CN111826284A

CN111826284A - High-flux culture plate, high-flux multi-organ co-culture chip and application thereof

Info

Publication number: CN111826284A
Application number: CN201910320158.XA
Authority: CN
Inventors: 肖荣荣; 周宇
Original assignee: Beijing Da Xiang Technology Co ltd
Current assignee: Beijing Da Xiang Technology Co ltd
Priority date: 2019-04-19
Filing date: 2019-04-19
Publication date: 2020-10-27

Abstract

The application discloses a high-flux culture plate, a high-flux multi-organ co-culture chip and application thereof, belonging to the field of biological tissue engineering. The suspension culture plate is provided with a plurality of convex columns on one side surface of a plate body, and a convex pattern is formed on the end surface of each convex column. A culture chip comprising a plate body having a plurality of culture wells formed therein and a fluid handling channel configured to allow fluid within the culture wells to be manipulated via the fluid handling channel. The co-culture chip is characterized in that the suspension culture plate is arranged on the culture chip in a mode that a plurality of convex columns of the suspension culture plate are correspondingly suspended in a plurality of culture holes of the culture chip. The co-culture chip is used for constructing a multi-organ co-culture model. Can simultaneously culture at least two organ cells, construct a multi-organ co-culture model, and provide a platform for large-scale metabolic drug screening and related mechanism research.

Description

High-flux culture plate, high-flux multi-organ co-culture chip and application thereof

Technical Field

The application relates to the technical field of biological tissue engineering, for example to a high-flux culture plate, a high-flux multi-organ co-culture chip and application thereof.

Background

Organ chip technology is an emerging frontier technology in the field of biotechnology, and is listed as one of ten emerging technologies by the dawss world economic forum in 2016. The micro-processing technology is used for constructing an organ physiological micro-system, generally comprises key elements of organ micro-environments such as living cells, tissue interfaces, biological fluids, mechanical acting force and the like, and can reflect the main structural and functional characteristics of tissues and organs.

The liver is an organ mainly having metabolic functions in the body, and has the effects of complete decomposition and biotransformation of drugs. The metabolism of the drug in the liver changes the chemical structure of the drug and increases or decreases the activity of the drug. In the process of drug development, it is difficult to accurately predict liver damage of developed drugs, and the liver damage is difficult to recover once occurring. Therefore, the in vitro construction of the liver organ model has great significance for researching and predicting the in vivo liver injury of the medicament, screening the activity of metabolic medicaments in vitro and the like.

Disclosure of Invention

The embodiment of the disclosure provides a culture plate, a high-flux multi-organ co-culture chip and application thereof. The following presents a simplified summary in order to provide a basic understanding of some aspects of the disclosed embodiments. This summary is not an extensive overview and is intended to neither identify key/critical elements nor delineate the scope of such embodiments. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

According to a first aspect of embodiments of the present disclosure, a suspended culture plate is provided.

In some embodiments, the suspension culture plate comprises a plate body, wherein a plurality of convex columns are arranged on one side surface of the plate body, and a convex pattern is formed on the end surface of each convex column.

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

In some embodiments, the culture chip comprises a plate body having a plurality of culture wells and a fluid handling channel formed therein, the fluid handling channel being configured to allow fluid within the culture wells to be manipulated via the fluid handling channel.

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

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

the first chip is the suspension culture plate of any one of claims 1 to 3;

the second chip, which is the culture plate of any one of claims 4 to 7;

the first chip is arranged on the second chip in a mode that the plurality of convex columns of the first chip are correspondingly suspended in the plurality of culture holes of the second chip.

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

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

inoculating a mixed cell suspension containing first organ cells and a matrix material on the end face of the convex column of the first chip, culturing at 37 ℃, and gelatinizing to obtain a gelatinized first chip;

adding a mixed cell suspension containing second organ cells and a matrix material into the culture hole of the second chip, culturing at 37 ℃, and gelatinizing to obtain a gelatinized second chip;

adding a culture medium into the culture holes of the gelatinized second chip, and then correspondingly extending the gelatinized first chip into the plurality of culture holes of the gelatinized second chip by the convex columns, wherein the gelatinized first chip is arranged on the gelatinized second chip to form a multi-organ co-culture model;

and culturing the multi-organ co-culture model at 37 ℃ to complete the construction of the multi-organ co-culture model.

The technical scheme provided by the embodiment of the disclosure can have the following beneficial effects:

the suspension culture plate of the embodiment of the disclosure can be used for suspension culture and is simple to prepare. The culture chip can be used for 3D cell culture and is simple to prepare. The two can play functions respectively, and the high-flux multi-organ co-culture chip obtained by combination can simultaneously culture at least two organ cells to construct a multi-organ co-culture model.

The high-flux multi-organ co-culture chip disclosed by the embodiment of the disclosure can be used for biomimetically constructing a 3D in-vitro organ model combined with metabolic drug high-flux drug screening, and is used for scientific research and drug screening of related drugs.

The high-throughput organ multi-organ co-culture chip disclosed by the embodiment of the disclosure is convenient in liquid operation, is suitable for high-throughput operation and characterization, and does not need external equipment.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.

Drawings

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 disclosure.

FIG. 1 is a schematic diagram of a suspended growth plate according to an exemplary embodiment;

FIG. 2 is an enlarged schematic view of the structure at A in FIG. 1;

FIG. 3 is a schematic diagram of a suspended growth plate according to another exemplary embodiment;

FIG. 4 is a schematic illustration of a partial structure of a suspended growth plate according to another exemplary embodiment;

FIG. 5 is a schematic illustration of a partial structure of a suspended growth plate according to another exemplary embodiment;

FIG. 6 is a schematic diagram showing a structure of a culture chip according to an exemplary embodiment;

FIG. 7 is a schematic view of an exploded structure of a culture chip according to another exemplary embodiment;

FIG. 8 is a schematic view of an exploded structure of a culture chip according to another exemplary embodiment;

FIG. 9 is a schematic view of an exploded structure of a culture chip according to another exemplary embodiment;

FIG. 10 is a schematic view of an exploded structure of a culture chip according to another exemplary embodiment;

FIG. 11 is a schematic cross-sectional view of a suspended growth plate, according to an exemplary embodiment;

FIG. 12 is a schematic sectional view of a culture chip shown in FIG. 6;

FIG. 13 is a schematic sectional view of a culture chip shown in FIG. 7;

FIG. 14 is a schematic sectional view of a culture chip shown in FIG. 8;

FIG. 15 is a schematic sectional view of a culture chip shown in FIG. 9;

FIG. 16 is a schematic sectional view showing one culture chip shown in FIG. 10;

FIG. 17 is a schematic cross-sectional view of a multi-organ co-culture chip according to an exemplary embodiment;

FIG. 18 is a schematic cross-sectional view of a multi-organ co-culture chip according to an exemplary embodiment;

FIG. 19 is a schematic diagram illustrating an exploded structure of a multi-organ co-culture chip according to an exemplary embodiment;

FIG. 20 is a schematic diagram showing a stepped cross-sectional structure of a multi-organ co-culture chip according to an exemplary embodiment.

FIG. 21 is a bar graph of cell culture time versus fluorescence intensity for 3D liver cells;

FIG. 22 is a bar graph comparing cell culture time versus fluorescence intensity for single and co-cultured 3D tumor cells;

FIG. 23 is a bar graph comparing cell culture time versus fluorescence intensity for single and co-cultured 3D tumor cells.

FIG. 24 is a graph showing the effect of different concentrations of CPT-11, a metabolic anti-tumor drug, on the activity of MCF-7 cells on a co-culture platform of liver and breast cancer tumors;

FIG. 25 is a graph showing the effect of different concentrations of the metabolic antineoplastic agent CPT-11 on the activity of HCT116 cells on a co-culture platform of liver and colon cancer tumors;

description of reference numerals:

10. suspending the culture plate; 11. a convex column; 110. a raised pattern; 111. a non-closed geometric pattern; 112. a ring shape; 12. a first through hole; 20. culturing the chip; 21. a culture well; 211. a lower culture microporous part; 212. an upper layer liquid storage hole part; 22. a fluid handling channel; 221. a second through hole; 222. a third through hole; 223. a transverse channel; 224. a fourth via hole; 201. a liquid storage layer; 202. a 3D culture layer; 2021. a 3D culture sublayer; 2022. a floor layer; 203. and a channel layer.

Detailed Description

The technical solutions of the present disclosure will be described clearly and completely below with reference to embodiments of the present disclosure, and it should be understood that the described embodiments are only a part of the embodiments of the present disclosure, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments disclosed herein without making any creative effort, shall fall within the protection scope of the present disclosure.

The embodiment of the present disclosure discloses a suspension culture plate 10, as shown in fig. 1 to 5, the suspension culture plate 10 includes a plate body, a plurality of convex pillars 11 are disposed on one side surface of the plate body, and a convex pattern 110 is formed on an end surface of each convex pillar 11.

In the suspension culture plate 10 according to the embodiment of the present disclosure, the surface of the convex pattern 110 has hydrophobicity, and the cell suspension is inoculated on the convex pattern 110, and the cell suspension can form a smooth hemisphere on the convex pattern 110, and after gelling, the cell suspension is suspended downward in a culture solution to perform hanging drop culture. Through the design to the arrangement mode of projection 11 on the suspension culture plate 10, can be compatible with commercial application of sample equipment, detector (e.g., ELIASA, high content imaging system etc.), can realize high flux application of sample and detect. E.g., 96-well, 384-well, etc.

In the embodiment of the present disclosure, the thickness of the suspension culture plate 10 is not limited, and in addition, the suspension culture plate is not easily deformed according to the adopted material with good biocompatibility, and the droplet shape on the convex pillar 11 is not affected when the operations such as taking and clamping are performed.

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

In some embodiments, the diameter of the convex column 11 is 1-5 mm. Optionally, the diameter of the convex column 11 is 2-3 mm. Optionally, the diameter of the stud 11 is 2.5 mm.

In some embodiments, the height of the studs 11 is 0.8-2 mm. Optionally, the height of the studs 11 is 1 mm.

In the embodiment of the present disclosure, the shape of the protrusion pattern 110 is not limited as long as the protrusion is formed on the end surface of the stud 11, and may be a regular geometric shape or an irregular geometric shape. The size of the protrusion pattern 110 is also not limited as long as a cell suspension can be used to form a droplet thereon.

In some embodiments, the height of the raised pattern 110 is 0.1-0.5 mm. Optionally, the height of the protrusion pattern 110 is 0.2 mm.

In some embodiments, as shown in fig. 1 and 2, the raised pattern 110 is a non-closed geometric pattern 111, e.g., a cross, a "C" shape, an "X" shape, etc.

In some embodiments, as shown in fig. 4, the raised pattern 110 is a ring 112, such as a ring with a regular geometry, e.g., a circular ring, a square ring, etc., or an irregular geometry.

In some embodiments, as shown in fig. 5, the raised pattern 110 is a non-closed geometric pattern 111 and a ring shape 112, the non-closed geometric pattern 111 being located within the ring shape 112.

In some embodiments, as shown in FIG. 3, the plate body of the culture plate 10 is further formed with a first through-hole 12. The suspension culture plate 10 is convenient for operating the culture solution during the suspension culture process, such as adding the culture solution or removing the culture solution. As in the multi-organ co-culture chip described below, the first through-hole 12 may form a fluid handling channel for handling fluid together with the fluid handling channel 22 on the second chip 20, facilitating handling of the culture solution in the culture well 21 during co-culture.

In the embodiment of the present disclosure, the suspension culture plate 10 may be formed by integral injection molding or laser etching.

Alternatively, the suspension culture plate 10 is fabricated by pouring a mold and turning over a polydimethylsiloxane prepolymer (PDMS prepolymer). Wherein the mold is fabricated by conventional techniques depending on the structure of the suspended growth plate 10. Alternatively, a polymethylmethacrylate mold (PMMA mold) is used. Namely, one type of suspension culture plate 10 is a PDMS micro-column plate. And performing a prepolymer monomer dissolving and sterilizing treatment, the dissolving and sterilizing treatment comprising: soaking in ethanol for ultrasound, soaking in pure water for ultrasound, and drying. Optionally, soaking in ethanol and ultrasonic treating for 30 min. Optionally, soaking in pure water for 30 min. Optionally, drying at 50 deg.C.

The present disclosure discloses a culture chip 20, and as shown in fig. 6 to 16, the culture chip 20 includes a plate body, on which a plurality of culture wells 21 and a fluid operation channel 22 are formed, the fluid operation channel 22 being configured to operate fluid in the culture wells 21 via the fluid operation channel 22.

The culture chip 20 of the embodiment of the present disclosure adds a structure of the fluid operation channel 22, which facilitates the operation of the fluid (e.g., culture solution) in the culture well 21, such as filling the culture solution, or removing the culture solution. Through the design to the mode of arranging of cultivateing hole 21 on cultivating chip 20, can be compatible with commercial application of sample equipment, detector (for example, ELIASA, high content imaging system etc.), can realize high flux application of sample and detect. E.g., 96-well, 384-well, etc.

In this embodiment, the fluid in the culture well 21 can be operated through the fluid operation channel 22, and it can be seen that the first end of the fluid operation channel 22 is communicated with the culture well 21, and the second end is located on the surface (the surface facing upward during culture) of the culture chip 20, which is convenient for operation. The number of the fluid handling channels 22 is not limited, and one fluid handling channel 22 may be provided corresponding to one culture well 21 (see FIG. 7), each fluid handling channel 22 having a first end and a second end. It is also possible to group a plurality of culture wells, each of which is provided with one fluid handling channel 22, in such a structure that each fluid handling channel 22 has a plurality of first ends, one second end, which are respectively communicated with the plurality of culture wells 21. Wherein, each group of culture holes comprises two or more culture holes 21, the specific number is not limited, and the culture holes are set according to actual conditions.

In some embodiments, as shown in FIGS. 12 to 16, the fluid handling channel 22 includes a vertical through hole and a lateral channel that communicate, the lateral channel communicates with the culture well 21, and an opening of the vertical through hole is formed on the surface of the culture chip 20. Optionally, the transverse channel is a microchannel. Alternatively, the dimensions of the transverse channels are 0.05-2mm wide and 10-500 μm deep. Alternatively, the dimensions of the lateral channels are 1mm wide and 200 μm deep.

In some embodiments, each culture well 21 on the culture chip 20 includes a lower culture micro-well portion 211 and an upper reservoir portion 212 (see FIG. 12). The lower culture micro-hole 211 is used for 3D cell culture, and the upper reservoir 212 is used for storing culture medium, so the shape and pore size of the holes of the lower culture micro-hole 211 and the upper reservoir 212 may or may not be the same. If the pore diameters are not uniform, the pore diameter of the lower culture microporous section 211 is smaller than the pore diameter of the upper liquid storage pore section 212. That is, optionally, the pore diameter of the lower culture microporous portion 211 is smaller than or equal to the pore diameter of the upper liquid storage pore portion 212.

In some embodiments, the communication port of the fluid handling channel 22 in communication with the culture well 21 is located in the upper reservoir portion 212. The operation of the fluid is convenient.

In some embodiments, culture well 21 is a stepped well. The pore diameter of the lower culture microporous portion 211 is smaller than the pore diameter of the upper liquid storage pore portion 212.

In some embodiments, the pore size of the upper reservoir portion 212 is 1-6 times the pore size of the lower culture microporous portion 211. Alternatively, the pore diameter of the upper reservoir portion 212 is 2 to 3 times the pore diameter of the lower culture microporous portion 211. Alternatively, the pore diameter of the upper layer reservoir portion 212 is 2.5 times the pore diameter of the lower layer culture microporous portion 211.

In some embodiments, the upper culture microporous portion 212 and/or the lower culture microporous portion 211 are straight pores, i.e., have uniform pore sizes. For example, when the upper layer liquid storage hole portion 212 has a circular cross section, the straight hole is a cylindrical hole. The straight hole has the advantages of simple focusing of a fluorescence microscope, simple screening and imaging of high content drugs and the like, and can also be a tapered hole with gradually changed depth.

In some embodiments, the shape of the upper layer liquid storage hole portion 212 and/or the lower layer culture micro-hole portion 211 can be a circular hole, a polygonal hole, or the like, or can be a hole with an irregular geometric shape. When the hole diameter is a round hole, the hole diameter is the diameter of the circle; when the hole is a polygon, the hole diameter is the diameter of an inscribed circle of the polygon; when a pore of irregular geometry, the pore diameter is the maximum width of the irregular geometry.

In some embodiments, the upper reservoir portion 212 has a depth of 2-15 mm. Optionally, the upper reservoir portion 212 has a depth of 3-8 mm. Optionally, the upper layer reservoir portion 212 is 6mm deep.

In some embodiments, the upper reservoir portion 212 has a bore diameter of 3-8 mm. Optionally, the aperture of the upper layer liquid storage hole portion 212 is 6 mm.

In an alternative embodiment, the depth of the lower culture micro-holes 211 is 0.5-2 mm. Alternatively, the depth of the lower culture micro-hole part 211 is 1 mm.

In an alternative embodiment, the aperture of the lower culture micro-hole part 211 is 2-4 mm. Alternatively, the pore diameter of the lower culture microporous part 211 is 2.5 mm.

In this embodiment, the upper layer of the liquid storage hole portion 212 is used for storing culture solution such as cell culture medium or drug diluent and transferring substances to the bionic micro-tissue, so as to provide nutrients necessary for growth or drugs to be tested for the whole system. The upper-layer liquid storage hole portion 212 has various structures and installation manners, and the above-described object can be achieved by forming the upper-layer liquid storage hole portion 212. The lower culture micro-hole part 211 can be used for culturing organ cells mixed with a three-dimensional matrix and can simulate a microenvironment for in vivo cell growth.

In the embodiment of the present disclosure, the formation manner of the culture wells 21 is not limited, and the culture chip 20 may be integrally formed (e.g., integrally injection-molded or laser-etched) to form the culture wells 21 in one piece. Alternatively, the layers may be processed in layers, and the liquid storage hole portion and the culture micro-hole portion may be formed in the layers in a stacked manner, respectively, and the culture holes 21 may be formed after the layers are stacked.

In some embodiments, as shown in FIGS. 6 and 11, the culture chip 20 is integrally formed. E, opening a mold to perform integral injection molding; alternatively, laser etching. Wherein the fluid handling channel 22 comprises a vertical through hole and a lateral channel communicating with the culture well 21.

In some embodiments, as shown in fig. 7 and 13, the culture chip 20 comprises a liquid storage layer 201 and a 3D culture layer 202 arranged in layers.

The liquid storage layer 201 has a plurality of liquid storage through holes (i.e., upper liquid storage hole portions 212) for storing a culture liquid.

The 3D culture layer 202 has a plurality of culture wells (i.e., lower culture wells 211) for 3D cell culture.

The liquid storage through holes correspond to the culture micropores one by one to form culture holes 21; and a fluid handling channel 22 is formed on the reservoir layer 201.

In this embodiment, the structure and size of the liquid storage through hole are the same as those of the liquid storage hole portion 212 on the upper layer, and the structure and size of the culture micro-hole are the same as those of the culture micro-hole portion 211 on the lower layer, which are not described herein again.

In this embodiment, the fluid operation channel 22 formed on the liquid storage layer 201 can operate the fluid in the culture well 21. Optionally, a plurality of second through holes 221 (i.e., vertical channels) and a plurality of transverse channels 223 are formed on the liquid storage layer 201, the plurality of second through holes 221 correspond to the plurality of liquid storage through holes one to one, two ends of the transverse channels 223 communicate the second through holes 221 and the liquid storage through holes, and openings of the second through holes 221 are formed on the surface of the liquid storage layer 201. Of course, the fluid operation channel 22 may have other configurations, and may be configured to operate the culture solution in the culture well 21.

In some embodiments, the liquid storage layer 201 may be made of polymethyl methacrylate (PMMA) or Polystyrene (PS), but is not limited to the listed materials, and may be implemented by laser perforation or one-time injection molding.

In some embodiments, the reservoir layer 201 is a reservoir plate, a plurality of reservoir through holes are formed in the reservoir plate, and a plurality of fluid handling channels 22 (including the second through holes 221 and the transverse channels 223, see the above description) are formed, and the plurality of second through holes 221 correspond to the plurality of reservoir through holes one to one, as shown in the reservoir layer 201 in fig. 7 and 13. In this embodiment, the depth of the liquid storage through hole is the thickness of the liquid storage plate.

Alternatively, as shown in fig. 8 to 10, the liquid storage hole of the liquid storage layer 201 is a liquid storage column hole (i.e., the liquid storage hole has a column part 2120 with a convex surface), and the second through hole 221 is also a column hole. That is, the liquid storage hole is formed by forming a through hole on a convex pillar (i.e., the pillar portion 2120) of the convex surface. At this time, the depth of the liquid storage column hole is the sum of the height of the column part 2120 and the thickness of the base body of the liquid storage layer 201.

In this embodiment, when the liquid storage through hole is in the form of a liquid storage column hole, the thickness of the substrate of the liquid storage layer 201 is not limited as long as the substrate has sufficient strength to support the liquid storage column holes arranged thereon. The wall thickness of the boss 2120 of the reservoir bore is also not limited.

The 3D culture layer 202 may be made of polymethyl methacrylate (PMMA) or Polystyrene (PS), but is not limited to the listed materials, and may be implemented by laser perforation or one-time injection molding.

In this embodiment, the culture micro-holes in the 3D culture layer 202 are blind holes, so the culture holes 21 may be holes formed on the plate (laser etching or mold-open integral injection molding), as shown in fig. 8, and are integrally formed 3D culture layer 202. Or may be formed by a laminated panel.

In some embodiments, as shown in fig. 9 and 10, the 3D culture layer 202 includes a 3D culture sub-layer 2021 and a bottom plate layer 2022, a through hole (i.e., the lower culture micro-hole 211) is opened on the 3D culture sub-layer 2021, the 3D culture sub-layer 2021 is stacked on the bottom plate layer 2022, and the through hole and the surface of the bottom plate layer form the culture micro-hole.

In some embodiments, referring to fig. 10 and 16, the culture chip 20 further comprises a channel layer 203; the channel layer 203 is positioned between the liquid storage layer 201 and the 3D culture layer 202. The channel layer 201 is provided with a third through hole 222, and the liquid storage layer 201 is provided with a plurality of second through holes 221; the third through holes 222 correspond to the liquid storage through holes one by one; the lateral passage 223 is configured to communicate the third through-hole 222 with the second through-hole 221. The lateral channel 223 may be formed on the side of the liquid storage layer 201 connected to the channel layer 203, or may be formed on the side of the channel layer 203 connected to the liquid storage layer 201.

Optionally, the liquid storage through hole, the third through hole 221 and the culture micro-hole are coaxially stacked to form a coaxial stepped hole. The liquid storage through hole and the third through hole 221 may together form the upper liquid storage hole portion 212 for storing fluids such as culture solution.

Optionally, the lateral channel 223 is formed on the channel layer 203. Alternatively, the lateral channel 223 is formed on the side of the channel layer 203 connected to the reservoir layer 201, as shown with reference to fig. 16.

In some embodiments, as shown in fig. 10 and 16, on the basis of the third through hole 222 and the transverse channel 223 formed in the foregoing, a fourth through hole 224 is further formed in the channel layer 203, the fourth through hole 224 corresponds to the second through hole 221 one by one, and the transverse channel 223 is configured to communicate the third through hole 222 and the fourth through hole 224. In this embodiment, the transverse channels 223 are disposed on the channel layer 203. Alternatively, the lateral channel 223 is formed on the side of the channel layer 203 connected to the 3D culture layer 202, and may also be formed on the side connected to the liquid storage layer 201.

Alternatively, the lateral channels 223 are formed on the side of the channel layer 203 that is connected to the reservoir layer 201, as shown in fig. 16. The fluid can be prevented from directly acting on the culture micropores of the lower layer.

Alternatively, the second through hole 221 and the fourth through hole 224 are coaxially stacked, forming a coaxial stepped hole.

The thickness of the channel layer 203 is not limited as long as the lateral channels 223 can be formed thereon.

In some embodiments, the transverse channels 223 are microchannels, in which fluid exchange is slow, reducing interference with cultured cells. The dimensions of the transverse channels are 0.05-2mm wide and 10-500 μm deep. Alternatively, the dimensions of the lateral channels are 1mm wide and 200 μm deep.

In some embodiments, the culture chip 20 is formed by stacking a layered structure, such as a liquid storage layer 201 and a 3D culture layer 202; or the liquid storage layer 201, the 3D culture sublayer 2021 and the bottom plate layer 2022; or a liquid storage layer 201, a channel layer 203, a 3D culture layer 202 and the like. Adjacent layers may have a connecting relationship therebetween, e.g., bonding; or in a lapped relationship, for example, three layers are sequentially stacked and then clamped and fixed by a clamp.

Optionally, when the two adjacent layers of the culture chip 20 have a connection relationship, a first adhesive layer (not shown) is further included, and the first adhesive layer is located between the liquid storage layer 201 and the 3D culture layer 202.

Optionally, when the two adjacent layers of the culture chip 20 have a connection relationship, the culture chip further includes a first adhesive layer (not shown) and a second adhesive layer (not shown), the first adhesive layer is located between the liquid storage layer 201 and the 3D culture sublayer 2021, and the second adhesive layer is located between the 3D culture sublayer 2021 and the bottom plate layer 2022.

Optionally, when the two adjacent layers of the culture chip 20 have a connection relationship, the culture chip further includes a first adhesive layer (not shown), a third adhesive layer (not shown), and a second adhesive layer (not shown), the first adhesive layer is located between the liquid storage layer 201 and the channel layer 203, the third adhesive layer is located between the channel layer 203 and the 3D culture sublayer 2021, and the second adhesive layer is located between the 3D culture sublayer 2021 and the bottom plate layer 2022.

The first adhesive layer may be integrally formed on the liquid storage layer 201 or the connecting side surface of the 3D culture layer 202, or may be adhesively provided on the liquid storage layer 201 or the connecting side surface of the 3D culture layer 202 when the liquid storage layer 201 and the 3D culture layer 202 are connected. Similarly, the second bonding layer and the third bonding layer are arranged in the same manner as the first bonding layer.

Alternatively, the first adhesive layer and the second adhesive layer may be double-sided adhesive tape, or PDMS liquid adhesive tape.

When the culture chip 20 of the disclosed embodiment adopts a layered structure, there is provided a method for preparing the culture chip 20, comprising,

the layers (the liquid storage layer and the 3D culture layer, or the liquid storage layer, the channel layer and the 3D culture layer, wherein the 3D culture layer is the integrally formed 3D culture layer, or comprises the 3D sub-culture layer and the bottom plate layer) of the culture chip 20 are respectively cleaned and then dried.

And (3) superposing and bonding the layers in sequence by using double-sided adhesive tapes to complete the preparation of the culture chip 20, thus obtaining the culture chip.

In some embodiments, the cleaning comprises soaking in deionized water for 12-24h, and then soaking in ethanol for disinfection. Alternatively, 75% by volume of ethanol is used as ethanol.

In some embodiments, the drying conditions are such that the drying temperature is from 45 ℃ to 65 ℃, optionally 50 ℃.

In some embodiments, the double-sided adhesive tape is integrally formed on the bonding side of each layer.

The embodiment of the disclosure also provides a high-flux multi-organ co-culture chip. Referring to fig. 17 to 20, the high-throughput multi-organ co-culture chip includes a first chip and a second chip, wherein the first chip is the suspension culture plate 10 and the second chip is the culture chip 20. The first chip 10 is disposed on the second chip 20 in such a manner that the plurality of protruding pillars 11 of the first chip 10 are suspended in the plurality of culture holes 21 of the second chip 20.

The high-throughput multi-organ co-culture chip can be used for simultaneously culturing at least two organ cells, constructing a multi-organ co-culture model and providing a platform for large-scale metabolic drug screening and related mechanism research. Moreover, the micro consumption of cells, matrix materials, reagents and medicines can be realized, and the problem of high cost in the large-scale medicine screening process is solved. The method can also be applied to models with high function integration and strong bionic ability. In addition, through the design of the arrangement mode of the convex columns 11 on the first chip 10 and the culture holes 21 on the second chip 20, the device can be compatible with commercial sample adding equipment and detectors (such as an microplate reader, a high-content imaging system and the like), and high-throughput sample adding and detection can be realized. E.g., 96-well, 384-well, etc.

The high-throughput multi-organ co-culture chip can construct a 3D liver-tumor co-culture model, can be used for researching the metabolism and biotransformation process of the prodrug, finds and evaluates the drug components, can effectively predict the hepatotoxicity of the drug, and reduces the failure rate of later clinical tests and the harm to human bodies. Of course, the method is not limited to the method, and can also be used for constructing and researching co-culture models of other different types of organs.

In order to facilitate manipulation of a fluid, e.g., a culture solution, in the culture wells 21 during co-culture using the multi-organ co-culture chip, in some embodiments, when the first through holes 12 are formed in the first chip 10, the first through holes 12 correspond to the second through holes 221 one by one. Namely, the first through-hole 12, the second through-hole 221, the third through-hole 222 and the lateral passage 223 constitute a fluid operation passage for operating the culture solution in 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 lateral passage 223 constitute a fluid operation passage for operating the culture solution in the culture well 21.

The high-throughput multi-organ co-culture chip of the present embodiment is not limited to the structure shown in FIGS. 17 and 18, and may be constituted by any combination of the aforementioned suspension culture plate 10 and culture chip 20.

The disclosed embodiments provide applications of high-throughput multi-organ co-culture chips for constructing multi-organ co-culture models.

The method for constructing the multi-organ co-culture model based on the high-throughput multi-organ co-culture chip comprises the following steps:

s11, inoculating a first mixed cell suspension containing first organ cells and a matrix material on the end face of the convex column 11 of the first chip 10, culturing at 37 ℃, and gelatinizing to obtain a gelatinized first chip;

s12, adding a second mixed cell suspension containing second organ cells and matrix materials into the culture hole 21 (lower culture microporous part 211) of the second chip 20, culturing at 37 ℃, and gelatinizing to obtain a gelatinized second chip;

s13, adding a culture medium into the culture holes 21 of the gelatinized second chip, and arranging the gelatinized first chip on the gelatinized second chip in a manner that the convex columns 11 correspond to and are suspended in the plurality of culture holes 21 of the gelatinized second chip to form a multi-organ co-culture model;

and (3) culturing the multi-organ co-culture model at 37 ℃ to complete the construction of the multi-organ co-culture model.

The steps S11 and S12 are not executed successively, and may be executed simultaneously, or S11 may be performed first and then S12 may be performed second and then S12 may be performed second and then S11 may be performed first.

In some embodiments, the matrix material in the first mixed cell suspension is collagen, agarose, gelatin, or PEG. Optionally, the matrix material is collagen, the concentration of collagen being 1-3 mg/mL. Alternatively, the concentration of collagen is 2 mg/mL. When the matrix material is agarose, gelatin or PEG, the concentration is determined according to the respective physical and chemical properties, and the colloid is ensured to be formed. This is not to be taken as an example.

In some embodiments, the first organ cells in the first mixed cell suspension are not limited and may be selected according to the particular study. Such as primary liver cells, liver tumor cells, or any liver cells, mixture of liver cells and endothelial cells, liver microsomes, etc., that have drug metabolism function, which are involved in drug metabolism studies. The concentration of the single cells in the first organ cells is not limited, and the concentration can be determined according to actual factors such as the inoculation amount, the inoculation volume and the like. Optionally, the concentration of cells in the first organ in the first mixed cell suspension is 1X 10⁶～2×10⁶cell/mL (pieces/mL).

In some embodiments, the first mixed cell suspension comprises a first organ single cell suspension and a matrix material, and the volume ratio of the first organ single cell suspension to the matrix material is 1.5-4: 1. Optionally, the volume ratio of the first organ single cell suspension to the matrix material is 1.5: 1.

The pH value of the first mixed cell suspension is 6.5-7.5, which is beneficial to cell culture. In some embodiments, the pH of the first mixed cell suspension is adjusted with an alkaline solution. Alternatively, the alkaline solution can be NaOH or NaHCO₃NaOH and NaHCO₃One or more of the above.

In step S11, the volume of the first mixed cell suspension seeded on the end surface of the convex column 11 of the first chip 10 is not limited, and the seeding volume may be determined according to the single cell concentration and the seeding amount of the first mixed cell suspension.

Optionally, the inoculum size of each convex column 11 is 1000-10000 cells.

Optionally, the seeding volume of the first mixed cell suspension is 1-10 μ L. The inoculation amount of the mixed cell suspension is microliter grade, and the dosage is small.

Alternatively, in step 11, the incubation at 37 ℃ is carried out in order to ensure gelling, and the hanging drop of hydrogel seeded on the stud 11 is solidified so as to be stably suspended on the stud 11. The culture time is not limited, and the gelatinization is ensured. Optionally, culturing at 37 deg.C for 5-15 min, optionally 10 min.

In some embodiments, the first mixed cell suspension is obtained by:

s111, digesting and centrifuging first organ cells: in situ tissue or cell lines (2D cultured) were digested into single cells with 0.25 (vt.)% pancreatin and resuspended by centrifugation to a single cell suspension of the first organ (e.g., 1.43X 10)⁶cell/mL). The first organ may be selected and determined according to an organ model to be actually constructed, for example, liver tumor cells and liver primary cells such as hepG2 liver tumor cells in a liver organ model.

S112, mixing the first organ single cell suspension with the matrix material, and adjusting the pH value to 6.5-7.5 to obtain a first mixed cell suspension.

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. Alternatively, the concentration of collagen is 1.5 mg/mL. As above, when the matrix material is agarose, gelatin or PEG, the concentration is determined according to the respective physical and chemical properties, and the colloid is ensured. This is not to be taken as an example.

In some embodiments, the second mixed cell suspension is not limited to cells of a second organ, e.g., lung tumor cells, colonA cancer cell. The concentration of the single cell in the second organ cell is not limited, and the concentration is determined according to actual factors such as the inoculation amount, the inoculation volume and the like. Optionally, the concentration of cells in the second organ in the second mixed cell suspension is 1X 10⁶～2×10⁶×10⁶cell/mL (pieces/mL).

In some embodiments, the second mixed cell suspension comprises 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 1.5-4: 1. Optionally, the volume ratio of the second organ single cell suspension to the matrix material is 3.5: 1.5.

The pH value of the second mixed cell suspension is 6.5-7.5, which is beneficial to cell culture. In some embodiments, the pH of the second mixed cell suspension is adjusted with an alkaline solution. Alternatively, the alkaline solution can be NaOH or NaHCO₃NaOH and NaHCO₃One or more of the above.

In step S12, the volume of the second mixed cell suspension to be seeded into each of the culture wells 21 of the second chip 20 is not limited, and the seeding volume may be determined based on the concentration of the second mixed cell suspension and the amount of seeding per well.

Optionally, the inoculum size in each culture well 21 is 1000-10000 cells.

Optionally, the seeding volume of the second mixed cell suspension is 1-10 μ L. The inoculation amount of the mixed cell suspension is microliter grade, and the dosage is small.

Optionally, in step 12, the incubation is at 37 ℃ to ensure gelling. The culture time is not limited, and the gelatinization is ensured. Optionally, culturing at 37 deg.C for 5-15 min, optionally 10 min.

In some embodiments, the second mixed cell suspension is obtained by:

s121, digesting and centrifuging cells of a second organ: in situ tissue or cell lines (2D cultured) were digested into single cells with 0.25 (vt.)% pancreatin and resuspended by centrifugation to a single cell suspension of the second organ (e.g., 1.43X 10)⁶cell/mL). Wherein the second organ is selected according to the organ model to be actually constructedThus, for example, various tumor cells such as breast cancer cell MCF-7 and colon cancer cell HCT116 can be used in the anti-tumor drug screening model.

S122, mixing the second organ single cell suspension with the matrix material, and adjusting the pH value to 6.5-7.5 to obtain a second mixed cell suspension.

The liver-tumor co-culture chip that can be used for screening metabolic antitumor drugs was constructed based on the high-throughput multi-organ co-culture chip shown in FIG. 19. Among them, the culture well 21 in the high-throughput multi-organ co-culture chip shown in FIG. 19 is 96-well or 384-well.

Among them, the high-throughput multi-organ co-culture chip (referred to as co-culture chip sample for short) shown in FIG. 19 includes,

the first chip, i.e., the suspended culture plate 10, is provided with a plurality of posts 11 on one side surface of the plate body, and a protrusion pattern 110 formed on an end surface of each post 11 is a cross-shaped non-closed geometric pattern 111. The convex column 11 is a cylinder with the diameter of 2.5mm and the height of 1 mm. The height of the protrusion pattern 110 is 0.2 mm. A plurality of first through holes 12 are also formed.

The second chip, i.e., the culture chip 20, includes a liquid storage layer 201, a channel layer 203, a 3D sub-culture layer 2021, and a bottom plate layer 2022. Wherein the content of the first and second substances,

the liquid storage layer 201 has a plurality of liquid storage through holes (i.e. the upper liquid storage hole portion 212), and a plurality of second through holes 221 are formed on the liquid storage layer 201, and the plurality of second through holes 221 correspond to the plurality of first through holes 12 on the first chip 10 one to one. The shape of stock solution through-hole is cylindrical, and the diameter is 6mm, and the degree of depth is 3 mm. The second through hole 221 has the same size as the liquid storage through hole.

The channel layer 203 is provided with a plurality of third through holes 222, a plurality of transverse channels 223 and a plurality of fourth through holes 224, and two ends of one transverse channel 223 are respectively communicated with one third through hole 222 and one fourth through hole 224; the third through holes 222 correspond to the liquid storage through holes one by one, and the fourth through holes 224 correspond to the second through holes one by one. The thickness of the channel layer 203 is not limited as long as the lateral channels 223 can be formed thereon. For example 3 mm.

The dimensions of the transverse passage 223 are: a width of 1mm and a depth of 200 μm, and a length determined according to a distance between the third through hole 222 and the fourth through hole 24.

The 3D seed culture layer 2021 has a plurality of culture micro-holes (i.e., lower culture micro-holes 211), and the culture micro-holes are disposed corresponding to the third through-holes 222 and the liquid storage through-holes.

The bottom plate layer 2022, the glass plate, and the thickness are not limited, and may be selected according to actual needs such as imaging effect. Such as 0.1 to 0.5 mm.

The method for constructing the liver-tumor co-culture chip based on the co-culture chip sample comprises the following steps:

and (3) sterilization treatment: the first chip 10 and the second chip 20 are sterilized for not less than 1 hour, wherein the sterilization can be ultraviolet sterilization.

3D liver organ model construction: 2D-cultured liver HepG2 cells were digested into single cells with 0.25 (vt.)% of pancreatic enzyme and then resuspended at a cell density of 1.43X 10⁶cell/mL of single cell suspension in the first organ. To illustrate, using 2mg collagen as an example, in a 1.5ml EP tube, the ratio of the first organ single cell suspension to the collagen solution by volume was 3: 2, adding a certain volume of 5mg/mL collagen material and the first organ single cell suspension to make the final concentration of collagen be 2mg/mL, obtaining a first mixed cell suspension, and ensuring that the 3D material forms a good three-dimensional structure under the concentration. The mixture was blown and evenly divided into 8 portions by a pipetting gun, and transferred to the convex pillars 11 of the first chip 10 by the pipetting gun, and 10. mu.L of cell hydrogel mixture (first mixed cell suspension) existed in each convex pillar, so that the gel formed a smooth hemisphere on the top surface of the pillar. And (3) placing the first chip 10 into a clean wet box, standing for 15min at 37 ℃ to fully solidify the collagen hydrogel. The gel may then be firmly suspended on the studs 11 of the first chip 10, resulting in a glued first chip.

Tumor organ model construction: the obtained 2D-cultured HCT-116 tumor cell line (I) or MCF-7 tumor cell line (II) was digested with 0.25 (vt.)% of pancreatic enzyme to prepare a single cell suspension, which was then centrifuged to prepare a suspension, and the suspension was further resuspended at a density of 1.43X 10 as required⁶cell/mL of single cell suspension in the second organ. In a 1.5ml EP tube, the ratio of the single cell suspension in the second organ to the collagen solution by volume was 3.5: 1.5According to the proportion, a certain volume of 5mg/mL collagen material and a second organ single cell suspension are added, so that the final concentration of collagen is 1.5mg/mL, a second mixed cell suspension is obtained, and the 3D material can form a good three-dimensional structure under the concentration. The liquid transfer gun is blown and beaten uniformly and evenly divided into 8 parts, and the liquid transfer gun is used for fast transferring and inoculating the liquid transfer gun into the lower culture micropores 211 of the culture holes 21 in a high-flux manner, wherein each hole is 5 mu L according to the size of the micropore; after the cell planting is finished, the cell is placed at 37 ℃ for ten minutes, so that the collagen matrix material can be well gelatinized. Thus, a gelled second chip I (seeded with HCT-116) and a gelled second chip II (seeded with MCF-7) were obtained.

Co-culturing: the wells 21 of the gelled second chips (I and II) were filled with 60. mu.L of medium per well. Arranging the gelatinized first chip on the gelatinized second chip to form a multi-organ co-culture model (a co-culture model I and a co-culture model II) in a manner that the convex columns 11 are correspondingly suspended in a plurality of culture holes 21 of the gelatinized second chip; the multi-organ CO-culture model was incubated at 37 ℃ with 5% CO₂Culturing under the condition, and constructing to obtain the liver-tumor co-culture model.

In order to detect the performance of the co-culture model conveniently, different co-culture models are obtained according to different culture time in the co-culture process. And culturing for 24h to obtain a co-culture model I-1 and a co-culture model II-1. And culturing for 72h to obtain a co-culture model I-2 and a co-culture model II-2. In the co-culture model I and the co-culture model II, the 3D liver cells cultured on the convex column 11 of the first chip 10 are the same.

In the examples of the present disclosure, for comparison, a single-culture comparative example was performed.

Single-culture 3D liver organ model (single-culture liver model) is prepared by combining the first gel-forming chip with blank liquid storage plate, placing at 37 deg.C and 5% CO₂Culturing under the condition to construct a single culture model I.

Single-culture 3D tumor organ models (Single-culture tumor models I and II) are prepared by adding culture medium into culture well 21 of the above gel-forming second chip (I and II), placing 80 μ L of culture medium in each well, standing at 37 deg.C,5％CO₂culturing under the condition, and constructing single culture models I and II.

Also, in order to compare with the co-culture model, different single-culture models were obtained depending on the culture time during the single-culture process. Culturing for 24h to obtain a single-culture liver model-1, a single-culture tumor model I-1 and a single-culture tumor model II-1. Culturing for 72h to obtain a single-culture liver model-2, a single-culture tumor model I-2 and a single-culture tumor model II-2.

Next, the performance of the liver-tumor co-culture model constructed above was examined.

1. Co-culture cell proliferation assay

1.1 proliferation detection method of 3D liver cells on the convex column of the first chip: the first chip model in the co-cultured organ models I and II was removed and then combined with a reservoir plate, respectively. The liquid storage plate comprises a liquid storage layer 201 and a bottom plate layer 2022 as shown in fig. 9, and the liquid storage layer 201 and the bottom plate layer 2022 are overlapped and connected to form the liquid storage plate. Wherein, after mixing the cell titer Blue test solution with the culture medium at a ratio of 1:5, 40 μ L of each well is added into the liquid storage well of the liquid storage plate. Inserting the first chip model into the liquid storage plate in a way that the convex columns 11 correspond to the liquid storage holes of the liquid storage plate one by one, and keeping the temperature at 37 ℃ and 5% CO₂After incubation for 1.5h in the incubator of (1), the fluorescence intensity was measured at 560/590(ex/em), as shown in FIG. 21. As can be seen from fig. 21, 3D liver cells cultured on the posts 11 of the first chip 10 had a good growth state and were able to proliferate stably.

2.2 detection method of proliferation of 3D tumor cells in culture well 21 of second chip 20: mixing Cell titer Blue detection solution with complete culture medium 1:5, adding 40 μ L per well into culture well 21 of second chip model (3D tumor organ model), and culturing at 37 deg.C with 5% CO₂After incubation for 1.5h in the incubator of (1), the fluorescence intensity was measured at 560/590 (ex/em).

Tumor cell proliferation assays were performed according to the assay methods described previously for co-culture model I-1 and co-culture model I-2, and single-culture tumor model I-1 and single-culture tumor model I-2, respectively, inoculated with HCT-116 tumor cells, as shown in FIG. 22. As is clear from fig. 22, the co-culture system of HCT116 and liver exhibited a good growth state of HCT116, and the growth was stable as compared with the culture system of HCT116 alone, and the drug inhibitory activity test was possible.

The detection of tumor cell proliferation was carried out according to the aforementioned detection method for the co-culture model II-1 and co-culture model II-2 inoculated with MCF-7 tumor cells, and for the single-culture tumor model II-1 and single-culture tumor model II-2, respectively, as shown in FIG. 23. As is clear from FIG. 23, the co-culture system of MCF-7 and liver exhibited a good growth state of MCF-7, and the growth was stable as compared with the culture system of MCF-7 alone, and the drug inhibitory activity test was possible.

2. High-pass screening of antitumor drugs

Taking Irinotecan hydrochloride as an example for explanation, Irinotecan hydrochloride (Irinotecan, CPT-11) is a semi-synthetic water-soluble camptothecin derivative. The product and its metabolite SN38 are DNA topoisomerase I inhibitors, and the complex formed by the product, topoisomerase I and DNA can cause DNA single strand break, prevent DNA replication and inhibit RNA synthesis, and is cell cycle S phase specificity. The high-throughput bionic liver-tumor co-culture model has the effects on the metabolism of irinotecan hydrochloride serving as an anticancer prodrug and the killing effect of a 3D tumor model.

In the co-culture process of the method for constructing the liver-tumor co-culture chip, after the culture time reaches 24 hours, the first chip is taken down, namely the convex column 11 leaves the culture hole 21, and the first chip is placed in a wet box to prevent the collagen microspheres from dehydrating. Then diluting the DMSO stock solution of 10mmol/L irinotecan hydrochloride by different times by using complete culture medium to obtain multiple groups of irinotecan hydrochloride solutions (CPT solutions) with the concentrations of 3 mu mol/L, 10 mu mol/L, 30 mu mol/L, 100 mu mol/L and 300 mu mol/L respectively, adding the obtained multiple groups of CPT solution complete exchange solutions into the culture holes 21 of the second chip respectively, after ensuring that bubbles are completely eliminated, re-inserting the convex columns of the first chip into the liquid storage holes in the second chip, and carrying out 5% CO at 37 ℃ to obtain the solution₂The culture box of (2) for 48 h. Each 96-well plate needs to be provided with a blank group (cell-free group) and a negative control group (co-culture chip without drug group), and each group is provided with 6 secondary wells.

Drug sensitive outcome detection

The co-culture organ chip drug screening system of the embodiment of the disclosure can use the existing drug sensitive detection means to perform characterization and cell metabolic capability evaluation system. For example, after the first chip is removed, 40. mu.L of Cell pigment blue is added to the Cell culture well of the second chip, and the effect of the drug is evaluated by comparing the metabolic capacity of the cells. After 48h drug stimulation, the media with drug was removed and Cell titer blue stock was added: complete medium 1:5 mixture, incubation at 37 ℃ for 1.5h, detection wavelength: 560em/590ex nm. The results show that on the co-culture platform, different concentrations of the metabolism drug CPT-11 show significant inhibition effect on MCF-7 (figure 24) and HCT-116 (figure 25) after liver metabolism, and the inhibition effect is enhanced with the increase of the concentration. Wherein, in fig. 24 and 25, the abscissa is the logarithm of the concentration C of the CPT solution; the value of the cell activity on the ordinate is relative to the cell activity of the control group.

In addition, ATP and fluorescein in 3D cultured cells can be evaluated using Cell titer glo and Steady glo. The high content imaging technology is used for imaging and representing the number of living and dead cells, and evaluating the anti-tumor drugs according to the size change of the tumor growth collagen bolus and the like. The present embodiment is applicable to, but not limited to, the above characterization methods.

It should be understood that the above description is only a specific embodiment of the present application, but the scope of the present application is not limited thereto, and any person skilled in the art can easily conceive of changes or substitutions within the technical scope of the present application, and all the changes or substitutions should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. The utility model provides a suspension culture plate which characterized in that, includes the plate body, be provided with a plurality of projections on one side of plate body, form protruding pattern on the terminal surface of every projection.

2. The culture plate of claim 1, wherein the raised pattern comprises a non-closed geometric pattern and/or a ring shape.

3. The culture plate of claim 1 or 2, wherein the plate body further has a first through hole formed therein.

4. A culture chip comprising a plate body having a plurality of culture wells and a fluid handling channel formed therein, the fluid handling channel being configured to allow fluid within the culture wells to be handled therethrough.

5. The culture chip according to claim 4, wherein the culture well includes a lower culture micro-well portion and an upper reservoir well portion.

6. The culture chip of claim 4, wherein the culture plate comprises a liquid storage layer and a 3D culture layer arranged in layers;

the liquid storage layer is provided with a plurality of liquid storage through holes, and the liquid storage through holes are used for storing culture liquid;

the 3D culture layer is provided with 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 culture holes; and forming the fluid handling channel on the reservoir layer.

7. The culture chip of claim 6, further comprising a channel layer; the channel layer is positioned between the liquid storage layer and the 3D culture layer; the channel layer is provided with a third through hole and a transverse channel, and the liquid storage layer is provided with a plurality of second through holes; the third through holes correspond to the liquid storage through holes one by one; the lateral passage is configured to communicate the third through-hole with the second through-hole.

8. A high-flux multi-organ co-culture chip is characterized by comprising a first chip and a second chip;

the first chip is the suspension culture plate of any one of claims 1 to 3;

the second chip, which is the culture plate of any one of claims 4 to 7;

9. Use of the high throughput multi-organ co-culture chip of claim 8 for constructing a multi-organ co-culture model.

10. Use according to claim 9, in a method for constructing a multi-organ co-culture model based on a high-throughput multi-organ co-culture chip according to claim 8, comprising:

adding a culture medium into the culture holes of the gelatinized second chip, and then suspending the gelatinized first chip in a plurality of culture holes of the gelatinized second chip in a manner that the convex columns correspondingly suspend, wherein the gelatinized first chip is arranged on the gelatinized second chip to form a multi-organ co-culture model;