Grid embedded cell co-culture device and method

Abstract

The present invention provides a grid-based embedded cell co-culture device and method, wherein the device includes a dish body, a dish cover, a first culture plate and a second culture plate; the first culture plate and the second culture plate respectively include a plurality of culture grooves distributed in a mesh-like interval and a plurality of hollow parts arranged between adjacent culture grooves; the hollow parts of the first culture plate correspond to the positions of the culture grooves of the second culture plate, and the hollow parts of the second culture plate correspond to the positions of the culture grooves of the first culture plate; the side wall height of the culture groove is less than the side wall height of the dish body; the dish cover cooperates with the dish body; when the first culture plate and the second culture plate are staggered and plugged to form a whole, the culture grooves of the first culture plate and the second culture plate are staggered; the dish body can accommodate the staggered plugged first culture plate and the second culture plate. A grid-based embedded cell co-culture device and method of the present invention can realize multi-cell free-proportion grid-based non-contact co-culture, and the production process is simple and efficient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a grid-based embedded cell co-culture device and method. Background Art

[0002] Tumors are one of the most important factors threatening human life, so people have been trying to study tumors in depth to achieve the goal of eliminating tumors. Tumors are in a highly informationized microenvironment in the human body, but most of the current research on tumors is focused on studying the biological information of tumor cells when they exist alone. The research results of tumor research separated from the microenvironment may be different from the situation of tumors in the human body. Many drugs have good killing effects in in vitro experiments, but their tumor killing effects in vivo are not as good as expected, which further proves the above conclusion. Therefore, in order to establish a tumor culture system that is more in line with the in vivo environment, people try to make the in vitro tumor research environment consistent with the in vivo tumor environment as much as possible, so that tumor cells can communicate with each other, and cell co-culture technology came into being.

[0003] There are two main methods of cell co-culture: direct contact co-culture and non-contact co-culture. Contact co-culture co-cultures different cells in different proportions. Because different cells are in direct contact and it is difficult to completely separate them for subsequent research and analysis, it is not widely used in scientific research. Another co-culture method is non-contact co-culture, which isolates different cells in different ways to achieve the purpose of being in the same culture system without direct contact. At present, the Chinese patent application number is: CN 112920992 A, and the patent name is "Method and application of co-culture of bone marrow mesenchymal stem cells and nucleus pulposus cells", but this co-culture device has obvious disadvantages: 1. Expensive: The price of Transwell culture chamber is high; 2. It is impossible to co-culture more than two adherent cells; 3. It is inconvenient to observe the state of the cells in the lower chamber. In addition, the Chinese patent application number is: CN 104164365 B, and the patent name is "An in vitro cell contact co-culture device and its culture operation method", but this co-culture device also has obvious disadvantages: 1. It is impossible to co-culture more than two adherent cells. 2. After the co-culture is completed, it is impossible to perform subsequent experimental operations on a single cell. In addition, the Chinese patent application number is: CN201545843 U, and the patent name is "A non-direct contact cell co-culture device", but this device cannot achieve co-culture of more than two types of cells and freely set the ratio for co-culture. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a grid-based embedded cell co-culture device and method, which can realize multi-cell free-proportion grid-based non-contact co-culture, and has a simple production process and high efficiency.

[0005] In order to achieve the above-mentioned purpose, the present invention provides a grid-based embedded cell co-culture device, comprising a dish body, a dish cover, a first culture plate and a second culture plate; the first culture plate and the second culture plate respectively include a plurality of culture grooves distributed in a mesh-like manner and a plurality of hollow portions arranged between adjacent culture grooves; the hollow portions of the first culture plate correspond to the positions of the culture grooves of the second culture plate, and the hollow portions of the second culture plate correspond to the positions of the culture grooves of the first culture plate; the side wall height of the culture groove is less than the side wall height of the dish body; the dish cover cooperates with the dish body; when the first culture plate and the second culture plate are staggered and plugged together to form a whole, the culture grooves of the first culture plate and the second culture plate are staggered; the dish body can accommodate the first culture plate and the second culture plate after staggered plugging.

[0006] Preferably, the first culture plate and the second culture plate are staggered and plugged into each other to form a rectangle.

[0007] Preferably, the culture tank is rectangular.

[0008] Preferably, the side wall of the culture tank is 5 mm high; the side wall of the dish body is 23 mm high.

[0009] Preferably, the distance between two adjacent culture grooves of the first culture plate and the second culture plate that are staggered is greater than or equal to 2 mm.

[0010] Preferably, the dish body, the dish cover, the first culture plate and the second culture plate are formed by 3D printing.

[0011] A grid-based embedded cell co-culture method based on the grid-based embedded cell co-culture device of the present invention comprises the following steps:

[0012] S1: cell plating step;

[0013] The S1 step further comprises the steps of:

[0014] S11: inserting the first culture plate and the second culture plate together alternately and placing them into the dish body;

[0015] S12: Add 200ul of cell suspension into the culture tank, wherein the cell density of the cell suspension is less than 80%; avoid shaking the dish vigorously;

[0016] S13: The cell suspension was incubated at 37°C and 5% CO 2 Incubate for at least 4 hours in an environment until the cells in the cell suspension adhere to the wall;

[0017] S2: step of forming a co-culture environment;

[0018] The S2 step further comprises the steps of:

[0019] S21: discarding the original culture medium in the culture tank;

[0020] S22: adding a sufficient amount of new culture medium so that the new culture medium covers each of the culture tanks and connects the culture tanks into one;

[0021] S23: The cells were incubated at 37°C and 5% CO 2 Incubate for a period of time in an environment;

[0022] S3: End the co-culture environment step;

[0023] The S3 step further comprises the steps of:

[0024] S31: discarding the new culture medium in the dish body and the culture tank;

[0025] S32: separating the first culture plate and the second culture plate;

[0026] S33: Collect the cells in different culture tanks respectively.

[0027] Preferably, in the step S12, the cell suspensions of different cells are cultured at different culture ratios.

[0028] The present invention adopts the above technical solution, so it has the following beneficial effects:

[0029] 1. Through the structure of the gridded staggered culture troughs of the first culture plate and the second culture plate, multi-cell free-proportion gridded non-contact co-culture can be achieved, and the production process is simple and efficient.

[0030] 2. Different plating schemes can be used, such as surrounding culture, which is more in line with the living environment of cells in the body, avoiding the potential disadvantages of studying cells without biological information exchange between cells, and greatly improving the reproducibility and stability of the experiment

[0031] 3. The first culture plate and the second culture plate are provided with a plurality of culture grooves, and culture modes of different proportions can be freely set, which is an ideal cell co-culture model.

[0032] 4. The 2mm interval between each culture tank avoids the error caused by the mixing of different types of cells due to cell migration, resulting in impure cells in the small holes.

[0033] 5. The most prominent advantage is that when the demand for multi-cell free-proportion co-culture cannot be fully met in actual work due to the limitations of supporting equipment and technologies such as transwell co-culture, the co-culture mode provided by the present invention will simulate the real survival state of cells in the body to the greatest extent; at the same time, the present invention is suitable for various adherent cells, which greatly improves the scope of application, recognition and accuracy of the present invention in scientific research.

[0034] The multi-cell free-proportion non-contact co-culture technology provided by the present invention can be used for in vitro and in vivo research on various cells, molecular mechanism research, as well as pharmacological mechanism and drug treatment evaluation research, etc., providing a solid tool for research in the field of medical biology; it has broad application prospects and potential social and economic value in basic research and preclinical drug research. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic structural diagram of a grid-based embedded cell co-culture device according to an embodiment of the present invention;

[0036] Figure 2 This is a schematic structural diagram of a first culture plate according to an embodiment of the present invention;

[0037] Figure 3 Schematic diagram of the structure of the second culture plate of an embodiment of the present invention. DETAILED DESCRIPTION

[0038] The following is based on the attached Figure 1 to Figure 3 , give the preferred embodiments of the present invention, and describe them in detail so that the functions and features of the present invention can be better understood.

[0039] See also Figure 1 to Figure 3 A grid-based embedded cell co-culture device according to an embodiment of the present invention comprises a dish body 1, a dish cover, a first culture plate 2 and a second culture plate 3; the first culture plate 2 and the second culture plate 3 respectively comprise a plurality of culture grooves 4 distributed in a mesh-like manner and a plurality of hollow portions 5 arranged between adjacent culture grooves 4; the hollow portions 5 of the first culture plate 2 correspond to the positions of the culture grooves 4 of the second culture plate 3, and the hollow portions 5 of the second culture plate 3 correspond to the positions of the culture grooves 4 of the first culture plate 2; the side wall height of the culture groove 4 is less than the side wall height of the dish body 1; the dish cover cooperates with the dish body 1; when the first culture plate 2 and the second culture plate 3 are staggered and plugged together to form a whole, the culture grooves 4 of the first culture plate 2 and the second culture plate 3 are staggered; the dish body 1 can accommodate the staggered first culture plate 2 and the second culture plate 3.

[0040] The first culture plate 2 and the second culture plate 3 are staggered and inserted into each other to form a rectangle.

[0041] The culture tank 4 is rectangular.

[0042] The side wall of the culture tank 4 is 5 mm high; the side wall of the dish body 1 is 23 mm high.

[0043] The distance between two adjacent culture grooves 4 that are staggered between the first culture plate 2 and the second culture plate 3 is greater than or equal to 2 mm.

[0044] The dish body 1, the dish cover, the first culture plate 2 and the second culture plate 3 are formed by 3D printing.

[0045] A grid-based embedded cell co-culture method based on the grid-based embedded cell co-culture device of the embodiment of the present invention comprises the following steps:

[0046] S1: cell plating step;

[0047] The S1 step further comprises the steps of:

[0048] S11: inserting the first culture plate 2 and the second culture plate 3 together in an alternating manner and placing them into the dish body 1;

[0049] S12: Add 200ul of cell suspension into the culture tank 4, the cell density of the cell suspension is <80%; avoid shaking the dish 1 vigorously;

[0050] S13: Incubate the cell suspension at 37°C and 5% CO 2 Incubate for at least 4 hours in an environment until the cells in the cell suspension adhere to the wall;

[0051] S2: step of forming a co-culture environment;

[0052] The S2 step further comprises the steps of:

[0053] S21: discarding the original culture medium in the culture tank 4;

[0054] S22: adding a sufficient amount of new culture medium so that the new culture medium covers each culture tank 4 and connects the culture tanks 4 into one;

[0055] S23: Incubate the cells at 37°C and 5% CO 2 Incubate for a period of time in an environment;

[0056] S3: End the co-culture environment step;

[0057] The S3 step further includes the steps of:

[0058] S31: discard the new culture medium in the dish body 1 and the culture tank 4;

[0059] S32: separating the first culture plate 2 and the second culture plate 3;

[0060] S33: Collect cells from different culture tanks 4 respectively.

[0061] In step S12, cell suspensions of different cells may be cultured at different culture ratios.

[0062] Different culture modes, such as surrounding co-culture and cross-co-culture, can be achieved by regulating the arrangement of the cell culture tanks 4 .

[0063] Different culture modes can be used to explore the factors affecting the proliferation, differentiation, migration and apoptosis of target cells in the co-culture system.

[0064] After the co-culture is completed, the first culture plate 2 and the second culture plate 3 can be easily separated, and the first culture plate 2 and the second culture plate 3 can be collected separately for subsequent experiments, such as cell cycle detection, molecular protein detection, etc.

[0065] The method provided by the present invention can not only realize the co-culture of two or more types of cells in different proportions, but also easily avoid the errors caused by the complexity of subsequent flow cytometer separation during contact co-culture.

[0066] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A grid-based embedded cell co-culture device, comprising a dish body, a dish cover, a first culture plate and a second culture plate; Features: The first culture plate and the second culture plate respectively include a plurality of culture grooves spaced apart in a mesh pattern and a plurality of hollow portions disposed between adjacent culture grooves; the hollow portions of the first culture plate correspond to the positions of the culture grooves of the second culture plate, and the hollow portions of the second culture plate correspond to the positions of the culture grooves of the first culture plate; The height of the side wall of the culture groove is smaller than the height of the side wall of the dish body; the dish cover is matched with the dish body; when the first culture plate and the second culture plate are staggered and plugged to form a whole, the culture grooves of the first culture plate and the second culture plate are staggered; the dish body can accommodate the staggered first culture plate and the second culture plate; A space is reserved between two adjacent culture grooves of the first culture plate and the second culture plate that are staggered.

2. The grid-based embedded cell co-culture device according to claim 1, It is characterized in that The first culture plate and the second culture plate are staggered and plugged together to form a rectangle.

3. The grid-based embedded cell co-culture device according to claim 2, It is characterized in that The culture tank is rectangular.

4. The grid-based embedded cell co-culture device according to claim 3, It is characterized in that The side wall of the culture tank is 5 mm high; the side wall of the dish body is 23 mm high.

5. The grid-based embedded cell co-culture device according to claim 4, It is characterized in that The distance between two adjacent culture grooves that are staggered between the first culture plate and the second culture plate is greater than or equal to 2 mm.

6. The grid-based embedded cell co-culture device according to claim 5, It is characterized in that The dish body, the dish cover, the first culture plate and the second culture plate are formed by 3D printing.

7. A grid-based embedded cell co-culture method based on the grid-based embedded cell co-culture device according to claim 1, comprising the steps of: S1: cell plating step; The S1 step further comprises the steps of: S11: inserting the first culture plate and the second culture plate together alternately and placing them into the dish body; When the first culture plate and the second culture plate are staggered and plugged together to form a whole, the culture grooves of the first culture plate and the second culture plate are staggered; A space is reserved between two adjacent culture grooves of the first culture plate and the second culture plate that are staggered; S12: Add 200ul of cell suspension into the culture tank, wherein the cell density of the cell suspension is less than 80%; avoid shaking the dish vigorously; S13: The cell suspension was incubated at 37°C and 5% CO 2 Incubate for at least 4 hours in an environment until the cells in the cell suspension adhere to the wall; S2: step of forming a co-culture environment; The S2 step further comprises the steps of: S21: discarding the original culture medium in the culture tank; S22: adding a sufficient amount of new culture medium so that the new culture medium covers each of the culture tanks and connects the culture tanks into one; S23: The cells were incubated at 37°C and 5% CO 2 Incubate for a period of time in an environment; S3: End the co-culture environment step; The S3 step further comprises the steps of: S31: discarding the new culture medium in the dish body and the culture tank; S32: separating the first culture plate and the second culture plate; S33: Collect the cells in different culture tanks respectively.

8. The grid-embedded cell co-culture method according to claim 7, It is characterized in that In the step S12, the cell suspensions of different cells are cultured at different culture ratios.

Images