CN117025391A

CN117025391A - Hanging drop culture plate and hanging drop culture device

Info

Publication number: CN117025391A
Application number: CN202210508468.6A
Authority: CN
Inventors: 勾洪磊
Original assignee: Beijing Baitu Zhijian Technology Service Co ltd
Current assignee: Baitu Shengke Beijing Intelligent Technology Co ltd
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2023-11-10

Abstract

The application discloses a hanging drop culture plate and a hanging drop culture device using the same, wherein the hanging drop culture plate comprises a plate body and at least one hanging drop culture hole penetrating through the plate body, and the hanging drop culture hole comprises a liquid drop hanging hole and a liquid adding hole which is arranged above the liquid drop hanging hole and communicated with the liquid drop hanging hole; wherein the aperture of the drop hanging hole gradually increases from the upper edge along the direction away from the liquid adding hole. The hanging drop culture plate provided by the application has stronger drop hanging capacity.

Description

Hanging drop culture plate and hanging drop culture device

Technical Field

The application belongs to the field of cell culture devices, and particularly relates to a hanging drop culture plate and a hanging drop culture device.

Background

The 3D cell culture is proved to better simulate the physiological environment in vivo compared with the cell monolayer culture of a two-dimensional plane, so that the physiological characteristics and behaviors which are closer to the actual physiological characteristics and behaviors of an organism are displayed, and therefore, the 3D cell culture technology is increasingly applied to the fields of medicine research and development, regenerative medicine, disease models, accurate medical treatment and the like. Currently 3D cell cultures mainly include scaffolds (scaffold-based) and non-scaffolds (scaffold-free) cultures. Among them, the scaffold culture system needs to use natural extracellular matrix (ECM) or artificial matrix (such as hydrogel, etc.) of cells as a support material for culturing cells, and the cells often need to be encapsulated in the scaffold material to provide a 3D environment for cell growth, and these matrices often have complex components and are expensive, and the subsequent transfer and treatment of the grown 3D cell culture is inconvenient. The bracket-free culture does not need to use the matrix materials, and the cells are mutually agglomerated and adhered mainly by the self gravity of the cells or a certain external force so as to form a 3D cell culture. In stentless culture, based on low adhesion to the bottom or low adhesionThe cell culture method by forming microwells on the attachment material is most commonly used. The former uses the low adhesion property of the substrate to make cells unable to grow by adhesion and can only agglomerate and adhere to each other by the self gravity of the cells to form cell spheroids, while the latter uses the liquid drop inversion hanging mode to make the cells in the liquid drop sink to the gas-liquid ellipsoidal interface at the bottom of the hanging drop by gravity to form agglomerates. The low adhesion approach allows for the simultaneous generation and cultivation of a large number of 3D cell spheroids, but often requires more complex chemical modifications or expensive equipment to be made to the material to produce surfaces or shapes with low adhesion properties, greatly increasing the cost of further 3D cell cultivation. Hanging drop cell culture is the earliest 3D cell culture method (1907, by Ross Granville Harrison), and the initial method is to drop cell suspension on the surface of a culture plate, then invert the culture plate to hang the liquid drop, and promote cell sedimentation and agglomeration by means of gravity, so as to obtain the cell spheroid with a three-dimensional structure. The method is simple to operate, does not depend on special equipment, and is widely used. But it has the following drawbacks: (1) Due to the very limited droplet size that can be suspended, it is insufficient to support longer and greater numbers of cell growth; (2) The liquid drops are easy to fall off due to vibration, movement and the like in the operation process because of inverted suspension of the liquid drops, and the liquid drop suspension surface is spread; (3) The operation of changing the liquid is very troublesome, the operation is needed to be carried out after the culture plate is placed in the normal position, and the culture plate is turned over again after the liquid is changed, so that not only the problems of liquid drop displacement, dripping, mixing and the like are easily caused, but also cells and spheres are possibly adsorbed on the surface of the culture plate and then attached to the surface of the culture plate, and the 3D culture cannot be continued; (4) In addition, because the hanging drop position can not be fixed and the operations such as overturning are involved, batch or automatic culture with large scale and high flux can not be basically realized, and a great deal of manpower and time are consumed; (5) In addition, since the droplet suspension culture, individual observation and recovery of single cell spheroids after the culture are not easy. Thus, later techniques improved the hanging drop culture method in these respects. The basic strategy is to realize cell sample injection and liquid exchange operation by introducing through holes while avoiding the overturning process, and simultaneously matching with a 96 or 384-well plate to realize the cell sample injection and liquid exchange operationImproving flux to meet the operation requirement of automatic hanging drop culture, such as Perfecta 3D of 3D Biomatrix company in the United states ^TM Hanging Drop Plates. However, perfecta 3D ^TM The hanging drop culture plate adopts a cylindrical straight through hole, the hanging drop capability is very limited, and generally, only 10-20uL culture drops can be hung and maintained, the hanging drops are easy to drop, and the normal culture of cell spheres can be maintained only under the condition of higher liquid exchange frequency; in addition, the hanging drop culture plates are required to be independently configured, cannot be directly matched with conventional culture pore plates, and are relatively high in manufacturing difficulty and relatively high in cost.

In view of the foregoing, there is a need to provide a new hanging drop culture plate with higher drop hanging capabilities.

Disclosure of Invention

The application aims to provide a hanging drop culture plate which has stronger capability of hanging drops.

The application provides the following technical scheme for solving the technical problems:

the first aspect of the application provides a hanging drop culture plate, which comprises a plate body and at least one hanging drop culture hole penetrating through the plate body, wherein the hanging drop culture hole comprises a liquid drop hanging hole and a liquid adding hole which is arranged above the liquid drop hanging hole and communicated with the liquid drop hanging hole; the diameter of the liquid drop hanging hole gradually increases from the upper edge of the liquid drop hanging hole along the direction away from the liquid adding hole.

The second aspect of the application provides a hanging drop culture device, which comprises the hanging drop culture plate and the cell culture plate provided by the first aspect of the application, wherein the cell culture plate comprises a bottom plate and an upper cover, a plurality of bottom plate holes are formed in the bottom plate, the bottom ends of the bottom plate holes are closed, the hanging drop culture plate is arranged between the bottom plate and the upper cover, and the hanging drop culture holes in the hanging drop culture plate are in one-to-one correspondence with the bottom plate holes.

In the hanging drop culture plate, the aperture of the drop hanging hole gradually increases along the direction away from the liquid adding hole, and compared with the existing cylindrical straight through hole, the drop hanging hole has stronger drop hanging capability, and the volume of the drop which can be stably hung by the drop hanging hole can be more than 50uL at maximum.

Drawings

FIG. 1 is a schematic diagram of a hanging drop culture plate according to the present application.

FIG. 2 is a physical view of a hanging-drop culture plate and a hanging-drop culture apparatus of the present application.

FIG. 3 is a graph showing the results of drop suspension experiments on hanging drop plates according to example 1 of the present application.

FIG. 4 is a graph showing the results of cell pellet uniformity culture experiments in a hanging-drop culture plate according to example 1 of the present application.

FIG. 5 is a graph showing the results of various initial cell culture experiments on hanging-drop plates according to example 1 of the present application.

FIGS. 6A and 6B are photomicrographs of cell spheres cultured and recovered from the primary well site using the hanging-drop culture plate of example 1 of the present application.

FIG. 7 is a fluorescent micrograph of a cell pellet cultured on a hanging-drop culture plate according to example 1 of the present application.

Detailed Description

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is apparent that the drawings in the description below are only one embodiment of the present application, and other embodiments may be obtained according to these drawings by those skilled in the art.

Definition of the definition

As used herein, the terms "a" and "an" and "the" and similar referents refer to the singular and the plural, unless the context clearly dictates otherwise.

As used herein, the terms "about," "substantially" and "similar to" refer to an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on the manner in which the value is measured or determined, or on the limitations of the measurement system.

The first aspect of the application provides a hanging drop culture plate, as shown in fig. 1, which comprises a plate body 5 and at least one hanging drop culture hole penetrating through the plate body 5, wherein the hanging drop culture hole comprises a liquid drop hanging hole 1 and a liquid adding hole 2 arranged above the liquid drop hanging hole 1; wherein the aperture of the drop hanging hole 1 gradually increases from the upper edge thereof in a direction away from the liquid adding hole 2.

In the present application, the cross-sections of the drop suspension holes 1 and the liquid adding holes 2 are substantially circular. The cross section is a plane in which the liquid drop hanging hole 1 or the liquid charging hole 2 is cut by any plane parallel to the bottom surface of the liquid drop hanging hole 1. In the present application, the upper edge of the drop hanging hole is understood to be the end of the drop hanging hole away from the bottom surface of the drop hanging hole or the end near the sample loading hole.

The inventors found in the study that since the aperture of the drop hanging hole 1 of the present application increases gradually from the top thereof in a direction away from the liquid charging hole 2, the liquid carrying capacity thereof is significantly enhanced, and when the aperture of the cylindrical straight through hole in the prior art is the same as the diameter of the bottom surface of the drop hanging hole of the present application, the drop hanging hole of the present application can stably hang a drop volume significantly larger than the suspension volume of the cylindrical through hole. In addition, the hanging drop culture plate can stably hang a larger volume of culture medium, so that long-time culture of cells and culture of a large number of cells can be realized; the suspended liquid drop volume is increased, so that the liquid change times can be reduced, and the drop falling caused by vibration and movement in the cell culture process can be obviously reduced.

The plate body 5 in the present application comprises an upper surface 3 and a lower surface 4, in some embodiments, the liquid adding hole 2 is disposed on the upper surface, the liquid drop hanging hole 1 is disposed on the lower surface, and the liquid adding hole 2 is communicated with the liquid drop hanging hole 1 to form the hanging drop culture hole. When the hanging-drop culture plate of the application is used for culturing cells, a liquid drop 6 formed by a culture medium is hung in the liquid drop hanging hole 1, and a cell ball 7 is contained in the liquid drop 6. During cell culture, the culture medium is added or sucked out through the liquid adding hole 2, so that the operations such as overturning and the like can be avoided, and further, large-scale and high-flux culture can be realized. In some embodiments, the upper surface 3 and the lower surface 4 of the plate body 5 are flat, which is advantageous for stable placement on a cell culture plate.

In some embodiments, the drop hanging hole 1 is conical, frustoconical, spherical, ellipsoidal, or ellipsoidal. The inventors have found in the study that the liquid carrying capacity is further improved when the side wall of the drop hanging hole is rectilinear (e.g. conical, frustoconical) and more preferably has an inwardly smooth recess (e.g. spherical segment, spherical mesa, ellipsoidal segment or ellipsoidal mesa). The term "inwardly" recess in the present application is understood to mean a recess such that the sidewall is away from the bottom surface of the drop hanging hole, such as a sphere (as shown in fig. 1), sphere, ellipsoid or ellipsoid. It is understood that in the present application, the truncated cone shape is understood to be a shape formed by cutting a conical shape by a plane parallel to the bottom surface; the segment shape is understood to be the shape of a sphere truncated by one face; the spherical table shape is understood to be a shape formed by a sphere truncated by two parallel faces; the ellipsoid shape is understood to be a shape in which the ellipsoid is truncated by a plane parallel to the major or minor axis; the ellipsoid is understood to be a shape in which an ellipsoid is truncated by two planes parallel to the major or minor axis.

The inventors have found that the diameter of the bottom surface of the drop hanging hole 1 influences the volume of the hanging drop, and that a person skilled in the art can select a suitable bottom surface diameter according to the desired drop volume. The inventor also found that the diameter of the bottom surface of the existing cylindrical through hole is usually not more than 2mm, namely stable liquid drops cannot be formed when the diameter of the bottom surface is more than 2mm, while the diameter of the liquid drop hanging hole of the application is gradually increased along the direction away from the liquid adding hole 2, and stable hanging drops can still be formed when the diameter of the bottom surface is more than 2mm, so that larger hanging drop volume is obtained; more unexpectedly, when the side wall of the drop hanging hole 1 has an inward smooth recess (such as a spherical segment, a spherical table, an ellipsoidal segment or an ellipsoidal table), the drop hanging can be stably suspended when the bottom diameter reaches 5mm or more, thereby obtaining a larger hanging drop volume. The inventors also found that the larger the diameter of the bottom surface, the larger the volume of the liquid drop which can be hung, but when the diameter of the bottom surface is too large, for example, larger than 5mm, the spherical radian of the liquid drop hung by the bottom surface is too large, the aggregation effect of cells is affected, and the spherical morphology of the cell sphere is further affected; the diameter of the bottom surface is too small, for example, less than 0.5mm, the formed hanging drop is too small, and the cell ball does not have enough growth space; thus in some embodiments of the application, the diameter of the bottom surface of the drop hanging hole 1 is 0.5-5mm; furthermore, in order to be compatible with conventional 96-well plates for cell culture, the diameter of the drop hanging well 1 is typically not greater than 4mm, otherwise oversized drops tend to stick to the side walls of the well plate, and thus in some preferred embodiments the drop hanging well 1 bottom surface is 2-4mm in diameter.

In some embodiments, the height of the drop hanging hole 1 is 0.5-4mm, preferably 1-4mm. The inventors found that the height of the drop hanging hole 1 affects the capacity of the drop hanging hole, and when the diameter D of the bottom surface of the drop hanging hole is constant and the heights H and D of the drop hanging hole 1 meet 0.4 D.ltoreq.H.ltoreq.0.6D, the volume of the drop hanging hole hanging the drop is larger. In the present application, the "height of the droplet hanging hole" is understood as a distance between a communicating portion of the droplet hanging hole and the liquid adding hole and a bottom surface of the droplet hanging hole.

In some embodiments, the drop hanging hole is in the shape of a sphere. The inventors have unexpectedly found that when the droplet hanging holes of different shapes have the same bottom surface diameter and the same height are the same, the droplet hanging hole having the shape of a sphere is the largest in volume of the droplet that can be stably hung.

In some embodiments, the high H of the segment shape meets h=0.25R to 1.1R with the radius R of the sphere; preferably, h=r, in which case the segment is hemispherical. The inventors have found that a hemispherical drop hanging hole can stably hang the largest drop volume with the same bottom diameter.

The inventors found in the study that the smaller the aperture of the liquid adding hole, the stronger the drop hanging stability of the drop hanging hole, and the inventors believe that this is probably due to the siphoning phenomenon generated by the liquid adding hole with small aperture, so that the drop is more stably hung in the drop hanging hole; however, the aperture is too small, which is not beneficial to the liquid changing operation; thus, in some embodiments, the filler opening has a pore size of 0.5-2mm, preferably 1-2mm.

Too large a depth of the filling hole increases the thickness of the plate body on the one hand and on the other hand is detrimental to the liquid exchange of the hanging drop culture cells, so that in some embodiments the filling hole has a depth of 0-2mm, preferably 0.5-1mm.

In the present application, the "depth of the liquid filling hole" is understood as a distance between a communicating portion of the liquid filling hole and the liquid drop hanging hole and an upper surface of the plate body. In some embodiments, the filling hole is cylindrical, and the depth of the filling hole may be understood as the height of the cylinder.

In some embodiments, the aperture of the filler hole is not less than half the depth of the filler hole. When the size of the liquid adding hole meets the proportion relation, the liquid changing operation is convenient.

In some embodiments, the centerline of the charging hole coincides with the centerline of the drop hanging hole.

In some embodiments, the thickness of the plate body is 0.5-6mm.

In some embodiments, the plate body may be a solid structure or a hollow structure.

In some embodiments, the material of the plate body includes at least one of polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate (PC), polyethylene (PE), polyester (PET), polydimethylsiloxane (PDMS), preferably PDMS. The hanging drop culture plate prepared from the material can be used once, can also be reused after high-temperature high-pressure sterilization or alcohol sterilization, and the manufacturing and using costs can be greatly reduced.

In some embodiments, the hanging-drop culture well is formed directly in the plate body. The hanging drop culture plate is simple in structure, the sample adding holes and the drop hanging holes of the hanging drop culture holes are directly formed in the plate body, other structures are not needed to form or support the drop hanging holes, the processing technology is simple, and the production cost is low.

The second aspect of the application provides a hanging drop culture device, which comprises the hanging drop culture plate and the cell culture plate provided by the first aspect of the application, wherein the cell culture plate comprises a bottom plate and an upper cover, a plurality of bottom plate holes are formed in the bottom plate, the bottom ends of the bottom plate holes are closed, the hanging drop culture plate is arranged between the bottom plate and the upper cover, and the hanging drop culture holes on the hanging drop culture plate are in one-to-one correspondence with the bottom plate holes, so that the whole transfer of the hanging drop culture device can be realized. In some embodiments, the cell culture plate may be a 96-well plate, 384-well plate, or the like, and one skilled in the art may select the size of the droplet hanging hole in the hanging-drop culture plate based on the aperture of the bottom plate hole in the cell culture plate. A physical diagram of the hanging drop culture device is shown in fig. 2, wherein the cell culture plate is a 96-well plate. FIG. 2A shows a hanging-drop culture plate of the present application, B shows a partial enlarged view of FIG. A, and C shows the hanging-drop culture plate of the present application disposed on a bottom plate of a cell culture plate. The hanging drop culture device can be directly placed on common cell culture plates such as 96-well plates, 384-well plates and the like to be matched for use, does not need special cell culture plates, is simple to operate, and obviously reduces the production cost.

In some embodiments, the hanging-drop culture plate is removably attached to the cell culture plate.

The hanging-drop culture plates of the present application are described in detail below in connection with specific examples.

Example 1 hanging drop culture plate

The hanging drop culture plate used in this example is made of PDMS, the hanging hole of the liquid drop is hemispherical, the radius of the bottom surface is about 2.1mm, the hole capacity is about 20. Mu.L, the aperture of the liquid adding hole is 1.5mm, and the depth is about 0.5mm.

Experimental example 1 drop suspension experiment

Drop hanging experiments were performed using the hanging drop culture plates of example 1 of the present application, and the ability of the hanging drop culture plates of the present application to stably hang drops was verified, as shown in FIG. 3, with a drop volume of 20. Mu.L in FIG. 3 in panel A, 30. Mu.L in panel B, 40. Mu.L in panel C, 50. Mu.L in panel D, and examples of drops indicated by arrows in the figure. It can be seen that the volume of the stably suspended water drops can reach 50 mu L by using the hanging drop culture plate.

In general, due to the abundance of ingredients in cell culture media, which tend to have a greater surface tension than water, when hanging media using hanging drop culture plates of the present application, a greater suspension volume than water drops is often obtained.

Cell culture experiments

Experimental example 2 cell pellet uniformity culture experiment

The hanging drop culture plate of the embodiment 1 is adopted to carry out a HepG2 liver cancer cell sphere uniformity culture experiment.

And (3) cells: hepG2 liver cancer cell

Culture medium: DMEM medium containing 15% fetal bovine serum;

culture medium suspension volume: 40. Mu.L;

initial cell number: 4000;

culture conditions: 5% CO ₂ Culturing at 37deg.C for 4 days

As can be seen from fig. 4, after 4 days of culture, the cell growth state was good; the cell growth was uniform among the different droplets.

Experimental example 3 cell culture experiments with different initial numbers

Cell culture experiments were performed with different initial numbers using hanging drop culture plates of example 1 of the present application.

And (3) cells: hepG2 liver cancer cell

Culture medium: DMEM medium containing 15% fetal bovine serum;

culture medium suspension volume: 40. Mu.L;

initial cell number: 800, 2000, 4000, 10000, 20000;

culture conditions: 5% CO ₂ Culturing at 37℃for 7 days, sucking out 7.5. Mu.L of the medium every 2-3 days, and supplementing 10. Mu.L of the medium.

FIG. 5 shows the growth state of the cell spheres after culturing HepG2 liver cancer cells of different initial numbers for 7 days by using the hanging-drop culture plate of the application, and it can be seen that the larger the initial cell number is, the larger the diameter of the cell spheres obtained after 7 days is, and the good the growth state of the cells, the initial concentration of the cells cultured by the hanging-drop culture plate of the application can reach 20000/40 mu L.

Experimental example 4HepG2 liver cancer cell sphere primary hole site recovery experiment

The hanging drop culture plate of the embodiment 1 is adopted to carry out HepG2 liver cancer cell sphere primary hole site recovery experiments.

And (3) cells: hepG2 liver cancer cell

Culture medium: DMEM medium containing 15% fetal bovine serum;

culture medium suspension volume: 40. Mu.L;

initial cell number: 10000;

After 7 days of cultivation, 20-50uL of PBS solution or culture medium was added to the liquid-adding hole corresponding to the hole site to be recovered by a pipette, and the hanging liquid drop containing the cell ball was dropped from the hanging drop cultivation hole to the cultivation hole corresponding to the lower side, and then microscopic observation was carried out, and the result is shown in FIG. 6A, and FIG. 6B is a bright field micrograph (20-fold objective lens) of the cell ball recovered from the original hole site by using a Nikon inverted microscope.

As can be seen from fig. 6A, the shape of the recovered cell pellet is complete, indicating that the shearing and impact generated during the dripping process have little effect on the cell pellet. As can be seen from FIG. 6B, the diameter of the HepG2 liver cancer cell pellet cultured by the hanging-drop culture plate can reach 375 mu m, which is obviously larger than the cell culture size of the existing hanging-drop culture plate.

The recovery mode of the original hole site can realize selective independent recovery without affecting hanging drops of other hole sites. The recovered cell balls can be further fixed and subjected to staining imaging observation, and can also be continuously cultured in the original hole site.

After the recovered pellet was fixed with paraformaldehyde solution, it was stained with PhenoVue Cell Painting Kit using a dye containing PhenoVue Fluor 555-WGA, phenoVue Fluor 488-Concanavalin A, phenoVue Fluor 568-Phlloidin, phenoVue Hoechst 33342 nucleic acid stand, and the stained pellet was subjected to high content imaging with Opera Phenix Plus High-Content Screening System to give a fluorescent micrograph of the pellet as shown in FIG. 7.

As can be seen from FIG. 7, the HepG2 liver cancer cell pellet cultivated by the hanging-drop culture plate of the application has uniform sphere shape and compact whole structure. PhenoVue Hoechst 33342 nucleic acid stand staining cell nucleus, phenoVue Fluor 555-WGA staining cell membrane, phenoVue Fluor 488-Concanavalin A staining cell endoplasmic reticulum, phenoVue Fluor 568-Phalidin staining actin filaments forming cell skeleton, it is obvious from fluorescence micrograph that the outer cell state of the cell ball is good, the adhesion is tight, and the characteristics of structural characteristics of liver parenchymal tumor cell ball and active proliferation of outer cell are met.

The foregoing description of the preferred embodiments of the application is not intended to limit the application to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the application are intended to be included within the scope of the application.

Claims

1. The hanging drop culture plate is characterized by comprising a plate body and at least one hanging drop culture hole penetrating through the plate body, wherein the hanging drop culture hole comprises a liquid drop hanging hole and a liquid adding hole which is arranged above the liquid drop hanging hole and communicated with the liquid drop hanging hole; wherein the aperture of the drop hanging hole gradually increases along the direction away from the liquid adding hole.

2. The hanging-drop culture plate of claim 1, wherein the drop hanging hole is conical, truncated cone, spherical segment, ellipsoidal segment, or ellipsoidal segment in shape.

3. Hanging-drop culture plate according to claim 1, characterized in that the diameter of the bottom surface of the drop hanging hole is 0.5-5mm, preferably 2-4mm.

4. Hanging-drop culture plate according to claim 1, characterized in that the height of the drop hanging-off holes is 0.5-4mm, preferably 1-4mm.

5. The hanging-drop culture plate of claim 1, wherein the drop hanging-drop hole is in the shape of a sphere segment.

6. The hanging-drop culture plate of claim 5, wherein the high H of the segment shape and the radius R of the sphere satisfy H = 0.25R-1.1R; preferably, h=r.

7. Hanging drop culture plate according to claim 1, characterized in that the aperture of the filling hole is 0.5-2mm, preferably 1-2mm.

8. Hanging drop culture plate according to claim 1, characterized in that the depth of the filling hole is 0-2mm, preferably 0.5-1mm.

9. The hanging-drop culture plate of claim 1, wherein the aperture of the filling hole is not less than half the depth of the filling hole.

10. The hanging-drop culture plate of claim 1, wherein a centerline of the liquid-adding hole coincides with a centerline of the drop hanging-hole.

11. The hanging-drop culture plate of claim 1, wherein the plate body has a thickness of 0.5-6mm.

12. The hanging-drop culture plate of claim 1, wherein the material of the plate body comprises at least one of PMMA, PS, PC, PE, PET, PDMS.

13. The hanging-drop culture plate of claim 1, wherein the hanging-drop culture well is formed directly in the plate body.

14. A hanging-drop culture device, comprising the hanging-drop culture plate and the cell culture plate according to any one of claims 1-13, wherein the cell culture plate comprises a bottom plate and an upper cover, a plurality of bottom plate holes are formed in the bottom plate, the bottom ends of the bottom plate holes are closed, the hanging-drop culture plate is arranged between the bottom plate and the upper cover, and the hanging-drop culture holes on the hanging-drop culture plate are in one-to-one correspondence with the bottom plate holes.

15. The hanging-drop culture device of claim 14, wherein the hanging-drop culture plate is removably coupled to the cell culture plate.