Cell culture insert and cell co-culture system comprising same

Abstract

The present invention relates to a cell culture insert including a side wall portion, a bottom surface portion, and a bottom portion, and to a cell co-culture system including the cell culture insert and a well plate. The cell co-culture system is configured such that material exchange between the cell culture insert and a lower well plate is carried out through a side wall opening having a completely open structure, and when the cell co-culture system having a bottom portion without pores is used, cell differentiation effectively occurs, such that the cell-co-culture system can be effectively used for cell culture and cell differentiation research, and the like.

Description

Cell culture insert and cell co-culture system comprising the same

[0001] The present invention relates to a cell culture insert comprising a side wall portion, a bottom portion, and a bottom portion; and a cell co-culture system comprising the cell culture insert and a multiwell plate.

[0002]

[0003] Existing cell co-culture systems utilize cell culture inserts composed of porous membranes, which facilitate the exchange of materials between cells through porous membranes of 0.4, 3.0, or 8.0 μm, and have been utilized to study cell signaling, cell motility, and chemotaxis. In particular, cell co-culture systems have been utilized for cell differentiation research. However, some cells have difficulty differentiating well in porous membranes, limiting research utilizing co-culture technology.

[0004] Understanding the interactions between muscle cells and surrounding tissue cells is crucial for studying the regeneration process of damaged muscle. Myokines secreted by muscle cells during differentiation are known to play a crucial role in activating progenitor cells within the muscle. However, Transwell®, a cell co-culture system with a porous membrane, is not suitable for culturing cells that are influenced by fine nano-topographic structures, such as muscle cell differentiation.

[0005] Accordingly, the inventors of the present invention have completed the present invention by developing a cell culture insert having a bottom made of glass or polystyrene (PS) material without a porous membrane (pore) and a cell co-culture system advantageous for cell differentiation.

[0006]

[0007] An object of the present invention is to provide a cell culture insert comprising: one or more side wall portions; one or more side wall openings; a bottom portion connected to the lower ends of the side wall portions and the side wall openings; and a bottom portion joined to the bottom portions.

[0008] Another object of the present invention is to provide a cell co-culture system comprising the cell culture insert and a multiwell plate capable of accommodating the cell culture insert.

[0009]

[0010] To achieve the above object, the present invention provides a cell culture insert comprising: one or more side wall portions; one or more side wall openings; a bottom portion connected to the lower ends of the side wall portions and the side wall openings; and a bottom portion connected to the bottom portions.

[0011] In one embodiment of the present invention, the side wall opening may have a completely open structure.

[0012] In one embodiment of the present invention, material exchange in the cell culture insert may be accomplished through a side wall opening.

[0013] In one embodiment of the present invention, the upper end of the side wall portion may have a structure that is placed on a cell culture plate.

[0014] In one embodiment of the present invention, the upper end of the side wall portion may be one or more side wall portions connected to each other.

[0015] In one embodiment of the present invention, the upper end of the side wall portion may be one or more side wall portions separated from each other.

[0016] In one embodiment of the present invention, the bottom portion may be an integral part that is joined to the lower surface portion.

[0017] In one embodiment of the present invention, the bottom portion may be a detachable type that can be detached from the bottom portion.

[0018] In one embodiment of the present invention, the bottom portion may have a tweezers groove that can detach the bottom portion.

[0019] In one embodiment of the present invention, the side wall portion and the bottom portion may be made of polylactic acid (PLA) and may be manufactured through extrusion molding or 3D printing.

[0020] In one embodiment of the present invention, the bottom portion may be pore-free, and preferably, the bottom portion may be made of glass or polystyrene (PS).

[0021] In one embodiment of the present invention, the cell culture insert may be for cell co-culture for cell differentiation, but is not limited thereto.

[0022] In addition, the present invention provides a cell co-culture system including the cell culture insert and a multi-well plate capable of accommodating the cell culture insert.

[0023] In one embodiment of the present invention, the multiwell plate may be selected from the group consisting of a 6-well plate, a 12-well plate, a 24-well plate, and a 96-well plate, but is not limited thereto.

[0024]

[0025] The cell culture insert and cell co-culture system according to the present invention have a side wall opening having a completely open structure through which material exchange occurs between the cell culture insert and the lower multi-well plate, and when the cell co-culture system having a bottom without pores is used, cell differentiation is well achieved, so that it can be usefully used for cell culture and cell differentiation research, etc.

[0026]

[0027] Figure 1 illustrates a cell co-culture system according to the present invention.

[0028] Figure 2 is a perspective view and a front view of a cell culture insert according to the present invention.

[0029] Figure 3 shows a plan view (center), a BB cross-section (left), and an enlarged view of part A (right) of a cell culture insert according to the present invention.

[0030] Figure 4 shows a cell culture insert according to the present invention (left), a cell culture insert with a bottom part joined (center), and a cross-section of the cell culture insert cut (right).

[0031] Figure 5 shows a cell co-culture system in which a cell culture insert according to the present invention is applied to a 6-well plate.

[0032] Figure 6 shows a cell culture insert according to the present invention manufactured through 3D printing.

[0033] Figure 7 shows the results of fluorescence staining after cell co-culture using a commercially available Transwell® insert with a porous membrane applied (red: non-specific fluorescence staining result; and white: fluorescence staining result of normal cells).

[0034] Figure 8 compares a method for inducing differentiation of muscle cells using a commercially available Transwell® chamber system with a 0.4 μm porous membrane (left) and a cell co-culture system according to the present invention (right).

[0035] Figure 9 shows the results of comparing the degree of differentiation of muscle cells after co-culturing cells using a commercially available Transwell® chamber system with a 0.4 μm porous membrane (left) and a cell co-culturing system according to the present invention (right).

[0036] Figure 10 shows the results of comparing the degree of differentiation from preadipocytes into adipocytes using BODIPY staining after co-culturing cells using a commercially available Transwell® chamber system with a 0.4 μm porous membrane (left) and a cell co-culture system according to the present invention (right).

[0037]

[0038] Hereinafter, preferred embodiments of the present invention, which can specifically achieve the above objectives, will be described with reference to the attached drawings. In describing the embodiments of the present invention, the same names and symbols will be used for identical components, and additional and redundant explanations thereof will be omitted.

[0039] First, referring to FIGS. 1 to 6, an embodiment of a cell culture insert according to the present invention and a cell co-culture system including the same will be described. Here, FIG. 1 is a schematic diagram showing a cell co-culture system according to the present invention and an actually manufactured cell culture insert, FIGS. 2 to 4 show a cell culture insert according to the present invention, FIG. 5 shows a cell co-culture system in which a cell culture insert according to the present invention is applied to a 6-well plate, and FIG. 6 shows a cell culture insert manufactured through 3D printing.

[0040] Referring to FIG. 1, a cell co-culture system (10) according to the present invention includes a cell culture insert (100); a bottom portion (200) included in the cell culture insert; and a multi-well plate (300) capable of accommodating the cell culture insert.

[0041] Referring to FIG. 2, a cell culture insert (100) according to the present invention includes one or more side wall portions (110), one or more side wall openings (120), a bottom portion (130) in which the lower ends of the side wall portions (110) and the side wall openings (120) are connected; and a bottom portion (200) coupled to the bottom portion (130).

[0042] In one embodiment of the present invention, the side wall opening may have a completely open structure. Accordingly, the bottom portion (130), where the side wall portion and the lower end of the side wall opening are connected, may be positioned below the bottom portion. That is, the bottom portion of the cell culture insert may be coupled above the bottom portion (see the left panel of FIG. 3).

[0043] In one embodiment of the present invention, material exchange in the cell culture insert may occur through a side wall opening. The material may be a substance secreted by cells for intercellular signaling, or a substance contained in a cell culture medium, but is not particularly limited thereto.

[0044] In particular, the gap between the bottom of the cell culture insert and the multiwell plate may be manufactured to be 2 to 5 mm, and preferably 2 to 3 mm. Due to this gap, cells cultured on the bottom of the cell culture insert and cells cultured in the multiwell plate are structured to enable signal transmission between cells through material exchange through the side wall openings.

[0045] In one embodiment of the present invention, the upper end of the side wall portion may have a structure that is mounted on a cell culture plate. In addition, the upper end of the side wall portion may be one or more side wall portions connected to each other (see FIG. 2 or FIG. 4), or one or more side wall portions separated from each other (see FIG. 1).

[0046] In one embodiment of the present invention, the bottom part may be an integral type that is connected to the bottom part, or a detachable type that can be detached from the bottom part, and the detachable bottom part includes a tweezers groove (140) that can detach the bottom part. The tweezers groove is manufactured to facilitate detachment of the bottom part, and can ensure that the bottom part is stably fixed when mounted through the tweezers groove.

[0047] In one embodiment of the present invention, the side wall portion and the bottom portion may be manufactured from an environmentally friendly medical polymer material, preferably polylactic acid (PLA). Furthermore, the side wall portion and the bottom portion may be manufactured through extrusion molding or 3D printing, but are not limited thereto.

[0048] In one embodiment of the present invention, the bottom portion is made of a pore-free material, i.e., unlike existing Transwell® inserts, it is not a porous membrane. Preferably, the bottom portion may be made of pore-free glass or polystyrene (PS).

[0049] In one embodiment of the present invention, the cell culture insert may be for cell co-culture for cell differentiation, but is not limited thereto.

[0050] The multiwell plate included in the cell co-culture system according to the present invention may be selected from the group consisting of a 6-well plate, a 12-well plate, a 24-well plate, and a 96-well plate, but is not limited thereto. The well plate may be made of polystyrene (PS) and may be a commercially available product.

[0051] In particular, the cell culture insert capable of accommodating a 6-well plate is composed of 6 pieces, and the bottom of the cell culture insert can be manufactured to a size that can be combined with the bottom of the cell culture insert, preferably having a diameter of 20 to 24 mm and an area of ​​4.5 to 4.7 cm. 2 It may be. In addition, the cell culture insert that can accommodate a 12-well plate may be composed of 12, and the bottom of the cell culture insert may be manufactured in a size that can be combined with the bottom of the cell culture insert, preferably having a diameter of 10 to 12 mm and an area of ​​0.9 to 1.2 cm. 2 It may be. In addition, the cell culture insert that can accommodate a 24-well plate may be composed of 24 pieces, and the bottom of the cell culture insert may be manufactured to a size that can be combined with the bottom of the cell culture insert, preferably having a diameter of 6 to 6.5 mm and an area of ​​0.3 to 0.4 cm. 2 It could be.

[0052] Referring to Figure 7, it shows that non-specific fluorescent staining occurs when cells are co-cultured using Transwell® inserts with a commercially available porous membrane. More specifically, when cells are co-cultured using Transwell® inserts and fluorescent staining is performed, non-specific fluorescent staining (red) is observed in addition to the fluorescent staining of normal cells (white), confirming that the analysis of cells (top) cultured on existing Transwell® insert membranes has limitations.

[0053] Referring to FIGS. 8 and 9, the cell co-culture system according to the present invention can induce cell differentiation better than the Transwell® chamber system with a 0.4 μm porous membrane applied to the existing commercially available system. More specifically, it is known that the differentiation pattern and shape of muscle cells may vary depending on the topographic characteristics of the cultured plate. When muscle cells were cultured on a Transwell® insert with a 0.4 μm porous membrane applied to the commercially available system and differentiation was induced, it was confirmed that muscle cell differentiation was hardly achieved (see the left panel of FIG. 9). In order to solve this problem, the cell co-culture system according to the present invention was developed, and when muscle cells were cultured using the cell co-culture system with the cell culture insert applied to the present invention and differentiation was induced, it was confirmed that muscle cell differentiation was very well achieved (see the right panel of FIG. 9).

[0054] Referring to Fig. 10, the cell co-culture system according to the present invention did not show non-specific fluorescent staining compared to the Transwell® chamber system to which a commercially available 0.4 μm porous membrane was applied, and it was confirmed that differentiation from preadipocytes into adipocytes was well achieved. More specifically, after cell co-culture using the Transwell® chamber system to which a commercially available 0.4 μm porous membrane was applied, fluorescent staining was performed to confirm the degree of differentiation from preadipocytes into adipocytes. As a result, in the Transwell® chamber system, non-specific fluorescent staining of the membrane made it difficult to accurately analyze the degree of adipocyte differentiation (see the left panel of Fig. 10). However, in the case of using the cell co-culture system according to the present invention, almost no non-specific fluorescent staining was found, and it was confirmed that differentiation of adipocytes was also well achieved (see the right panel of Fig. 10). Accordingly, it was confirmed that the cell co-culture system of the present invention enables more accurate analysis by using a cell culture insert including a bottom without pores, thereby hardly detecting non-specific fluorescent staining.

[0055]

[0056] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0057] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0058]

[0059] [Explanation of symbols]

[0060] 10: Cell co-culture system

[0061] 100: Cell culture insert

[0062] 110: Side wall

[0063] 120: Side wall opening

[0064] 130: Bottom

[0065] 140: Tweezer Home

[0066] 200: Bottom

[0067] 300: Multiwell Plate

Claims

1. One or more side walls; One or more side wall openings; A bottom surface portion connected to the side wall portion and the lower portion of the side wall opening; and A cell culture insert comprising a bottom portion coupled to the above-described lower surface portion.

2. In paragraph 1, A cell culture insert wherein the side wall opening has a completely open structure.

3. In paragraph 1, A cell culture insert wherein material exchange in the cell culture insert occurs through side wall openings.

4. In paragraph 1, A cell culture insert, wherein the upper part of the side wall has a structure that is placed on a cell culture plate.

5. In paragraph 1, A cell culture insert, wherein the upper part of the side wall portion is formed by connecting one or more side walls to each other.

6. In paragraph 1, A cell culture insert, wherein the upper portion of the side wall portion is formed by one or more side walls separated from each other.

7. In paragraph 1, A cell culture insert in which the bottom portion is an integral part connected to the lower surface portion.

8. In paragraph 1, A cell culture insert wherein the bottom portion is a detachable type that can be detached from the bottom surface.

9. In paragraph 8, A cell culture insert having a bottom portion having a tweezers groove for detaching the bottom portion.

10. In paragraph 1, A cell culture insert, wherein the side wall and bottom portion are made of polylactic acid (PLA).

11. In paragraph 1, A cell culture insert, wherein the side wall and bottom portion are manufactured through extrusion molding or 3D printing.

12. In paragraph 1, A cell culture insert wherein the bottom portion has no pores.

13. In paragraph 1, A cell culture insert, wherein the bottom is made of glass or polystyrene (PS).

14. In paragraph 1, The above cell culture insert is a cell culture insert for cell co-culturing for cell differentiation.

15. A cell culture insert according to any one of claims 1 to 14; and A cell co-culture system comprising a multiwell plate capable of accommodating the above cell culture insert.

16. In paragraph 15, A cell co-culture system, wherein the above multiwell plate is selected from the group consisting of a 6-well plate, a 12-well plate, a 24-well plate, and a 96-well plate.

Images