Cell co-culture device

Abstract

The invention discloses a cell co-culture device which comprises a culture container and at least one support assembly, the culture container comprises at least one relatively independent chamber used for storing a culture solution, at least one hole is formed in a top cover of the culture container, and the at least one hole and the at least one chamber are arranged in a one-to-one correspondence mode; the scaffold assembly comprises at least two cell culture scaffolds with different sizes and specifications, the at least two cell culture scaffolds with different sizes and specifications are separably nested together, and a nanofiber layer is arranged at one end of each cell culture scaffold. And the nanofiber layers on the at least two cell culture scaffolds with different sizes and specifications are arranged at intervals and are suspended at different liquid levels of the culture solution. According to the invention, an environment closer to a physiological state can be provided, a researcher can conveniently separate cells and analyze respective state changes, the reliability and flexibility of an experiment are improved, a culture solution is saved, and pollution is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a cell co-culture device. Background Art

[0002] Cell co-culture technology is a method of culturing two or more different types of cells in the same system at the same time. It can simulate the cell microenvironment in the body and study the interaction between different cells and their impact on biological processes. The 3D co-culture system can simulate the in vivo environment, show cell activity and cell differentiation, protein expression and other reactions, realize real cell biology and function, and provide a reliable method for studying complex cell interactions, such as the synergistic and protective effects between various neural cells, and the specific mechanism of crosstalk between fibroblasts and immune cells.

[0003] Existing direct 3D co-culture scheme: add 3D hydrogel to Transwell chamber, co-encapsulate different cells in the gel, and make them grow and interact in a three-dimensional environment. It is used to study the growth, differentiation and interaction of cells in three-dimensional space, which is closer to the cell microenvironment in vivo. Although this scheme can directly observe the interaction between cells, it may cause cell mixing and make it difficult to separate and analyze later.

[0004] The existing indirect 3D co-culture scheme is to grow various cells separately in different culture chambers or culture dishes, and interact with each other through secreted signal molecules, avoiding direct contact between cells and facilitating subsequent separation. However, it is wasteful of culture fluid and prone to contamination. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a cell co-culture device that can provide an environment closer to the physiological state, facilitate the study of cell interactions, and enable researchers to easily separate cells and analyze their respective state changes, thereby improving the reliability and flexibility of the experiment, while saving culture fluid and reducing pollution.

[0006] The present invention is achieved through the following technical solutions:

[0007] A cell co-culture device, comprising:

[0008] A culture container, wherein the culture container comprises at least one relatively independent chamber for storing culture fluid, and the top cover of the culture container is provided with at least one hole, and the at least one hole is arranged in a one-to-one correspondence with the at least one chamber;

[0009] At least one bracket component, at least one of the bracket components is arranged in a one-to-one correspondence with at least one of the holes, and the bracket component can be detachably placed in the hole; wherein,

[0010] The support assembly comprises at least two cell culture supports of different sizes and specifications, and the at least two cell culture supports of different sizes and specifications can be detachably stacked together. A nanofiber layer is arranged at one end of the cell culture support, and the nanofiber layers on the at least two cell culture supports of different sizes and specifications are arranged at intervals and suspended at different liquid levels of the culture solution.

[0011] Furthermore, the spacing between adjacent nanofiber layers on the cell culture scaffold is 0.9-1.1 mm.

[0012] Furthermore, the distance between the uppermost nanofiber layer in the support assembly and the surface of the culture solution is 0.8-1.0 cm, and the distance between the lowermost nanofiber layer in the support assembly and the inner bottom wall of the culture container is 0.1-0.3 cm.

[0013] Furthermore, the at least two cell culture scaffolds of different specifications include:

[0014] A first cell culture support, wherein the first cell culture support is arranged in the hole, the first cell culture support comprises a first through hole arranged along the axial direction thereof, a first nanofiber layer is adhered to an axial end of the first cell culture support, and a projection of the first nanofiber layer on the first cell culture support is at least partially located in a cross section of the first through hole;

[0015] a second cell culture support, the second cell culture support being arranged in the first through hole, the second cell culture support comprising a second through hole extending through the second cell culture support in an axial direction thereof, a second nanofiber layer being adhered to an axial end of the second cell culture support, and a projection of the second nanofiber layer on the second cell culture support being at least partially located in a cross section of the second through hole;

[0016] Wherein, the first nanofiber layer and the second nanofiber layer are arranged at intervals and suspended at different liquid levels of the culture solution.

[0017] Furthermore, one end of the first cell culture support away from the first nanofiber layer protrudes radially outward to form a first carrier, and the first carrier abuts against the outer surface of the top cover.

[0018] Furthermore, one end of the second cell culture support away from the second nanofiber layer protrudes radially outward to form a second carrier, and a first support for supporting the second carrier is protruded on the inner side wall of the first through hole.

[0019] Furthermore, the upper surface of the first carrier is coplanar with the upper surface of the second carrier.

[0020] Furthermore, the cell culture support is in a bowl shape, and the nanofiber layer is arranged close to the small opening end of the cell culture support.

[0021] Furthermore, the cell culture support is integrally formed and air holes are provided on the outer peripheral wall of the cell culture support.

[0022] Furthermore, the at least two cell culture scaffolds of different sizes and specifications include three cell culture scaffolds of different sizes and specifications, and the three cell culture scaffolds of different sizes and specifications can be detachably stacked together.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] By designing at least two cell culture scaffolds of different sizes to be detachably stacked together, and providing a nanofiber layer at one end of the cell culture scaffold, and the nanofiber layers on the at least two cell culture scaffolds of different sizes are arranged at intervals and suspended at different liquid levels of the culture solution, the following beneficial effects can be achieved:

[0025] (1) More reliable: The bottom of the cell culture scaffold is a nanofiber layer that can mimic the structure and biological function of the natural extracellular matrix (ECM). The stackable cell culture scaffold provides a true 3D co-culture system that simulates the in vivo environment and provides a reliable method for studying complex cell interactions.

[0026] (2) Flexibility in experimental design: In some experimental designs, one cell may respond to a signaling factor secreted by another cell, and the transmission of these signaling factors depends on their spatial arrangement. In a stackable cell culture scaffold, the positions of different cells in co-culture can be interchanged. This flexibility allows researchers to explore the mechanisms and dynamics of interactions between different cells.

[0027] (3) Convenience of operation: It is easy to operate and replace the scaffold, and different types of cells can be separated in different layers to avoid direct contact. This indirect co-culture method allows researchers to easily separate cells and analyze their respective state changes, and is also suitable for other 3D cell cultures such as dynamic culture in the future.

[0028] (4) Save culture fluid and reduce pollution: The nested cell culture scaffold can make more efficient use of culture fluid and reduce waste. It can also meet the use of different or the same culture media at the same time. If a single culture unit is abnormal, it can be quickly replaced, thereby reducing the risk of contamination and helping to achieve long-term stable cell culture.

[0029] (5) Diverse applications: Different models of culture scaffolds can choose different pore sizes and membrane materials, or surface modifications according to experimental requirements, and are suitable for a variety of cell types and experimental conditions. When not used as a co-culture experiment, the system can also achieve the culture of the same cell type in different well plates, thereby enabling the simultaneous comparison of the interactions between different cell densities under the same conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of the structure of the culture container;

[0031] Figure 2 is a schematic diagram of the structure of the bracket assembly;

[0032] Figure 3 The exploded view of the bracket assembly;

[0033] Figure 4 A cross-sectional view of the bracket assembly.

[0034] 1. Culture container; 11. Top cover; 12. Holes; 2a. First cell culture support; 20a. First through hole; 21a. First carrier; 22a. First support; 23. Air holes; 2b. Second cell culture support; 20b. Second through hole; 21b. Second carrier; 22b. Second support; 2c. Third cell culture support; 20c. Third through hole; 21c. Third carrier; 3a. First nanofiber layer; 3b. Second nanofiber layer; 3c. Third nanofiber layer. DETAILED DESCRIPTION

[0035] The following is a further non-restrictive detailed description of the technical solution of the invention in conjunction with the preferred embodiments and the accompanying drawings. In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate the orientation or position relationship based on the orientation or position relationship shown in the accompanying drawings. In addition, the terms "first" and "second" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and cannot be understood as limitations on the present invention.

[0036] like Figure 1As shown, a cell co-culture device according to an embodiment of the present invention includes a culture container 1, the culture container 1 includes at least one relatively independent chamber for storing culture fluid, and at least one hole 12 is provided on the top cover 11 of the culture container 1, and at least one hole 12 is arranged in a one-to-one correspondence with at least one chamber. Among them, the culture container 1 can adopt a 12-well cell culture plate, a 24-well cell culture plate, and a 48-well cell culture plate.

[0037] The cell co-culture device further includes at least one support component, which is disposed in a one-to-one correspondence with at least one hole 12 , and the support component can be detachably placed in the hole 12 .

[0038] The scaffold assembly includes at least two cell culture scaffolds of different sizes and specifications, at least two cell culture scaffolds of different sizes and specifications can be detachably stacked together, a nanofiber layer is arranged at one end of the cell culture scaffold, and the nanofiber layers on at least two cell culture scaffolds of different sizes and specifications are arranged at intervals and suspended at different liquid levels of the culture solution. By arranging the nanofiber layer at the bottom of the cell culture scaffold, the nanofiber layer can mimic the structure and biological function of the natural extracellular matrix (ECM), and the stackable cell culture scaffold provides a true 3D co-culture system, simulating the in vivo environment, and providing a reliable method for studying complex cell interactions. In addition, by culturing different cells on nanofiber layers located at different liquid levels, in some experimental designs, one cell may respond to a signal factor secreted by another cell, and the transmission of these signal factors depends on their spatial arrangement. By in the cell culture scaffold that can be stacked up and down, the positions of different cells in co-culture can be interchanged. This flexibility allows researchers to explore the mechanism and dynamics of interactions between different cells. Secondly, different types of cells can be separated in nanofiber layers at different liquid levels to avoid direct contact. This indirect co-culture method allows researchers to easily separate cells and analyze their respective state changes. It is also suitable for other 3D cell cultures such as dynamic culture in the later stage. In addition, the cell culture scaffolds are stacked together and stored in the same culture space, which can make more efficient use of the culture fluid and reduce waste. It can also meet the use of different or the same culture media at the same time. If a single culture unit is abnormal, it can be quickly replaced, thereby reducing the risk of contamination and helping to achieve long-term stable cell culture. It is worth noting that cell culture scaffolds of different sizes can choose nanofiber layers with different pore sizes, different membrane materials, or surface modifications according to experimental requirements to suit a variety of cell types and experimental conditions; of course, when not used as a co-culture experiment, the system can also realize the culture of the same cell type in different well plates, and thus can make synchronous comparisons of the interactions between different cell densities under the same conditions.

[0039] In this embodiment, the spacing between the nanofiber layers on adjacent cell culture supports is 0.9-1.1 mm, preferably 1 mm. By separating different types of cells in nanofiber layers at different liquid levels to avoid direct contact, this indirect co-culture method allows researchers to easily separate cells and analyze their respective state changes, and is also suitable for other 3D cell cultures such as dynamic culture in the future.

[0040] The distance between the top nanofiber layer in the support assembly and the surface of the culture solution is 0.8-1.0 cm, preferably 0.9 cm; the distance between the bottom nanofiber layer in the support assembly and the inner bottom wall of the culture container 1 is 0.1-0.3 mm, preferably 0.2 cm. The nanofiber layer at the bottom of the cell culture support is suspended in the middle of the culture solution, so that the cells can absorb nutrients from both top and bottom, and the cells can penetrate deeper into the nanofiber layer, so that the cell yield is higher and long-term culture can be supported.

[0041] In this embodiment, the height of the cell culture support is between 13.30 mm and 16.00 mm, and is optionally compatible with 12-well cell culture plates, 24-well cell culture plates, and 48-well cell culture plates.

[0042] like Figure 2-Figure 4 As shown, when two cell culture supports of different specifications are specifically used, at least two cell culture supports of different specifications include a first cell culture support 2a and a second cell culture support 2b, the first cell culture support 2a is arranged in the hole 12, the first cell culture support 2a includes a first through hole 20a arranged along its axial direction, the axial end of the first cell culture support 2a is adhered with a first nanofiber layer 3a, and the projection of the first nanofiber layer 3a on the first cell culture support 2a is at least partially located in the cross-section of the first through hole 20a; the second cell culture support 2b is arranged in the first through hole 20a, the second cell culture support 2b includes a second through hole 20b arranged along its axial direction, the axial end of the second cell culture support 2b is adhered with a second nanofiber layer 3b, and the projection of the second nanofiber layer 3b on the second cell culture support 2b is at least partially located in the cross-section of the second through hole 20b; wherein, the first nanofiber layer 3a and the second nanofiber layer 3b are arranged at intervals and suspended at different liquid levels of the culture solution. When in use, different types of cells can be placed in the first nanofiber layer 3a and the second nanofiber layer 3b at different liquid levels to avoid direct contact between different types of cells. This indirect co-culture method allows researchers to easily separate cells and analyze their respective state changes, and will also be suitable for other 3D cell cultures such as dynamic culture in the future.

[0043] One end of the first cell culture support 2a away from the first nanofiber layer 3a radially protrudes outward to form a first carrier 21a, which abuts against the outer surface of the top cover 11, so that the first cell culture support 2a can be conveniently placed on or removed from the culture container 1.

[0044] One end of the second cell culture support 2b away from the second nanofiber layer 3b protrudes radially outward to form a second carrier 21b, and a first support 22a for supporting the second carrier 21b is protruded on the inner wall of the first through hole 20a. The upper surface of the first carrier 21a is coplanar with the upper surface of the second carrier 21b.

[0045] Of course, at least two cell culture supports of different sizes and specifications can also use three or more cell culture supports of different sizes and specifications. When used, these cell culture supports can also be detachably stacked together. When three cell culture supports of different sizes and specifications are specifically used, at least two cell culture supports of different specifications also include a third cell culture support 2c, and the third cell culture support 2a is embedded in the second through hole 20b of the second cell culture support 2b in the same way. Specifically, the third nanofiber layer 3c includes a third through hole 20c that is arranged along its axial direction, and the axial end of the third cell culture support 2c is adhered with the third nanofiber layer 3c, and the projection of the third nanofiber layer 3c on the third cell culture support 2c is at least partially located in the cross section of the third through hole 20c, and the end of the third cell culture support 2c away from the third nanofiber layer 3c protrudes radially outward to form a third carrier 21c, and the inner side wall of the second through hole 20b is convexly provided with a second support 22b for supporting the third carrier 21c. Theoretically, when the number of cell culture supports is more than three, they can be infinitely stacked in the above manner. Of course, considering the limited nutrition of the culture solution in the chamber, in this embodiment, the number of cell culture supports is preferably two or three.

[0046] In this embodiment, the cell culture support is bowl-shaped, and the nanofiber layer is arranged near the small end of the cell culture support. A vent hole 23 is provided on the outer wall of the cell culture support to facilitate the mutual internal and external conduction of the mutually nested cell culture supports and to facilitate easy access to the support.

[0047] In this embodiment, the cell culture scaffold is made of polystyrene, and is integrally formed by means including but not limited to 3D printing, machining, injection molding, etc. In addition, those skilled in the art can change the material of the cell culture scaffold to polyurethane, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, polyethylene terephthalate, epoxy resin, silicone resin, etc. according to the actual needs and processing requirements. These materials are preferably medical grade raw materials, or are easily sterilized by common processes.

[0048] In this embodiment, the nanofiber layer is composited with a plurality of different spinning raw materials by a layer-by-layer alternating spinning technique to obtain a layer-by-layer stacked nanofiber film material, wherein the structure of the nanofiber layer can be any one of a random arrangement, a parallel orientation arrangement, a grid arrangement, and a macroporous structure arrangement. In addition, the nanofiber layer is prepared by an electrostatic spinning process and fixed to the bottom of the cell culture scaffold by a hot pressing process to make it integral.

[0049] The thickness of the nanofiber layer is 0.5-50 μm.

[0050] The diameter of the nanofibers in the nanofiber layer 4 is 50-900 nm, and the components of the nanofibers are polycaprolactone, polylactic acid, collagen, gelatin; other materials that can be used include one or more combinations of polylactic acid-glycolic acid, polyglycolic acid, polyhydroxybutyrate, polyamide, polyacrylonitrile, polyurethane, polystyrene, polyvinyl butyral, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene oxide, chitosan, silk protein, cellulose acetate, etc. Natural raw materials and synthetic biodegradable polymer raw materials are preferred.

[0051] The preparation process of the three-dimensional cell culture device of the present invention is as follows:

[0052] Medical grade polylactic acid (PLA) was dissolved in hexafluoroisopropanol to prepare a 5% PLA solution as spinning solution A; medical grade polycaprolactone (PCL) was dissolved in hexafluoroisopropanol to prepare an 8% PCL solution as spinning solution B.

[0053] Use a jet electrospinning device with two electrospinning devices, which are respectively set on both sides of the roller receiver. Use a syringe to extract spinning solution A and spinning solution B respectively, and place them in the liquid supply pump position of the two electrospinning devices and fix them. Connect a metal spinning nozzle to the front end of the syringe and connect it to the electrostatic positive high voltage.

[0054] A layer of silicone oil paper loading film is spread on the surface of the rotating roller receiver to receive the nanofiber layer.

[0055] The spinning distance from the spinning nozzle to the receiver is set to 18 cm, the liquid supply rate of spinning liquid A is 0.08 mm / min, the positive high voltage is 15 kV, the liquid supply rate of spinning liquid B is 0.12 mm / min, the positive high voltage is 12.5 kV, the receiver speed is set to 40 rpm, and the electrostatic negative high voltage is connected, and the negative high voltage is set to -3.0 kV.

[0056] Under the above conditions, electrospinning is carried out, and the jets of spinning solution A and spinning solution B do not affect each other, and are respectively deposited on one side of the receiver. As the receiver rotates, layer-by-layer alternating spinning is achieved to obtain a layer-by-layer nanofiber structure.

[0057] The obtained fiber membrane layer and silicone oil paper are cut into discs with the same diameter as the circular base, placed on the circular base, and adhered to the circular bottom of the cell culture scaffold through a hot pressing process. The silicone oil paper is then removed, and the remaining nanofiber layer is adhered to the circular bottom of the cell culture scaffold to form a cell culture scaffold with a nanofiber structure.

[0058] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A cell co-culture device, characterized in that: include: A culture container (1), the culture container (1) comprising at least one relatively independent chamber for storing culture fluid, and at least one hole (12) is provided on a top cover (11) of the culture container (1), and at least one hole (12) is arranged in a one-to-one correspondence with at least one chamber; At least one bracket component, at least one bracket component is arranged in a one-to-one correspondence with at least one hole (12), and the bracket component can be detachably placed in the hole (12); wherein, The support assembly comprises at least two cell culture supports of different sizes and specifications, and the at least two cell culture supports of different sizes and specifications can be detachably stacked together. A nanofiber layer is arranged at one end of the cell culture support, and the nanofiber layers on the at least two cell culture supports of different sizes and specifications are arranged at intervals and suspended at different liquid levels of the culture solution.

2. The cell co-culture device according to claim 1, characterized in that: The spacing between adjacent nanofiber layers on the cell culture scaffold is 0.9-1.1 mm.

3. The cell co-culture device according to claim 1, characterized in that: The distance between the nanofiber layer located at the top of the support assembly and the surface of the culture solution is 0.8-1.0 cm, and the distance between the nanofiber layer located at the bottom of the support assembly and the inner bottom wall of the culture container (1) is 0.1-0.3 cm.

4. The cell co-culture device according to claim 1, characterized in that: The at least two cell culture scaffolds of different specifications include: A first cell culture support (2a), the first cell culture support (2a) being arranged in the hole (12), the first cell culture support (2a) comprising a first through hole (20a) penetrating along its axial direction, a first nanofiber layer (3a) being adhered to an axial end of the first cell culture support (2a), and a projection of the first nanofiber layer (3a) on the first cell culture support (2a) being at least partially located within a cross section of the first through hole (20a); A second cell culture support (2b), the second cell culture support (2b) being arranged in the first through hole (20a), the second cell culture support (2b) comprising a second through hole (20b) penetrating along the axial direction thereof, a second nanofiber layer (3b) being adhered to the axial end of the second cell culture support (2b), and a projection of the second nanofiber layer (3b) on the second cell culture support (2b) being at least partially located in the cross section of the second through hole (20b); Wherein, the first nanofiber layer (3a) and the second nanofiber layer (3b) are arranged at intervals and suspended at different liquid levels of the culture liquid.

5. The cell co-culture device according to claim 4, characterized in that: One end of the first cell culture support (2a) away from the first nanofiber layer (3a) protrudes radially outward to form a first carrier (21a), and the first carrier (21a) abuts against the outer surface of the top cover (11).

6. The cell co-culture device according to claim 5, characterized in that: One end of the second cell culture support (2b) away from the second nanofiber layer (3b) protrudes radially outward to form a second carrier (21b), and a first support (22a) for supporting the second carrier (21b) is protruded on the inner side wall of the first through hole (20a).

7. The cell co-culture device according to claim 6, characterized in that: The upper surface of the first carrier (21a) is coplanar with the upper surface of the second carrier (21b).

8. The cell co-culture device according to claim 1, characterized in that: The cell culture support is in a bowl shape, and the nanofiber layer is arranged close to the small opening end of the cell culture support.

9. The cell co-culture device according to claim 1, characterized in that: The cell culture support is integrally formed and an air permeable hole (23) is provided on the outer peripheral wall of the cell culture support.

10. The cell co-culture device according to claim 1, characterized in that: The at least two cell culture supports of different sizes and specifications include three cell culture supports of different sizes and specifications, and the three cell culture supports of different sizes and specifications can be detachably stacked together.

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