Co-culture bottle for different types of cells

Abstract

The invention discloses a co-culture bottle for different types of cells, which comprises a bottle body, a separation membrane is arranged in the bottle body, the interior of the bottle body is divided into a first culture area and a second culture area by the separation membrane, the bottle body is provided with a first bottle opening and a second bottle opening, the first bottle opening is communicated with the first culture area, and the second bottle opening is communicated with the second culture area. And the second bottle opening is communicated with the second culture area. The bottle body is divided into the first culture area and the second culture area which are independent from each other through the separation membrane, so that physical isolation of different types of cells can be realized, target cell purity reduction caused by mutual confusion of the cells is avoided, free exchange of small molecular substances such as a culture medium, cell metabolites and growth factors can be allowed by utilizing a microporous structure, and the cell culture efficiency is improved. The requirement of interaction between cells through substance signals in co-culture is met, and the in-vivo cell microenvironment is simulated to the maximum extent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cell culture, and particularly relates to a co-culture bottle for different kinds of cells. BACKGROUND

[0002] In the field of cell culture, trophoblast cells are often used as feeder layers to support the growth, proliferation and differentiation of target cells by providing necessary growth factors, extracellular matrix components and intercellular signals. Therefore, co-culture of different kinds of cells is of great significance in life science research and related applications.

[0003] In the prior art, cell co-culture is mostly dependent on culture plates of 6-well, 12-well, 24-well and the like, and cell isolation is achieved by embedding culture chambers. However, such culture devices have obvious limitations: firstly, the culture chamber and the culture plate are of a separate structure, and the opening is large, so the risk of contamination is high during operation; secondly, the culture area is small, and it is only suitable for small-scale experimental research, and it is difficult to meet the needs of commercial large-scale cell production; thirdly, some devices are difficult to balance between isolation effect and material exchange efficiency, and either the exchange of cell metabolic products and nutrients is insufficient due to excessive isolation, which affects the co-culture effect, or the purpose cells are mixed due to insufficient isolation, which leads to a decrease in the purity of the purpose cells. In addition, the operation convenience of the existing devices is poor, and operations such as pipetting and cell scraping are easily limited by the structure, and there are operation dead angles, which affect the experimental efficiency and accuracy.

[0004] Therefore, it is a technical problem to be solved in the field to develop a co-culture device that can effectively isolate cells, ensure efficient exchange of materials, reduce the risk of contamination, adapt to large-scale culture and be convenient to operate.

[0005] The purpose of the present application is to provide a co-culture bottle for different kinds of cells to solve the above problems in the prior art.

[0006] To this end, the present application provides, in a first aspect, a co-culture bottle for different kinds of cells, comprising a bottle body, a separation membrane is arranged in the bottle body, the separation membrane separates the interior of the bottle body into a first culture area and a second culture area, a first bottle opening and a second bottle opening are arranged on the bottle body, the first bottle opening communicates with the first culture area, and the second bottle opening communicates with the second culture area.

[0007] Preferably, the separation membrane is a microporous membrane to enable cells to absorb or permeate molecules from the micropores.

[0008] Preferably, the pore size of the micropores on the separation membrane ranges from 0.2 to 12 microns.

[0009] Preferably, the separation membrane and the bottle body are fixedly connected by ultrasonic welding or heat melting.

[0010] Preferably, the first bottle mouth and the second bottle mouth are arranged obliquely.

[0011] Preferably, the sidewall of the bottle body is provided with at least one air-permeable area.

[0012] Preferably, the air-permeable area is composed of a plurality of air-permeable holes arranged in a matrix.

[0013] Preferably, the air-permeable area is attached with an air-permeable film on the inner wall of the bottle body.

[0014] Preferably, the first bottle mouth is detachably connected with a first bottle cap, and the second bottle mouth is detachably connected with a second bottle cap.

[0015] Preferably, the first bottle cap and the second bottle cap are provided with through holes, and are provided with a hydrophobic air-permeable film inside.

[0016] Beneficial effects:

[0017] The application provides a co-culture bottle for different kinds of cells, which is divided into two independent first culture areas and second culture areas by a separation membrane, can realize physical isolation of different kinds of cells, avoid mutual confusion of cells to cause decrease of purity of target cells, can utilize microporous structure to allow free exchange of culture medium, cell metabolites and growth factors and other small molecule substances, meet the demand of mutual interaction of cells through material signals in co-culture, and maximize simulation of in-vivo cell microenvironment. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only show some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 It is a structure schematic view of a co-culture bottle for different kinds of cells in the present application.

[0020] Figure 2 It is a structure schematic view of a bottle body of a co-culture bottle for different kinds of cells in the present application.

[0021] Figure 3 It is a sectional view of a bottle body of a co-culture bottle for different kinds of cells in the present application.

[0022] In the diagram, 1-bottle body, 11-first culture zone, 12-second culture zone, 13-first bottle opening, 14-second bottle opening, 15-ventilation zone, 16-ventilation hole, 2-septum membrane, 3-first bottle cap, 4-second bottle cap. Detailed Implementation

[0023] The invention will be more readily understood by referring to the following detailed description of preferred embodiments and included examples. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the definitions in this specification shall prevail.

[0024] like Figures 1-3 As shown, the first aspect of this embodiment provides a co-culture flask for different types of cells, comprising:

[0025] The bottle body 1 has a separator membrane 2 inside, which divides the interior of the bottle body 1 into a first culture zone 11 and a second culture zone 12. The bottle body 1 has a first bottle opening 13 and a second bottle opening 14. The first bottle opening 13 is connected to the first culture zone 11, and the second bottle opening 14 is connected to the second culture zone 12.

[0026] The bottle body 1 is made of optically transparent polystyrene (PS), providing high optical clarity and mechanical strength, facilitating observation of the cell culture process. A separator membrane 2 divides the interior of the bottle body 1 into a first culture zone 11 and a second culture zone 12. The separator membrane 2 allows for the exchange of substances between cells, preventing cell mixing and ensuring the purity of the target cells, thus achieving the purpose of co-culture. A first bottle opening 13 and a second bottle opening 14 are used to add samples to the first culture zone 11 and the second culture zone 12 within the bottle body 1. Optionally, the first bottle opening 13 and the second bottle opening 14 can be integrally injection molded with the bottle body 1, providing better sealing performance and mechanical strength, and avoiding operational contamination.

[0027] A first cap 3 is detachably connected to the first bottle opening 13, and a second cap 4 is detachably connected to the second bottle opening 14. Both the first cap 3 and the second cap 4 have through holes, and a hydrophobic and breathable membrane is disposed inside both caps. The first cap 3 and the first bottle opening 13 can optionally be threaded together, as can the second cap 4 and the second bottle opening 14. The first cap 3 and the second cap 4 can optionally be made of high-density polyethylene. The through holes on the caps allow for ventilation, and the hydrophobic and breathable membrane covering the inside of the caps helps to prevent liquid exchange while maintaining ventilation, thus reducing the risk of contamination. The hydrophobic and breathable membrane can optionally be made of polyester fiber or hydrophobic polyethersulfone (PES).

[0028] The first bottle mouth 13 and the second bottle mouth 14 are arranged obliquely. The first bottle mouth 13 and the second bottle mouth 14 are arranged obliquely, in line with the principle of ergonomics, so that when the experimenter performs pipetting, cell scraping and other operations, the hand and the operation tool such as a pipette, a cell scraper can naturally extend into the bottle along the oblique angle without deliberately adjusting the holding angle or exerting additional force, effectively reducing hand fatigue during the operation. At the same time, the oblique structure avoids the right angle at the connection between the neck and the bottle body 1 in the traditional straight bottle mouth, so that the tool can easily reach all the cell growth surfaces in the culture bottle, including the area close to the edge of the bottle wall, completely solving the problem of incomplete cell scraping and residual culture solution caused by the difficulty of the tool to reach the corner in the traditional straight bottle mouth design, significantly shortening the single operation time and improving the experimental efficiency. The oblique first bottle mouth 13 and the second bottle mouth 14 make the transition of the neck and the bottle body 1 smoother, and the culture solution can flow smoothly along the inclined inner wall during addition, shaking or replacement, uniformly covering all the cell growth areas, ensuring that every cell can obtain consistent nutrient supply and metabolic environment, which is beneficial to the cultivation of a uniform cell population. At the same time, increasing the contact area between the bottle body 1 and the outside air improves the gas exchange efficiency and the adaptability of the culture environment.

[0029] The separation membrane 2 is a microporous membrane to allow cells to absorb or permeate molecules from the micropores. The bidirectional permeability of the separation membrane 2 can simulate the in vivo physiological environment. In the in vivo physiological environment, cells do not exist in isolation, but are attached to the extracellular matrix through the basal surface, and exchange substances with the surrounding tissue fluid, adjacent cell secretions, etc. through the apical surface, which is crucial for cell growth, differentiation and function maintenance. The microporous membrane of the culture bottle allows cells to absorb nutrient molecules or permeate metabolic products from their basal surface (the side close to the membrane) and apical surface (the side away from the membrane), thereby simulating this bidirectional substance exchange mode of in vivo cells. For example, when the feeder layer cells in the first culture area 11 secrete growth factors, the factors can diffuse to the target cells in the second culture area 12 through the microporous membrane; at the same time, the metabolic products of the target cells can also permeate to the area where the feeder layer cells are located in the opposite direction, forming a dynamic substance circulation, making the co-culture system more close to the real in vivo cell microenvironment, avoiding the problem of unbalanced nutrition or metabolic gradient caused by the traditional one-way permeable structure, and providing more physiological growth conditions for cells.

[0030] The distance between the microporous membrane of the culture bottle and the bottom layer is 0.2 mm, which reduces the lower culture volume and further increases the upper cell culture space and culture space utilization, which is more conducive to the expansion of target cells.

[0031] The material of the separation film 2 can be polyurethane or polycarbonate or polyethylene terephthalate. The pore size of the micropores on the separation film 2 ranges from 0.2 to 12 microns. The pore size can be selected according to the size of the cells and whether the cells need to pass through the membrane pores. For example, a 0.4-um pore size membrane can be used for general cell co-culture, and a 3-, 5-, or 8-um pore size membrane can be used for chemotaxis experiments, cell migration, and invasion experiments.

[0032] The separation film 2 is fixedly connected to the bottle body 1 by ultrasonic welding or hot melting. By fixing the separation film 2 to the bottle body 1 by ultrasonic welding or hot melting, no additional components are added, and the risk of unknown leachables is reduced.

[0033] The side wall of the bottle body 1 is provided with at least one gas permeable area 15, which is composed of a plurality of matrix-distributed gas permeable holes 16. The gas permeable area 15 is attached with a gas permeable film 17 on the inner wall of the bottle body 1. Specifically, in some disclosed embodiments, the gas permeable area 15 is provided with four, arranged in a 2x2 matrix. Each gas permeable area 15 is a square of 4cm x 4cm. The gas permeable area 15 is provided with gas permeable holes 16 for gas permeation. When the upper layer of cells exceeds the upper layer of holding space, it is necessary to realize gas permeation without overflowing, so the gas permeable film has hydrophobicity and can permeate gas. The material of the gas permeable film can be polyester fiber or hydrophobic polyether sulfone (PES).

[0034] Example 1

[0035] In Example 1, a bottle body 1 is provided with a separation film 2 inside the bottle body 1, which separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12. The bottle body 1 is provided with a first bottle mouth 13 and a second bottle mouth 14. The first bottle mouth 13 is in communication with the first culture area 11, and the second bottle mouth 14 is in communication with the second culture area 12. The material of the separation film 2 is polyurethane. In cell co-culture experiments, the pore density of the polyurethane film affects the interaction between cells and the efficiency of material exchange. Higher pore density can promote signal transmission and nutrient sharing between cells

[0036] Example 2

[0037] In Example 2, a bottle body 1 is provided with a separation film 2 inside the bottle body 1, which separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12. The bottle body 1 is provided with a first bottle mouth 13 and a second bottle mouth 14. The first bottle mouth 13 is in communication with the first culture area 11, and the second bottle mouth 14 is in communication with the second culture area 12. The material of the separation film 2 is polycarbonate. The separation film 2 of polycarbonate has uniform pore size and good chemical stability, and is commonly used in Corning Transwell, BeyoGold TMCell chamber products, etc. The pore size is generally 0.2 μm, 0.4 μm, 3 μm, 5 μm, 8 μm, etc. The 0.4 μm pore size PC film is usually used for cell co-culture experiments, which can effectively prevent cell passage while allowing free exchange of small molecules. The 3, 5 or 8 μm pore size membrane is suitable for chemotaxis experiments, cell migration and invasion experiments;

[0038] Example 3:

[0039] In Example 3, a bottle body 1 is provided, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, the bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14, the first bottle opening 13 is in communication with the first culture area 11, the second bottle opening 14 is in communication with the second culture area 12, and the material of the separation film 2 is polyethylene terephthalate. Polyethylene terephthalate has good flexibility and chemical stability, and its surface can be modified in various ways, such as coating treatment, to meet different cell culture needs, and is suitable for microscopy and photography, cell invasion experiments, cell migration experiments, etc. which require higher observation requirements or specific gas exchange environment;

[0040] Comparative Example 1:

[0041] In Comparative Example 1, a bottle body 1 is provided, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, the bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14, the first bottle opening 13 is in communication with the first culture area 11, the second bottle opening 14 is in communication with the second culture area 12, and the material of the separation film 2 is polyester fiber.

[0042] Comparative Example 2:

[0043] In Comparative Example 2, a bottle body 1 is provided, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, the bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14, the first bottle opening 13 is in communication with the first culture area 11, the second bottle opening 14 is in communication with the second culture area 12, and the material of the separation film 2 is polyvinylidene fluoride.

[0044] Comparative Example 3:

[0045] In Comparative Example 3, a bottle body 1 is provided, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, the bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14, the first bottle opening 13 is in communication with the first culture area 11, the second bottle opening 14 is in communication with the second culture area 12, and the material of the separation film 2 is hydrophobic polyether sulfone.

[0046] The examples 1-3 and the comparative examples 1-3 were tested to determine the effect on cell culture, and the test results are as follows:

[0047]

[0048] According to the test results, it can be seen that the separation membranes 2 of examples 1-3 all exhibit good hydrophilicity, which can promote the adsorption of extracellular matrix components and cell adhesion, meeting the growth needs of adherent cells; at the same time, the cell adhesion is strong or suitable for specific culture scenarios, ensuring that the cells can grow stably on the membrane surface or on both sides of the membrane, avoiding the problem of cell detachment caused by too strong hydrophobicity of the material. The three materials have no particle shedding, which can avoid contamination of the culture system; the mechanical strength and chemical stability are good, which can withstand temperature changes, pH fluctuations of the culture solution and operation friction during the culture process, ensuring the stability of the membrane structure during the co-culture period; at the same time, the polyurethane and PET have good air permeability, which can assist in ensuring the gas exchange on both sides of the membrane, and although the PC has relatively poor air permeability, it can be compensated by the independent air permeable area 15 of the culture bottle, such as the air permeable hole 16 and the air permeable membrane on the upper surface of the bottle body 1, which does not affect the overall gas environment.

[0049] Example 4:

[0050] In example 4, a bottle body 1 is provided, and a separation membrane 2 is arranged in the bottle body 1, which separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12. The bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14, the first bottle opening 13 communicates with the first culture area 11, and the second bottle opening 14 communicates with the second culture area 12. The separation membrane 2 is fixedly connected with the bottle body 1 by ultrasonic welding.

[0051] Example 5:

[0052] In example 5, a bottle body 1 is provided, and a separation membrane 2 is arranged in the bottle body 1, which separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12. The bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14, the first bottle opening 13 communicates with the first culture area 11, and the second bottle opening 14 communicates with the second culture area 12. The separation membrane 2 is fixedly connected with the bottle body 1 by a hot melting process.

[0053] Comparative example 4:

[0054] In comparative example 4, a bottle body 1 is provided, and a separation membrane 2 is arranged in the bottle body 1, which separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12. The bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14, the first bottle opening 13 communicates with the first culture area 11, and the second bottle opening 14 communicates with the second culture area 12. The separation membrane 2 is fixedly connected with the bottle body 1 by AB glue bonding.

[0055] Comparative example 5:

[0056] In Comparative Example 5, a bottle body 1 is included, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, a first bottle mouth 13 and a second bottle mouth 14 are arranged on the bottle body 1, the first bottle mouth 13 communicates with the first culture area 11, the second bottle mouth 14 communicates with the second culture area 12, and the separation film 2 is fixedly connected with the bottle body 1 by PYU hot melt adhesive bonding.

[0057] Comparative Example 6:

[0058] In Comparative Example 6, a bottle body 1 is included, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, a first bottle mouth 13 and a second bottle mouth 14 are arranged on the bottle body 1, the first bottle mouth 13 communicates with the first culture area 11, the second bottle mouth 14 communicates with the second culture area 12, and the separation film 2 is fixedly connected with the bottle body 1 by UV adhesive bonding.

[0059] Comparative Example 7:

[0060] In Comparative Example 7, a bottle body 1 is included, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, a first bottle mouth 13 and a second bottle mouth 14 are arranged on the bottle body 1, the first bottle mouth 13 communicates with the first culture area 11, the second bottle mouth 14 communicates with the second culture area 12, and the separation film 2 is fixedly connected with the bottle body 1 by laser welding.

[0061] Comparative Example 8:

[0062] In Comparative Example 8, a bottle body 1 is included, a separation film 2 is arranged in the bottle body 1, the separation film 2 separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12, a first bottle mouth 13 and a second bottle mouth 14 are arranged on the bottle body 1, the first bottle mouth 13 communicates with the first culture area 11, the second bottle mouth 14 communicates with the second culture area 12, and the separation film 2 is integrally formed with the bottle body 1 by injection molding.

[0063] The bonding effect, sealing property, and stability of Comparative Examples 4-5 and Comparative Examples 4-8 are tested, and the test results are as follows:

[0064] Serial number Film fusion process Bonding effect Sealing test Stability Example 4 Ultrasonic welding Good Yes Good Example 5 Hot melt Good Yes Good Comparative Example 4 AB glue Poor Yes Poor Comparative Example 5 PYU hot melt glue Poor Yes Poor Comparative Example 6 UV glue Good Yes Poor Comparative Example 7 Laser welding Poor No Poor Comparative Example 8 Injection molding Poor No Good

[0065] Examples 4-5 showed good adhesion effect, sealing compliance, and good stability. The core advantage is that no external substances such as adhesives need to be added, the film and the bottle body 1 contact surface are locally fused and combined by physical means, avoiding the risk of contamination by adhesive dissolution; the connection part after welding or fusion is tight and seamless, can withstand long-term immersion in culture solution, temperature changes and external forces, ensuring stable sealing during co-culture period; it adapts to the connection needs of thin film materials, and can avoid film damage by precise control of energy while ensuring connection strength, fully meeting the stringent requirements of cell co-culture without contaminants, reliable sealing, and stable structure.

[0066] In the comparative examples, the adhesion effect of AB glue and PYU hot melt glue is poor, and the stability is poor. Such process relies on chemical adhesives to connect the film and the bottle body 1 (polystyrene PS), and the glue layer is easily swollen and aged by immersion in culture solution and temperature changes, resulting in separation of the film and the bottle body 1. Not only can it not guarantee long-term sealing, but it also has the risk of culture solution leakage or external contamination, and it may also contaminate the culture system with unknown substances dissolved by the adhesive, which does not meet the core requirement of "no external contaminants" for cell culture. Although UV glue has good adhesion effect and sealing compliance, its stability is poor. The curing relies on ultraviolet irradiation, and the glue layer is prone to embrittlement and cracking after curing when it is in contact with the culture solution or temperature fluctuations for a long time, resulting in sealing failure in the later period, and the composition of the glue layer may have potential cytotoxicity, which is not suitable for long-term co-culture scenarios. For laser welding and injection molding processes, both processes show poor adhesion effect and sealing compliance. Because the filter membrane, which is a micro-porous membrane suitable for cell co-culture, needs to consider both light transmission and material exchange efficiency, the thickness is usually thin, and high temperature during laser welding can easily cause damage or burning of the membrane, which cannot form an effective seal; injection molding is difficult to achieve uniform fusion of the film and the bottle body 1 due to the large difference in melting temperature between the film and PS, and it is easy to form gaps at the connection site, resulting in sealing failure, so it cannot meet the process requirements.

[0067] Combining the core requirements of cell co-culture such as avoiding external contamination, ensuring long-term sealing, and tolerating the culture environment, ultrasonic welding and hot melt process are the best choices. Both of them solve the connection problem of the film and the bottle body 1 by physical fusion, and show excellent adhesion effect, sealing compliance, and stability, and no additional chemicals are added, which can minimize the risk of contamination of the culture system, and adapt to different types of cell co-culture for long-term use scenarios.

[0068] Example 6:

[0069] The bottle body 1 is provided with a separation film 2, which separates the inside of the bottle body 1 into a first culture area 11 and a second culture area 12. The bottle body 1 is provided with a first bottle opening 13 and a second bottle opening 14. The first bottle opening 13 communicates with the first culture area 11, and the second bottle opening 14 communicates with the second culture area 12. The material of the separation film 2 is polycarbonate. The separation film 2 is fixedly connected with the bottle body 1 through ultrasonic welding. The bottle body 1 is provided with a gas permeable area 15. The volume of the bottle body 1 is 175 cm 2 .

[0070] Comparative Example 9:

[0071] T175 commercial culture bottles were used.

[0072] Example 6 and Comparative Example 9 were subjected to a first group of experiments. Cell expansion culture comparative experiments were carried out on lung epithelial cells (adherent cells) and NK cells (suspended cells).

[0073] In the experimental group A of Example 6, lung epithelial cells were inoculated in the second culture area 12, and feeder layer 3T3 cells were inoculated in the first culture area 11 (cells were attached to gelatin 3D microcarriers, Huagang Biotechnology). In the experimental group B, lung precursor cells were inoculated in the second culture area 12, and no cells were inoculated in the first culture area 11. The control group of Comparative Example 9 used T175 commercial culture bottles to inoculate lung precursor cells. The same culture medium was used for co-culture for 5 days, and the cells were harvested at 1 day, 3 days and 5 days of culture for cell number comparison.

[0074] The cells used for testing were from ATCC, and the complete culture medium for cell culture was DMEM medium (Gibco) + 10% FBS (Gibco) + 1% P / S (Yuan Sheng Biotechnology), T175 culture bottles (PS material, TC treated, Corning), and the pipettes and centrifuge tubes used in the culture were from NEST.

[0075] The equipment used for testing was a desktop normal-temperature low-speed centrifuge (SCILOGEX), a low-speed desktop refrigerated centrifuge (Hunan Kecheng Instrument and Equipment Co., Ltd.), a constant-temperature water bath kettle (Changzhou Langyue), a carbon dioxide incubator (ESCO), a refrigerator (Haier), a biological safety cabinet (Sunjing Antai), a cell counter (CountStar), and an inverted microscope (OLYMPUS).

[0076] The experimental results are shown below.

[0077]

[0078] Compared with the control group, the experimental group A of Example 6 has obvious advantages in cell proliferation rate on the third day of culture under the stimulation of 3T3 feeder cells, and the cell proliferation capacity on the fifth day of culture is 2.23 times that of the control group. The experimental group B has obvious advantages in cell proliferation rate on the fifth day of culture, which is 1.33 times that of the control group. Thus, the comparative example 9 has obvious advantages in adherent cell culture.

[0079] A second group of experiments was conducted on Example 6 and Comparative Example 9. The experimental group A of Example 6 inoculated MSC cells in the second culture area 12 and inoculated NK cells in the first culture area 11. The experimental group B inoculated NK cells in the first culture area 11 and did not inoculate cells in the second culture area 12. Comparative Example 9 inoculated NK cells in a conventional culture by using a T175 commercial culture bottle. The same culture medium was used for co-culture for 7 days, and the cells were harvested for cell number comparison at 3 days, 5 days and 7 days of culture, respectively.

[0080] The cells used for testing were from ATCC, and the co-culture medium was from Suzhou Huachen Biological Technology Co., Ltd. The pipettes and centrifuge tubes used in the culture were from NEST.

[0081] Test equipment: benchtop low-speed centrifuge (SCILOGEX), low-speed benchtop refrigerated centrifuge (Hunan Kecheng Instrument and Equipment Co., Ltd.), constant-temperature water bath (Changzhou Langyue), carbon dioxide incubator (ESCO), refrigerator (Haier), biological safety cabinet (Suzhou Jingantai), cell counter (CountStar), inverted microscope (OLYMPUS).

[0082] The experimental results are shown as follows,

[0083]

[0084] Compared with the control group, the experimental group A of Example 6 has obvious advantages in cell proliferation rate on the third day of culture under the stimulation of MSC feeder cells, and the cell proliferation capacity on the fifth day of culture is 1.61 times that of the control group. The experimental group B has obvious advantages in cell proliferation rate on the seventh day of culture, which is 1.13 times that of the control group. Thus, the comparative example 9 has obvious advantages in adherent cell culture.

[0085] The above-described examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A co-culture flask for different kinds of cells, characterized by, The application relates to a bottle body, which is provided with a separation film inside the bottle body, the separation film separates the inside of the bottle body into a first culture area and a second culture area, the bottle body is provided with a first bottle opening and a second bottle opening, the first bottle opening communicates with the first culture area, and the second bottle opening communicates with the second culture area. The separation film is a microporous film to enable cells to absorb or permeate molecules from micropores.

2. The co-culture flask for different kinds of cells according to claim 1, wherein, The pore size of the micropores on the separation film ranges from 0.2 to 12 microns.

3. The co-culture flask for different kinds of cells according to claim 2, wherein, The separation film is fixedly connected with the bottle body in a mode of ultrasonic welding or hot melting.

4. The co-culture flask for different kinds of cells according to claim 1, wherein, The first bottle opening and the second bottle opening are arranged in an inclined mode.

5. The co-culture flask for different kinds of cells according to claim 1, wherein, At least one air-permeable area is arranged on the side wall of the bottle body.

6. The co-culture flask for different kinds of cells according to claim 1, wherein, The air-permeable area is composed of a plurality of air-permeable holes arranged in a matrix mode.

7. The co-culture flask for different kinds of cells according to claim 6, wherein, An air-permeable film is attached to the inner wall of the bottle body.

8. The co-culture flask for different kinds of cells according to claim 6, wherein, A first bottle cap is detachably connected to the first bottle opening, and a second bottle cap is detachably connected to the second bottle opening.

9. The co-culture flask for different kinds of cells according to claim 1, wherein, Holes are arranged on the first bottle cap and the second bottle cap, and hydrophobic air-permeable films are arranged in the first bottle cap and the second bottle cap.

10. The co-culture flask for different kinds of cells according to claim 1, wherein, ​

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