. Specifically, the present invention relates to an intestinal organ-on-a-chip with enhanced barrier function and its applications. Background Art

[0002] The intestine is an important digestive and absorptive organ in the human body, participating in processes such as nutrient absorption, drug metabolism, immune system maturation, and defense against pathogenic bacteria. Research shows that the intestine also plays a central role in the normal functions of other organs and the etiology of many diseases. The barrier function of the intestine is important in restricting the entry and exit of compounds from the digestive system, blocking the passage of harmful substances, and the spread of pathogens; the intestine is also an important part of the immune and endocrine systems. Digestive, immune, or endocrine defects in the intestine can lead to the occurrence of various organ diseases outside the intestine. Therefore, the research on the intestine has very important significance and value.

[0003] Currently, in the research of human intestinal physiology and drug development, the commonly used models are still in vitro cell culture models and mammalian models. Although in vitro cell culture technology has made considerable progress, the existing two-dimensional cell culture models still cannot fully simulate the intestinal structure and intestinal microenvironment, and it is difficult to achieve the complex physiological functions of the intestine. At the same time, animal experiments require a long period, high costs, and there are significant differences in the physiological states between animals and humans, making it difficult to accurately predict the physiological responses of the human intestine. To better simulate the complex structure and functions of the intestine and overcome the limitations of existing cell culture and animal models, currently, microsystem engineering and microfabrication technologies are applied to cell biology research, and a human intestinal physiological system is constructed in a microfluidic organ-on-a-chip, developing the intestinal chip technology.

[0004] However, most of the existing chips currently are mainly for culturing 2D cell lines. Although they are simple to operate and low in cost, in drug screening, due to the lack of tissue structure and its complexity in 2D culture, the constructed models have insufficient physiological relevance and are relatively limited in simulating the real disease environment and complex cell interactions. This may be an important reason why the drug screening results cannot be reproduced in the in vivo environment many times. In addition, the existing chips are extremely prone to problems such as curling and shedding of the monolayer intestinal epithelial cells or intestinal organoids at the edges on the porous membrane during the experimental operation process, and are extremely prone to leakage, resulting in an incomplete intestinal barrier being constructed, causing imperfect experimental data such as subsequent toxicity tests and inaccurate experimental results.

[0005] Therefore, there is an urgent need to develop a chip that can highly simulate in vivo tissues and ensure the integrity of the intestinal barrier. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] Based on this, the present invention designs a double-channel enhanced barrier function intestinal organoid chip with a wider upper part and a narrower lower part. The enhanced intestinal organoid chip uses human intestinal organoids to construct a model in the upper sub-chip, which can simulate real organs to the greatest extent in terms of structure and function, simulate in-vivo tissues and physiological functions. The intestinal organoids can also generate microstructures such as intestinal crypts and villus-like epithelia, and can reproduce physiological responses with high fidelity in vitro. At the same time, endothelial cells are inoculated in the lower sub-chip to simulate a monolayer blood vessel, integrating a vascularized intestinal model in vitro. This chip can simulate the interaction between pathogens and the human intestine in vitro, can be used to study the process of pathogen infection and pathogenic mechanisms, and provides a research platform with higher simulation degree for developing new therapies for intestinal-related diseases. At the same time, it is more suitable for studying the intestinal metabolism of drugs and can better predict human pharmacokinetic parameters.

[0008] Therefore, in the first aspect of the present invention, the present invention proposes an enhanced intestinal organoid chip. According to an embodiment of the present invention, the chip includes at least one structural unit, and the structural unit includes: an upper sub-chip, a porous membrane, and a lower sub-chip; wherein, the upper sub-chip includes: a first flow channel, and the first flow channel is adapted to accommodate intestinal organoids and a first culture medium; the lower sub-chip includes: a second flow channel, and the second flow channel is adapted to accommodate endothelial cells and a second culture medium; the porous membrane is disposed above the second flow channel and covers at least a part of the bottom of the first flow channel; the projection of the first flow channel towards the lower sub-chip passes through the porous membrane and at least partially overlaps with the second flow channel.

[0009] The structural unit of the enhanced intestinal organ-on-a-chip according to an embodiment of the present invention includes two layers of sub-chips, which are separated by a porous membrane. The upper sub-chip is used for culturing intestinal organoids. By using the intestinal organoid construction model, it can simulate real organs to the greatest extent in terms of structure and function, simulate in-vivo tissues and physiological functions. The intestinal organoids can also generate microstructures such as intestinal crypts and villus-like epithelia, and can reproduce physiological responses in vitro with high fidelity; the lower sub-chip is used for culturing endothelial cells to construct a single-layer blood vessel; the design of the porous membrane can, on the one hand, play a supporting role, enabling the intestinal organoids and endothelial cells to grow on both sides of the porous membrane respectively. On the other hand, the porous membrane allows soluble molecules to be transported between the simulated intestine and blood vessel. Thus, by using the enhanced intestinal organ-on-a-chip of the present invention, a vascularized intestinal model can be obtained in vitro, which can simulate the interaction between pathogens and the human intestine in vitro, and can be used to study the pathogen infection process and pathogenic mechanism, providing a research platform with higher simulation degree for developing new therapies for intestine-related diseases. At the same time, it is more suitable for studying the intestinal metabolism of drugs and can better predict human pharmacokinetic parameters. Moreover, the enhanced intestinal organ-on-a-chip of the present invention can be designed with multiple structural units to achieve high-throughput implementation. The enhanced intestinal organ-on-a-chip of the present invention enables the chip sampling to be matched with automated equipment through an open design, realizing automated sampling and being suitable for large-scale popularization and application.

[0010] According to an embodiment of the present invention, the enhanced intestinal organ-on-a-chip may further include at least one of the following additional technical features:

[0011] According to an embodiment of the present invention, the width of the first flow channel is greater than the width of the second flow channel.

[0012] According to an embodiment of the present invention, the width of the first flow channel is 1.2 mm to 1.6 mm, and the width of the second flow channel is 0.9 mm to 1.1 mm.

[0013] According to an embodiment of the present invention, the upper sub-chip further includes a first flow channel inlet and a first flow channel outlet, and the first flow channel inlet and the first flow channel outlet are respectively located at both ends of the first flow channel.

[0014] According to an embodiment of the present invention, the lower sub-chip further includes a second flow channel inlet and a second flow channel outlet, and the second flow channel inlet and the second flow channel outlet are respectively located at both ends of the second flow channel.

[0015] According to an embodiment of the present invention, the upper sub-chip and the lower sub-chip respectively include at least two bonding positioning holes, and the projection of the bonding positioning holes of the upper sub-chip facing the lower sub-chip corresponds one by one to the bonding positioning holes on the lower sub-chip.

[0016] According to an embodiment of the present invention, the porous membrane is selected from a PETE porous membrane, and the pore density is (1-10)×10 5 pores / cm 2 .

[0017] In a second aspect of the present invention, the present invention provides an enhanced intestinal organoid construction system. According to an embodiment of the present invention, the system includes: the enhanced intestinal organoid chip described in the first aspect; a first liquid storage device connected to the enhanced intestinal organoid chip and adapted to inject a third culture solution into the first flow channel; a second liquid storage device connected to the enhanced intestinal organoid chip and adapted to inject a fourth culture solution into the second flow channel.

[0018] Using the enhanced intestinal organoid construction system of the present invention, the prepared enhanced intestinal organoids can simulate real organs to the greatest extent in terms of structure and function, and simulate in vivo tissues and physiological functions. The prepared enhanced intestinal organoid model can simulate the interaction between pathogens and the human intestine in vitro, and can be used to study the process of pathogen infection and pathogenic mechanisms, providing a research platform with higher simulation degree for the development of new therapies for intestinal-related diseases.

[0019] According to an embodiment of the present invention, the enhanced intestinal organoid construction system may further include at least one of the following additional technical features:

[0020] According to an embodiment of the present invention, the first liquid storage device is connected to the first flow channel inlet and / or the second liquid storage device is connected to the second flow channel inlet through a pump.

[0021] According to an embodiment of the present invention, the pump includes a peristaltic pump.

[0022] According to an embodiment of the present invention, the enhanced intestinal organoid chip construction system further includes: a shaking culture device adapted to shake the enhanced intestinal organoid chip. Preferably, the shaking culture device includes a shaking table.

[0023] In a third aspect of the present invention, the present invention provides a method for constructing an enhanced intestinal organoid chip using the enhanced intestinal organoid chip described in the first aspect or the enhanced intestinal organoid construction system described in the second aspect. According to an embodiment of the present invention, the method includes: injecting endothelial cells and a second culture solution into the second flow channel and performing a first culture treatment; after the human umbilical vein endothelial cells form blood vessels, injecting human intestinal organoids and a first culture solution into the first flow channel and performing a second culture treatment.

[0024] The enhanced intestinal organoids constructed by using the present invention can highly simulate real intestinal organs in terms of structure and function, and can generate microstructures such as intestinal crypts and villus-like epithelia, so as to highly reproduce the physiological responses of the intestine in vitro. This research platform with higher simulation degree can simulate the interaction between pathogens and the human intestine in vitro, providing a more reliable and real platform for studying the pathogen infection process and pathogenic mechanism, as well as developing new therapies for intestinal-related diseases.

[0025] According to an embodiment of the present invention, after the second culture treatment, it further includes: after the second culture treatment ends, discard the first culture medium, inject a fifth culture medium into the first flow channel, and perform a third culture treatment; after the third culture treatment ends, discard the fifth culture medium, inject a sixth culture medium into the first flow channel, and perform a fourth culture treatment; after the fourth culture treatment ends, discard the sixth culture medium, inject a seventh culture medium into the first flow channel, and perform a fifth culture treatment.

[0026] According to an embodiment of the present invention, during the first culture treatment, the third culture treatment, the fourth culture treatment, and the fifth culture treatment, start the shaking culture device to shake the enhanced organoid chip; and / or start the peristaltic pump between the first liquid storage device and the first flow channel to circulate and inject a proliferation culture medium, a shaping culture medium or a maturation culture medium into the first flow channel and / or start the peristaltic pump between the second liquid storage device and the second flow channel to circulate and inject a second culture medium into the second flow channel.

[0027] In the fourth aspect of the present invention, the present invention provides an enhanced intestinal organoid. According to an embodiment of the present invention, the enhanced intestinal organoid is obtained by the method described in the third aspect. The intestinal organoids of the present invention can highly simulate real intestinal organs in terms of structure and function, and can generate microstructures such as intestinal crypts and villus-like epithelia, so as to highly reproduce the physiological responses of the intestine in vitro. This research platform with higher simulation degree can simulate the interaction between pathogens and the human intestine in vitro, providing a more reliable and real platform for studying the pathogen infection process and pathogenic mechanism, as well as developing new therapies for intestinal-related diseases.

[0028] In the fifth aspect of the present invention, the present invention provides the application of the enhanced intestinal organoid chip described in the first aspect, the enhanced intestinal organoid construction system described in the second aspect, or the enhanced intestinal organoid described in the fourth aspect in constructing an intestinal model, drug screening, pathological model simulation, biomaterial evaluation, immune co-culture, tumor infiltration or migration.

[0029] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0030] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0031] Figure 1 is a schematic diagram of the structural unit of an enhanced intestinal organoid chip according to an embodiment of the present invention;

[0032] Figure 2 is a schematic diagram of the modular and high-throughput well-plate design of the enhanced intestinal organoid chip unit according to an embodiment of the present invention;

[0033] Figure 3 is a schematic diagram of the enhanced intestinal organoid chip simulating the intestinal structure and function according to an embodiment of the present invention;

[0034] Figure 4 is a physical diagram of the supporting fluid device shaker according to an embodiment of the present invention;

[0035] Figure 5 is a diagram of the intestinal model constructed using an enhanced upper-wide and lower-narrow intestinal organoid chip according to an embodiment of the present invention;

[0036] Figure 6 is a diagram of the villous structure and immunofluorescence staining of the intestinal organoid according to an embodiment of the present invention;

[0037] Figure 7 is a diagram of the intestinal model constructed using a chip with the same width of the upper and lower flow channels according to an embodiment of the present invention. Detailed Description of the Embodiments

[0038] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as limiting the present invention.

[0039] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0040] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0041] In this text, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0042] In this text, it should be understood that the orientation or positional relationship indicated by terms such as "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0043] In this text, unless otherwise clearly specified and defined, terms such as "installed", "connected", "joined", "fixed", etc. should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements or the interaction relationship between two elements, unless otherwise clearly defined. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0044] In this text, unless otherwise clearly specified and defined, the first feature being "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature can be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature has a higher horizontal height than the second feature. The first feature being "under", "beneath" and "underneath" the second feature can be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature has a lower horizontal height than the second feature.

[0045] The present invention provides an enhanced intestinal organ-on-a-chip, an enhanced intestinal organ system, a method for constructing an enhanced intestinal organ-on-a-chip, an enhanced intestinal organ, and their applications, which will be described in detail below respectively.

[0046] Enhanced intestinal organ-on-a-chip

[0047] The present invention provides an enhanced intestinal organ-on-a-chip. According to an embodiment of the present invention, refer to Figure 1, the enhanced intestinal organ-on-a-chip includes at least one structural unit, and the structural unit includes: an upper sub-chip 100, a porous membrane 300, and a lower sub-chip 200, which will be described in detail below.

[0048] In some embodiments of the present invention, the upper sub-chip 100 includes: a first flow channel 110, and the first flow channel 110 is used to accommodate intestinal organs and a first culture medium. Among them, the first culture medium is used to culture intestinal organs, and it mainly provides nutrients for intestinal organs. In the present invention, no specific limitation is made on the first culture medium, as long as it can culture intestinal organs.

[0049] In some embodiments of the present invention, the lower sub-chip 200 is bonded to the upper sub-chip 100; the lower sub-chip 200 includes: a second flow channel 210, and the second flow channel is used to accommodate endothelial cells and a second culture medium. Among them, the second culture medium is used to culture endothelial cells, and it mainly provides nutrients for endothelial cells. In the present invention, no specific limitation is made on the second culture medium, as long as it can culture endothelial cells.

[0050] In some embodiments of the present invention, the porous membrane 300 is disposed between the first flow channel 110 and the second flow channel 210 and covers at least a part of the bottom of the first flow channel 110, and the projection of the first flow channel towards the lower sub-chip passes through the porous membrane and at least partially overlaps with the second flow channel. The porous membrane can support intestinal organs to achieve adherent growth of intestinal organs, and at the same time, it can also allow some small molecule substances to enter the second flow channel through the porous membrane.

[0051] Thus, by using the enhanced intestinal organ-on-a-chip of the present invention, an enhanced intestinal model can be obtained in vitro, which can simulate the interaction between pathogens and the human intestine in vitro, can be used to study the process of pathogen infection and pathogenic mechanism, and provides a research platform with higher simulation degree for the development of new therapies for intestinal-related diseases. At the same time, it is more suitable for studying the intestinal metabolism of drugs, can better predict human pharmacokinetic parameters, and may be applied to patients through personalized drugs in the future. And, the enhanced intestinal organ-on-a-chip of the present invention can be designed with multiple structural units to achieve high-throughput implementation. The enhanced intestinal organ-on-a-chip of the present invention enables the chip sampling to be matched with automated equipment through an open design, realizes automated sampling, and is suitable for large-scale popularization and application.

[0052] In some embodiments of the present invention, the width of the first flow channel 110 is greater than the width of the second flow channel 210. Therefore, it can avoid the problems of edge curling and falling off of intestinal organs on the porous membrane during the culture process, improve the barrier function of the enhanced intestinal organ-on-a-chip, prevent the occurrence of non-physiological leakage, and improve the success rate of constructing the intestinal organ model.

[0053] In some embodiments of the present invention, the width of the first flow channel is 1.2 mm to 1.6 mm, and the width of the second flow channel is 0.9 mm to 1.1 mm. For example, the width of the first flow channel is 1.3 mm to 1.5 mm, and the width of the second flow channel is 0.9 mm to 1.1 mm. Specifically, the width of the first flow channel is 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, and the width of the second flow channel is 0.9 mm, 1 mm, 1.1 mm.

[0054] In some embodiments of the present invention, the upper sub-chip 100 further includes a first flow channel inlet 120 and a first flow channel outlet 130, and the first flow channel inlet 120 and the first flow channel outlet 130 are respectively located at both ends of the first flow channel 110. The intestinal organoids and the first culture medium can be injected into the first flow channel 110 through the first flow channel inlet 120, and when there is liquid overflowing from the first flow channel outlet 130, the injection is stopped.

[0055] In some embodiments of the present invention, the lower sub-chip 200 further includes a second flow channel inlet 220 and a second flow channel outlet 230, and the second flow channel inlet 220 and the second flow channel outlet 230 are respectively located at both ends of the second flow channel. The endothelial cells and the second culture medium can be injected into the second flow channel 210 through the second flow channel inlet 220, and when there is liquid overflowing from the second flow channel outlet 230, the injection is stopped.

[0056] In some embodiments of the present invention, the upper sub-chip 100 and the lower sub-chip 200 respectively include at least two bonding positioning holes, and the projection of the bonding positioning holes 140 of the upper sub-chip towards the lower sub-chip corresponds to the bonding positioning holes 240 on the lower sub-chip one by one. Thus, it is convenient to accurately position the upper sub-chip and the lower sub-chip, and after the positioning is completed, bonding is carried out.

[0057] In some embodiments of the present invention, the porous membrane is selected from a PETE porous membrane, and the pore density is (1 to 10)×10 5 pores / cm 2 . Thus, it can not only support the intestinal organoids and facilitate their adherent growth, but the PETE material will not affect the normal physiological functions of the cells. Using the above pore density can not only avoid cell exudation, but also allow some small molecule substances to permeate into the second flow channel.

[0058] In some embodiments of the present invention, referring to Figure 2 the right figure, the enhanced intestinal organoid chip includes multiple structural units, and thus, high-throughput detection can be achieved. In addition, from Figure 2As can be seen from the left figure, after the upper sub-chip and the lower sub-chip are bonded, the first flow channel inlet and the second flow channel inlet face different ends of the enhanced intestinal organoid chip respectively, and the first flow channel outlet and the second flow channel outlet also face different ends of the enhanced intestinal organoid chip respectively. Only the middle parts of the first flow channel and the second flow channel overlap.

[0059] Enhanced intestinal organoid construction system

[0060] The present invention provides an enhanced intestinal organoid construction system. According to an embodiment of the present invention, the enhanced intestinal organoid system includes: the aforementioned enhanced intestinal organoid chip; a first liquid storage device connected to the enhanced intestinal organoid chip and adapted to inject a third culture medium into the first flow channel; a second liquid storage device connected to the enhanced intestinal organoid chip and adapted to inject a fourth culture medium into the second flow channel.

[0061] Therefore, it is possible to culture intestinal organoids in the upper sub-chip to simulate the physiological intestinal lumen microenvironment, and to culture endothelial cells in the lower sub-chip to construct a single-layer blood vessel. By using the system of the present invention, an enhanced intestinal model can be constructed, which more realistically simulates the intestinal structure and provides a research platform with higher simulation degree for developing new therapies for intestinal-related diseases. At the same time, it is more suitable for studying the intestinal metabolism of drugs and can better predict human pharmacokinetic parameters.

[0062] It should be noted that the "third culture medium" can be a first culture medium containing intestinal organoids or other culture media, such as a proliferation medium, a differentiation medium, or a maturation medium, etc. Specifically, it can be specifically selected according to the required culture medium during the culture of intestinal organoids; the "fourth culture medium" can be a second culture medium containing endothelial cells or a proliferation medium, etc. Specifically, it can be specifically selected according to the required culture medium during the culture of endothelial cells.

[0063] In some embodiments of the present invention, the first liquid storage device is connected to the first flow channel inlet and / or the second liquid storage device is connected to the second flow channel inlet through a pump. Injecting the required culture medium into the first flow channel through the pump helps to achieve dynamic culture and is beneficial to the growth of intestinal organoids. Injecting the required culture medium into the second flow channel through the pump is beneficial to the growth of vascular endothelial cells.

[0064] In some embodiments of the present invention, the pump includes a peristaltic pump.

[0065] In some embodiments of the present invention, the enhanced intestinal organ-on-a-chip construction system further comprises: a shaking culture device, which is adapted to shake the enhanced intestinal organ-on-a-chip. Preferably, the shaking culture device comprises a shaker. The shaking culture device is beneficial to simulate blood flow in vivo and the fluid shear stress received by the cell surface. Thus, the intestinal organoids constructed by using the system of the present invention can simulate real organs to the greatest extent in terms of structure and function, simulate in vivo tissues and physiological functions. The intestinal organoids can also generate microstructures such as intestinal crypts and villus-like epithelia, and can reproduce physiological responses in vitro with high fidelity.

[0066] In some embodiments of the present invention, the chip structure of the present invention is designed by using AutoCAD software, and the processing of the two-layer chip mold is carried out by 3D printing, and then the PDMS chip is made by casting. Both the upper and lower layers are PDMS with a length of 48 mm and a width of 45 mm. The thickness of the upper layer is 3 mm, and the thickness of the lower layer is 2 mm. A PETE porous membrane is spaced between the two-layer structure, and the pore density of the porous membrane is 6×10 5 pores / cm 2 . The bonding is carried out by using the plasma treatment method and the MPTMS chemical bonding method. First, a 0.2% solution of (3-mercaptopropyl)trimethoxysilane, i.e., the MPTMS reagent, is prepared. The PETE membrane is cut into a suitable size, immersed in the MPTMS reagent for 2 s, washed in deionized water, taken out, dried, and then pasted to the upper layer chip structure of the PDMS to cover its central structure, so that the membrane and the upper layer chip are bonded. Finally, the upper sub-chip and the lower sub-chip are put into the plasma together for plasma bonding, and the intestinal chip is completed. As Figure 1As shown, the width of the first flow channel is 1.4 mm, and the small holes at both ends are the inlets and outlets for cell seeding (the first flow channel inlet and the first flow channel outlet). This structure is used for seeding human intestinal organoids. The PETE porous membrane covers the upper structure and serves as the bottom of the upper layer. The seeded human intestinal organoids adhere to this membrane. A liquid storage device is added above the small holes at both ends of the flow channel and a shaker device is equipped, or directly connected to a peristaltic pump for continuous perfusion of cell culture medium to achieve dynamic culture. The width of the second flow channel is 1 mm and is used for seeding HUVEC cells. The small holes at both ends are the inlets and outlets for seeding cells (the second flow channel inlet and the second flow channel outlet). The chip of the present invention is equipped with a shaker device. After endothelial cells are seeded in the lower sub-chip, culture medium is perfused in the liquid storage device, and it is placed on the shaker. Fluid flow is driven by gravity, and the shear force generated in the micro-structure promotes the formation of a single-layer blood vessel. After blood vessels are formed in the second flow channel, intestinal organoids can be seeded in the first flow channel of the upper sub-chip to construct an intestinal organoid model. On this basis, this chip model can be further used to simulate the interaction between pathogens and the human intestine in vitro, study the pathogen infection process and pathogenic mechanism, etc., providing a research platform with a higher degree of simulation for the development of new therapies for intestinal-related diseases. At the same time, it is more suitable for studying the intestinal metabolism of drugs and can better predict human pharmacokinetic parameters.

[0067] Method for constructing enhanced intestinal organoids

[0068] The present invention proposes a method for constructing enhanced intestinal organoids. According to an embodiment of the present invention, the method for constructing intestinal organoids is implemented using the aforementioned enhanced intestinal organoid chip or the aforementioned enhanced intestinal organoid construction system. The method includes: injecting endothelial cells and a second culture solution into the second flow channel and performing a first culture treatment; after the endothelial cells form blood vessels, injecting intestinal organoids and a first culture solution into the first flow channel and performing a second culture treatment to obtain enhanced intestinal organoids. As Figure 3 shown, both the intestinal organoids and endothelial cells adhere to the porous membrane for growth. Among them, endothelial cells will generate a single-layer blood vessel in the lower layer of the porous membrane (in the second flow channel), and intestinal organoids will simulate the intestinal microenvironment in the upper layer of the porous membrane (in the first flow channel). Thus, the intestinal organoids constructed by the method of the present invention can simulate real organs to the greatest extent in terms of structure and function, simulate in vivo tissues and physiological functions. The intestinal organoids can also generate micro-structures such as intestinal crypts and villus-like epithelia, and can reproduce physiological responses in vitro with high fidelity.

[0069] In some embodiments of the present invention, further included after the second culture treatment is: after the endothelial cells adhere to the wall, discard the second culture solution, inject a proliferation medium into the second flow channel, and perform a sixth culture to form a single-layer blood vessel.

[0070] In some embodiments of the present invention, after the second culture treatment, it further includes: after the second culture treatment ends, discard the first culture medium, inject a fifth culture medium into the first flow channel, and perform a third culture treatment; after the third culture treatment ends, discard the fifth culture medium, inject a sixth culture medium into the first flow channel, and perform a fourth culture treatment; after the fourth culture treatment ends, discard the sixth culture medium, inject a seventh culture medium into the first flow channel, and perform a fifth culture treatment. Thus, the intestinal organoids can differentiate in the first flow channel, possess the functional characteristics of the physiological intestinal lumen microenvironment, and can also generate microstructures such as villus-like epithelia, and can simulate real organs to the greatest extent in terms of structure and function, and simulate in vivo tissues and physiological functions.

[0071] In some embodiments of the present invention, the time of the first culture treatment is 5 to 7 days.

[0072] In some embodiments of the present invention, the time of the second culture treatment is 20h to 30h.

[0073] In some embodiments of the present invention, the time of the third culture treatment is 40h to 50h. Among them, after the third culture treatment ends, the cell confluence should reach 100%.

[0074] In some embodiments of the present invention, the time of the fourth culture treatment is 40h to 50h.

[0075] In some embodiments of the present invention, the time of the fifth culture treatment is 40h to 50h.

[0076] It should be noted that the "fifth culture medium" described in the present invention is a proliferation medium, which helps the growth and amplification of intestinal organoids; the "sixth culture medium" is a shaping medium, which helps the preliminary differentiation of intestinal organoids; the "seventh culture medium" is a maturation medium, which can differentiate intestinal organoids, so that intestinal organoids possess the characteristics of multiple functional cells.

[0077] In some embodiments of the present invention, during the first culture treatment, the third culture treatment, the fourth culture treatment, and the fifth culture treatment, start the shaking culture device (see Figure 4 ) to shake the enhanced organoid chip; and / or start the peristaltic pump between the first liquid storage device and the first flow channel to circulate and inject the required culture medium into the first flow channel and / or the peristaltic pump between the second liquid storage device and the second flow channel to circulate and inject the required culture medium into the second flow channel. Thus, dynamic culture can be achieved to simulate blood flow in vivo and the fluid shear stress received by the cell surface.

[0078] Application

[0079] The present invention provides the application of the aforementioned enhanced intestinal organoid chip or enhanced intestinal organoid construction system in constructing enhanced intestinal organoids, drug screening pathological models, determining the pharmacological properties of drugs, drug screening, in vitro simulation of intestinal diseases, biomaterial evaluation, immune co-culture, tumor infiltration or migration.

[0080] The present invention provides the application of the aforementioned enhanced intestinal organoids in constructing intestinal models, drug screening pathological models, in vitro simulation of intestinal diseases, determining the pharmacological properties of drugs or screening drugs.

[0081] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in the art or according to the product specifications are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0082] The specific components of the culture medium used in the present invention are as follows:

[0083] Proliferation medium: 10 nM Gastrin, 1×B27, 1×N2, 2 mM GlutaMax, 1% double antibody, 10 mM HEPES, 1 mM Nace, 50 ng / ml EGF, 100 ng / ml Noggin, 1 μg / ml R-spondin 1, 100 ng / ml Wnt3A, 500 nM A83-01, 10 mM Nico, 10 μM SB202190;

[0084] Differentiation medium: 10 nM Gastrin, 1×B27, 1×N2, 2 mM GlutaMax, 1% double antibody, 10 mM HEPES, 1 mM Nace, 50 ng / ml EGF, 100 ng / ml Noggin, 1 μg / ml R-spondin 1, 100 ng / ml Wnt3A, 500 nM A83-01, 100 ng / ml IGF-1, 50 ng / ml FGF-2;

[0085] Maturation medium: 1×B27, 1×N2, 2 mM GlutaMax, 1% double antibody, 10 mM HEPES, 1 mM Nace, 50 ng / ml EGF, 100 ng / ml Noggin, 1 μg / ml R-spondin 1, 5 ng / ml Wnt3A, 500 nM A83-01;

[0086] Among them, the specific components in each culture medium can be obtained by purchase.

[0087] Example 1: For generating a single-layer blood vessel

[0088] When the primary human umbilical vein endothelial cells (HUVECs) in the culture dish grow to 85% - 90%, they are then trypsinized, centrifuged, and counted. According to a cell density of 2×10 6 cells / mL, the primary human umbilical vein endothelial cells are inoculated into the lower microchannel (the second flow channel) of the enhanced intestinal chip of the present invention, and then inverted and statically cultured in an incubator overnight to make the cells evenly adhere to the periphery of the lumen. Then the chip is placed upright, the peristaltic pump is turned on, and the proliferation medium is perfused at a flow rate of 1 μL / min. After 3 days of culture, the physiological characteristic indexes of vascular endothelial cells are detected by immunofluorescence staining. The enhanced intestinal chip is taken out, and conventional immunofluorescence staining is performed. The endothelial cell markers CD31, VE-cadherin, vWF, and the cytoskeletal proteins F-actin, HS, and SOD1 are observed under a fluorescence microscope and a confocal microscope. Thus, a single-layer blood vessel model is formed in the enhanced intestinal chip.

[0089] Example 2: For constructing intestinal organoids

[0090] 1. One day before inoculating the intestinal organoids into the enhanced intestinal chip that forms a single-layer blood vessel model in Example 1, first, DPBS, Matrigel (purchased from Corning), and Laminin (purchased from sigma) are mixed in a certain ratio (DPBS:Matrigel:Laminin = 977:13:10) according to a dosage of 300 μL / chip to prepare an intestinal chip coating solution. The function of the coating solution is to make the subsequently inoculated intestinal organoids adhere more easily. The enhanced intestinal chip after ultraviolet sterilization is subjected to 2 min of hydrophilic treatment with a PLASMA vacuum plasma processor. The intestinal chip is coated in a biosafety cabinet. 300 μL of the intestinal chip coating solution is added to the upper flow channel (the first flow channel) of each enhanced intestinal chip and incubated overnight in a cell culture incubator.

[0091] 2. On the day of inoculating the intestinal chip, the small intestinal organoids (cultured by conventional methods) separately cultured in a well plate are placed on a rotary mixer at 4°C to degum for 45 min. After the small intestinal organoids finish degumming, they are centrifuged, the supernatant is removed, and they are resuspended with the proliferation medium to obtain an organoid suspension.

[0092] 3. The coating solution in the upper flow channel of the intestinal chip in step 1 is aspirated and discarded, and the residual coating solution is washed with the proliferation medium, taking care not to have air bubbles in the flow channel.

[0093] 4. Use a pipette to aspirate 50 μL of the organoid suspension in step 2, insert it into the upper flow channel of the intestinal chip, and gently inject the organoid suspension into the upper flow channel. Then, gently move the intestinal chip to under the microscope to observe the cell distribution. The cells should be evenly distributed in the flow channel.

[0094] 5. Place the intestinal chip inoculated with the above organoids into the cell culture incubator and let it stand overnight. The next day, change the proliferation medium in the upper flow channel, place the intestinal chip on a programmable Bluetooth shaker, set the angle to 3°, and the interval to 1 h. Change the medium every two days.

[0095] 6. Two days later, the cell confluence should reach 100%. Change the intestinal organoid shaping medium in the first flow channel and continue to culture it on the shaker with the settings unchanged. Change the medium every two days.

[0096] 7. Two days later, discard the intestinal organoid shaping medium, add the intestinal organoid maturation medium, continue to culture it on the shaker with the settings unchanged, change the medium every day, and culture for 3 days. After 3 days, test the barrier tightness and cell type identification of the intestinal chip quality to obtain enhanced intestinal organoids.

[0097] The experimental results are as Figure 5 and Figure 6 shown. As can be seen from Figure 5 , for the intestinal organoids cultured with the enhanced intestinal chip with a wider upper part and a narrower lower part of the present invention, their barriers are completely and tightly connected, which is convenient for the subsequent growth and differentiation of organoids. In addition, as can be seen from Figure 6 , the small intestinal organoids on the chip formed an intestinal epithelium with villus structures ( Figure 6 A) under fluid stimulation, simulating the multi-lineage differentiation of the natural human intestine. It was confirmed by immunostaining cell quantitative analysis that all major differentiated intestinal epithelial cell types were present, including absorptive enterocytes (villin), goblet cells (mucin2), Paneth cells (lyz), and enteroendocrine cells (ChgA), etc. ( Figure 6 B, 1 - 5), and important drug transporters MDR1, PEPT1, and the main small intestinal drug-metabolizing enzyme CYP3A4 could be expressed ( Figure 6 B, 6 - 8).

[0098] Comparative Example 1

[0099] In this Comparative Example 1, the method described in Examples 1 and 2 was used to construct intestinal organoids. The difference was that a commercially available (purchased from Emulate) chip with the same width of the upper and lower flow channels was used for culture. Intestinal organoids were cultured in the upper layer and endothelial cells were cultured in the lower layer. After the culture was completed, the barrier tightness was tested. The results are as Figure 7 shown. For the chip with the same upper and lower widths, the phenomenon of matrix gel shrinkage and curling occurred before the formation of villus-like structures, resulting in an incomplete barrier.

[0100] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0101] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An enhanced intestinal organoid chip, characterized in that: It comprises at least one structural unit, and the structural unit comprises: an upper sub-chip, a porous membrane and a lower sub-chip; wherein, The upper sub-chip includes: a first flow channel, the first flow channel being suitable for containing intestinal organoids and a first culture solution; The lower sub-chip comprises: a second flow channel, the second flow channel being suitable for accommodating endothelial cells and a second culture fluid; The porous membrane is disposed above the second flow channel and covers at least a portion of the bottom of the first flow channel; The projection of the first flow channel toward the lower sub-chip passes through the porous membrane and at least partially overlaps with the second flow channel; The width of the first flow channel is greater than the width of the second flow channel.

2. The enhanced intestinal organoid chip according to claim 1, characterized in that: The width of the first flow channel is 1.2 mm to 1.6 mm, and the width of the second flow channel is 0.9 mm to 1.1 mm.

3. The enhanced intestinal organoid chip according to claim 1, characterized in that: The upper sub-chip further includes a first flow channel inlet and a first flow channel outlet, wherein the first flow channel inlet and the first flow channel outlet are respectively located at two ends of the first flow channel; The lower sub-chip further includes a second flow channel inlet and a second flow channel outlet, and the second flow channel inlet and the second flow channel outlet are respectively located at two ends of the second flow channel.

4. The enhanced intestinal organoid chip according to claim 1, characterized in that: The upper sub-chip and the lower sub-chip respectively include at least two bonding positioning holes, and the projection of the bonding positioning holes of the upper sub-chip toward the lower sub-chip corresponds one-to-one to the bonding positioning holes on the lower sub-chip.

5. The enhanced intestinal organoid chip according to claim 1, characterized in that: The porous membrane is selected from PETE porous membrane, and the pore density is (1-10)×10 5 Hole / cm 2 .

6. An enhanced intestinal organoid construction system, characterized in that: include: The enhanced intestinal organoid chip according to any one of claims 1 to 5; a first liquid storage device, the first liquid storage device being connected to the enhanced intestinal organ chip and being suitable for injecting a third culture solution into the first flow channel; A second liquid storage device, wherein the second liquid storage device is connected to the enhanced intestinal organ chip and is suitable for injecting a fourth culture solution into the second flow channel.

7. The enhanced intestinal organoid construction system according to claim 6, characterized in that: The upper sub-chip further includes a first flow channel inlet, the lower sub-chip further includes a second flow channel inlet, and the first liquid storage device and the first flow channel inlet and / or the second liquid storage device and the second flow channel inlet are connected via a pump.

8. The enhanced intestinal organoid construction system according to claim 7, characterized in that: The pump comprises a peristaltic pump.

9. The enhanced intestinal organoid construction system according to claim 8, characterized in that: Further including: A shaking culture device, wherein the shaking culture device is suitable for shaking the enhanced intestinal organoid chip.

10. The enhanced intestinal organoid construction system according to claim 9, characterized in that: The shaking culture device includes a shaking table.

11. A method for constructing enhanced intestinal organoids using the enhanced intestinal organoid chip according to any one of claims 1 to 5 or the enhanced intestinal organoid construction system according to any one of claims 6 to 10, characterized in that: include: injecting endothelial cells and a second culture fluid into the second flow channel to perform a first culture treatment; After the endothelial cells form blood vessels, the intestinal organoids and the first culture fluid are injected into the first flow channel, and a second culture treatment is performed to obtain the enhanced intestinal organoids.

12. The method according to claim 11, characterized in that After the second culture treatment, the method further comprises: after the second culture treatment is completed, discarding the first culture solution, injecting a fifth culture solution into the first flow channel, and performing a third culture treatment; After the third culture treatment is completed, the fifth culture solution is discarded, and the sixth culture solution is injected into the first flow channel to perform a fourth culture treatment; After the fourth culture treatment is completed, the sixth culture solution is discarded, and the seventh culture solution is injected into the first flow channel to perform the fifth culture treatment.

13. The method according to claim 12, characterized in that During the first culture treatment, the third culture treatment, the fourth culture treatment and the fifth culture treatment, a shaking culture device is started to shake the enhanced intestinal organoid chip; and / or a peristaltic pump is started.

14. An enhanced intestinal organoid, characterized in that The enhanced intestinal organoid is obtained by the method according to any one of claims 11 to 13.

15. Use of the enhanced intestinal organoid chip according to any one of claims 1 to 5, the enhanced intestinal organoid construction system according to any one of claims 6 to 10, or the enhanced intestinal organoid according to claim 14 in constructing intestinal models, drug screening pathological models, biomaterial evaluation, immune co-culture, tumor infiltration or migration.

Images