CN114149921A

CN114149921A - Tumor complex microenvironment cell culture device

Info

Publication number: CN114149921A
Application number: CN202111500465.XA
Authority: CN
Inventors: 杨舒雅; 何治一; 赵巍; 甘婧; 杨琨; 孙元杰; 王静
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2022-03-08
Anticipated expiration: 2041-12-09
Also published as: CN114149921B

Abstract

The invention belongs to the technical field of cell culture devices, and particularly relates to a cell culture device for a tumor complex microenvironment. The cell culture dish comprises a dish body, a plurality of culture holes are formed in the bottom of the dish body, a channel is formed in the position, corresponding to each culture hole, of the side wall of the dish body, and an opening in the bottom of the channel is communicated with the culture holes; the liquid adding device comprises a container bottle, an injection tube, a piston head and a pressing rod, the bottom of the container bottle is in one-to-one correspondence with and detachably connected with the top opening of the culture hole, one end of the injection tube is communicated with the bottom of the container bottle, the other end of the injection tube is communicated with the top opening of the channel, the piston head can move up and down in the container bottle, and the pressing rod is installed at the top of the piston head. The invention realizes the liquid adding operation in a closed environment, has better closure and greatly reduces the risk of cell pollution.

Description

Tumor complex microenvironment cell culture device

Technical Field

The invention belongs to the technical field of cell culture devices, and particularly relates to a cell culture device for a tumor complex microenvironment.

Background

Cell culture is an important tool for studying cell viability, cell morphology, and the performance of cellular agents, involving a variety of cell types, media types, and reagent types. Taking tumor cell research as an example, the research of antitumor drugs is a common operation of tumor cell culture, and the general means of tumor cell culture is to place tumor cells in a cell culture plate (48-well plate, 96-well plate, etc.), add a culture medium with a proper formula, incubate for a period of time under proper culture conditions, after the cells are attached, replace the new culture medium and add test drugs, continue to incubate for a period of time, detect the activity of the cells, and evaluate the activity of the test drugs by comparing the cell activity difference between a control group without adding the test drugs and an experimental group with the test drugs.

Since tumor cells can exist in a plurality of body parts such as cervix, the microenvironment around the actual growth of the cells is relatively complex and may contain a plurality of different cytokines, proteins, water and the like, so the formula requirements on the culture medium are often relatively complex and are easily infected by microorganisms and the like in the environment, and attention needs to be paid to preventing the pollution of environmental pollutants in the culture process. In the prior art, a dual-antibody type containing streptomycin is generally adopted to prevent the infection of microorganisms in the environment, but the two antibiotics are used for a long time, drug-resistant microorganisms possibly exist in the environment, and the microorganisms are not sensitive to the dual-antibody and cannot play a good anti-pollution effect in the cell culture process. Furthermore, since most tumor cells are malignant proliferating cells, the operator must take great care during the tumor cell culture to prevent the environmental microbial contamination and the splashing of the cells into the environment.

However, the cell culture plate of the prior art is only a nearly flat plate structure containing multiple wells, and in some possible culture boxes, regardless of the structure, the "wells" need to be exposed to the environment when the culture medium is added or the test drug is added, which increases the risk of cell contamination, and therefore, a cell culture device which can reduce the risk of contamination needs to be developed.

Disclosure of Invention

In order to solve the technical problems, the invention provides a cell culture device with a complex tumor microenvironment, which realizes liquid adding operation in a relatively closed environment, has good closure and greatly reduces the risk of cell pollution.

The invention aims to provide a cell culture device for a tumor complex microenvironment, which comprises a cell culture dish and a liquid adding device;

the cell culture dish comprises a dish body, a plurality of culture holes are formed in the bottom of the dish body, a channel is formed in the position, corresponding to each culture hole, of the side wall of the dish body, and an opening in the bottom of the channel is communicated with the culture holes;

the liquid adding device comprises a container bottle, an injection tube, a piston head and a pressing rod, the bottom of the container bottle corresponds to the top opening of the culture hole one by one and is detachably connected with the top opening of the culture hole, one end of the injection tube is communicated with the bottom of the container bottle, the other end of the injection tube is communicated with the top opening of the channel, the piston head is located in the container bottle and can move up and down in the container bottle, and the pressing rod is installed at the top of the piston head.

Preferably, above-mentioned complicated microenvironment cell culture device of tumour, the bottom outer wall of container bottle is equipped with the invagination groove, the open-top of cultivateing the hole be equipped with the protruding groove that the invagination groove matches.

Preferably, in the above tumor complex microenvironment cell culture device, the bottom opening of the channel extends to the bottom of the dish body, and the top opening of the channel is located on the side wall of the dish body.

Preferably, above-mentioned complicated microenvironment cell culture device of tumour, the intercommunication of culture hole lateral wall upper end is provided with the one end in atmospheric pressure balanced pore, the other end in atmospheric pressure balanced pore extends to ware body outer wall to with external intercommunication, and this end intussuseption is filled with the ventilative aseptic stopper of strain fungus.

Preferably, in the above tumor complex microenvironment cell culture device, the injection tube is a zigzag tube, and has an upper horizontal tube, a lower horizontal tube and a longitudinal tube, after the injection tube is communicated with the container bottle, a U-shaped communicating vessel with the upper horizontal tube is formed, and the bottom of the upper horizontal tube is higher than the channel.

Preferably, above-mentioned complicated microenvironment cell culture device of tumour, every the culture hole corresponds and is connected with a plurality of the liquid feeding device, all keeping away from of atmospheric pressure balance pore the tip of culture hole gathers on a house steward, then house steward extends to the ware body outer wall to with external intercommunication, the port that house steward and external intercommunication is filled up and is had the ventilative aseptic stopper of strain.

Preferably, the tumor complex microenvironment cell culture device further comprises a liquid pumping device, wherein the liquid pumping device has the same structure as the liquid adding device, and the bottom opening of the channel corresponding to the liquid pumping device extends into the culture hole.

Preferably, the tumor complex microenvironment cell culture device further comprises a porous plate and a control switch, wherein the porous plate is provided with a plurality of through holes connected with the container bottles; the perforated plate is arranged in the dish body cultivate on the hole, control switch includes support column, a plurality of press plates, a plurality of press switch and a plurality of press head, the vertical setting of support column, its bottom is fixed on the perforated plate, center on the upper end periphery of support column is equipped with a plurality of press plates, installs on every press plate press the depression bar, every press the top of the press plate and all be equipped with press switch, every press the switch top and install press head.

Preferably, the above tumor complex microenvironment cell culture device, the pressing switch includes at least two switch claws, the bottom of the switch claw is connected with the pressing plate, the top of the supporting column is provided with a convex plate, the bottom of the convex plate is provided with a vertical convex rod above each pressing plate, each switch claw of the pressing switch is arranged around the corresponding convex rod, and the switch claw can clamp the convex rod.

Preferably, in the tumor complex microenvironment cell culture device, a culture tube is arranged in the culture hole, and a mouthpiece is arranged on the side wall of the upper end of the culture tube; a channel tube is laid in the channel, one end of the channel tube penetrates through the hole wall of the culture hole and is detachably communicated with the connector tube, and the other end of the channel tube penetrates out of the top opening of the channel and is detachably communicated with the injection tube.

Compared with the prior art, the invention has the following beneficial effects:

1. in the invention, the cell culture dish is used for culturing cells in a relatively closed environment, and the liquid adding device is used for adding liquid into the cell culture dish in the relatively closed environment. The container bottle has the advantages that one function is to contain a liquid to be added, the other function is to seal the culture hole, so that the liquid and cells in the culture hole are in a closed environment, the third function is to enable the liquid to sequentially flow through the injection tube and the channel from the container bottle and finally enter the culture hole, and the liquid adding process is carried out in the closed environment. In the process of adding liquid by using the device, the liquid is not contacted with the external environment, the sealing performance is good, the probability of contamination is greatly reduced, and the device does not need to be moved to the outside of the incubator, so that the operation steps of adding the sample are simplified, the use times of a sterile operation table are reduced, and the device is energy-saving and environment-friendly. The priming volume is known by the distance the piston head is moved downward.

2. In the invention, the injection tube adopts the zigzag tube, the zigzag tube is communicated with the container bottle to form the U-shaped communicating vessel with the upper end transverse tube, the relation between the downward movement distance of the piston head and the volume of the liquid entering the upper end transverse tube is made into a data corresponding table, and an operator can know the volume of the liquid added into the culture hole only by paying attention to the downward movement distance of the piston head, thereby realizing convenient quantitative liquid adding operation.

3. In the invention, one culture hole can be correspondingly provided with a plurality of liquid adding devices and channels, so that a plurality of different liquids can be added into cells in the culture hole, and the culture hole is suitable for cell culture experiments with complicated liquid adding.

4. In the invention, the arrangement of the control switch structure can prevent the piston head from rebounding upwards while quantitatively adding liquid, thereby reducing the risk of liquid suck-back.

5. In the invention, in order to facilitate the cleaning of the device, a culture tube is arranged in the culture hole, and a channel tube is laid in the channel.

Drawings

FIG. 1 is a schematic diagram of a longitudinal cross-sectional structure of a tumor complex microenvironment cell culture device of example 1;

FIG. 2 is a perspective view of a vessel body according to embodiment 1;

FIG. 3 is a bottom structural view of a dish body according to embodiment 1;

FIG. 4 is a perspective view of the cell culture apparatus for complex microenvironment of tumor in example 2;

FIG. 5 is a schematic diagram of the longitudinal cross-sectional structure of the cell culture device of example 2

FIG. 6 is a schematic perspective view showing the connection between a supporting column and a pressing plate according to example 2;

FIG. 7 is a schematic view showing the connection between the culture well and the channel in example 3.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention to be implemented, the present invention will be further described with reference to the following specific embodiments and accompanying drawings.

In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention.

The terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature; in the description of the present invention, "a plurality" means two or more unless otherwise specified.

In the description of the present invention, unless otherwise expressly specified or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral part; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

In the description of the present invention, unless otherwise explicitly specified or limited, a first feature "on" or "under" a second feature may be directly contacting the first feature or the second feature through intervening media. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.

Example 1

A cell culture device with a complex tumor microenvironment, as shown in figures 1-3, comprises a cell culture dish for culturing cells in a relatively closed environment and a liquid adding device for adding liquid, which can be culture medium or test medicine, into the cell culture dish in the relatively closed environment.

Cell culture dish includes the dish body 1, the dish body 1 is hollow cube or cylindrical, bottom and lateral wall all have certain thickness in it, preferred thickness is 0.5-1.5cm, a plurality of cultivation holes 11 have been seted up to 1 bottom of the dish body, be used for adding the cell in the cultivation hole 11, culture medium and test medicine etc. passageway 12 has all been seted up to the position that the 1 lateral wall of dish body corresponds every cultivation hole 11, the one end of passageway 12 extends to 1 bottom of the dish body, and rather than the cultivation hole 11 upper end intercommunication that corresponds, the other end opening of passageway 12 is located 1 lateral wall of dish body.

The liquid adding device comprises container bottles 2, injection pipes 21, piston heads 22 and pressing rods 23, the number of the container bottles 2 is the same as that of the culture holes 11, the container bottles are correspondingly arranged in positions, the bottoms of the container bottles 2 are detachably connected with the top openings of the culture holes 11, the bottoms of the container bottles 2 can be correspondingly embedded into the top openings of the culture holes 11, specifically, the outer wall of the bottom of each container bottle 2 is provided with an invagination groove, and the top openings of the culture holes 11 are provided with convex grooves which are matched and connected with the invagination grooves; one end of the injection tube 21 is communicated with the bottom of the container bottle 2, and the other end is communicated with an opening of the channel 12 positioned on the side wall of the dish body 1; the piston head 22 is located in the container bottle 2 and can move up and down in the container bottle 2, and the piston head 22 acts as a piston and can press the liquid in the container bottle 2 into the injection tube 21 and then enter the culture hole 11 through the channel 12. The pressing rod 23 is attached to the top of the piston head 22, and the piston head 22 is moved toward the bottom of the container bottle 2 by pressing the pressing rod 23 downward. In order to make the pressing operation smoothly carried out, the upper end of the side wall of the culture hole 11 is communicated with one end provided with an air pressure balance hole channel 13, the other end of the air pressure balance hole channel 13 extends to the outer wall of the dish body 1 and is communicated with the outside, and a port of the air pressure balance hole channel 13 communicated with the outside is filled with a sterile plug with a bacteria filtering and ventilating function, wherein the sterile plug can be sterile cotton. Because the length of the air pressure balance pore canal 13 is longer, and the diameter is far smaller than the diameter of the traditional hole of a 48-hole plate or a 96-hole plate, for example, the diameter of the air pressure balance pore canal 13 is set to be 2-4mm, the contact area of the air pressure balance pore canal 13 and the external environment is greatly reduced, and the risk of contaminating mixed bacteria in the external environment is almost eradicated at the air pressure balance pore canal 13 due to the arrangement of a sterile plug.

It should be noted that, when there are a plurality of culture holes 11 and their corresponding container bottles 2 and air pressure balance pore channels 13, the ends of all the air pressure balance pore channels 13 far from the culture holes 11 are collected onto one header pipe, then the header pipe extends to the outer wall of the dish body 1 and is communicated with the outside, and the port of the header pipe communicated with the outside is plugged with a sterile plug with a bacteria-filtering and ventilating function.

One effect of container bottle 2 is to hold the liquid body of waiting to add, and another effect is to seal culture hole 11, makes its inside liquid and cell be in confined environment, and the third effect is to make liquid follow container bottle 2 and flow through injection tube 21 and passageway 12 in proper order to finally enter into culture hole 11 in, this liquid adding process is gone on under the confined environment, reducible dish body 1's external environment's pollution.

Preferably, the injection tube 21 is a zigzag tube having an upper end horizontal tube, a lower end horizontal tube and a longitudinal tube, after the injection tube 21 is communicated with the container bottle 2, a U-shaped communicating vessel with the upper end horizontal tube is formed, the upper end horizontal tube is the horizontal tube at the upper end of the zigzag tube, the bottom height of the upper end horizontal tube is higher than the highest height of the channel 12, referring to fig. 1, when the piston head 22 is not embedded in the container bottle 2, the height of the liquid added into the container bottle 2 is the same as the bottom height of the upper end horizontal tube, under the action of the U-shaped communicating vessel, the liquid level in the container bottle 2 and the bottom height of the upper end horizontal tube are always the same, at this time, the piston head 22 is installed in the container bottle 2, after the piston head 22 moves downwards, the liquid in the container bottle 2 is squeezed into the upper end horizontal tube, and finally enters the culture hole 11, it should be noted that the length of the upper end horizontal tube should be less than 0.5cm, and the closing of the upper end horizontal tube is smooth, in the liquid moving process, the volume of the residual liquid in the liquid moving process is almost 0, the downward moving distance of the piston head 22 corresponds to the volume of the liquid entering the upper end transverse pipe in the container bottle 2, experiments and measurement are carried out in advance, the relation between the downward moving distance of the piston head 22 and the volume of the liquid entering the upper end transverse pipe is made into a data corresponding table, and an operator can know the volume of the liquid added into the culture hole 11 only by paying attention to the downward moving distance of the piston head 22, so that convenient quantitative liquid adding operation is realized.

The working principle of the embodiment is as follows: respectively sterilizing a cell culture dish and a liquid adding device, then putting a device to be used, reagents such as cell sap and the like into an aseptic operation table, and carrying out cell inoculation and liquid adding preparation work in the aseptic operation table, on one hand, adding a culture medium or a test medicine solution and the like required by cell culture into a container bottle 2, and then installing a corresponding injection tube 21, a piston head 22 and a pressing rod 23, wherein the piston head 22 plays a role of a container cover, and is dustproof and anti-pollution; on the other hand, cells to be cultured are added to the culture well 11; then, the bottom of the container bottle 2 is embedded into the opening at the upper end of the culture hole 11 filled with cells, and the culture hole 11 is sealed, so that the container bottle is dustproof and anti-pollution; the number of cells in the culture well 11 that need to be cultured, and the number of devices that are assembled, avoids the waste of sterile devices. Then, the device containing the cells and the liquid to be added is placed into an incubator with a suitable environment, after the cells are cultured for a period of time and the cells are adhered to the wall, the door of the incubator is opened, the pressing rod 23 corresponding to the liquid to be added is pressed downwards, the piston head 22 is forced to move downwards, the container bottle 2 is squeezed into the injection tube 21, and finally the container bottle enters the culture hole 11 through the channel 12. In the whole liquid feeding process, liquid does not contact with the external environment, the leakproofness is good, greatly reduced the probability of contracting bacteria, and need not to remove the device outside to the incubator, simplified application of sample operating procedure, reduced aseptic technique platform's use number of times, energy-concerving and environment-protective. The priming volume is known by the distance the piston head 22 is moved downward.

It should be noted that, in order to observe the cell culture state conveniently, all parts of the cell culture dish are made of transparent materials, and all parts of the liquid adding device are made of transparent materials as much as possible.

It should be noted that, a plurality of liquid adding devices and channels 12 can be correspondingly arranged in one culture hole 11, each liquid adding device is correspondingly arranged with one channel 12, and one culture hole 11 can be correspondingly connected with a plurality of channels 12, so that a plurality of different liquids can be added into cells in the culture hole 11, and the liquid adding device is suitable for cell culture experiments with complicated liquid adding.

It should be noted that, for the cell culture experiment that the previous liquid needs to be discarded before the liquid is added, a liquid pumping device may be further provided, and the liquid pumping device has a structure substantially the same as that of the liquid adding device, for example, the liquid pumping device includes a liquid pumping container bottle, a liquid pumping injection tube, a liquid pumping piston head, and a liquid pumping pressing rod, and the liquid pumping device is different from the liquid adding device in that a bottom opening of the channel 12 corresponding to the liquid pumping device extends into the culture well 11, and the position of the bottom of the tip may be set according to experience, for example, after most of cell adherent culture is finished, the height of the cell is 0.5mm from the bottom in the specific culture well 11, and the distance from the tip to the bottom in the culture well 11 may be set to be 0.7-1 mm; when the pressing rod for drawing liquid of the liquid drawing device is pulled upwards, the piston head for drawing liquid moves upwards to draw the liquid in the culture hole 11 away and temporarily store the liquid in the container bottle for drawing liquid of the liquid drawing device, and the volume of the container bottle for drawing liquid of the liquid drawing device can be set to be larger so as to meet the liquid drawing operation of a cell culture experiment.

Example 2

A tumor complex microenvironment cell culture device, which has the same structure as that of the embodiment 1, except that, referring to fig. 4-6, the liquid adding device further comprises a porous plate 24, the porous plate 24 is provided with a plurality of through holes connected with the container bottle 2, the porous plate 24 is arranged on the culture holes 11 of the dish body 1, namely, arranged at the bottom of the dish in the dish body 1; the tumor complex microenvironment cell culture device further comprises a control switch, the control switch comprises a support column 3, a plurality of press plates 31, a plurality of press switches 32 and a plurality of press heads 33, the support column 3 is vertically arranged, the bottom of the support column is fixed at the center of the porous plate 24, a plurality of press plates 31 are arranged around the upper end periphery of the support column 3, each press plate 31 is correspondingly provided with one press rod 23, each press switch 32 is correspondingly arranged at the top of one press plate 31, each press head 33 is correspondingly arranged at the top of one press switch 32, and under the action of the press force of the press head 33, the press switches 32 can be opened, under the action of the press force without the press head 33, the press switches 32 can be clamped on the outer wall of the support column 3.

Under the control switch structure of this embodiment, through opening and closing of the push switch 32, the push plate 31 can be positioned at different positions of the support column 3, and then the push rod 23 correspondingly connected with the push plate 31 can also be positioned at different heights, and the piston head 22 correspondingly connected with the push rod 23 can be positioned at different heights in the container bottle 2, so that when liquid is quantitatively added, the piston head 22 can be prevented from rebounding upwards, and the risk of liquid suck-back is reduced.

The outer wall of support column 3 is equipped with the scale mark of vertical arrangement, then measurable quantity press plate 31's displacement, and this distance can correspond the liquid volume size of following container bottle 2 and discharging, then realized visual quantitative liquid feeding process, need not additionally to measure the descending distance of piston head 22.

Preferably, in this embodiment, the pressing switch 32 includes at least two switch claws 321, the bottom of the switch claw 321 is connected to the pressing plate 31, the top of the supporting column 3 is provided with a convex plate, a vertical convex rod 322 is arranged at the bottom of the convex plate and above each pressing plate 31, the switch claw 321 of each pressing switch 32 is arranged around its corresponding convex rod 322, and the switch claw 321 tightly clamps the convex rod 322 without the action of pressing force, and under the action of pressing force, the switch claw 321 moves outwards and separates from the convex rod 322, at this time it can move downwards along with the pressing plate 31. The pressing head 33 is mounted on the protruding rod 322 in a penetrating manner, and is movable up and down along the axial direction of the protruding rod 322 to contact the switching claw 321.

Preferably, the switch pawl 321 can be made of an elastic plate with an obtuse angle structure and elasticity and rigidity, and can move in a direction away from the protruding rod 322; or, the switch claws 321 are made of a non-elastic material, at this time, the bottom of the switch claw 321 is hinged with the top of the pressing plate 31, springs are arranged between every two switch claws 321 of the same pressing switch 32, under the condition that the pressing force is not available, the elastic force of the springs draws each pressing switch 32 towards the center, so that the switch claws 321 clamp the convex rods 322, and when the pressing force is generated, the switch claws 321 are dispersed from the center to the periphery and separated from the convex rods 322.

Example 3

A tumor complex microenvironment cell culture device has substantially the same structure as that of example 1, except that, referring to FIG. 7, a culture tube 111 is provided in a culture well 11, and a mouthpiece is provided on the upper end side wall of the culture tube 111 for facilitating the cleaning of the device. A channel tube 121 is laid in the channel 12, one end of the channel tube 121 penetrates through a through hole formed in the hole wall of the culture hole 11 and is detachably communicated with the mouthpiece, and the other end of the channel tube 121 penetrates out of an opening in the top of the channel 12 on the side wall of the dish body 1 and is detachably communicated with the injection tube 21.

When the culture device is used, the channel tube 121 is installed in the above mode, liquid adding operation is carried out, after the culture device is used, the channel tube 121 is detached to be independently cleaned, and due to the fact that in the liquid adding process, the dish body 1 is not directly contacted with cells or liquid, cleanliness is high, and cleaning operation is easy. The passage tube 121 may alternatively be prepared as a disposable tube.

It should be noted that, the connection relation of the components not specifically mentioned in the present invention is the default of the prior art, and the connection relation of the structures is not described in detail since it does not relate to the invention point and is a common application of the prior art.

It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A tumor complex microenvironment cell culture device is characterized by comprising a cell culture dish and a liquid adding device;

the cell culture dish comprises a dish body (1), a plurality of culture holes (11) are formed in the bottom of the dish body (1), a channel (12) is formed in the position, corresponding to each culture hole (11), of the side wall of the dish body (1), and an opening in the bottom of each channel (12) is communicated with each culture hole (11);

the liquid adding device comprises a container bottle (2), an injection tube (21), a piston head (22) and a pressing rod (23), wherein the bottom of the container bottle (2) corresponds to the top opening of the culture hole (11) in a one-to-one mode and is detachably connected with the top opening of the culture hole, one end of the injection tube (21) is communicated with the bottom of the container bottle (2), the other end of the injection tube is communicated with the top opening of the channel (12), the piston head (22) is located in the container bottle (2) and can move up and down in the container bottle (2), and the pressing rod (23) is installed at the top of the piston head (22).

2. The tumor complex microenvironment cell culture device of claim 1, wherein the outer wall of the bottom of the container bottle (2) is provided with an inward groove, and the top opening of the culture hole (11) is provided with a convex groove matching with the inward groove.

3. Tumor complex microenvironment cell culture device according to claim 1, characterized in that the bottom opening of the channel (12) extends to the bottom of the dish body (1) and the top opening of the channel (12) is located on the side wall of the dish body (1).

4. The tumor complex microenvironment cell culture device according to claim 3, wherein the upper end of the side wall of the culture hole (11) is communicated with one end provided with an air pressure balance hole (13), the other end of the air pressure balance hole (13) extends to the outer wall of the dish body (1) and is communicated with the outside, and the end is filled with a sterile plug with air permeability and bacteria filtration.

5. The tumor complex microenvironment cell culture device of claim 4, wherein the injection tube (21) is a zigzag tube having an upper horizontal tube, a lower horizontal tube and a longitudinal tube, and the injection tube (21) is communicated with the container bottle (2) to form a U-shaped communicator having the upper horizontal tube, and the bottom of the upper horizontal tube is higher than the channel (12).

6. The tumor complex microenvironment cell culture device according to claim 4, wherein each culture hole (11) is correspondingly connected with a plurality of the liquid adding devices, the end parts of all the air pressure balance pore channels (13) far away from the culture holes (11) are gathered to a main pipe, then the main pipe extends to the outer wall of the dish body (1) and is communicated with the outside, and the port of the main pipe communicated with the outside is filled with a sterile plug with bacteria filtration and ventilation.

7. The tumor complex microenvironment cell culture device of claim 6, further comprising a liquid pumping device, wherein the liquid pumping device is identical to the liquid adding device in structure, and the bottom opening of the channel (12) corresponding to the liquid pumping device extends into the culture hole (11).

8. The tumor complex microenvironment cell culture device of claim 6, further comprising a multi-well plate (24) and a control switch, wherein the multi-well plate (24) has a plurality of perforations connected to the container bottle (2); perforated plate (24) are arranged in the dish body (1) on culture hole (11), control switch includes support column (3), a plurality of pressing plate (31), a plurality of press switch (32) and a plurality of press head (33), the vertical setting of support column (3), its bottom is fixed on perforated plate (24), centers on the upper end periphery of support column (3) is equipped with a plurality of pressing plate (31), installs on every pressing plate (31) press pole (23), every press plate (31) top all is equipped with press switch (32), every press switch (32) top is installed press head (33).

9. The tumor complex microenvironment cell culture device of claim 8, wherein the push switch (32) comprises at least two switch claws (321), the bottom of the switch claw (321) is connected to the push plates (31), the top of the support column (3) is provided with a convex plate, the bottom of the convex plate and above each push plate (31) is provided with a vertical convex rod (322), the switch claw (321) of each push switch (32) is arranged around the corresponding convex rod (322), and the switch claw (321) can clamp the convex rod (322).

10. The tumor complex microenvironment cell culture device of claim 1, wherein a culture tube (111) is disposed in the culture hole (11), and a mouthpiece is disposed on the upper end side wall of the culture tube (111); a channel tube (121) is laid in the channel (12), one end of the channel tube (121) penetrates through the hole wall of the culture hole (11) and is detachably communicated with the mouthpiece, and the other end of the channel tube (121) penetrates out of the top opening of the channel (12) and is detachably communicated with the injection tube (21).