3D Multi-Organ Co-Culture Chip

Abstract

This application relates to the field of bio-tissue engineering technology, and discloses a 3D multi-organ co-culture chip, which includes: a chip body, on which one or more groups of culture modules are provided; wherein, each group of culture modules includes: a liquid storage hole, one end of which is open and located on the upper surface of the chip body; a first culture micro-hole, located below the liquid storage hole and communicating with it; a second culture micro-hole, located below the first culture micro-hole and communicating with it; a plurality of second fluid operation holes, one end of which is open and located on the upper surface of the chip body, and the other ends are respectively communicated with the second culture micro-holes through channels. The 3D multi-organ co-culture chip provided by the embodiments of the present disclosure has a simple structure. By connecting the second fluid operation holes with the second culture micro-holes through channels, and by operating the culture solution in the fluid operation holes, the change of the culture environment in the second culture micro-holes can be realized, which is convenient for operation.

Description

Technical Field

[0001] This application relates to the technical field of bio-tissue engineering, for example, to a 3D multi-organ co-culture chip. Background Art

[0002] Conventional 2D cell culture has been developed for nearly a century and has contributed great value to the biomedical field. However, the 2D culture method cannot achieve tissue specificity and differentiation functions of multiple cell types, or accurately predict tissue functions and drug activities in vivo. In vitro constructed 3D cell models can better represent the spatial and chemical complexity models of living tissues. Compared with traditional 2D models, 3D cell models have great advantages in studying the molecular mechanisms of tissue functions, collecting signal pathways, and drug responses to certain diseases. However, traditional 3D models also have some limitations. For example, organoids have variable sizes and shapes, and it is difficult to fix cells in a consistent position for further analysis. In addition, many 3D model systems lack microscale structures and tissue-tissue interfaces for multi-organ co-culture. For example, the vascular endothelial cell layer and the surrounding connective tissue and parenchymal cells are crucial for all organs. Moreover, cells are usually not exposed to normal mechanical signals such as fluid shear stress, tension, and compression, which can affect organ development and function. The lack of fluid flow will affect the interaction between cultured tissue cells, circulating blood cells, and immune cells.

[0003] Microfluidic organ-on-a-chip technology is an emerging technology that can overcome these limitations. Organ-on-a-chip includes cell culture channels that continuously perfuse and simulate tissue and organ-level physiological structures, which can reproduce multicellular structures, tissue-tissue interfaces, physicochemical microenvironments, and vascular perfusion of the body. These devices can reproduce the functional levels of tissues and organs. Moreover, high-resolution, real-time imaging of living cells can be achieved, as well as monitoring of biochemical, genetic, and metabolic activities. This technology has great potential to promote the research of tissue development, organ physiology, and disease pathology. In the process of drug discovery and development, organ-on-a-chip technology plays an important role in lead compound research, drug toxicity testing, and biomarker identification. Organ-on-a-chip technology can construct low-cost in vitro models that reproduce tissue and organ-level functions.

[0004] In the process of implementing the embodiments of the present disclosure, it is found that there are at least the following problems in the related art: The existing co-culture microfluidic organ-on-a-chip has a narrow application range, poor universality, complex model construction operations, and complex fluid control methods. Summary of the Invention

[0005] To provide a basic understanding of some aspects of the disclosed embodiments, a simple summary is given below. This summary is not a general review, nor is it intended to identify key / important elements or delineate the scope of protection of these embodiments, but rather serves as a preface to the detailed description that follows.

[0006] Embodiments of the present disclosure provide a 3D multi-organ co-culture chip to solve the problems of narrow application range, poor universality, complex model construction operation, and complex fluid control method in existing co-culture microfluidic organ chips.

[0007] In some embodiments, the 3D multi-organ co-culture chip includes: a chip body, on which one or more groups of culture modules are provided; wherein each group of culture modules includes:

[0008] A liquid storage hole, one end of which is open and located on the upper surface of the chip body;

[0009] A first culture micro-hole, located below the liquid storage hole and communicating with the liquid storage hole;

[0010] A second culture micro-hole, located below the first culture micro-hole and communicating with the first culture micro-hole;

[0011] A plurality of second fluid operation holes, one end of which is open and located on the upper surface of the chip body, and the other ends are respectively communicated with the second culture micro-holes through channels.

[0012] The 3D multi-organ co-culture chip provided by the embodiments of the present disclosure can achieve the following technical effects:

[0013] The 3D multi-organ co-culture chip provided by the embodiments of the present disclosure has a simple structure. The second fluid operation holes are communicated with the second culture micro-holes through channels (for example, through channels). Simple operations are carried out through the liquid storage holes and fluid operation holes, which is convenient for cell seeding, medium replacement, sampling and other operations. The operation is simple and does not require professional technicians, expanding the application range of the culture chip and improving the universality. The first and second culture micro-holes can flexibly achieve 2D and 3D organ single or co-culture. Gravity-based fluid driving is a simple and accurate fluid control method, which can realize real-time dynamic update of the culture environment in the second culture micro-hole. Moreover, long-term in vitro dynamic co-culture of multiple organs can be achieved. Multiple culture modules are arranged in a high-throughput manner according to the multi-well plate spacing and are compatible with high-throughput and automated equipment. Physical forces including fluid shear force, cyclic pressure and mechanical compression force related to physiological levels can also be combined, which can achieve organ-specific responses such as aggregation of circulating immune cells, response to drugs, toxins and other environmental disturbances. Moreover, long-term in vitro dynamic co-culture of multiple organs can be achieved.

[0014] The above general description and the following description are only exemplary and explanatory and are not used to limit the present application. Description of the Drawings

[0015] One or more embodiments are exemplarily illustrated by corresponding drawings. These exemplary illustrations and the drawings do not constitute a limitation on the embodiments. Elements with the same reference numerals in the drawings are shown as similar elements. The drawings do not constitute a scale limitation, and wherein:

[0016] Figure 1 is an exploded view of the structure of a culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0017] Figure 2 is an exploded view of the structure of another culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0018] Figure 3 is a top view structural diagram of a culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0019] Figure 4 is an exploded view of the structure of another culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0020] Figure 5 is Figure 4 a top view structural diagram of the shown culture module;

[0021] Figure 6 is a partial structural diagram of another culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0022] Figure 7 is a partial structural diagram of another culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0023] Figure 8 is an exploded view of the structure of another culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0024] Figure 9 is Figure 8 a top view structural diagram of the shown culture module;

[0025] Figure 10 is an exploded view of the structure of another culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0026] Figure 11 is Figure 10 a top view structural diagram of the shown culture module;

[0027] Figure 12 is an exploded view of the structure of another culture module of a 3D multi-organ co-culture chip provided by an embodiment of the present disclosure;

[0028] Figure 13 is Figure 12 a top - view structural schematic diagram of the shown culture module.

[0029] Reference numerals:

[0030] 11. First liquid storage layer; 12. First culture layer; 13. Second culture layer; 14. Film layer; 21. Liquid storage hole; 22. First culture micro - hole; 23. Second culture micro - hole; 231. First side; 232. Second side; 233. Third side; 234. Fourth side; 24. Second fluid operation hole; 2401. First - side second fluid operation hole; 2402. Second - side second fluid operation hole; 241. Second operation hole I; 242. Second operation hole II; 243. Second operation hole III; 25. Through - channel; 251. First - side through - channel; 252. Second - side through - channel; 26. Bypass channel; 261. Vertical fence; 262. Vertical micro - channel; 263. Horizontal fence; 264. Horizontal micro - channel; 27. First fluid operation hole; 271. First - side first fluid operation hole; 272. Second - side first fluid operation hole; 28. First communication channel; 29. Side - edge fluid operation hole; 291. Side - edge operation hole I; 292. Side - edge operation hole II; 293. Side - edge operation hole III. Detailed implementation manners

[0031] In order to be able to understand the features and technical content of the embodiments of the present disclosure in more detail, the implementation of the embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. The attached drawings are for reference and illustration only and are not used to limit the embodiments of the present disclosure. In the following technical description, for the sake of explanation, multiple details are provided to provide a full understanding of the disclosed embodiments. However, one or more embodiments can still be implemented without these details. In other cases, well - known structures and devices can be shown in a simplified manner to simplify the drawings.

[0032] The terms "first", "second", etc. in the description and claims of the embodiments of the present disclosure and the above - mentioned drawings are used to distinguish similar objects and do not necessarily describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so as to implement the embodiments of the present disclosure described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non - exclusive inclusion.

[0033] In the embodiments of the present disclosure, the orientation or positional relationship indicated by terms such as "upper", "lower", "inner", "middle", "outer", "front", "rear", etc. is based on the orientation or positional relationship shown in the drawings. These terms are mainly used to better describe the embodiments of the present disclosure and their embodiments, and are not used to limit that the indicated devices, elements or components must have a specific orientation, or be constructed and operated in a specific orientation. Moreover, in addition to being able to represent the orientation or positional relationship, some of the above terms may also be used to represent other meanings. For example, the term "upper" may also be used to represent a certain attachment relationship or connection relationship in some cases. For those of ordinary skill in the art, the specific meanings of these terms in the embodiments of the present disclosure can be understood according to specific circumstances.

[0034] In addition, the terms "arranged", "connected", "fixed" should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral structure; it can be a mechanical connection or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, or there is an internal connection between two devices, elements or components. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present disclosure can be understood according to specific circumstances.

[0035] Unless otherwise specified, the term "plurality" means two or more.

[0036] It should be noted that, without conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0037] Combined with Figure 1-11 As shown, the embodiments of the present disclosure provide a 3D multi-organ co-culture chip, including a chip body, on which one or more culture modules are arranged. Among them, each culture module includes a liquid storage hole 21, a first culture micro-hole 22, a second culture micro-hole 23 and a plurality of second fluid operation holes 24. One end of the liquid storage hole 21 is open and located on the upper surface of the chip body. The first culture micro-hole 22 is used for culturing first organ cells; it is located below the liquid storage hole 21 and is communicated with the liquid storage hole 21. The second culture micro-hole 23 is used for culturing second organ cells; it is located below the first culture micro-hole 22 and is communicated with the first culture micro-hole 22. One end of each of the plurality of second fluid operation holes 24 is open and located on the upper surface of the chip body, and the other ends are respectively communicated with the second culture micro-hole 23 through channels.

[0038] The 3D multi-organ co-culture chip provided by the embodiments of the present disclosure has a simple structure. The second fluid operation hole is communicated with the second culture micro-hole through a channel (for example, a through-channel). Simple operations are carried out through the liquid storage hole and the fluid operation hole, facilitating operations such as cell seeding, medium replacement, and sampling. The operation is simple and does not require professional technicians, expanding the application scope of the culture chip and improving the universality. The first and second culture micro-holes can flexibly realize 2D and 3D organ culture alone or in co-culture. Gravity-based fluid driving is a simple and precise fluid control method, which can realize real-time dynamic update of the culture environment in the second culture micro-hole. Moreover, long-term in vitro dynamic co-culture of multiple organs can be realized. Multiple culture modules are arranged in a high-throughput manner according to the multi-well plate spacing and are compatible with high-throughput and automated equipment. Physical forces including fluid shear force, cyclic pressure, and mechanical compression force related to physiological levels can also be combined, enabling organ-specific responses such as aggregating circulating immune cells, responding to drugs, toxins, and other environmental disturbances. Moreover, long-term in vitro dynamic co-culture of multiple organs can be realized.

[0039] The 3D multi-organ co-culture chip of the embodiments of the present disclosure can be used to construct a multi-organ model in vitro. Among them, the second organ cells cultured in the second culture micro-hole 23 can be one or more cells related to the same organ. For example, 2, 3, or even more kinds. The co-culture chip can realize in-situ 2D or 3D co-culture of multiple organ types. Models for studying the interaction of multiple organs can be constructed, such as vascular-tumor models, blood-brain barrier-tumor models, etc., for studying angiogenesis, drug penetration, cell polarization, cell migration, and drug activity evaluation.

[0040] In the embodiments of the present disclosure, in the culture module, the liquid storage hole 21 contains a culture medium or a drug dilution solution, etc., providing the required culture solution or the drug to be tested for the first organ cells. The first organ cells can be 3D cultured in the first culture micro-hole 22. The second organ cells are cultured in the second culture micro-hole 23, that is, the liquid storage hole 21, the first culture micro-hole 22, and the second culture micro-hole 23 are coaxially communicated. The second fluid operation hole 24 and the channel realize microfluidic control of the culture solution in the second culture micro-hole 23. At the same time, fluid operations are carried out through the liquid storage hole 21 and the second fluid operation hole 24, which is a simple and precise fluid control method.

[0041] In the embodiments of the present disclosure, the shapes of the liquid storage hole 21, the first culture micro-hole 22, the second culture micro-hole 23, and the second fluid operation hole 24 are not limited and can be geometric shapes such as round holes, ellipses, squares, rectangles, sectors, or polygons (such as hexagons, octagons, etc.). Under the condition of meeting the design requirements, the shapes of the liquid storage hole 21 and the second fluid operation hole 24 are designed based on the principle of containing as much culture medium or drug dilution solution as possible, and the shapes of the first culture micro-hole 22 and the second culture micro-hole 23 are designed based on facilitating growth.

[0042] Optionally, the shapes of the liquid storage holes 21 and the second fluid operation holes 24 include circular or oval shapes.

[0043] Optionally, the shape of the first culture micro-well 22 is circular.

[0044] Optionally, the shape of the second culture micro-well 23 is square, rectangular or hexagonal.

[0045] In addition, the seeding and culturing of the first organ cells in the first culture micro-well 22 are carried out in an open through-hole manner, which is convenient for subsequent on-machine detection and cell recovery for analysis such as RNA and protein extraction. The number and arrangement form of the culture modules provided on the chip body are not limited and can be determined according to needs.

[0046] In the embodiments of the present disclosure, the second culture micro-well 23 can be a through-hole or a groove.

[0047] Optionally, the second culture micro-well 23 is a through-hole. Then the liquid storage holes 21, the first culture micro-wells 22 and the second culture micro-wells 23 form through-holes, and when the chip body is used, it can be used in cooperation with the bottom plate.

[0048] Optionally, the second culture micro-well 23 is a groove. Then the liquid storage holes 21, the first culture micro-wells 22 and the second culture micro-wells 23 form non-through-holes and do not need to be used in cooperation with the bottom plate.

[0049] Optionally, the number of the multiple culture modules provided on the chip body is 12, 24, 36, 96, etc., and the arrangement mode in the multiple culture modules is compatible with existing commercial sample adding devices and detectors (such as enzyme labelers, high-content imaging systems, etc.). High-throughput organ culture and detection are realized.

[0050] In some embodiments, the cross-sectional area of the liquid storage hole 21 is larger than the cross-sectional area of the first culture micro-well 22; the cross-sectional area of the second culture micro-well 23 is larger than or equal to the cross-sectional area of the first culture micro-well 22. In this embodiment, the cross-sectional area of the liquid storage hole 21 is larger than the cross-sectional area of the first culture micro-well 22, so that the liquid changing process does not affect the cells in the first culture micro-well 22. The cross-sectional area of the second culture micro-well 23 is larger than the cross-sectional area of the first culture micro-well 22, so that the cells growing on the edge of the second culture micro-well 23 do not contact or rarely contact the cells in the first culture micro-well 22.

[0051] In the embodiments of the present disclosure, the relative sizes of the cross-sectional areas of the liquid storage hole 21 and the second culture micro-well 23 are not limited and can be set according to actual needs.

[0052] Optionally, when the cross-sectional area of the liquid storage hole 21 is larger than the cross-sectional area of the first culture micro-well 22, the cross-sectional area of the liquid storage hole 21 is made larger than the cross-sectional area of the second culture micro-well 23. It is convenient for culture operation and observation.

[0053] In the embodiments of the present disclosure, in the culture module, the specific dimensional parameters of the liquid storage hole 21, the first culture micro-hole 22, and the second culture micro-hole 23 can be determined according to actual needs and are not limited herein.

[0054] In the embodiments of the present disclosure, the liquid storage hole 21 adopts a columnar hole design to reduce the risk of cross-contamination between holes and reduce the edge effect caused by evaporation.

[0055] In the embodiments of the present disclosure, the first culture micro-hole 22 communicates with the second culture micro-hole 23. The communication here is understood in a broad sense, including not only the direct communication between the first culture micro-hole 22 and the second culture micro-hole 23 (as shown in Figure 1 ), but also the communication between the two through a diaphragm. For example, as shown in Figure 2 , a thin film layer 14 is provided between the first culture micro-hole 22 and the second culture micro-hole 23, but micro-nano-sized holes are distributed on the thin film layer 14. Through these micro-nano-sized holes, the culture media in the first culture micro-hole 22 and the second culture micro-hole 23 can exchange substances, which is also considered that the first culture micro-hole 22 communicates with the second culture micro-hole 23. Different organ models can be constructed according to whether the first culture micro-hole 22 and the second culture micro-hole 23 are directly connected or connected through a diaphragm for relevant research.

[0056] Optionally, the communication mode between the first culture micro-hole 22 and the second culture micro-hole 23 is a direct connection, that is, no diaphragm is provided. The 3D multi-organ co-culture chip composed of this culture module is defined as a membrane-free chip. For this membrane-free chip, when organ cells are planted, the two organs are physically isolated by a 3D matrix, and the first organ and the second organ cells and secreted factors in 3D culture can interact through the matrix. The organ model constructed by the membrane-free chip can be used to study the interaction between cells from two organs, the induced migration of cells, and the growth and budding of blood vessels during the co-culture of blood vessels with various tissues and organs.

[0057] Optionally, the communication mode between the first culture micro-well 22 and the second culture micro-well 23 is diaphragm connection. The 3D multi-organ co-culture chip composed of this kind of culture module is defined as a chip with a membrane. For this kind of chip with a membrane, the first organ cells in the first culture micro-well 22 communicate with the second organ cells in the second culture micro-well 23 through the diaphragm, which can not only achieve physical isolation but also achieve the co-communication of biological and chemical factors. The organ model constructed by this kind of chip with a membrane can be used to simulate the interaction between the barrier-like model and other organs, such as the blood-brain barrier. The diaphragm layer provides mechanical support for the adhesion of human brain microvascular endothelial cells. Channels (for example, through channels) can achieve fluid flow to provide the necessary shear force for barrier formation. It is used for screening and related research of nerve drugs and tumor drugs based on the blood-brain barrier, for example, anti-glioma compounds for the blood-brain barrier.

[0058] In some embodiments, in combination with Figure 3 As shown, the second fluid operation hole 24 is a stepped hole, and from the upper surface of the chip body to the inside, the aperture of the stepped hole becomes smaller. This reduces the sedimentation of the inoculated cells at the bottom of the second fluid operation hole 24. Optionally, the stepped hole includes a first-order stepped hole or a second-order stepped hole. It can be determined according to actual needs. For specific content about the stepped hole in a specific structural form of a 3D multi-organ co-culture chip, please refer to the following.

[0059] In some embodiments, the center distance between the liquid storage hole 21 and the second fluid operation hole 24 is 1 - 10 mm. It is compatible with the electrodes of a transmembrane resistance meter and can measure the TEER value in real time. Among them, the center distance refers to the distance between the center of the liquid storage hole 21 and the center of the second fluid operation hole 24.

[0060] Optionally, the center distance between the liquid storage hole 21 and the second fluid operation hole 24 is 3 - 8 mm.

[0061] In some embodiments, in the culture module, multiple second fluid operation holes 24 are arranged in pairs; each pair of second fluid operation holes 24 is connected through a through-channel 25; the through-channel 25 includes a first-side through-channel 251 and a second-side through-channel 252, and the first-side through-channel 251 and the second-side through-channel 252 are oppositely arranged on opposite sides of the second culture micro-hole 23. In the embodiments of the present disclosure, the second fluid operation hole 24 connected to the first-side through-channel 251 is defined as the first-side second fluid operation hole 2401, and the second fluid operation hole 24 connected to the second-side through-channel 252 is defined as the second-side second fluid operation hole 2402. During the 3D organ culture process, the first-side second fluid operation hole 2401 and the first-side through-channel 251 can be used as the sample injection channels, and the second-side second fluid operation hole 2402 and the second-side through-channel 252 can be used as the sample output channels; they can also be exchanged, which is not limited. On the upper surface of the chip body, the culture solution in the first-side second fluid operation hole 2401 or the second-side second fluid operation hole 2402 can be operated, so that the culture solution in the first-side through-channel 251 (or the second-side through-channel 252) flows into the second culture micro-hole 23, and the culture solution in the second culture micro-hole 23 then flows into the second-side through-channel 252 (or the first-side through-channel 251), realizing the connection and flow of the culture solution in the second culture micro-hole 23.

[0062] In some embodiments, as Figure 1 and Figure 2 shown, the first type of culture module includes a liquid storage hole 21, a first culture micro-hole 22, a second culture micro-hole 23, two second fluid operation holes 24 (a pair of second fluid operation holes 24), and a through-channel 25; the liquid storage hole 21, the first culture micro-hole 22, and the second culture micro-hole 23 are sequentially connected and coaxially arranged from top to bottom; the two second fluid operation holes 24 are respectively located on both sides of the liquid storage hole 21; the first-side second fluid operation hole 2401 is connected to the second culture micro-hole 23 through the first-side through-channel 251, and the second-side second fluid operation hole 2402 is connected to the second culture micro-hole 23 through the second-side through-channel 252.

[0063] Of course, on the basis of the foregoing first type of culture module, the number of through-channels 25 can be increased, and the number of pairs of second fluid operation holes 24 can be correspondingly increased, that is, one through-channel 25 corresponds to a pair of second fluid operation holes 24, to obtain different deformed culture modules, which will not be elaborated here.

[0064] In some embodiments, in the culture module, there are further a plurality of side fluid operation holes 29 and a bypass channel 26. One end of each side fluid operation hole 29 is open and located on the upper surface of the chip body. The two ends of the bypass channel 26 are respectively communicated with one side fluid operation hole 29, and the middle part is communicated with the second culture micro-hole 23. In the embodiments of the present disclosure, other types of cells belonging to the same organ as the cells in the second culture micro-hole 23 can be added into the bypass channel 26, and co-culture is realized at the connection between the bypass channel 26 and the second culture micro-hole 23. And the side fluid operation holes 29 at the two ends of one bypass channel 26 can be filled with cell culture medium, and microfluidic culture is realized.

[0065] Optionally, the connection between the bypass channel 26 and the second culture micro-hole 23 is provided with one or more micro-channels. Here, the micro-channels can be formed by arranging one or more fences at the communication ports at the connection. The size of the micro-channels is not limited, based on the ability to prevent the cells in the bypass channel 26 from entering the second culture micro-hole 23 but enabling material exchange. Physical separation of the cells in the bypass channel 26 from the cells in the second culture micro-hole 23 is realized, and at the same time, exchange of cell secretory factors between the bypass channel 26 and the second culture micro-hole 23 is realized.

[0066] Combined Figure 4 and Figure 5 As shown, a plurality of fences are provided at the connection between the bypass channel 26 and the second culture micro-hole 23, forming a plurality of micro-channels. The construction methods of the plurality of fences and the micro-channels are not limited.

[0067] Optionally, as Figure 4 and Figure 5 shown, one or more vertical fences 261 are arranged at a set interval on the communication port between the side wall on one side of the middle part of the bypass channel 26 and the second culture micro-hole 23, dividing the communication port into a plurality of vertical and parallel vertical micro-channels 262. The independent growth of the cells or 3D matrix in the second culture micro-hole 23 and the bypass channel 26 can be controlled while maintaining factor exchange by controlling the height and width of the vertical micro-channels.

[0068] Optionally, one or more horizontal fences 263 are arranged horizontally on the communication port between the side wall on one side of the middle part of the bypass channel 26 and the second culture micro-hole 23. When one horizontal fence 263 is arranged horizontally, the one horizontal fence is arranged in the middle in the vertical direction of the communication port, that is, a horizontal micro-channel 264 is formed above and below the one horizontal fence respectively. When a plurality of horizontal fences are arranged horizontally, they can be arranged at a set interval.

[0069] Combined Figure 7As shown, the connection between the bypass channel 26 and the second culture micro-well 23 is configured as a micro-channel. The construction method of a micro-channel is not limited. Optionally, a transverse barrier 263 is horizontally arranged along the upper edge of the connection port between one side wall in the middle of the bypass channel 26 and the second culture micro-well 23, covering the lower part (as shown in Figure 7 shown) or the upper part of the connection port, so as to form a transverse micro-channel 264 above (as shown in Figure 7 shown) or below the transverse barrier 263.

[0070] In some embodiments, the bypass channel 26 is arranged on both sides or one side of the other two opposite sides of the second culture micro-well 23 that is not connected to the through-channel 25. Then, correspondingly, the number of pairs of second fluid operation holes 24 equal to the number of bypass channels 26 is increased, and they are respectively connected to the two ports of the bypass channel 26.

[0071] Optionally, as shown in Figure 4 and Figure 5 shown, the second culture module, on the basis of the first culture module, adds two bypass channels 26, which are respectively arranged on the other two opposite sides of the second culture micro-well 23 that is not connected to the through-channel 25; and the connection ports at the connection between the bypass channel 26 and the second culture micro-well 23 are separated by a plurality of vertical barriers 261 into a plurality of vertical micro-channels 262. And correspondingly, two pairs of side fluid operation holes 29 are added, which are respectively connected to the four ports of the two bypass channels 26.

[0072] Optionally, a variation of the second culture module, on the basis of the first culture module, only adds one bypass channel 26, which is arranged on one side of the other two opposite sides of the second culture micro-well 23 that is not connected to the through-channel 25; and the connection ports at the connection between the bypass channel 26 and the second culture micro-well 23 are separated by a plurality of vertical barriers 261 into a plurality of vertical micro-channels 262. And correspondingly, one pair of side fluid operation holes 29 is added, which is connected to the two ports of one bypass channel 26. Refer to Figure 4 and Figure 5 , only adding one bypass channel 26 on one of the side edges is enough.

[0073] In some embodiments, as shown in Figure 7 shown, the third culture module, on the basis of the second culture module, sets the connection port at the connection between the bypass channel 26 and the second culture micro-well 23 as a micro-channel. The construction of this micro-channel refers to the relevant content above and will not be elaborated here.

[0074] In some embodiments, refer to Figures 4 to 7As shown, the extension direction of the bypass channel 26 is the same as that of the through channel 25. The flow of the culture medium in the second culture micropores 23 is regular, and the culture effect is good. For example, bidirectional flow or unidirectional drive flow driven by gravity is utilized.

[0075] Optionally, as Figure 7 shown, the second culture micropores are square, having a first side 231, a second side 232, a third side 233, and a fourth side 234. Among them, the first side 231 and the second side 232 are opposite sides, and the third side 233 and the fourth side 234 are opposite sides; a first side through channel 251 of the through channel 25 is provided on the first side 231 side, a second side through channel 252 of the through channel 25 is provided on the second side 232 side, and a communication port is opened on the third side 233 and / or the fourth side 234 for communicating with the middle part of the bypass channel 26.

[0076] In some embodiments, in combination with Figures 8 to 13 shown, the culture module further includes a plurality of first fluid operation holes 27; one ends of the plurality of first fluid operation holes 27 are open and located on the upper surface of the chip body, and the other ends are respectively communicated with the liquid storage holes 21 through first communication channels 28. Through the first communication channels 28, the fluid in the liquid storage holes 21 and the first fluid operation holes 27 is communicated and flows, so that by operating the culture medium in the first fluid operation holes 27, the flow of the culture medium in the liquid storage holes 21 can be realized, that is, the culture environment in the first culture micropores 22 surrounded by the culture medium in the liquid storage holes 21 is in a flowing state, forming a dynamic culture environment.

[0077] In the embodiments of the present disclosure, the setting positions and numbers of the plurality of first fluid operation holes 27 are not limited, as long as the fluid in the liquid storage holes 21 can be realized by operating the culture medium therein. Optionally, the plurality of first fluid operation holes 27 are divided into two groups and are respectively arranged on opposite sides of the liquid storage hole 21. That is, the plurality of first fluid operation holes 27 are divided into a first side first fluid operation hole 271 and a second side first fluid operation hole 272. Herein, the first side and the second side may be the same as the first side and the second side of the second fluid operation hole 24 part described above.

[0078] Optionally, in the culture module, the plurality of first fluid operation holes 27 are arranged in pairs, and each pair of first fluid operation holes 27 is respectively arranged on opposite sides of the liquid storage hole 21.

[0079] Optionally, in the culture module, two first fluid operation holes 27 are included and are respectively arranged on opposite sides of the liquid storage hole 21. As Figures 10 to 13 shown, the first fluid operation holes 27 are arranged on opposite sides of the diameter of the liquid storage hole 21 and are parallel to the through channel 25. Or, as Figure 8 and Figure 9As shown, the central connection lines of the two first fluid operation holes 27 and the liquid storage hole 21 form a set angle, that is, the first fluid operation holes 27 are arranged on the opposite sides that are not across the diameter. Among them, the set angle is not limited. That is, the arrangement positions of the first fluid operation holes 27 in the embodiments of the present disclosure are not limited.

[0080] In the embodiments of the present disclosure, on the basis of the foregoing first culture module, second culture module, and third culture module, a plurality of first fluid operation holes 27 are respectively added, and they are respectively communicated with the liquid storage hole 21 through the first communication channels 28 to form corresponding fourth culture modules, fifth culture modules, and sixth culture modules.

[0081] In the 3D multi-organ co-culture chip of the embodiments of the present disclosure, as long as it has one or more of the foregoing culture modules, the specific structural form is not limited. The following gives a specific structural form of a 3D multi-organ co-culture chip, but it is not limited to this specific structural form.

[0082] In some embodiments, the chip body includes:

[0083] The first liquid storage layer 11, on which there are arranged a liquid storage hole 21 and a second operation hole I 241 of the second fluid operation hole 24;

[0084] The first culture layer 12, on which there are arranged first culture micro-holes 22 and a second operation hole II 242 of the second fluid operation hole 24; the first culture layer 12 is stacked below the first liquid storage layer 11, and the first culture micro-holes 22 are communicated with the liquid storage hole 21; the second operation hole II 242 is communicated with the second operation hole I 241;

[0085] The second culture layer 13, on which there are arranged second culture micro-holes 23 and channels (for example, through channels 25); the second culture layer 13 is stacked below the first culture layer 12, and the second culture micro-holes 23 are communicated with the first culture micro-holes 22, and the channels are communicated with the second culture micro-holes 23 and the second operation hole II 242.

[0086] In the embodiments of the present disclosure, the chip body includes three-layer chip structures, and the three-layer chip structures can be stacked and connected in sequence. Various sealing processes such as double-sided tape, ultrasonic, thermal bonding, plasma, and hot pressing can be used to bond and assemble each layer of the chip together. Among them, the first liquid storage layer 11 and the first culture layer 12 can be combined and set as one-layer structure for processing, which can be determined according to the actual situation.

[0087] In the embodiments of the present disclosure, the materials of the chip structures of each layer in the chip body are one or several of polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polycarbonate (PC), PS, COC, COP, etc. The production of each layer structure can be obtained by soft lithography, molding method, laser etching, machining, LIGA or one-time injection molding and other methods to obtain the chip structures of each layer.

[0088] Optionally, the second fluid operation hole 24 is a stepped hole; and the aperture of the first second operation hole 241 is larger than the aperture of the second second operation hole 242. The second fluid operation hole 24 of this embodiment is a first-order stepped hole.

[0089] In the embodiments of the present disclosure, the channel (penetrating channel 25) on the second culture layer 13 communicates with the second second operation hole 242 on the first culture layer 12. The communication method is not limited. Optionally, one end of the channel extends into the second second operation hole 242 to make the two communicate.

[0090] Optionally, a hole groove is further provided on the second culture layer 13, and one port of the channel is arranged on the side wall of the hole groove, that is, the hole groove can be regarded as the third second operation hole 243 of the second fluid operation hole 24.

[0091] Optionally, the aperture of the first second operation hole 241 is larger than the aperture of the second second operation hole 242, and the aperture of the second second operation hole 242 is larger than the aperture of the third second operation hole 243. Reduce the residence of cells in the hole when inoculating cells. The second fluid operation hole 24 of this embodiment is a second-order stepped hole.

[0092] Optionally, the aperture of the first second operation hole 241 is equal to the aperture of the second second operation hole 242, and the aperture of the second second operation hole 242 is larger than the aperture of the third second operation hole 243. The first second operation hole 241 and the second second operation hole 242 are of the same size, reducing the generation of bubbles. The aperture of the third second operation hole 243 is the smallest, reducing the deposition of cells in the hole when inoculating cells. The second fluid operation hole 24 of this embodiment is a first-order stepped hole.

[0093] In some embodiments, when the culture module includes the side fluid operation hole 29, the first side operation hole 291 of the side fluid operation hole 29 is further provided on the first liquid storage layer 11, the second side operation hole 292 of the side fluid operation hole 29 is further provided on the first culture layer 12, and the second side operation hole 292 communicates with the first side operation hole 291; a bypass channel 26 is further provided on the second culture layer 13, and the bypass channel 26 communicates the second culture micro-hole 23 with the second side operation hole 292.

[0094] In the embodiments of the present disclosure, the bypass channel 26 on the second culture layer 13 communicates with the side operation hole II 292 on the first culture layer 12. The communication method is not limited. Optionally, one end of the bypass channel 26 extends into the side operation hole II 292 to make the two communicate.

[0095] Optionally, a hole groove is further provided on the second culture layer 13, and one port of the bypass channel 26 is arranged on the side wall of the hole groove, that is, the hole groove can be regarded as the side operation hole III 293 of the side fluid operation hole 29.

[0096] In the embodiments of the present disclosure, the structural form of the side fluid operation hole 29 is the same as that of the aforementioned second fluid operation hole 24. Optionally, the side fluid operation hole 29 is a stepped hole; the composition form of the stepped hole can refer to the second fluid operation hole 24.

[0097] In some embodiments, when the culture module includes the first fluid operation hole 27, the first fluid operation hole 27 and the first communication channel 28 are further provided on the first liquid storage layer 11.

[0098] Optionally, the first fluid operation hole 27 is a straight hole.

[0099] Optionally, the first communication channel 28 is provided through on the side wall between the liquid storage hole 21 and the first fluid operation hole 27.

[0100] In some embodiments, the chip body further includes a thin film layer 14 with micro-nano sized holes distributed thereon; the thin film layer 14 is arranged between the first culture micro-holes 22 and the second culture micro-holes 23. That is, on the basis of the aforementioned three-layer chip body, the thin film layer 14 is added to form a four-layer chip body. In the embodiments of the present disclosure, the thin film layer 14 can be a transparent polyester (polyethylene terephthalate, PET) film with apertures. When assembling the four-layer chip body of the embodiments of the present disclosure, the thin film layer 14 can be first assembled with the first liquid storage layer 11 and the first culture layer 12, and then assembled with the second culture layer 13 as a whole. In the embodiments of the present disclosure, the thin film layer 14 needs to avoid the communication part between the second fluid operation hole 24 and the micro-channels (the through channel 25 and the bypass channel 26).

[0101] In the embodiments of the present disclosure, the 3D multi-organ co-culture chip is divided into a film-free chip and a film chip (the same as the aforementioned film-free chip and film chip) according to whether the thin film layer 14 is provided between the first culture micro-holes 22 and the second culture micro-holes 23.

[0102] In some embodiments, the liquid storage holes 21 and the second operation holes I 241 provided on the first liquid storage layer 11 are columnar holes. When the culture module includes side fluid operation holes, the side operation holes I provided on the first liquid storage layer are columnar holes. When the culture module includes first fluid operation holes, the first fluid operation holes are columnar holes. In this embodiment, the first liquid storage layer 11 has a plurality of columnar holes protruding from its surface, avoiding the mutual influence of fluids in different holes and avoiding contamination.

[0103] An application of the foregoing 3D multi-organ co-culture chip according to an embodiment of the present disclosure in vitro constructing a multi-organ model. The following gives a specific method for in vitro constructing a multi-organ model for reference only. When the 3D multi-organ co-culture chip according to the embodiment of the present disclosure is used for in vitro constructing a multi-organ model, it is not limited to the following construction method.

[0104] The construction method of the co-culture chip according to the embodiment of the present disclosure for constructing a 3D blood vessel and tumor multi-organ model includes the following steps:

[0105] Sterilize the chip by ultraviolet light. After the sterilization is completed, add 20 µL of low-concentration rat tail type I collagen to the second fluid operation hole 24, incubate at 37 °C, then discard it, and wash it three times with PBS. Add 2 uL of fibrin (2.5 mg / mL)-collagen (0.2 mg / mL) gel along the first culture micro-hole 22 to the second culture micro-hole 23 for culture, ensuring that the gel can only fill the second culture micro-hole 23, and solidify at 37 °C for 0.5 h.

[0106] Digest and centrifuge HUVECs, resuspend them with complete medium into a single-cell suspension with a certain concentration (1×10 6 cells / mL), and add 20 µL of endothelial cell suspension at the port (which can be defined as the injection port) of the first side through-channel 251 (including the through-channel 25 and the bypass channel 26). Tilt the chip to allow the endothelial cells to fully adhere to the surface of the solidified fibrin and collagen gel.

[0107] On the second day after the inoculation of HUVECs, digest and centrifuge U251 cells, resuspend them with a medium containing 10% serum into a single-cell suspension of 200w / ml. In a 1.5 ml EP tube, add a specific volume of other matrix materials such as 5 mg / mL collagen or Matrigel according to the ratio, ensuring that a good three-dimensional structure is formed by the 3D material at this concentration. Then add the cell suspension according to the ratio, and pipette and mix evenly, and quickly transfer and inoculate it into the first culture micro-hole 22 in a high-throughput manner, 6-12 µL per hole according to the micro-hole size; after the cell seeding is completed, transfer the whole chip to a 37 °C incubator for culture.

[0108] The above description and drawings fully disclose embodiments of the present disclosure so that those skilled in the art can practice them. Other embodiments may include structural and other changes. Embodiments merely represent possible variations. Unless explicitly required, individual components and functions are optional, and the order of operations may vary. Parts and features of some embodiments may be included in or substituted for parts and features of other embodiments. Embodiments of the present disclosure are not limited to the structures described above and shown in the drawings, and various modifications and changes can be made without departing from its scope. The scope of the present disclosure is limited only by the appended claims.

Claims

1. A 3D multi-organ co-culture chip, characterized in that, it includes: a chip body, on which multiple groups of culture modules are arranged; the multiple groups of culture modules are arranged in a high-throughput layout according to the well spacing of a multi-well plate; wherein, each group of the culture modules includes: a liquid storage hole, one end of which is open and located on the upper surface of the chip body; a first culture micro-hole, located below the liquid storage hole and communicated with the liquid storage hole; a second culture micro-hole, located below the first culture micro-hole and communicated with the first culture micro-hole; a plurality of second fluid operation holes, one end of which is open and located on the upper surface of the chip body, and the other end is communicated with the second culture micro-hole through a channel; the plurality of second fluid operation holes are arranged in pairs; each pair of the second fluid operation holes is communicated through a through-channel; the through-channel includes a first side through-channel and a second side through-channel, and the first side through-channel and the second side through-channel are oppositely arranged on opposite sides of the second culture micro-hole; the liquid storage hole and the second fluid operation hole are used for containing a culture medium or a drug dilution solution; the cross-sectional area of the liquid storage hole is larger than the cross-sectional area of the first culture micro-hole; the cross-sectional area of the second culture micro-hole is larger than the cross-sectional area of the first culture micro-hole; the cross-sectional area of the liquid storage hole is larger than the cross-sectional area of the second culture micro-hole; the center distance between the liquid storage hole and the second fluid operation hole is 1-10 mm.

2. The 3D multi-organ co-culture chip according to claim 1, characterized in that, the culture module further includes: a plurality of side fluid operation holes, one end of which is open and located on the upper surface of the chip body; a bypass channel, the two ends of which are respectively communicated with one of the side fluid operation holes, and the middle part of which is communicated with the second culture micro-hole.

3. The 3D multi-organ co-culture chip according to claim 2, characterized in that, one or more micro-channels are arranged at the connection of the bypass channel and the second culture micro-hole.

4. The 3D multi-organ co-culture chip according to any one of claim 1, characterized in that, the culture module further includes: a plurality of first fluid operation holes, one end of which is open and located on the upper surface of the chip body, and the other ends are respectively communicated with the liquid storage hole through a first communication channel.

5. The 3D multi-organ co-culture chip according to claim 1, characterized in that, the center distance between the liquid storage hole and the second fluid operation hole is 3-8 mm.

6. The 3D multi-organ co-culture chip according to claim 1, characterized in that, the liquid storage hole adopts a column hole.

7. The 3D multi-organ co-culture chip according to claim 1, characterized in that, the second fluid operation hole is a stepped hole, and the aperture of the stepped hole becomes smaller from the upper surface of the chip body to the inside.

8. The 3D multi-organ co-culture chip according to claim 1, characterized in that, one or more vertical fences are arranged at a set interval on the communication port of one side sidewall in the middle of the bypass channel and the second culture micro-hole, separating the communication port into a plurality of vertical and parallel vertical micro-channels; or, one or more horizontal fences are arranged horizontally on the communication port of one side sidewall in the middle of the bypass channel and the second culture micro-hole; or, A transverse fence is horizontally arranged along the upper edge of the communication port between the side wall on one side in the middle of the bypass channel and the second culture micro-well, covering the lower or upper part of the communication port, and a transverse micro-channel is formed above or below the transverse fence.

9. The 3D multi-organ co-culture chip according to claim 1, characterized in that the bypass channel is arranged on both sides or one side of the other two opposite sides of the second culture micro-well not communicating with the through-channel.

10. The 3D multi-organ co-culture chip according to claim 1, characterized in that the second culture micro-well is square, having a first side, a second side, a third side and a fourth side, wherein the first side and the second side are opposite sides, and the third side and the fourth side are opposite sides; a first side through-channel of the through-channel is arranged on the first side, a second side through-channel of the through-channel is arranged on the second side, and communication ports are opened on the third side and / or the fourth side for communicating with the middle of the bypass channel.

11. The 3D multi-organ co-culture chip according to any one of claims 1 to 10, characterized in that the chip body includes: a first liquid storage layer, on which the liquid storage hole and the second operation hole I of the second fluid operation hole are arranged; a first culture layer, on which the first culture micro-well and the second operation hole II of the second fluid operation hole are arranged; the first culture layer is stacked below the first liquid storage layer, and the first culture micro-well is communicated with the liquid storage hole; the second operation hole II is communicated with the second operation hole I; a second culture layer, on which the second culture micro-well and the channel are arranged; the second culture layer is stacked below the first culture layer, and the second culture micro-well is communicated with the first culture micro-well, and the channel communicates the second culture micro-well with the second operation hole II; wherein, when the culture module includes side fluid operation holes and a bypass channel, a side operation hole I of the side fluid operation hole is further arranged on the first liquid storage layer, a side operation hole II of the side fluid operation hole is further arranged on the first culture layer, and the side operation hole II is communicated with the side operation hole I; a bypass channel is further arranged on the second culture layer, and the bypass channel communicates the second culture micro-well with the side operation hole II; when the culture module includes a first fluid operation hole and a first communication channel, the first fluid operation hole and the first communication channel are further arranged on the first liquid storage layer.

12. The 3D multi-organ co-culture chip according to claim 11, characterized in that the chip body further includes: a thin film layer, on which micro-nano sized holes are distributed; arranged between the first culture micro-well and the second culture micro-well.

13. The 3D multi-organ co-culture chip according to claim 11, characterized in that the liquid storage hole and the second operation hole I arranged on the first liquid storage layer are column holes; when the culture module includes side fluid operation holes, the side operation hole I arranged on the first liquid storage layer is a column hole; When the culture module includes the first fluid operation hole, the first fluid operation hole is a columnar hole.

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