WO2013047974A1 - Three dimensional cell culturing apparatus and three dimensional culturing method for cells using same - Google Patents

Abstract

The present invention relates to a three dimensional cell culturing apparatus and to a three dimensional culturing method for cells using same. In the three dimensional cell culturing device and the three dimensional cell culturing method using same of the present invention, cells are subjected to basic culturing in a culture fluid comprising nanoparticles, magnetic nanoparticles are introduced into the cells or the cell membranes, and magnetism is applied to the cells into which the magnetic nanoparticles have been introduced, such that spheroids can be formed in a state of suspension within culture fluids without any separate surface processing.

Description

Three-Dimensional Cell Culture Tool and Three-Dimensional Culture Method

The present invention relates to a three-dimensional cell culture apparatus and a three-dimensional culture method of cells using the same.

Today, in the development of drugs, cells cultured by cell culture techniques are used for the pharmacological activity evaluation of drugs and the simulator of toxicity tests.

However, since cells cultured by conventional cell culture techniques are monolayer cells spread in two-dimensional directions, three-dimensional tissues equivalent to those in vivo cannot be constructed, and the specific functions of the cells in the body can be maintained for a long time. Therefore, there is a problem that the accuracy of the simulation is not guaranteed.

Recently, in order to solve such a problem, the cultivation of spheroid, which is a three-dimensional tissue having a function equivalent to in vivo, has attracted attention. In particular, with the development of stem cell technology, various methods have been attempted for three-dimensional culturing embryonic stem cells and applying them to studies of various differentiation mechanisms.

For example, culturing spheroids by seeding a plurality of single cells in a funnel having a bottom surface and agglomerating and dividing the single cells on the bottom surface (Japanese Patent Laid-Open No. 6-327462 (paragraph 0016) 1)) or culturing spheroids on a honeycomb structure obtained by condensing water on the surface of the cast liquid of a predetermined polymer mixture and evaporating the microdroplets produced by the condensation (Japanese Patent Laid-Open No. 2002-335949). Publication p3 (FIG. 1)).

However, according to Japanese Unexamined Patent Application Publication No. 6-327462, there is no spheroid site produced by culture, and it is difficult to fix the spheroid to the cell culture construct or the cell culture vessel. Therefore, during the medium exchange, there was a problem that the spheroids disappeared with the medium, or the medium exchange work became very complicated, and it was difficult to maintain a good culture environment. In addition, since a plurality of cells aggregate to form spheroids, even when attempting to simulate the dynamics of cancer cells, the characteristics of the tumor and the spheroids formed by dividing from one cancer cell in vivo are strictly different. There was a problem of poor precision.

In addition, there is a problem that it is difficult to precisely control the size of the honeycomb structure, and it is difficult to make the structure a shape other than the honeycomb structure having a circular shape in the planar direction. In addition, it takes a considerable time to manufacture the honeycomb structure, there is a problem that it is expensive and not suitable for mass production.

Another method of three-dimensional culture of cells is a hanging drop culture.

Figure 2 is a conceptual diagram showing a general method of three-dimensional culture of cells through a hanging drop culture (hanging drop culture). In the method of three-dimensional culture of cells through a hanging drop culture, first, the number of collected cells is counted as shown in FIG. 2 (a), and then about 300 to 500 cells are collected using a pipette or the like. The cover 11 of the 100 is dispensed in the form of drops and inverted to incubate. Then, as shown in Figure 1 (b), the hanging cells are collected at the end of the drop by gravity to form an embryonic cell mass, which is generally referred to as embryoid body.

Figure 3 A, B is another form of this cultivation method developed by 3d biomatrix, using a cover plate with a predetermined size of holes, the cell culture solution is accommodated in the cover plate, the cells from the hole by gravity Represents the form of three-dimensional culture.

However, in the case of the hanging drop culture, a cell is dispensed on the cover of the culture vessel by using a pipette or the like, and thus, physical damage to the cell occurs during the transfer process or serious contamination due to microbial contamination. There was a fear of problems.

It is an object of the present invention to provide a three-dimensional cell culture tool for three-dimensional culture of cells that can solve the problems of the conventional three-dimensional culture technology of such cells.

Another object of the present invention is to provide a method for three-dimensional culture of cells using the three-dimensional cell culture tool.

The present invention to solve the above problems

A culture plate unit including a well for culturing cells;

A cover part covering the culture plate part; And

It provides a three-dimensional cell culture tool comprising a; protruding portion extending from the surface in contact with the culture plate portion of the cover portion into the well of the culture plate portion.

In the present invention, the protrusion is characterized in that the magnetic.

In the present invention, a method of causing the protrusion to show magnetic properties is not particularly limited, and in one embodiment of the present invention, the protrusion may be made of a permanent magnet material.

In the present invention, the protrusion includes an electromagnet, the control unit for controlling the size and supply time of the current supplied to the electromagnet and a power supply for supplying power to the electromagnet for a predetermined time by the control unit It is done.

In the present invention, the length of the protrusion is characterized in that 1/20 to 9/10 of the depth of the well.

In the present invention, the culture plate portion comprises a plurality of wells for culturing the cells, wherein the protrusions are characterized in that it is formed to include one per well for culturing the cells.

In the present invention, the protrusion is characterized in that it is formed to include a plurality per well for culturing the cells.

In the present invention, the end of the protruding portion is not particularly limited in shape, but is preferably formed sharply in order to concentrate the magnetism and efficiently culture the cells in three dimensions.

The present invention also provides

Basal culture of cells desired to be cultured in a spheroid form in a culture solution containing nanoparticles;

Receiving the cultured cells and the culture solution in the wells of the culture plate;

Covering the surface of the culture plate part with a cover part including the protrusion so that the protrusion is located inside the well; And

It provides a three-dimensional culture method of cells using the three-dimensional cell culture tool of the present invention comprising the step of three-dimensional culture of the cells by forming the spheroid by the magnetism of the protrusion.

In the present invention, the cells which are desired to be cultured in the spheroid form are first cultured in a culture solution containing nanoparticles, and are aggregated by external magnetism to be cultured in the spheroid form. Cells cultured basally in a culture medium containing nanoparticles contain the nanoparticles inside the cells, nanoparticles adhere to the cell membrane, or magnetic nanoparticles adhere to the cells by other mechanisms, thereby moving the cells by external magnetism. Will be induced.

In the present invention, the nanoparticles typically refer to very small particles having a diameter ranging from 1 nanometer to a few hundred micrometers. Dyes and pigments because of their small size; Aesthetic or functional coatings; Tools for biological research, medical imaging and treatment; Magnetic recording media; Quantum dots; And even and uniform nanoscale semiconductors.

Magnetic nanoparticles have been proposed to be used in various biomedical applications, which include magnetic resonance imaging, hyperthermic treatment of malignant cells, and drug delivery. Recently, the nanoparticles were grown by culturing the cells in a mixture of nanoparticles. Techniques for introducing into cells and for immobilizing cells into which magnetic particles have been introduced have been developed.

In the present invention, the magnetic nanoparticles are characterized in that one or more selected from the group consisting of magnetite, Fe ₃ O ₄ , γ-Fe ₂ O ₃ , manganese ferrite, cobalt ferrite and nickel ferrite.

In the present invention, the step of receiving the cultured cells and the culture solution in the well of the culture plate portion, characterized in that the cultured cells and culture medium is accommodated so as not to contact the protrusion.

In the present invention, the step of receiving the culture solution containing the cultured cells in the well of the culture plate portion is characterized in that the end of the protrusion is accommodated so as to contact the cultured cells and the culture solution. In this case, by appropriately treating the surface of the protrusion, it is possible to separate the three-dimensional cultured cells around the protrusion from the protrusion.

In the present invention, in the step of basal culture of the cells to be cultured in the spheroid form in the culture medium containing the nanoparticles are a plurality of cells to be cultured in the spheroid form, the plurality of types of spheroid Cells desired to be cultured in the form is mixed in a culture solution containing the nanoparticles, characterized in that the basal culture at the same time.

Three-dimensional culture method of the cells using the three-dimensional culture tool of the present invention, further comprising the step of basal culture of each of the first cell, the second cell to be cultured in the spheroid form separately in each culture solution containing nanoparticles;

Receiving a culture solution containing the cultured first cells in a well of a culture plate;

Covering the surface of the culture plate with a cover including the protrusion so that the protrusion is positioned inside the well; And

Culturing the first cells by forming a spheroid by the magnetism of the protrusions;

Supplying a culture solution including the cultured second cells to a well of the first cell spheroid-formed culture plate;

And culturing the first cell spheroid as a core part by the magnetism of the protrusion, and forming the spheroid forming the shell part with the second cell.

Three-dimensional culture method of the cells using the three-dimensional culture tool of the present invention, the step of culturing a plurality of types of cells to be cultured in the spheroid form separately in each culture medium containing nanoparticles;

Receiving a culture solution including the cultured plurality of cells in a well of a separate culture plate;

Covering the surface of the culture plate with a cover including the protrusion so that the protrusion is positioned inside the well;

Culturing the plurality of cell types while forming primary spheroids by magnetism of the protrusions;

Mixing the spheroids of each of the plurality of cultured cells; And

And a step of culturing the primary spheroid of the mixed plural types of cells to form a secondary spheroid while being aggregated by the magnetism of the protrusion.

Hereinafter, the present invention will be described in more detail.

Figure 4 shows a schematic diagram of the three-dimensional cell culture tool according to the present invention.

As shown in FIG. 4, the three-dimensional cell culture apparatus according to the present invention includes a culture plate part 10 including a plurality of wells 30 for culturing cells; A cover part 20 covering the culture plate part; And one or more protrusions 50 extending from the surface in contact with the culture plate of the cover part toward the inside of the well of the culture plate.

In the three-dimensional cell culture material of the present invention, the protrusion is characterized in that the magnetic, the method for this is not limited. Specifically, the protrusion may be made of a permanent magnet material, or may be made of an electromagnet material.

4 illustrates a case in which the entire protrusion is made of a permanent magnet material (51) and a case in which the permanent magnet is mounted at the end (52).

In the case of Figure 5 shows a schematic diagram when the protrusion is made of an electromagnet material. As shown in FIG. 5, when the three-dimensional cell culture tool of the present invention includes an electromagnet protrusion, a culture plate 110, a plurality of wells 130 included in the culture plate, and a cover for covering the culture plate. 120 and an electromagnet protrusion extending from the surface in contact with the culture plate of the cover portion into each well of the culture plate, and connected to each tip to adjust the magnetism of the electromagnet material. The external power supply unit 160 and the control unit 170 are included. In the present invention, whether the power applied to the external power source 160 is applied by the control unit 170, the intensity of the application, the application time, etc. are adjusted, and accordingly the magnetic force generated in the protrusion of the electromagnet material can be adjusted. Will be. Thereby, various forms of cell culture are possible. That is, it is possible to culture the cells by various methods such as periodically applying magnetism in the process of culturing the cells, or applying the magnetism only until the cells aggregate, and then not applying the magnetism.

The overall shape and the well shape of the culture plate 10 are not particularly limited. Specifically, not only round bottom flasks, but also 6-well, 96-well plates and the like are generally used in the art for culturing cells. It can select suitably according to the culture purpose. As a preferred embodiment of the present invention, a case in which the culture plate has a multiwell shape is shown in FIG. 6, and a case in which the culture plate has a circular plate shape is shown in FIG. 7.

In the three-dimensional cell culture material of the present invention, the shape and number of the tip may be appropriately selected by the capacity of the well for culturing the cells, and is not particularly limited, and the culture plate 10 as shown in FIG. ) Includes a plurality of wells 30 for cell culture, the protrusions may be formed to include one per well 30 for culturing the cells.

In addition, it is possible to include a plurality of protrusions in the well for culturing the cells as shown in FIG. As such, when several tips are installed in a well for culturing a single cell, spheroids of the same trait may be simultaneously formed in each portion where a protrusion is located in the well for culturing a cell, and then three-dimensional. Further growth of the cultured cells is also effective when the culture medium is replaced or separated at once.

In the three-dimensional cell culture material of the present invention, the length of the protrusion is not particularly limited, and is preferably 1/20 to 9/10 of the depth of the well. If the length of the protrusion is too long compared to the depth of the well, the number of cells in contact with the protrusion increases and the efficiency of the three-dimensional culture decreases. If the length of the protrusion is too short compared to the depth of the well, the magnetic influence range is small and the three-dimensional culture. The efficiency of will be reduced.

In the present invention, the tip is capable of three-dimensional culturing of the cells even without contact with the cell culture solution in the well, and three-dimensional culturing of the cells even in the contacted state. When culturing the cells in contact with the cell is preferable because the magnetic acts more directly, it is preferable to perform appropriate surface treatment on the surface of the protrusions because the cells adhere to the surface of the protrusions. At this time, the surface of the protrusion may be coated with a polymer material (a high water-repellent material such as PEG) that interferes with cell adhesion, or by overlaying a material that prevents direct contact with the cell.

8 shows a three-dimensional cell culture method using a three-dimensional cell culture tool according to the present invention. Three-dimensional cell culture method using the three-dimensional cell culture tool of the present invention comprises the steps of i) basal culture of the cells to be cultured in the spheroid form in the culture medium containing nanoparticles; ii) receiving a culture solution containing the cultured cells in a well of a culture plate; iii) covering the surface of the culture plate with a cover including the protrusion so that the protrusion is located inside the well; iv) culturing the cells three-dimensionally by forming the spheroids by the magnetism of the protrusions; It is configured to include.

In the three-dimensional cell culture method of the present invention, the cell is desired to be cultured in spheroid form in the culture medium containing the nanoparticles.

As such, the magnetic nanoparticles may be introduced into the cells by basal culture of the cells in the culture medium containing the nanoparticles, or attached to the cell membranes of the cells by surface modification of the nanoparticles.

A method of introducing magnetic nanoparticles into a cell may be a method generally used by those skilled in the art. The magnetic nanoparticles may be included in the cell culture medium, preferably at a concentration of 10 pg to 1000 pg / cell, The magnetic nanoparticles are introduced into the cells by culturing the cells in the included medium.

In the present invention, as the magnetic nanoparticles, superparamagnetic or ferromagnetic iron oxide nanoparticles are used. Preferably, the oxides included in the nanoparticles are ferro- or ferrimagnetic compounds such as magnetite, Fe ₃ O ₄ , γ-Fe ₂ O ₃ , manganese ferrite, cobalt ferrite and nickel Ferrite. More preferably, the iron-oxide contained in the superparamagnetic nanoparticles used in the present invention is Fe ₃ O ₄ or γ-Fe ₂ O ₃ , and most preferably Fe ₃ O ₄ .

Then, in consideration of the size of the well for culturing the cells of the three-dimensional cell culture tool of the present invention and the desired spheroid size for adjusting the volume and cell concentration of the culture medium containing the basal culture cells for culturing the cells It is accommodated in a well and covered with the cover portion so that the protrusion formed on the surface of the cover portion is located inside the well of the culture plate.

By culturing the cells in this way, the cells containing the magnetic nanoparticles are gathered close to the tip by the magnetism of the protrusion of the cover part, and as a result, three-dimensional cell culture in which the cells aggregate with each other is possible.

In later stages, the cells once aggregated can continue to be cultured while increasing the number of cells in the three-dimensional cell cultured spheroid, thereby further growing the size of the spheroid.

       In the present invention, it is possible to control the cell type in the basal culture, or to culture the cells in various forms in the spheroid formation step after basal culture.

       In the present invention, a possible spheroid production method is schematically shown in FIG.

Specifically, A) a method of randomly mixed spheroid form, ie, using a plurality of cell types in basal culture, forming spheroids by random mixing while agglomerating the plurality of types of cells, B) a core formed by sequential cell introduction. Core-shell spheroids, i.e., after forming a spheroid with a first cell, a second cell forms a shell part using the first cell spheroid as a core part, C) fusion between spheroids fusion, that is, it is possible to form various types of spheroids such as forming spheroids based on the first cell spheroid and the second cell spheroid. 20 schematically shows the cases of A), B) and C), respectively.

In the three-dimensional cell culture apparatus of the present invention and the three-dimensional cell culture method using the same, the magnetic nanoparticles are introduced into the cells, and the cells are inoculated with the cells by culturing the magnetic force using the protrusions representing the magnetic, thereby inoculating the cells on the cover part. Even without a separate treatment such as the like, the cells have the effect of forming a spheroid in a suspended state in the basal culture.

Figure 1 shows the shape of the plate bottom used for three-dimensional culture of conventional cells.

2 and 3 show the process and apparatus of the conventional culture method used for three-dimensional culture of conventional cells.

Figure 4 shows a cross-sectional view of a three-dimensional cell culture apparatus according to an embodiment of the present invention.

5 and 6 show a three-dimensional cell culture apparatus using an electromagnet according to another embodiment of the present invention.

Figure 7 shows a cross-sectional view of a three-dimensional cell culture apparatus according to another embodiment of the present invention.

Figure 8 shows a method of three-dimensional culture of cells by the three-dimensional cell culture method of the present invention.

Figure 9 shows a cover picture of the three-dimensional cell culture apparatus prepared according to an embodiment of the present invention.

10 to 12 show the results of culturing the cells using the three-dimensional cell culture tool of the present invention, introducing the fluorescent material into the cells and observed with a fluorescence microscope

Figure 13 shows the results of measuring the viability of cells cultured using the three-dimensional cell culture tool of the present invention.

FIG. 14 is a diagram illustrating a state of each cell by randomly extracting three-dimensional cultured cells formed in a cell culture tool in a case where the cell culture well includes a plurality of protrusions in one embodiment of the present invention.

Figure 15 shows the results of measuring the distribution of proteins involved in the skeleton within the cells in the case of cell culture using the three-dimensional culture tool of one embodiment of the present invention and in the two-dimensional culture comparative example.

Figure 16 shows the response of each cell to toxic substances in the case of culturing cells using the three-dimensional culture tool of one embodiment of the present invention and in the two-dimensional culture comparative example.

17 to 19 show the results of measuring the process of forming a fusion spheroid using the three-dimensional culture tool of an embodiment of the present invention.

Fig. 20 schematically shows a possible spheroid production method in the present invention.

Hereinafter, the present invention will be described in detail by the following examples. However, the present invention is not limited by the following examples.

<Example 1>

<Example 1-1> Preparation of a three-dimensional cell culture device comprising one protrusion per cell culture well

A 96-well plate was used as the culture plate, a tip-shaped protrusion was made of NdFeB, which is a permanent magnet material, and then attached to a cover of the 96-well plate to prepare a three-dimensional cell culture device. The cover part of the prepared cell culture device is shown in FIG. 9.

Example 1-2 Basic Culture

HeLa cells as cell lines and Fe as magnetic nanoparticles₃O₄end The cells were cultivated in the medium contained so as to introduce 800 pg of particles into the cells.

Example 1-3 Three-Dimensional Cell Culture

800 pg cells cultured in Example 1-2 were cultured to contain 500 cells per well of each of the three-dimensional cell culture apparatus prepared in Example 1. Cells were introduced with a substance that stains the nucleus of the cell (Hoechst) for easy observation.

Covering the culture plate with the cover part shown in FIG. 9 and incubating for 5 minutes while measuring the three-dimensional aggregation process of the cells with time changes with a fluorescence microscope over time, the results are shown in FIG.

As shown in FIG. 10, after 5 minutes of covering with the cover including the tip-shaped protrusion, the cells aggregated and three-dimensional culture.

Example 1-4 Three-Dimensional Cell Culture According to Culture Concentration

Per well of the culture plate prepared in Example 1-1, the cells cultured in each of the basal cultures in Example 1-2 were cultured to contain 125 to 1000 cells, respectively, and after 5 minutes, z under a confocal microscope After reconstructing the 3D image after -axis scanning, the result of confirming whether the cells were cultured in 3D and the result of confirming in 3D images are shown in FIG. 11.

As shown in Figure 11 it can be seen that as the number of cells initially cultured per well increases the size of the cells themselves cultured in three dimensions.

Example 1-5 3D Cell Culture According to the Concentration of Magnetic Nanoparticles Used in Basic Culture

In Example 1-2, the HeLa cells were basally cultured while changing the concentration of magnetic nanoparticles introduced into the cells during basal culture in the range of 200-800 pg / cell, and the culture plate prepared in Example 1-1 The wells were moved to 1000 cells / well, the covers were mounted, and the cells were incubated in three dimensions for 5 minutes.

In Figure 12 it can be seen the degree of cell culture for each concentration. Depending on the concentration of the magnetic nanoparticles per well in the culture plate it can be seen that the difference in the size of the spheroid, which is a three-dimensional cells produced.

Experimental Example 1 Confirmation of Viability of Three-Dimensional Cultured Cells

The cells cultured in 3D in Example 1-5 were subsequently cultured for 5 days, followed by fluorescence observation using the Live / DEAD viability kit, and the results are shown in FIG. 13.

In case of using Live / DEAD viability kit, after mixing the cells with Calcein AM (2 μM) and EthD-1 soultion (4 μM), the living cells are green and the dead cells are red fluorescence. As shown in FIG. 13, cell death was not observed until day 5, and it was confirmed that the cells proliferated in three dimensions.

Example 2 Cell Culture Wells Contain Multiple Projections

Example 2-1 Preparation of 3D Cell Culture Apparatus

In the case of including a plurality of protrusions in the well for culturing the cells, in order to check whether the three-dimensional cell culture, using a circular plate as a culture plate, and attaching a plurality of tip-shaped magnets at regular intervals on the cover of the circular plate To prepare a three-dimensional cell culture device of the form as shown in FIG.

Example 2-2 3D Cell Culture

800 pg of the cells cultured in Example 1-2 were cultured to include 500 cells per well of each of the culture plates prepared in Example 2-1.

After introducing the cells into which the magnetic particles prepared in Example 2-1 were introduced into the prepared three-dimensional cell culture device, the cover was mounted to allow the cells to be cultured in three dimensions, and the cultured three-dimensional cultured cells were randomized. The state of each cell was extracted as shown in FIG.

When one well includes a plurality of protrusions, it can be seen that several spheroids having the same biological properties and states formed in three dimensions at the position where the protrusions are formed can be obtained at once.

Comparative Example 2D Culture

HeLa cells and mesenchymal stem cells were suspended in a 96 well plate with culture medium, and then allowed to settle for a certain period of time so that the cells settled at the bottom of the plate, and the cells were cultured by a general culture method in an incubator.

Experimental Example 1 Actin Staining and Confocal Microscope Comparison

After culturing two kinds of HeLa and mesenchymal stem cells by 3D spheroid by the method of Example 1, comparing the protein distribution in the cultured spheroid and the protein distribution represented by the 2D cultured cells of the comparative example Actin was stained with Alexa 488-conjugated phalloidin for comparison and observed under a confocal microscope. The results are shown in FIG. 15.

As shown in FIG. 15, the three-dimensional cell cultured spheroid of Example 1 and the two-dimensional cultured cells of the comparative example have different protein distributions, and in the three-dimensional cell cultured spheroid of Example 1, between cells Organic influences tend to shift the distribution of proteins outward with the organic binding of actin.

Experimental Example 2 Comparison of Cytotoxicity to Staurosporine

After treating the 3D cell cultured spheroid of Example 1 and 500 uM of staurosporine, which is a toxic substance, to the 2D cultured cells of the comparative example for 6 hours, viability was performed using the Live / DEAD viability kit. Was confirmed by fluorescence observation and the results are shown in FIG. 16.

Red fluorescence in FIG. 16 means dead cells. As shown in Figure 16 in the case of the three-dimensional cell cultured spheroid of Example 1 of the present invention it can be seen that the response to the toxic drug appears from the outside of the spheroid, which is characterized by the characteristics of the three-dimensional cultured cells Shows.

Example 3-1 Fusion Spheroid Formation Between Spheroids

After two cultures with primary spheroids using mesenchymal stem cells, the two cultured stem cell spheroids were mixed and cultured with a secondary spheroid in the cell culture apparatus prepared in Example 1 above.

The process of forming the secondary spheroid according to the culture time is shown in FIG. 17. The cells forming one of the two primary spheroids were shown in red by lipid membrane staining, and the cells forming the other spheroid were shown in blue by nuclear staining. In FIG. 17, it can be seen that over time, two primary spheroids are fused to form a new secondary fusion spheroid.

Example 3-2 Core-Shell Type Spheroid Formation

After basal culture of fluorescent protein-containing HEK-293 cells, the spheroid culture was carried out first, and the cultured spheroids were spheroid cultured in a medium containing HeLa cells with GFP green color, and then core-shell type spores. Cultured with Lloyd, the results are shown in FIG. In Figure 18 it can be seen that the spheroid of the core shell structure is formed.

Example 3-3 Random Mixed Type Spheroid Formation

After basal culture of HeLa cells with GFP fluorescence and HEK-293 cells with Red fluorescent protein, respectively, they were separated and mixed, and spheroids were formed. The results are shown in FIG. 19. In Figure 19 it can be seen that the mixed cells form a spheroid.

Claims (16)

A culture plate unit including a well for culturing cells; A cover part covering the culture plate part; And And a protrusion extending from the surface in contact with the culture plate portion of the cover portion toward the inside of the well of the culture plate portion. The method of claim 1, Three-dimensional cell culture material, characterized in that the protrusions show a magnetic. The method of claim 2, The protrusion is a three-dimensional cell culture, characterized in that it comprises a permanent magnet. The method of claim 2, Three-dimensional cell culture material, characterized in that the protrusion comprises an electromagnet. The method of claim 4, wherein And a power supply unit for supplying power to the electromagnet for a time determined by the controller and a control unit for controlling the magnitude and supply time of the current supplied to the electromagnet. The method of claim 1, The length of the protrusion is three-dimensional cell culture, characterized in that 1/20 to 9/10 of the depth of the well. The method of claim 1, Three-dimensional cell culture equipment, characterized in that the protrusion is formed so as to include one per well for culturing the cells. The method of claim 1, Three-dimensional cell culture equipment characterized in that the protrusion is formed so as to include a plurality per well for culturing the cells. The method of claim 1, Three-dimensional cell culture equipment, characterized in that the end of the protrusion is formed pointed. Basal culture of cells desired to be cultured in a spheroid form in a culture solution containing nanoparticles; Receiving a culture solution containing the cultured cells in a well of a culture plate; Covering the surface of the culture plate with a cover including the protrusion so that the protrusion is positioned inside the well; And The three-dimensional culture method of the cells using the three-dimensional cell culture tool of any one of claims 1 to 8 comprising the step of culturing the cells forming a spheroid by the magnetism of the protrusions. The method of claim 10, In the step of accommodating the culture solution containing the cultured cells in the well of the culture plate portion 3 of the cells using the three-dimensional cell culture tool, characterized in that the end of the protrusion is accommodated so as not to contact the cultured cells and the culture medium Dimensional culture method. The method of claim 10, In the step of accommodating the culture solution containing the cultured cells in the well of the culture plate portion, the three-dimensional cell of the cell using a three-dimensional cell culture tool, characterized in that the end of the protrusion is accommodated in contact with the cultured cells and the culture medium Cultivation method. The method of claim 10, In the step of basing the cells to be cultured in the spheroid form in the culture solution containing the nanoparticles There are a plurality of types of cells to be cultivated in the spheroid form, the cells desired to be cultured in the plural kinds of spheroid form are mixed in the culture solution containing the nanoparticles and simultaneously cultured in three dimensions Three-dimensional culture method of cells using the culture tool. The method of claim 10, Basal culture of the first cell and the second cell, which are desired to be cultured in the spheroid form, separately in each culture solution containing the nanoparticles; Receiving a culture solution containing the cultured first cells in a well of a culture plate; Covering the surface of the culture plate with a cover including the protrusion so that the protrusion is positioned inside the well; Culturing the first cells by forming a spheroid by the magnetism of the protrusions; Supplying a culture solution including the cultured second cells to a well of the first cell spheroid-formed culture plate; And The second cell is cultured by forming the spheroid forming the shell part by using the first cell spheroid as a core part by the magnetism of the protrusion; three-dimensional culture of the cell using a three-dimensional culture tool including a Way. The method of claim 10, Basal culture of a plurality of cells desired to be cultured in a spheroid form separately in each culture solution containing nanoparticles; Receiving a culture solution including the cultured plurality of cells in a well of a separate culture plate; Covering the surface of the culture plate with a cover including the protrusion so that the protrusion is positioned inside the well; Culturing the plurality of cell types while forming primary spheroids by magnetism of the protrusions; Mixing the spheroids of each of the plurality of cultured cells; And And culturing the primary spheroid of the mixed plural types of cells while forming the secondary spheroid by the magnetism of the protrusions. 3. The method of claim 10, The magnetic nanoparticles are one or more selected from the group consisting of magnetite, Fe ₃ O ₄ , γ-Fe ₂ O ₃ , manganese ferrite, cobalt ferrite and nickel ferrite. 3D culture method.

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