Insertable culture container and kit for three-dimensional cell culture, and three-dimensional cell co-culture method using same

Abstract

The present invention relates to an insertable culture container and a kit for three-dimensional cell culture, and a three-dimensional cell co-culture method using the same, the insertable culture container for three-dimensional cell culture comprising: a cylindrical side wall having open upper and lower portions; at least one hook protruding outward from the upper side of the side wall; and at least one support protruding inward from the lower side of the side wall. The present invention is advantageous in that air required for a three-dimensional cell culture structure can be smoothly supplied since the cell is cultured at a position spaced apart from a bottom surface of the culture container, and an existing culture plate can be used without change due to the culture container configured as an insert type.

Claims

1. An insertable culture container for three-dimensional cell culture comprising: a cylindrical side wall having open upper and lower portions; at least one hook protruding outward from an upper side of the cylindrical side wall; and a plurality of supports protruding inward from a lower side of the cylindrical side wall and provided to support a three-dimensional structure inoculated with cells, wherein the plurality of supports are spaced apart from each other and spaced apart from a center of an inner space surrounded by the cylindrical side wall, wherein distances from the cylindrical side wall to ends of the plurality of supports are 15 to 30% of a diameter of the upper portion, and wherein the open lower portion of the container does not comprise a membrane.

2. The insertable culture container of claim 1, wherein the plurality of supports are located below a central portion of the cylindrical side wall.

3. The insertable culture container of claim 1, wherein the plurality of supports are disposed at equal intervals.

4. The insertable culture container of claim 1, wherein the insertable culture container has a cylindrical shape, in which the upper side of the cylindrical sidewall has a larger cross section than the lower side of the cylindrical sidewall.

5. A kit for three-dimensional cell culture comprising the insertable culture container of claim 1 and a plate into which the insertable culture container is inserted, wherein the plate includes a plurality of wells recessed in one direction; a connecting portion connecting the plurality of wells, wherein the insertable culture container is insertable into the plurality of wells of the plate such that the at least one hook of the culture container is caught on the connecting portion of the plate; and bottom surfaces of the plurality of wells spaced apart from a lower portion of the insertable culture container.

6. The kit of claim 5, wherein a separation distance of the bottom surfaces of the plurality of wells from the lower portion of the insertable culture container is 10 to 40% of depths of the plurality of wells.

7. A three-dimensional cell co-culture method comprising: a preparation step of preparing the kit of claim 5; an inoculation step of inoculating a first cell inside the plurality of wells of the plate; an exchange step of removing a supernatant and at least partially exchanging a culture solution after culturing the first cell; an insertion step of inserting the insertable culture container into the plurality of well of the plate; and a co-culture step of co-culturing cells by locating a three-dimensional structure inoculated with a second cell on the plurality of supports.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a perspective view illustrating an insertable culture container for three-dimensional cell culture of the present invention

(2) FIG. 2 is a cross-sectional view illustrating the insertable culture container for three-dimensional cell culture of the present invention

(3) FIG. 3 is a plan view illustrating the insertable culture container for three-dimensional cell culture of the present invention

(4) FIG. 4 is a perspective view illustrating a cell culture plate of an insertable culture container kit for three-dimensional cell culture of the present invention

(5) FIG. 5 is a perspective view illustrating a state in which an insertable culture container is introduced onto a plate in the insertable culture container kit for three-dimensional cell culture of the present invention

(6) FIG. 6 is a cross-sectional view illustrating a state in which the insertable culture container is introduced onto the plate in the insertable culture container kit for three-dimensional cell culture of the present invention

(7) FIG. 7 is a flowchart sequentially illustrating a three-dimensional cell co-culture method of the present invention

(8) FIG. 8 is a graph illustrating experimental results in which three-dimensional cell culture is performed by the insertable culture container kit for three-dimensional cell culture of the present invention

MODES OF THE INVENTION

(9) Hereinafter, one preferred embodiment of the present invention for an insertable culture container and a kit for three-dimensional cell culture, and a three-dimensional cell co-culture method using the same according to the present invention will be described in detail with reference to the accompanying drawings. The present invention can be better understood by the following examples, and the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of protection, which is limited by the attached claims.

(10) As illustrated in FIGS. 1 to 3, an insertable culture container 100 for three-dimensional cell culture of the present invention may include an upper portion 10, a lower portion 11, a side wall 12, and a support 13.

(11) First, the upper portion 10 means an open upper portion for introducing a three-dimensional structure, and along with the lower portion 11 and the side wall 12, may form a basic structure having a columnar shape which is opened vertically.

(12) The lower portion 11 is formed in an open form so as to co-culture heterogeneous cells with a culture solution.

(13) The ratio of a diameter of the upper portion 10 and a diameter of the lower portion may be 3:1 to 1.2:1, preferably 2:1 to 1.5:1, and more preferably 1.8:1 to 1.6:1, which is effective. In this range, an inclined sidewall that maximizes the interaction between the culture solution and the heterogeneous cells while enabling a three-dimensional structure to be inserted and removed into/from the upper portion, and is optimal for securing a structural bonding stability with the well, is formed.

(14) When the ratio is more than 3:1, the efficiency of circulating and supplying air from the lower portion, co-culturing two or more types of cells, and the like may remarkably deteriorate, and when the ratio is less than 1.2:1, the ease with which the three-dimensional structure is inserted and removed into/from the upper portion may be lowered, or the structural bonding stability with the lower well may deteriorate.

(15) The side wall 12 is configured to extend from the upper portion 10 to the lower portion 11, protects the three-dimensional structure, and forms a base surface for forming the support 13.

(16) Further, the support 13 is formed to protrude inward from the lower side of the side wall 12, and the number of supports 13 is preferably 2 to 5, more preferably 3 to 4, and most preferably 3, which is effective. There are problems in that when the number is less than 2, it is virtually impossible to support a three-dimensional cell structure, and when the number is more than 5, the connection space with the actual lower well is excessively narrow compared to the open space of the lower portion 12 due to the supports, so that the efficiency of circulating and supplying air, co-culturing two or more types of cells, and the like remarkably deteriorates.

(17) The at least one support is characterized by being disposed at equal intervals.

(18) The support is characterized by being located below a central portion of the side wall.

(19) The support 13 is spaced apart from the upper portion 10, and is located at a height which is preferably 0 to 40%, more preferably 5 to 25%, and most preferably 5 to 10% of the side wall 12 from the lower open end 11, which is effective. There is a problem in that when the support 13 is located at a height which is more than 40% of the side wall 12 from the lower open end 11, it is difficult to co-culture a three-dimensional cell structure which is put on the support 13 and other cells contained in the well.

(20) The distance from the side wall 12 to the end of the support 13 is preferably 15 to 30%, more preferably 20 to 25%, and most preferably 22 to 23% of the diameter of the upper portion 10, which is effective. There are problems in that when the distance is less than 15%, it is difficult to stably support a three-dimensional cell structure, and when the distance is more than 30%, the effect of being spaced apart from the lower portion 11 of the support 13 is lowered, that is, cell growth is adversely affected because it is difficult to circulate and supply air smoothly.

(21) The support 13 may extend to the lower portion 11. Since the support 13 is formed along the side wall 12 from the lower portion 11, there is an advantage in that the manufacturing process is simple and economic feasibility is high because injection molding as one body is facilitated. In addition, the support 13 is formed in a columnar form along the side wall 12, and thus has higher stability and durability.

(22) Furthermore, the hook 14 may be formed protruding outward from the upper side of the side wall 12.

(23) The upper portion 10, the lower portion 11, the side wall 12, the support 13, and the hook 14 may be formed of any material applied to cell culture, such as polystyrene (PS), polypropylene (PP), polyethylene (PE), polycarbonate (PC), and glass, and the side wall 12 and the support 13, which are formed of the same material, are suitable for a durable and integral side wall which is easily injection-moldable.

(24) Next, as illustrated in FIGS. 4 to 6, the insertable culture container kit for three-dimensional cell culture of the present invention may include an insertable culture container 100 for three-dimensional cell culture and a cell culture plate 200, that is, a plate 200 and an insertable culture container 100.

(25) Here, the plate 200 may include: at least one well 20 recessed in one direction; and a connecting portion 21 connecting the wells 20.

(26) As illustrated in FIGS. 5 and 6, an insertable culture container 100 may be introduced into the well 20 of the plate 200. When introduced, the hook 14 is caught on the connecting portion 21, and thus serves to support the insertable culture container 100.

(27) A bottom surface of the well 20 is spaced apart from the lower open end 11, and a separation distance of the bottom surface of the well from the lower open end 11 is preferably 10 to 40%, more preferably 15 to 25%, and most preferably 15 to 20%, of a depth of the well, which is effective. There are problems in that when the distance is less than 10%, it is difficult to smoothly circulate and supply air to cells in the lower portion, and when the distance is more than 40%, it is difficult to co-culture a three-dimensional cell structure which is put on the support 13 and other cells contained in the well.

(28) The number of wells 20 formed on the plate 200 is not limited, and the plate 200 may be configured to have various numbers of wells such as 16 wells and 96 wells.

(29) The three-dimensional cell co-culture method of the present invention may include: a preparation step S20; an inoculation step S21, an exchange step S22; an insertion step 23; and a co-culture step S24. This is a method which can effectively co-culture a first cell and a second cell using the insertable culture container structure for three-dimensional cell culture of the present invention.

(30) First, the preparation step S20 is a step of preparing a kit for three-dimensional cell culture, which includes an insertable culture container according to the present invention and a plate into which the culture container is introduced.

(31) The inoculation step S21 is a step of inoculating the first cell inside the well. That is, the first cell and the culture solution may be inoculated inside the well.

(32) Here, the first cell may be any cell that can be co-cultured with the second cell, but may be precisely an adherent cell, and may be at least one such as a mesenchymal cell or mesenchymal stem cell, a pre-adipocyte, an adipocyte, a smooth muscle cell (SMC), or a macrophage.

(33) The exchange step S22 is a step of removing a supernatant and at least partially exchanging a culture solution after culturing the first cell. Co-culture efficiency may be increased by removing a supernatant generated by culturing the first cell from the culture solution in the well and exchanging a part or all of the culture solution.

(34) The insertion step S23 is a step of inserting the insertable culture container into the well of the plate.

(35) The co-culture step S24 is a step of co-culturing cells by locating a three-dimensional structure inoculated with a second cell on the support of the culture container. Here, the three-dimensional structure is preferably in the form of a bead or support.

(36) Here, the second cell may be a culturable eukaryotic cell, and more specifically, at least one of an epithelial cell, a fibroblast, an osteoblast, a chondrocyte, a hepatocyte, an umbilical cord blood cell, an umbilical cord mesenchymal stem cell (UCMSC), an adipose-derived mesenchymal stem cell (AMSC), or a bone marrow-derived mesenchymal stem cell (BMMSC).

(37) Finally, the exchange step S24 is a step of exchanging the culture solution at an interval of 1 to 5 days after the co-culture step S24. More preferably, it is effective to exchange the culture solution at an interval of 2 to 3 days. This is to ensure the continuous growth of cells or tissues by supplying nutrient ingredients required for cell culture by and removing cell survival inhibitors produced during the culture.

Example 1) Manufacture and Sterilization of Culture Container

(38) As an example of the present development, a culture container was manufactured using a 3D printing technique in which layers are laminated one by one by an arbitrary solid freeform fabrication (SFF) method using a rapid prototyping (RP) device, and sterilization was performed using an electron beam accelerator.

Example 2) First Cell Culture

(39) As an experiment for measuring the effect of three-dimensional cell culture using the insertable culture container kit for three-dimensional cell culture of the present invention, an experiment was performed under the following conditions. Cells: 3T3-L1 (pre-adipocyte)(2.45×10.sup.6 cells/5 mL/Tube), adipose-derived mesenchymal stem cell (ADMSC) Culture period: 7 day culture Cell counting: cell counting on days 0, 3, and 7 Alginate mixture: 1.3% concentration

(40) The first cell was 3T3-L1(pre-adipocyte), and specifically, cryopreserved first cells were thawed in a constant-temperature water bath at 37° C. and transferred to a 15-ml tube, and then a basal medium (DMEM+1% antibiotics) was added thereto and centrifuged at 1,500 RPM for 5 minutes to remove a supernatant, and after cryopreservation solution ingredients were completely removed by adding the basal medium again and repeating the previous method, cells were inoculated along with a medium including DMEM+1% antibiotics+10% FBS into a 100-mm culture dish and cultured in a 5% carbon dioxide incubator at 37° C. and when the cells were sufficiently proliferated after 4 to 5 days, the cells were inoculated at a concentration of 1×10.sup.3 per well of the culture container by treating the cells with 0.05% trypsin-EDTA.

Example 3) Manufacture of Bead Including Second Cell

(41) The second cell was an adipose-derived mesenchymal stem cell (ADMSC), and a three-dimensional structure inoculated with the second cell was manufactured in the form of a bead.

(42) The bead was manufactured by a method including: a melting step of introducing a culture solution, alginate, and gelatin into a tube and melting the resulting solution at 65° C.; a stirring step of lowering the temperature of the tube to 37° C., introducing the second cell (2.45×10.sup.6 cells/5 mL/Tube) into the tube, and stirring the tube at 500 RPM for 2 minutes; and a manufacturing step of making the mixture in the tube in the form of a bead and manufacturing the bead by putting the mixture in a calcium chloride solution for cross-linking.

(43) After the stirring, bubbles were removed at 1500 RPM through centrifugation. In the manufacturing step, the concentration of the calcium chloride solution was set at 5%.

(44) A three-dimensional structure in the form of a bead inoculated with the second cell was manufactured by washing the cross-linked bead with phosphate buffered saline (PBS) and putting the bead into a culture solution containing 10% fetal bovine serum and culturing in a 5% carbon dioxide incubator at 37° C.

Example 4) Manufacture of Support and Sterilization Process

(45) A support was manufactured using polycaprolactone. First, after polycaprolactone was introduced into a tube and a pressure of 650 to 730 kPa was applied thereto, a three-dimensional support was manufactured using the suction function of an air controller, and a sterilization process was performed using electron beam acceleration.

Example 5) Three-Dimensional Cell Culture Using Support Including Second Cell

(46) For a three-dimensional cell culture using a support including a second cell, after 25 μl of the second cells was each inoculated onto 4 drop supports, a three-dimensional structure in the form of a support inoculated with the second cells was manufactured by culturing the second cell for 1 hour, adding 1 ml of a medium thereto, and culturing the second cells under 37° C. and 5% carbon dioxide conditions.

(47) As illustrated in FIG. 8, it can be seen that for the three-dimensional cell culture performed using the insertable culture container of the present invention, as the culture time elapses, the number of cells are is not decreased, but increased, unlike a second-dimensional cell culture using a general culture container. That is, it could be confirmed in an actual experiment that the three-dimensional cell culture could be performed more sustainably with the culture container of the present invention.

(48) While preferred embodiments of the present invention have been described, the present invention is susceptible to various changes, modifications and equivalents. It is clear that the embodiments of the present invention can be appropriately modified and equally applied. Therefore, the aforementioned description should be construed as not limiting the scope of the present invention defined by the following claims.

EXPLANATION OF REFERENCE NUMERALS AND SYMBOLS

(49) 100: Insertable culture container for three-dimensional cell culture 10: Upper portion 11: Lower portion 12: Side wall 13: Support 14: Hook 200: Cell culture plate 20: Well 21: Connecting portion

INDUSTRIAL APPLICABILITY

(50) The present invention relates to an insertable culture container and a kit for three-dimensional cell culture, and a three-dimensional cell co-culture method using the same, and is industrially applicable.