High-throughput culture plates, high-throughput multi-organ co-culture chips and their applications

Abstract

The present application discloses a high-throughput culture plate, a high-throughput multi-organ co-culture chip and its application, which belongs to the field of biological tissue engineering. The suspended culture plate is provided with a plurality of protrusions on one side of the plate body, and a protrusion pattern is formed on the end face of each protrusion. The culture chip includes a plate body, and a plurality of culture wells and a fluid operation channel are formed on the plate body, and the fluid operation channel is constructed to operate the fluid in the culture well through the fluid operation channel. The co-culture chip is arranged on the culture chip in such a way that the plurality of protrusions of the suspended culture plate correspond to the plurality of culture wells of the culture chip. The co-culture chip is used for the construction of a multi-organ co-culture model. It can culture at least two organ cells at the same time and construct a multi-organ co-culture model, which can provide a platform for large-scale metabolic drug screening and related mechanism research.

Description

Technical Field

[0001] The present application relates to the field of biological tissue engineering technology, for example, to a high-throughput culture plate, a high-throughput multi-organ co-culture chip and applications thereof. Background Art

[0002] Organ-on-a-chip technology is an emerging frontier in biotechnology, listed as one of the "Top 10 Emerging Technologies" by the 2016 World Economic Forum in Davos. It utilizes microfabrication techniques to construct physiological organ microsystems. These typically incorporate key elements of the organ microenvironment, such as living cells, tissue interfaces, biofluids, and mechanical forces, and can reflect the primary structural and functional characteristics of tissues and organs.

[0003] The liver is an organ in the body primarily responsible for metabolism, thoroughly breaking down and biotransforming drugs. Drug metabolism in the liver alters the drug's chemical structure, increasing or decreasing its activity. During drug development, it is difficult to accurately predict liver damage caused by a drug under development, and once such damage occurs, it is difficult to reverse. Therefore, constructing an in vitro liver organ model is of great significance for studying and predicting liver damage in vivo, as well as for screening the activity of metabolic drugs in vitro. Summary of the Invention

[0004] The disclosed embodiments provide culture plates, high-throughput multi-organ co-culture chips, and their applications. To provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is provided below. This summary is not intended to be a comprehensive review, identify key or important components, or delineate the scope of protection for these embodiments. Its sole purpose is to present some concepts in a simplified form, serving as a prelude to the detailed description that follows.

[0005] According to a first aspect of an embodiment of the present disclosure, a hanging culture plate is provided.

[0006] In some embodiments, the suspended culture plate includes a first plate body, a plurality of convex columns are provided on one side surface of the first plate body, and a convex pattern is formed on the end surface of each convex column.

[0007] According to a second aspect of the embodiments of the present disclosure, a culture chip is provided.

[0008] In some embodiments, the culture chip includes a second plate body, on which a plurality of culture wells and fluid operation channels are formed, and the fluid operation channels are configured to operate the fluid in the culture wells via the fluid operation channels.

[0009] According to a third aspect of the embodiments of the present disclosure, a high-throughput multi-organ co-culture chip is provided.

[0010] In some embodiments, the multi-organ co-culture chip comprises a first chip and a second chip;

[0011] The first chip is the aforementioned suspended culture plate;

[0012] The second chip is the aforementioned culture chip;

[0013] The first chip is arranged on the second chip in such a manner that the plurality of protrusions of the first chip are correspondingly suspended in the plurality of culture wells of the second chip.

[0014] According to a fourth aspect of the embodiments of the present disclosure, a high-throughput multi-organ co-culture chip is provided for use in constructing a multi-organ co-culture model.

[0015] In some embodiments, in the application, the method for constructing a multi-organ co-culture model based on the high-throughput multi-organ co-culture chip comprises:

[0016] Inoculating a mixed cell suspension comprising cells of the first organ and a matrix material onto the end surface of the convex column of the first chip, culturing at 37° C. to form a gel, thereby obtaining a gelled first chip;

[0017] adding a mixed cell suspension comprising cells of the second organ and a matrix material into the culture wells of the second chip, culturing at 37° C. to form a gel, thereby obtaining a gelled second chip;

[0018] Adding culture medium into the culture wells of the second gelled chip, and then placing the first gelled chip on the second gelled chip in a manner such that the protrusions extend into the multiple culture wells of the second gelled chip, thereby forming a multi-organ co-culture model;

[0019] The multi-organ co-culture model was cultured at 37° C. to complete the construction of the multi-organ co-culture model.

[0020] The technical solutions provided by the embodiments of the present disclosure may have the following beneficial effects:

[0021] The suspended culture plate of the disclosed embodiment enables suspension culture and is simple to prepare. The culture chip enables 3D cell culture and is simple to prepare. Both can function independently or be combined to create a high-throughput multi-organ co-culture chip capable of simultaneously culturing cells from at least two organs, creating a multi-organ co-culture model.

[0022] The high-throughput multi-organ co-culture chip of the disclosed embodiment can be used to biomimetically construct a 3D in vitro organ model combined with high-throughput drug screening of metabolic drugs, and can be used in scientific research and drug screening of related drugs.

[0023] The high-throughput organ multi-organ co-culture chip of the disclosed embodiment is convenient for liquid operation, suitable for high-throughput operation and characterization, and does not require external equipment.

[0024] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0026] Figure 1 is a schematic structural diagram of a suspended culture plate according to an exemplary embodiment;

[0027] Figure 2 yes Figure 1 Schematic diagram of the enlarged structure at A in the middle;

[0028] Figure 3 is a schematic structural diagram of a suspended culture plate according to another exemplary embodiment;

[0029] Figure 4 is a schematic diagram of a partial structure of a suspended culture plate according to another exemplary embodiment;

[0030] Figure 5 is a schematic diagram of a partial structure of a suspended culture plate according to another exemplary embodiment;

[0031] Figure 6 is a schematic structural diagram of a culture chip according to an exemplary embodiment;

[0032] Figure 7 is a schematic diagram of an exploded structure of a culture chip according to another exemplary embodiment;

[0033] Figure 8 is a schematic diagram of an exploded structure of a culture chip according to another exemplary embodiment;

[0034] Figure 9 is a schematic diagram of an exploded structure of a culture chip according to another exemplary embodiment;

[0035] Figure 10 is a schematic diagram of an exploded structure of a culture chip according to another exemplary embodiment;

[0036] Figure 11 is a schematic cross-sectional view of a suspended culture plate according to an exemplary embodiment;

[0037] Figure 12 yes Figure 6 Schematic diagram of the cross-sectional structure of a culture chip is shown;

[0038] Figure 13 yes Figure 7 Schematic diagram of the cross-sectional structure of a culture chip is shown;

[0039] Figure 14 yes Figure 8 Schematic diagram of the cross-sectional structure of a culture chip is shown;

[0040] Figure 15 yes Figure 9 Schematic diagram of the cross-sectional structure of a culture chip is shown;

[0041] Figure 16 yes Figure 10 Schematic diagram of the cross-sectional structure of a culture chip is shown;

[0042] Figure 17 is a schematic cross-sectional structural diagram of a multi-organ co-culture chip according to an exemplary embodiment;

[0043] Figure 18 is a schematic cross-sectional structural diagram of a multi-organ co-culture chip according to an exemplary embodiment;

[0044] Figure 19 is a schematic diagram of an exploded structure of a multi-organ co-culture chip according to an exemplary embodiment;

[0045] Figure 20 It is a schematic diagram of a stepped cross-sectional structure of a multi-organ co-culture chip according to an exemplary embodiment.

[0046] Figure 21 It is a histogram of cell culture time-fluorescence intensity of 3D liver cells;

[0047] Figure 22 It is a comparative bar graph of cell culture time-fluorescence intensity of single-cultured and co-cultured 3D tumor cells;

[0048] Figure 23 It is a bar graph comparing the cell culture time-fluorescence intensity of single-cultured and co-cultured 3D tumor cells.

[0049] Figure 24 This is a graph showing the effect of different concentrations of the metabolic anti-tumor drug CPT-11 on the activity of MCF-7 cells in a co-culture platform of liver and breast cancer tumors;

[0050] Figure 25 This is a graph showing the effect of different concentrations of the metabolic anti-tumor drug CPT-11 on the activity of HCT116 cells in a liver and colon cancer co-culture platform;

[0051] Description of reference numerals:

[0052] 10. Suspended culture plate; 11. convex column; 110. convex pattern; 111. non-closed geometric pattern; 112. ring; 12. first through hole; 20. culture chip; 21. culture well; 211. lower culture micropore portion; 212. upper liquid storage hole portion; 2120. column portion; 22. fluid operation channel; 221. second through hole; 222. third through hole; 223. transverse channel; 224. fourth through hole; 201. liquid storage layer; 202. 3D culture layer; 2021. 3D culture sublayer; 2022. bottom plate layer; 203. channel layer. DETAILED DESCRIPTION

[0053] The following will be combined with the embodiments of the present disclosure to clearly and completely describe the technical solutions of the present disclosure. Obviously, the embodiments described are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present disclosure.

[0054] The present disclosure discloses a suspension culture plate 10, combined with Figures 1 to 5 As shown, the suspended culture plate 10 includes a plate body (first plate body), a plurality of convex columns 11 are provided on one side surface of the plate body (first plate body), and a convex pattern 110 is formed on the end surface of each convex column 11.

[0055] The surface of the raised pattern 110 of the suspended culture plate 10 of the disclosed embodiment is hydrophobic. When the cell suspension is inoculated on the raised pattern 110, the cell suspension can form a smooth hemisphere on the raised pattern 110, culture, and after gelation, it is suspended downward in the culture medium for hanging drop culture. By designing the arrangement of the raised columns 11 on the suspended culture plate 10, it can be compatible with commercial sample loading equipment and detectors (such as microplate readers, high-content imaging systems, etc.), enabling high-throughput sample loading and detection. For example, 96-well, 384-well, etc.

[0056] In the disclosed embodiment, the thickness of the suspension culture plate 10 is not limited and depends on the biocompatible material used. In addition, it is necessary to ensure that the suspension culture plate is not easily deformed and does not affect the shape of the droplets on the protrusion 11 during operations such as picking up and clamping.

[0057] In the embodiment of the present disclosure, the shape and size of the protrusion 11 are not limited, as long as it has a certain end surface on which the protrusion pattern 110 can be formed. Optionally, the shape of the protrusion 11 is a cylindrical protrusion.

[0058] In some embodiments, the diameter of the protrusion 11 is 1 to 5 mm. Alternatively, the diameter of the protrusion 11 is 2 to 3 mm. Alternatively, the diameter of the protrusion 11 is 2.5 mm.

[0059] In some embodiments, the height of the protrusion 11 is 0.8-2 mm. Optionally, the height of the protrusion 11 is 1 mm.

[0060] In the disclosed embodiment, the shape of the protrusion pattern 110 is not limited, as long as it forms a protrusion on the end surface of the protrusion 11. It can be a regular or irregular geometric shape. The size of the protrusion pattern 110 is also not limited, as long as it can form droplets using the cell suspension.

[0061] In some embodiments, the height of the raised pattern 110 is 0.1-0.5 mm. Alternatively, the height of the raised pattern 110 is 0.2 mm.

[0062] In some embodiments, as Figure 1 and Figure 2 As shown, the raised pattern 110 is a non-closed geometric pattern 111, such as a cross, a "C", an "X", etc.

[0063] In some embodiments, as Figure 4 As shown, the raised pattern 110 is in the form of a ring 112, such as a ring with a regular geometric shape such as a circular ring or a square ring, or a ring with an irregular geometric shape.

[0064] In some embodiments, as Figure 5 As shown, the raised pattern 110 is a non-closed geometric pattern 111 and a ring 112 , and the non-closed geometric pattern 111 is located inside the ring 112 .

[0065] In some embodiments, as Figure 3 As shown, the plate body (first plate body) of the suspension culture plate 10 is further formed with a first through-hole 12. This facilitates manipulation of the culture fluid during the suspension culture process, such as adding or removing the culture fluid. For example, in the multi-organ co-culture chip described below, the first through-hole 12 can form a fluid manipulation channel with the fluid manipulation channel 22 on the second chip (culture chip 20) for fluid manipulation, facilitating manipulation of the culture fluid within the culture well 21 during the co-culture process.

[0066] In the disclosed embodiment, the suspended culture plate 10 can be obtained by integral injection molding or by laser etching.

[0067] Optionally, the suspended culture plate 10 is made by casting a polydimethylsiloxane prepolymer (PDMS prepolymer) mold. The mold can be made using conventional technical means based on the structure of the suspended culture plate 10. Optionally, a polymethyl methacrylate mold (PMMA mold) is used. That is, a suspended culture plate 10 is a PDMS microcolumn plate. The prepolymer monomer is dissolved and disinfected, and the dissolution and disinfection treatment includes: soaking in ethanol and ultrasonically, then soaking in pure water and ultrasonically, and then drying. Optionally, soaking in ethanol and ultrasonically for 30 minutes. Optionally, soaking in pure water and ultrasonically for 30 minutes. Optionally, drying at 50°C.

[0068] The present disclosure embodiment discloses a culture chip 20, combined with Figures 6 to 16 As shown, the culture chip 20 includes a plate body (second plate body) on which a plurality of culture wells 21 and a fluid operation channel 22 are formed. The fluid operation channel 22 is constructed so that the fluid in the culture wells 21 can be operated via the fluid operation channel 22.

[0069] The culture chip 20 of the disclosed embodiment has a fluid manipulation channel 22 structure, which facilitates manipulation of the fluid (e.g., culture fluid) within the culture wells 21, such as adding or removing culture fluid. The design of the arrangement of the culture wells 21 on the culture chip 20 makes it compatible with commercially available sample loading equipment and detectors (e.g., microplate readers, high-content imaging systems, etc.), enabling high-throughput sample loading and detection, such as 96-well and 384-well arrays.

[0070] In this embodiment, the fluid in the culture well 21 can be operated through the fluid operation channel 22. As can be seen, the first end of the fluid operation channel 22 is connected to the culture well 21, and the second end is located on the surface of the culture chip 20 (the surface facing upward during cultivation), which is convenient for operation. The number of fluid operation channels 22 is not limited, and one fluid operation channel 22 can be set for each culture well 21 (see Figure 7 As shown, each fluid operation channel 22 has a first end and a second end. Alternatively, multiple culture wells can be grouped, with each group of culture wells provided with a fluid operation channel 22. In this configuration, each fluid operation channel 22 has multiple first ends and one second end, with the multiple first ends communicating with multiple culture wells 21, respectively. Each culture well group includes two or more culture wells 21, and the specific number is not limited and can be set based on actual conditions.

[0071] In some embodiments, as Figures 12 to 16As shown, the fluid manipulation channel 22 includes a vertical through hole and a transverse channel. The transverse channel is connected to the culture well 21. The opening of the vertical through hole is formed on the surface of the culture chip 20. Optionally, the transverse channel is a microchannel. Optionally, the dimensions of the transverse channel are 0.05-2 mm in width and 10-500 μm in depth. Optionally, the dimensions of the transverse channel are 1 mm in width and 200 μm in depth.

[0072] In some embodiments, each culture well 21 on the culture chip 20 includes a lower culture micropore portion 211 and an upper liquid storage well portion 212 (see Figure 12 (As shown). The lower culture microporous portion 211 is used for 3D cell culture, and the upper liquid storage portion 212 is used to store culture fluid. Therefore, the shapes and pore sizes of the pores in the lower culture microporous portion 211 and the upper liquid storage portion 212 can be consistent or inconsistent. If the pore sizes are inconsistent, the pore size of the lower culture microporous portion 211 is smaller than the pore size of the upper liquid storage portion 212. In other words, optionally, the pore size of the lower culture microporous portion 211 is smaller than or equal to the pore size of the upper liquid storage portion 212.

[0073] In some embodiments, the communication port between the fluid operation channel 22 and the culture well 21 is located in the upper liquid storage hole portion 212, which facilitates the operation of the fluid.

[0074] In some embodiments, the culture wells 21 are stepped wells, and the pore size of the lower culture micropore portion 211 is smaller than the pore size of the upper liquid storage well portion 212 .

[0075] In some embodiments, the pore size of the upper liquid reservoir portion 212 is 1-6 times the pore size of the lower culture microporous portion 211. Alternatively, the pore size of the upper liquid reservoir portion 212 is 2-3 times the pore size of the lower culture microporous portion 211. Alternatively, the pore size of the upper liquid reservoir portion 212 is 2.5 times the pore size of the lower culture microporous portion 211.

[0076] In some embodiments, the upper reservoir portion 212 and / or the lower culture micropore portion 211 are straight pores, i.e., have a uniform pore diameter. For example, if the cross-section of the upper reservoir portion 212 is circular, the straight pores are cylindrical. Straight pores offer advantages such as simplified focusing for fluorescence microscopy and simplified imaging for high-content drug screening. Alternatively, the pores may be tapered pores with gradually varying depths.

[0077] In some embodiments, the upper liquid storage hole portion 212 and / or the lower culture microporous portion 211 can be shaped as a circular hole, a polygonal hole, or a hole of an irregular geometric shape. For a circular hole, the pore diameter is the diameter of the circle; for a polygonal hole, the pore diameter is the diameter of the inscribed circle of the polygon; for an irregularly shaped hole, the pore diameter is the maximum width of the irregular geometric shape.

[0078] In some embodiments, the depth of the upper liquid storage hole portion 212 is 2-15 mm. Alternatively, the depth of the upper liquid storage hole portion 212 is 3-8 mm. Alternatively, the depth of the upper liquid storage hole portion 212 is 6 mm.

[0079] In some embodiments, the pore size of the upper liquid storage hole portion 212 is 3-8 mm. Optionally, the pore size of the upper liquid storage hole portion 212 is 6 mm.

[0080] In an optional embodiment, the depth of the lower culture microporous portion 211 is 0.5-2 mm. Optionally, the depth of the lower culture microporous portion 211 is 1 mm.

[0081] In an optional embodiment, the pore size of the lower culture microporous portion 211 is 2-4 mm. Optionally, the pore size of the lower culture microporous portion 211 is 2.5 mm.

[0082] In this embodiment, the upper reservoir 212 is used to store culture fluids such as cell culture medium or drug diluents and deliver substances to the biomimetic microtissue, providing the entire system with nutrients necessary for growth or drugs to be tested. The structure and configuration of the upper reservoir 212 can be varied, as long as the upper reservoir 212 can be formed to achieve the aforementioned objectives. The lower microporous culture section 211 can be used to culture organ cells mixed with a three-dimensional matrix, more closely simulating the in vivo cell growth microenvironment.

[0083] In the disclosed embodiment, the formation method of the culture wells 21 is not limited. The culture chip 20 can be integrally formed (e.g., by integral injection molding or laser etching) to form an integral culture well 21. Alternatively, the culture wells 21 can be formed by layered processing, with each layer stacked sequentially to form corresponding liquid reservoir portions and culture micropore portions on each layer. After stacking, the culture wells 21 are formed.

[0084] In some embodiments, as Figure 6 and Figure 11 As shown, the culture chip 20 is integrally formed, for example, by injection molding or laser etching. The fluid manipulation channel 22 includes a vertical through hole and a transverse channel that are connected to the culture well 21.

[0085] In some embodiments, as Figure 7 and Figure 13 As shown, the culture chip 20 includes a liquid storage layer 201 and a 3D culture layer 202 arranged in layers.

[0086] The liquid storage layer 201 has a plurality of liquid storage through holes (ie, the upper liquid storage hole portion 212 ), and the liquid storage through holes are used to store culture fluid.

[0087] The 3D culture layer 202 has a plurality of culture microwells (ie, the lower culture microwell portion 211 ), which are used for 3D cell culture.

[0088] The liquid storage through holes correspond to the culture micropores one by one to form culture wells 21 ; and a fluid operation channel 22 is formed on the liquid storage layer 201 .

[0089] In this embodiment, the structure and size of the liquid storage through hole are the same as those of the upper liquid storage hole portion 212, and the structure and size of the culture micropores are the same as those of the lower culture micropore portion 211, which will not be repeated here.

[0090] In this embodiment, the fluid manipulation channel 22 formed on the liquid reservoir layer 201 can manipulate the fluid within the culture wells 21. Optionally, the liquid reservoir layer 201 comprises a plurality of second through-holes 221 (i.e., vertical channels) and a plurality of transverse channels 223. The plurality of second through-holes 221 correspond one-to-one with the plurality of liquid reservoir through-holes. The ends of the transverse channels 223 connect the second through-holes 221 and the liquid reservoir through-holes. The openings of the second through-holes 221 are formed on the surface of the liquid reservoir layer 201. Of course, the fluid manipulation channel 22 can also adopt other structural forms, as long as it can complete the structure of manipulating the culture fluid within the culture wells 21.

[0091] In some embodiments, the liquid storage layer 201 may be made of materials such as polymethylmethacrylate (PMMA) or polystyrene (PS), but is not limited to the listed materials. The liquid storage layer 201 may be implemented by laser perforation or one-time injection molding.

[0092] In some embodiments, the liquid storage layer 201 is a liquid storage plate, on which a plurality of liquid storage through holes are provided, and a plurality of fluid operation channels 22 (including second through holes 221 and transverse channels 223, see above) are formed, wherein the plurality of second through holes 221 correspond one to one with the plurality of liquid storage through holes, such as Figure 7 and Figure 13 The liquid storage layer 201 shown in FIG. In this embodiment, the depth of the liquid storage through hole is the thickness of the liquid storage plate.

[0093] Alternatively, as Figures 8 to 10 As shown, the liquid storage through-hole of the liquid storage layer 201 is a liquid storage column hole (i.e., the liquid storage through-hole has a columnar portion 2120 with a protruding surface), and the second through-hole 221 is also a columnar hole. In other words, the liquid storage column hole is formed by opening a through-hole in the protruding column (i.e., the columnar portion 2120). In this case, the depth of the liquid storage column hole is the sum of the height of the columnar portion 2120 and the thickness of the base of the liquid storage layer 201.

[0094] In this embodiment, when the liquid storage through holes are in the form of liquid storage column holes, the thickness of the substrate of the liquid storage layer 201 is not limited, as long as it has sufficient strength to support the liquid storage column holes arranged thereon. The wall thickness of the column portion 2120 of the liquid storage column hole is also not limited.

[0095] The 3D culture layer 202 may be made of materials such as polymethyl methacrylate (PMMA) or polystyrene (PS), but is not limited to the materials listed above. The 3D culture layer 202 may be formed by laser perforation or one-time injection molding.

[0096] In this embodiment, in the 3D culture layer 202, the culture micropores are blind holes. Therefore, the culture holes 21 can be holes formed on the plate (formed by laser etching or integral injection molding). Figure 8 As shown, it is an integrally formed 3D culture layer 202. It can also be formed by layering plates.

[0097] In some embodiments, as Figure 9 and Figure 10 As shown, the 3D culture layer 202 includes a 3D culture sublayer 2021 and a bottom plate layer 2022. A through hole (i.e., the lower culture micropore portion 211) is opened on the 3D culture sublayer 2021. The 3D culture sublayer 2021 is superimposed on the bottom plate layer 2022, and the through hole and the surface of the bottom plate layer form culture micropores.

[0098] In some embodiments, see Figure 10 and Figure 16 As shown, the culture chip 20 further includes a channel layer 203 located between the liquid reservoir layer 201 and the 3D culture layer 202. The channel layer 203 is provided with third through-holes 222, and the liquid reservoir layer 201 is provided with a plurality of second through-holes 221. The third through-holes 222 correspond one-to-one with the liquid reservoir through-holes. Transverse channels 223 are configured to connect the third through-holes 222 with the second through-holes 221. The transverse channels 223 can be formed on either the side of the liquid reservoir layer 201 that connects to the channel layer 203 or the side of the channel layer 203 that connects to the liquid reservoir layer 201.

[0099] Optionally, the liquid storage through hole, the third through hole 222 and the culture micropore are coaxially stacked to form a coaxial stepped hole. The liquid storage through hole and the third through hole 222 can together constitute the upper liquid storage hole portion 212 for storing fluids such as culture fluid.

[0100] Optionally, the transverse channel 223 is formed on the channel layer 203. Optionally, the transverse channel 223 is formed on the side of the channel layer 203 connected to the liquid storage layer 201, Figure 16 shown.

[0101] In some embodiments, as Figure 10 and Figure 16 As shown, in addition to the third through hole 222 and transverse channel 223 previously formed, the channel layer 203 further has a fourth through hole 224. The fourth through hole 224 corresponds one-to-one with the second through hole 221, and the transverse channel 223 is configured to connect the third through hole 222 and the fourth through hole 224. In this embodiment, the transverse channel 223 is provided on the channel layer 203. Optionally, the transverse channel 223 can be formed on the side of the channel layer 203 that connects to the 3D culture layer 202, or on the side that connects to the liquid reservoir layer 201.

[0102] Optionally, the transverse channel 223 is formed on the side of the channel layer 203 connected to the liquid storage layer 201, such as Figure 16 This can prevent the fluid from directly acting on the lower culture microwells.

[0103] Optionally, the second through hole 221 and the fourth through hole 224 are coaxially stacked to form a coaxial stepped hole.

[0104] The thickness of the channel layer 203 is not limited as long as the transverse channel 223 can be formed thereon.

[0105] In some embodiments, the transverse channel 223 is a microchannel, which slows fluid exchange and reduces interference with cultured cells. The transverse channel has a width of 0.05-2 mm and a depth of 10-500 μm. Optionally, the transverse channel has a width of 1 mm and a depth of 200 μm.

[0106] In some embodiments, the culture chip 20 is formed by stacking layers, such as a liquid reservoir layer 201 and a 3D culture layer 202; or a liquid reservoir layer 201, a 3D culture sublayer 2021, and a bottom plate layer 2022; or a liquid reservoir layer 201, a channel layer 203, and a 3D culture layer 202. Adjacent layers can be connected, such as by bonding, or simply overlapped, such as by stacking the three layers sequentially and then clamping them in place using a fixture.

[0107] Optionally, when two adjacent layers of the culture chip 20 are connected, a first adhesive layer (not shown) is further included. The first adhesive layer is located between the liquid storage layer 201 and the 3D culture layer 202 .

[0108] Optionally, when two adjacent layers of the culture chip 20 are connected, it also includes a first bonding layer (not shown) and a second bonding layer (not shown), the first bonding layer is located between the liquid storage layer 201 and the 3D culture sublayer 2021, and the second bonding layer is located between the 3D culture sublayer 2021 and the bottom plate layer 2022.

[0109] Optionally, when two adjacent layers of the culture chip 20 are connected, it also includes a first bonding layer (not shown), a third bonding layer (not shown) and a second bonding layer (not shown). The first bonding layer is located between the liquid storage layer 201 and the channel layer 203, the third bonding layer is located between the channel layer 203 and the 3D culture sublayer 2021, and the second bonding layer is located between the 3D culture sublayer 2021 and the bottom plate layer 2022.

[0110] The first adhesive layer can be integrally formed on the liquid reservoir layer 201 or on the connecting side of the 3D culture layer 202, or can be adhesively provided on the liquid reservoir layer 201 or on the connecting side of the 3D culture layer 202 when the liquid reservoir layer 201 and the 3D culture layer 202 are connected. Similarly, the second and third adhesive layers are provided in the same manner as the first adhesive layer.

[0111] Optionally, the first adhesive layer and the second adhesive layer may be made of double-sided adhesive or PDMS liquid adhesive.

[0112] When the culture chip 20 of the embodiment of the present disclosure adopts a layered structure, a preparation method of the culture chip 20 is provided, comprising:

[0113] Each layer of the culture chip 20 (liquid reservoir layer and 3D culture layer; or, liquid reservoir layer, channel layer and 3D culture layer; wherein the 3D culture layer is an integrally formed 3D culture layer, or includes a 3D culture sublayer and a bottom plate layer) is cleaned separately and then dried.

[0114] The layers are stacked and bonded in sequence using double-sided tape to complete the preparation of the culture chip 20 and obtain the culture chip.

[0115] In some embodiments, cleaning includes soaking in deionized water for 12-24 hours and then soaking in ethanol for disinfection. Optionally, the ethanol is 75% (volume) ethanol.

[0116] In some embodiments, the drying condition is a drying temperature of 45°C to 65°C, optionally 50°C.

[0117] In some embodiments, the double-sided tape is integrally formed on the bonding surfaces of each layer.

[0118] The present disclosure also provides a high-throughput multi-organ co-culture chip. Figures 17 to 20 As shown, the high-throughput multi-organ co-culture chip includes a first chip and a second chip. The first chip is the aforementioned suspended culture plate 10, and the second chip is the aforementioned culture chip 20. The first chip is arranged on the second chip in such a way that the plurality of protrusions 11 of the first chip are correspondingly suspended within the plurality of culture wells 21 of the second chip.

[0119] The high-throughput multi-organ co-culture chip of the present embodiment can culture at least two organ cells at the same time, build a multi-organ co-culture model, and can provide a platform for large-scale metabolic drug screening and related mechanism research. Moreover, it is possible to achieve micro-consumption of cells, matrix materials, reagents and drugs, solving the problem of high cost in large-scale drug screening processes. It can also be applied to models with highly integrated functions and strong biomimetic capabilities. In addition, by designing the arrangement of the convex column 11 on the suspension culture plate 10 (first chip) and the culture well 21 on the culture chip 20 (second chip), it is possible to be compatible with commercial sample loading equipment and detectors (such as microplate readers, high-content imaging systems, etc.), and high-throughput sample loading and detection can be achieved. Such as 96 wells, 384 wells, etc.

[0120] The high-throughput multi-organ co-culture chip of this embodiment can be used to construct a 3D liver-tumor co-culture model, which can be used to study the metabolism and biotransformation of prodrugs, discover and evaluate drug components, and effectively predict drug hepatotoxicity, reducing the failure rate of later clinical trials and harm to the human body. Of course, it is not limited to this and can also be used to construct and study co-culture models of other different organ types.

[0121] During co-culture using a multi-organ co-culture chip, to facilitate manipulation of the fluid, such as the culture fluid, within the culture well 21, in some embodiments, when a first through-hole 12 is formed on the first chip, the first through-hole 12 corresponds one-to-one with the second through-hole 221. That is, the first through-hole 12, the second through-hole 221, the third through-hole 222, and the transverse channel 223 constitute a fluid manipulation channel for manipulating the culture fluid within the culture well 21. Alternatively, the first through-hole 12, the second through-hole 221, the third through-hole 222, the fourth through-hole 224, and the transverse channel 223 constitute a fluid manipulation channel for manipulating the culture fluid within the culture well 21.

[0122] The high-throughput multi-organ co-culture chip of this embodiment is not limited to Figure 17 and Figure 18 The structure shown can be composed of any combination of the aforementioned suspended culture plate 10 and culture chip 20.

[0123] The disclosed embodiments provide an application of a high-throughput multi-organ co-culture chip for constructing a multi-organ co-culture model.

[0124] Among them, the method for constructing a multi-organ co-culture model based on a high-throughput multi-organ co-culture chip includes:

[0125] S11, inoculating a first mixed cell suspension comprising first organ cells and matrix material onto the end surface of the protrusion 11 of the first chip (suspension culture plate 10), culturing at 37° C. to form a gel, and obtaining a gelled first chip;

[0126] S12, adding a second mixed cell suspension containing cells of the second organ and a matrix material into the culture well 21 (lower culture microporous portion 211) of the second chip (culture chip 20), culturing at 37° C. to form a gel, thereby obtaining a gelled second chip;

[0127] S13, adding culture medium into the culture wells 21 of the second gelled chip, and then placing the first gelled chip on the second gelled chip in a manner such that the protrusions 11 are suspended in the multiple culture wells 21 of the second gelled chip, thereby forming a multi-organ co-culture model;

[0128] The multi-organ co-culture model was cultured at 37°C to complete the construction of the multi-organ co-culture model.

[0129] There is no particular order in which step S11 and step S12 should be executed. They can be performed simultaneously, with step S11 being performed first and then S12, or step S12 being performed first and then S11.

[0130] In some embodiments, the matrix material in the first mixed cell suspension is collagen, agarose, gelatin, or PEG. Optionally, the matrix material is collagen, and the concentration of the collagen is 1-3 mg / mL. Alternatively, the concentration of the collagen is 2 mg / mL. When agarose, gelatin, or PEG is used as the matrix material, the concentration is determined based on the respective physicochemical properties to ensure that the matrix material can form a gel. These are not listed here one by one.

[0131] In some embodiments, the first organ cells in the first mixed cell suspension are not limited and can be selected according to the specific research content. For example, primary liver cells, liver tumor cells, or any liver cells with drug metabolism function, a mixture of liver cells and endothelial cells, liver microsomes, etc. are involved in drug metabolism research. The single cell concentration in the first organ cells is not limited and can be determined based on actual factors such as inoculation amount and inoculation volume. Optionally, the concentration of the first organ cells in the first mixed cell suspension is 1×10 6 ~2×10 6 cell / mL (cells / ml).

[0132] In some embodiments, the first mixed cell suspension is a mixture comprising a first organ single cell suspension and a matrix material, wherein the volume ratio of the first organ single cell suspension to the matrix material is 1.5 to 4:1. Optionally, the volume ratio of the first organ single cell suspension to the matrix material is 1.5:1.

[0133] The pH of the first mixed cell suspension is between 6.5 and 7.5, which is conducive to cell culture. In some embodiments, the pH of the first mixed cell suspension is adjusted using an alkaline solution. Alternatively, the alkaline solution can be a mixture of one or more of NaOH, NaHCO3, NaOH, and NaHCO3.

[0134] In step S11 , the volume of the first mixed cell suspension inoculated on the end surface of the protrusion 11 of the first chip is not limited, and the inoculation volume can be determined according to the single cell concentration and inoculation amount of the first mixed cell suspension.

[0135] Optionally, the inoculation amount of each protrusion 11 is 1000 to 10000 cells.

[0136] Optionally, the inoculation volume of the first mixed cell suspension is 1 to 10 μL. The inoculation volume of the mixed cell suspension is at the microliter level, and the amount used is small.

[0137] Optionally, in step 11, the incubation at 37°C is to ensure gelation, allowing the hanging drop of hydrogel inoculated on the protrusion 11 to solidify and be securely suspended on the protrusion 11. The incubation time is not limited, as long as gelation is ensured. Optionally, the incubation at 37°C is for 5 to 15 minutes, optionally 10 minutes.

[0138] In some embodiments, the first mixed cell suspension is obtained by the following steps:

[0139] S111. Digestion and centrifugation of first organ cells: Digest the in situ tissue or cell line (2D culture) into single cells using 0.25 (vt.)% trypsin, centrifuge and resuspend into first organ single cell suspension (e.g., 1.43×10 6 The first organ can be selected and determined according to the actual organ model to be constructed, for example, in a liver organ model, liver tumor cells, primary liver cells, such as hepG2 liver tumor cells.

[0140] S112. Mix the first organ single cell suspension with the matrix material, and adjust the pH value to 6.5-7.5 to obtain a first mixed cell suspension.

[0141] In some embodiments, in step S12, the matrix material in the second mixed cell suspension is collagen, agarose, gelatin, or PEG. Optionally, the collagen matrix material is collagen, and the concentration of collagen is 1-3 mg / mL. Optionally, the concentration of collagen is 1.5 mg / mL. As above, when the matrix material is agarose, gelatin, or PEG, the concentration is determined based on the respective physicochemical properties to ensure that it can form a gel. These are not listed here one by one.

[0142] In some embodiments, the second organ cells in the second mixed cell suspension are not limited, such as lung tumor cells, colon cancer cells. The single cell concentration of the second organ cells is not limited and can be determined based on actual factors such as the inoculation amount and inoculation volume. Optionally, the concentration of the second organ cells in the second mixed cell suspension is 1×10 6 ~2×10 6 ×10 6 cell / mL (cells / ml).

[0143] In some embodiments, the second mixed cell suspension is a mixture of a second organ single cell suspension and a matrix material, wherein the volume ratio of the second organ single cell suspension to the matrix material is 1.5 to 4:1. Optionally, the volume ratio of the second organ single cell suspension to the matrix material is 3.5:1.5.

[0144] The pH of the second mixed cell suspension is 6.5 to 7.5, which is conducive to cell culture. In some embodiments, the pH of the second mixed cell suspension is adjusted using an alkaline solution. Alternatively, the alkaline solution can be a mixture of one or more of NaOH, NaHCO3, NaOH, and NaHCO3.

[0145] In step S12 , the volume of the second mixed cell suspension inoculated in the culture wells 21 of the second chip is not limited, and the inoculation volume can be determined according to the concentration of the second mixed cell suspension and the inoculation amount per well.

[0146] Optionally, the inoculation amount in each culture well 21 is 1,000 to 10,000 cells.

[0147] Optionally, the inoculation volume of the second mixed cell suspension is 1 to 10 μL. The inoculation volume of the mixed cell suspension is at the microliter level, and the amount used is small.

[0148] Optionally, in step 12, the culture is carried out at 37°C to ensure gel formation. The culture time is not limited, as long as gel formation is ensured. Optionally, the culture is carried out at 37°C for 5 to 15 minutes, and optionally, for 10 minutes.

[0149] In some embodiments, the second mixed cell suspension is obtained by the following steps:

[0150] S121. Digestion and centrifugation of second organ cells: Digest the in situ tissue or cell line (2D culture) into single cells using 0.25 (vt.)% trypsin, centrifuge and resuspend into a second organ single cell suspension (e.g., 1.43×10 6The second organ can be selected and determined based on the actual organ model to be constructed, for example, various types of tumor cells in the anti-tumor drug screening model, such as breast cancer cells MCF-7 and colon cancer cells HCT116.

[0151] S122. Mix the second organ single cell suspension with the matrix material, and adjust the pH value to 6.5-7.5 to obtain a second mixed cell suspension.

[0152] Below Figure 19 Based on the high-throughput multi-organ co-culture chip shown in , a liver-tumor co-culture chip that can be used for screening metabolic anti-tumor drugs was constructed. Figure 19 The culture well 21 in the high-throughput multi-organ co-culture chip shown in FIG is 96-well or 384-well.

[0153] in, Figure 19 The high-throughput multi-organ co-culture chip (referred to as the co-culture chip sample) shown includes:

[0154] The first chip, i.e., the suspension culture plate 10, has multiple protrusions 11 disposed on one side of the plate. Each protrusion 11 has a raised pattern 110 formed on its end face, forming a cross-shaped, non-closed geometric pattern 111. The protrusions 11 are cylindrical, 2.5 mm in diameter, and 1 mm in height. The raised pattern 110 is 0.2 mm in height. Multiple first through-holes 12 are also provided.

[0155] The second chip, i.e., the culture chip 20, includes a liquid storage layer 201, a channel layer 203, a 3D culture sublayer 2021, and a bottom plate layer 2022.

[0156] The liquid reservoir layer 201 has multiple liquid reservoir holes (i.e., the upper liquid reservoir portion 212). A plurality of second through-holes 221 are formed in the liquid reservoir layer 201. These second through-holes 221 correspond one-to-one with the plurality of first through-holes 12 on the first chip (suspension culture plate 10). The liquid reservoir holes are cylindrical, with a diameter of 6 mm and a depth of 3 mm. The dimensions of the second through-holes 221 are the same as those of the liquid reservoir holes.

[0157] The channel layer 203 has multiple third through holes 222, multiple transverse channels 223, and multiple fourth through holes 224. Each transverse channel 223 has two ends connected to a third through hole 222 and a fourth through hole 224, respectively. The third through holes 222 correspond one-to-one with the liquid storage through holes, and the fourth through holes 224 correspond one-to-one with the second through holes. The thickness of the channel layer 203 is not limited, as long as it can form the transverse channels 223. For example, 3 mm is sufficient.

[0158] The dimensions of the transverse channel 223 are: 1 mm in width, 200 μm in depth, and the length is determined by the distance between the third through hole 222 and the fourth through hole 24 .

[0159] The 3D culture sublayer 2021 has a plurality of culture micropores (ie, the lower culture micropore portion 211 ), and the culture micropores are arranged corresponding to the third through holes 222 and the liquid storage through holes.

[0160] The bottom plate layer 2022 is a glass plate, and its thickness is not limited and can be selected according to actual needs such as imaging effect, for example, 0.1 to 0.5 mm.

[0161] The method for constructing a liver-tumor co-culture chip based on the above-mentioned co-culture chip sample comprises:

[0162] Sterilization treatment: sterilize the first chip (suspension culture plate 10) and the second chip (culture chip 20) for no less than 1 hour, wherein the sterilization can be performed by ultraviolet sterilization.

[0163] Construction of 3D liver organ model: 2D cultured liver HepG2 cells were digested into single cells with 0.25 (vt.)% trypsin and resuspended to a cell density of 1.43 × 10 6 A single-cell suspension of the first organ is prepared at 500 μL / mL. Using 2 mg of collagen as an example, a 1.5 ml EP tube is filled with a volume of 5 mg / mL collagen material and the first organ single-cell suspension, with a volume ratio of 3:2 between the first organ single-cell suspension and the collagen solution, to achieve a final collagen concentration of 2 mg / mL. This creates a first mixed cell suspension, ensuring that the 3D material forms a well-defined three-dimensional structure at this concentration. Pipette the mixture thoroughly and evenly, then divide it into 8 equal portions. Use a pipette to transfer the mixture to the columns 11 of the first chip (suspension culture plate 10). Each column contains 10 μL of the cell-hydrogel mixture (first mixed cell suspension). The gel forms a smooth hemisphere on the top surface of the column. The first chip is placed in a clean, humidified chamber and allowed to stand at 37°C for 15 minutes to allow the collagen hydrogel to fully solidify. The gel can then be securely suspended on the columns 11 of the first chip, resulting in a gelled first chip.

[0164] Construction of tumor organ model: 2D cultured HCT-116 tumor cell line (I) or MCF-7 tumor cell line (II) were digested with 0.25 (vt.)% trypsin to form a single cell suspension, centrifuged and resuspended, and the suspension was prepared to a density of 1.43 × 10 6cell / mL of the second organ single cell suspension. In a 1.5ml EP tube, according to the volume ratio of the second organ single cell suspension to the collagen solution of 3.5:1.5, a certain volume of 5mg / mL collagen material and the second organ single cell suspension was added to make the final collagen concentration of 1.5mg / mL to obtain a second mixed cell suspension, ensuring that the 3D material forms a good three-dimensional structure at this concentration. Use a pipette to mix evenly, divide it into 8 equal parts, and use a pipette to quickly and high-throughput transfer and inoculate it into the lower culture micropore part 211 of the culture well 21, 5μL per well according to the micropore size; after the cell planting is completed, place it at 37℃ for ten minutes to ensure that the collagen matrix material can gel well. The gelled second chip I (inoculated with HCT-116) and the gelled second chip II (inoculated with MCF-7) were obtained.

[0165] Co-culture: Add culture medium to the culture wells 21 of the second gelled chip (I and II), 60 μL per well. The first gelled chip is placed on the second gelled chip, with the protrusions 11 correspondingly suspended within the multiple culture wells 21 of the second gelled chip, to form a multi-organ co-culture model (Co-culture Model I and Co-culture Model II). The multi-organ co-culture model is cultured at 37°C, 5% CO2, to establish a liver-tumor co-culture model.

[0166] To facilitate performance testing of the co-culture models, different co-culture models were obtained based on the incubation time. After 24 hours of incubation, co-culture models I-1 and II-1 were obtained. After 72 hours of incubation, co-culture models I-2 and II-2 were obtained. In both co-culture models I and II, the 3D liver cells cultured on the protrusions 11 of the first chip were identical.

[0167] As a comparison, in the examples disclosed herein, a comparative example of single culture was performed.

[0168] The monoculture 3D liver organ model (monoculture liver model) was constructed by combining the aforementioned gelled first chip with a blank liquid storage plate and culturing them at 37° C. and 5% CO 2 to construct monoculture model I.

[0169] The monoculture 3D tumor organ models (monoculture tumor models I and II) were constructed by adding culture medium to the culture well 21 of the aforementioned second gelled chip (I and II), with 80 μL of culture medium per well, and culturing at 37°C and 5% CO2 to construct monoculture models I and II.

[0170] Similarly, to compare with the co-culture model, different monoculture models were generated during the monoculture process based on different culture times. After 24 hours of culture, monoculture liver model-1, monoculture tumor model I-1, and monoculture tumor model II-1 were obtained. After 72 hours of culture, monoculture liver model-2, monoculture tumor model I-2, and monoculture tumor model II-2 were obtained.

[0171] Next, the performance of the liver-tumor co-culture model constructed above was tested.

[0172] 1. Co-culture cell proliferation detection

[0173] 1.1 Method for detecting proliferation of 3D liver cells on the convex pillars of the first chip: remove the first chip model from the co-culture organ model I and II, and then combine them with a liquid storage plate respectively. The liquid storage plate includes Figure 9 The liquid reservoir layer 201 and the bottom plate layer 2022 shown in the figure are stacked and connected to form a liquid reservoir plate. After the cell titer Blue test solution is mixed with the culture medium at a ratio of 1:5, 40 μL per well is added to the liquid reservoir wells of the liquid reservoir plate. The first chip model is inserted into the liquid reservoir plate in a one-to-one correspondence between the protrusions 11 and the liquid reservoir wells of the liquid reservoir plate. After incubation for 1.5 hours in an incubator at 37°C and 5% CO2, the fluorescence intensity is detected at 560 / 590 (ex / em), see Figure 21 As shown. Figure 21 It can be seen that the 3D liver cells cultured on the convex pillars 11 of the first chip are in good growth condition and can proliferate stably.

[0174] 2.2 3D tumor cell proliferation detection method in culture well 21 of the second chip (culture chip 20): After mixing Celltiter Blue detection solution with complete culture medium at a ratio of 1:5, 40 μL per well was added to culture well 21 of the second chip model (3D tumor organ model). After incubation for 1.5 hours in an incubator at 37°C and 5% CO2, the fluorescence intensity was detected at 560 / 590 (ex / em).

[0175] The co-culture model Ⅰ-1 and co-culture model Ⅰ-2 inoculated with HCT-116 tumor cells, as well as the single culture tumor model Ⅰ-1 and single culture tumor model Ⅰ-2, were respectively subjected to tumor cell proliferation detection according to the aforementioned detection method, such as Figure 22 As shown. Figure 22 It can be seen that in the co-culture system of HCT116 and liver, HCT116 grows well and proliferates stably compared to the HCT116 culture system alone, allowing for drug inhibitory testing.

[0176] The co-culture model II-1 and co-culture model II-2 inoculated with MCF-7 tumor cells, as well as the single culture tumor model II-1 and single culture tumor model II-2, were respectively subjected to tumor cell proliferation detection according to the aforementioned detection method, such as Figure 23 As shown. Figure 23 It can be seen that in the co-culture system of MCF-7 and liver, MCF-7 grows well and proliferates stably compared to the MCF-7 culture system alone, which allows for drug inhibitory testing.

[0177] 2. High-throughput screening of anti-tumor drugs

[0178] Taking irinotecan hydrochloride as an example, Irinotecan (CPT-11) is a semisynthetic, water-soluble camptothecin derivative. Irinotecan hydrochloride and its metabolite, SN38, are DNA topoisomerase I inhibitors. The complex they form with topoisomerase I and DNA can induce DNA single-strand breaks, preventing DNA replication and inhibiting RNA synthesis. This complex is specific for the S phase of the cell cycle. A high-throughput biomimetic liver-tumor co-culture model was used to investigate the metabolic effects of the anticancer prodrug irinotecan hydrochloride and its anti-tumor effect on a 3D tumor model.

[0179] During the co-culture process of the aforementioned method for constructing a liver-tumor co-culture chip, after 24 hours of incubation, the first chip was removed, i.e., the protrusions 11 were removed from the culture wells 21, and the first chip was placed in a humidified chamber to prevent dehydration of the collagen microspheres. A 10 mmol / L irinotecan hydrochloride stock solution in DMSO was then diluted with complete culture medium at various times to obtain multiple sets of irinotecan hydrochloride solutions (CPT solutions) with concentrations of 3 μmol / L, 10 μmol / L, 30 μmol / L, 100 μmol / L, and 300 μmol / L, respectively. These multiple sets of CPT solutions were then fully replaced and added to the culture wells 21 of the second chip. After ensuring that all bubbles were completely eliminated, the protrusions of the first chip were reinserted into the reservoir wells of the second chip, and the cells were co-cultured in a 37°C, 5% CO2 incubator for 48 hours. Each 96-well plate required a blank group (no cell group) and a negative control group (co-culture chip group without drug), with six replica wells for each group.

[0180] Drug sensitivity test results

[0181] The co-culture organ chip drug screening system of the disclosed embodiment can be characterized using existing drug sensitivity detection methods and a cell metabolic capacity evaluation system. For example, after the first chip is pulled out, 40 μL of Cell titer blue is added to the cell culture wells in the second chip to compare the cell metabolic capacity and evaluate the drug effect. After 48 hours of drug stimulation, the culture medium with the drug is removed, and a mixture with a volume ratio of Cell titer blue stock solution: complete culture medium = 1:5 is added, incubated at 37°C for 1.5 hours, and the detection wavelength is 560em / 590ex nm. The results show that on this co-culture platform, different concentrations of the metabolic drug CPT-11 have a significant effect on MCF-7 ( Figure 24 ) and HCT-116( Figure 25 ) showed a significant inhibitory effect, and its inhibitory effect increased with the increase of concentration. Figure 24 and Figure 25 In the figure, the horizontal axis is the logarithm of the concentration C of the CPT solution; the vertical axis shows the cell activity value relative to the cell activity of the control group, which is a relative value.

[0182] In addition, Cell titer glo and Steady glo can be used to evaluate ATP and fluorescein in 3D cultured cells. High-content imaging can be used to characterize the number of live and dead cells, and to evaluate anti-tumor drugs by imaging changes in the size of tumor-growing collagen aggregates. This embodiment is applicable to, but not limited to, the aforementioned characterization methods.

[0183] It should be understood that the above description is merely a specific embodiment of the present application, and the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A high-throughput multi-organ co-culture chip, characterized in that: comprising a first chip and a second chip; The first chip is a suspended culture plate; the suspended culture plate includes a first plate body, a side surface of which is provided with a plurality of protrusions, and an end surface of each protrusion is formed with a protrusion pattern; the diameter of the protrusion is 1 to 5 mm, the height of the protrusion is 0.8 to 2 mm, and the height of the protrusion pattern is 0.1 to 0.5 mm; the first plate body is also formed with a first through hole; The second chip is a culture chip; the culture chip includes a second plate body, on which are formed a plurality of culture wells and a fluid operation channel, the fluid operation channel being configured to operate the fluid in the culture wells via the fluid operation channel; a first end of the fluid operation channel is connected to the culture wells, and a second end is located on the surface of the culture chip; wherein the first end of the fluid operation channel is located inside the second plate body; the plurality of culture wells are arranged corresponding to the plurality of protrusions for suspending the culture plate; the fluid operation channel includes a vertical through hole and a transverse channel connected to each other, the transverse channel being connected to the culture wells, the opening of the vertical through hole being formed on the surface of the culture chip, the transverse channel being a microchannel, and having a size of 0.05 to 2 mm in width and 10 to 500 μm in depth; The first chip is disposed on the second chip in such a manner that the plurality of protrusions of the first chip are correspondingly suspended in the plurality of culture wells of the second chip; Wherein, when the multiple protrusions of the suspended culture plate are correspondingly suspended in the multiple culture holes of the culture chip, the fluid operation channel is correspondingly arranged with the first through hole of the suspended culture plate.

2. The high-throughput multi-organ co-culture chip according to claim 1, characterized in that: In the suspended culture plate, the raised pattern includes a non-closed geometric pattern and / or a ring.

3. The high-throughput multi-organ co-culture chip according to claim 2, characterized in that: In the suspended culture plate, the raised pattern is in the shape of a cross, "C" or "X"; or, the raised pattern is in the shape of a circular ring or a square ring; or, the raised pattern is in the shape of a non-closed geometric pattern and a ring, and the non-closed geometric pattern is located inside the ring.

4. The high-throughput multi-organ co-culture chip according to claim 1, characterized in that In the culture chip, the culture wells include a lower culture micropore portion and an upper liquid storage well portion.

5. The high-throughput multi-organ co-culture chip according to claim 4, characterized in that: The communication port for the fluid operation channel to communicate with the culture well is located in the upper liquid storage well portion.

6. The high-throughput multi-organ co-culture chip according to any one of claims 1 to 5, characterized in that: The culture chip includes a layered liquid storage layer and a 3D culture layer; The liquid storage layer has a plurality of liquid storage through holes, and the liquid storage through holes are used to store culture fluid; The 3D culture layer has a plurality of culture micropores, and the culture micropores are used for 3D cell culture; The liquid storage through holes correspond to the culture micropores one by one to form the culture pores; and the fluid operation channels are formed on the liquid storage layer.

7. The high-throughput multi-organ co-culture chip according to claim 6, characterized in that: It also includes a channel layer; the channel layer is located between the liquid storage layer and the 3D culture layer; a third through hole and a transverse channel are opened on the channel layer, and a plurality of second through holes are formed on the liquid storage layer; the third through holes correspond one-to-one to the liquid storage through holes; the transverse channel is constructed to connect the third through holes and the second through holes.

8. The high-throughput multi-organ co-culture chip according to claim 7, characterized in that: The transverse channel is formed on the side of the channel layer connected to the 3D culture layer; alternatively, the transverse channel is formed on the side of the channel layer connected to the liquid reservoir layer.

9. The high-throughput multi-organ co-culture chip according to claim 6, characterized in that: The liquid storage through hole of the liquid storage layer is a liquid storage column hole, and the second through hole is a column hole.

10. Use of the high-throughput multi-organ co-culture chip according to any one of claims 1 to 9 for constructing a multi-organ co-culture model.

11. The use according to claim 10, characterized in that A method for constructing a multi-organ co-culture model based on the high-throughput multi-organ co-culture chip according to any one of claims 1 to 9, comprising: Inoculating a mixed cell suspension comprising cells of the first organ and a matrix material onto the end surface of the convex column of the first chip, culturing at 37° C. to form a gel, thereby obtaining a gelled first chip; adding a mixed cell suspension comprising cells of the second organ and a matrix material into the culture wells of the second chip, culturing at 37° C. to form a gel, thereby obtaining a gelled second chip; Adding culture medium into the culture wells of the second gelled chip, and then suspending the first gelled chip in the plurality of culture wells of the second gelled chip with the protrusions corresponding thereto, so that the first gelled chip is arranged on the second gelled chip to form a multi-organ co-culture model; The multi-organ co-culture model was cultured at 37° C. to complete the construction of the multi-organ co-culture model.

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