KR20130033938A - 3 dimensional cell culture tool and cell culture method using the same - Google Patents

Abstract

PURPOSE: A three dimensional cell culture device and a three dimensional cell culture method using the same are provided to form a floating spheroid in a culture medium without a separate treatment such as inoculation of cells on a cover part. CONSTITUTION: A three dimensional cell culture device comprises a culture plate part, a cover part, and a protrusion part(50). The culture plate part has wells for culturing cells. The cover part covers the culture plate part. The protrusion part is extended toward the inside of the well from the surface which touches the culture plate part.

Description

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

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

In today's development of cells, cells cultured by cell culture techniques are being used for simulating pharmacological activity of drugs and 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 of cells having an equivalent function in vivo, has attracted attention. In particular, with the development of stem cell technology, various methods have been attempted for spheroid culture of embryonic stem cells in three dimensions and applied to the study of various differentiation mechanisms.

For example, seeding a plurality of single cells in a well having a funnel shape on the bottom surface, aggregating and dividing the single cells on the bottom surface, and culturing the spleoid (Japanese Patent Application Laid-Open No. 6-327462 , Fig. 1)), and a method in which spoil is cultured on a honeycomb structure obtained by dewatering water on the cast liquid surface of a predetermined polymer mixture and evaporating minute droplets generated by this condensation (JP-A-2002-335949 (P3, Fig. 1)).

However, according to Japanese Unexamined Patent Publication No. 6-327462, there is no space of sprooid produced by culturing, and it is difficult for the sphere to be fixed to the cell culture construct or the cell culture container. 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 if the behavior of the cancer cells is simulated, the nature of the tumor formed by division from one cancer cell in vivo and the properties of the spoloid are strictly different. There was a problem that accuracy was poor.

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

As another method of culturing cells in a three-dimensional spoloid, there is a hanging drop culture.

2 is a conceptual diagram showing a general method of three-dimensionally culturing cells through a hanging drop culture. In the method of three-dimensionally culturing cells by hanging drop culture, the number of collected cells is counted as shown in FIG. 2 (a), and about 300 to 500 cells are collected using a pipette, (11) of the culture dish (100) in a droplet form and adhere them in an inverted manner. Thereafter, as shown in FIG. 1 (b), the hanging cells gather at the end of the droplet by gravity to form an embryoid-like cell mass, which is generally referred to as a compensator.

Fig. 3A and 3B show another embodiment of the current culture method developed by the 3d biomatrix, which uses a cover plate having a hole with a predetermined size to accommodate the cell culture liquid in the cover plate, Is three-dimensionally cultured.

However, in the case of hanging drop culture, cells are placed on the cover of the culture container by using a pipette or the like, so that the cells are physically damaged during the transfer, There was a concern that the problem would be caused.

It is an object of the present invention to provide a three-dimensional cell culture apparatus for three-dimensional culture of cells which can overcome the problems of the conventional three-dimensional spheroid culture technology of cells.

The present invention also aims to provide a method for three-dimensionally culturing cells using the three-dimensional cell culture apparatus.

The present invention has been made to solve the above problems

A culture plate portion including a well for culturing the cells;

A cover portion covering the culture plate portion; And

And a protrusion extending from the surface of the cover portion in contact with the culture plate portion toward the inside of the well of the culture plate portion.

In the present invention, the projecting portion is characterized by exhibiting magnetism.

In the present invention, a method of causing the protrusions to exhibit magnetism is not particularly limited, and in one embodiment of the present invention, the protrusions can be made of a permanent magnet material.

In the present invention, the projecting portion may further include a control portion for controlling the magnitude and the supply time of the current supplied to the electromagnet, and a power portion for supplying the electromagnet with a power of a predetermined size for a predetermined period of time .

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

In the present invention, the culture plate portion includes a plurality of wells for culturing the cells, and the protrusions are formed to include one per well for culturing the cells. In the present invention, the protrusions are formed to include a plurality of cells per well for culturing the cells.

In the present invention, the shape of the distal end of the protruding portion is not particularly limited, but it is preferable that the protruding portion is sharply formed so as to concentrate magnetism and efficiently cultivate the cells in three dimensions.

The present invention also provides

Culturing the cells to be cultured in the form of spoil in a culture solution containing nanoparticles;

Receiving the cultured cells and a culture solution in a well of a culture plate portion;

Covering the surface of the culture plate portion with a cover portion including the protrusions so that the protrusions are positioned inside the wells; And

And three-dimensional culture of the cells using the three-dimensional cell culture apparatus of the present invention including the step of culturing the cells three-dimensionally while forming the spheroids by the magnetism of the protrusions.

In the present invention, cells to be cultured in the form of a sphere are firstly cultured in a culture solution containing nanoparticles. In the present invention, the basic culture method is not particularly limited, and can be basically cultured by shaking culture or other methods.

The cells cultured on the basis of the nanoparticle-containing culture can be prepared by allowing the nanoparticles to be accommodated in the cells, attaching nanoparticles to the cell membranes, or attaching the magnetic nanoparticles to the cells by other mechanisms, Lt; / RTI >

Magnetic nanoparticles have been proposed to be used in a variety of biomedical applications, including magnetic resonance imaging, high-temperature treatment of malignant cells, and drug delivery. In recent years, cells have been cultured in a mixture of nanoparticles, A technique of immobilizing the cells into the cells and fixing the cells into which the magnetic particles have been introduced is being developed.

In the present invention, the nanoparticle refers to a very small particle, typically ranging from one nanometer to a few hundred micrometers in diameter. Because of the small size dyes and pigments; Aesthetic or functional coating; Tools for biological research, medical imaging and therapy; A magnetic recording medium; Quantum dots; And uniform and constant nano-sized semiconductors.

In the present invention, the magnetic nanoparticles may be 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, in the step of accommodating the cultured cells and the culture solution in the well of the culture plate portion, the cultured cells and the culture solution are received so as not to contact the protrusions.

In addition, in the present invention, in the step of accommodating the culture solution containing the cultured cells in the well of the culture plate portion, the end of the projected portion is received in contact with the cultured cells and the culture solution. In this case, it is possible to separate the three-dimensionally cultured cells from the protrusions around the protrusions by appropriately treating the surface of the protrusions.

In the present invention, in the step of basically culturing the cells to be cultured in a spleod form in the culture solution containing the nanoparticles, there are a plurality of kinds of cells to be cultured in the spoloid form, The cells that are desired to be cultured in the form of nanoparticles are mixed in a culture solution containing the nanoparticles and simultaneously cultured.

The method for three-dimensional culture of cells using the three-dimensional culture apparatus of the present invention may further comprise the steps of: separately culturing the first and second cells, which are desired to be cultured in a spoloid form, in respective cultures containing nanoparticles;

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

Covering the surface of the culture plate portion with a cover portion 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 medium containing the cultured second cells to a well of the first cell spleen-formed culture plate portion; And

And culturing the first cell spleoid as a core part by magnetic force of the protruding part and forming the second cell as a shell part.

The method for three-dimensional culture of cells using the three-dimensional culture apparatus of the present invention may further comprise the steps of: separately culturing a plurality of types of cells desired to be cultured in a spoloid form in respective cultures containing nanoparticles;

Receiving a culture solution containing the cultured plural kinds of cells in a well of another culture plate portion;

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

Culturing the plurality of types of cells while forming primary spheredoids by the magnetism of the protrusions;

Mixing the first spheroids of each of the cultured plural kinds of cells; And

And culturing the mixed spherooids of the plurality of kinds of cells while being coagulated by the magnetism of the protrusions to form secondary spherooids.

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

4 is a schematic view of a three-dimensional cell culture apparatus 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 portion 10 including a plurality of wells 30 for culturing cells; A cover part (20) covering the culture plate part; And at least one protrusion (50) extending from the surface of the cover portion in contact with the culture plate toward the inside of the well of the culture plate.

In the three-dimensional cell culture equipment of the present invention, the projecting portion is characterized by exhibiting magnetism, and the method for this is not limited. Specifically, the protruding portion may be made of a permanent magnet material, or may be made of an electromagnet material.

Fig. 4 shows a case (51) in which the entire protrusion is made of a permanent magnet material, and a case (52) in which a permanent magnet is attached at the end.

FIG. 5 is a schematic view of a case where the protrusion is made of an electromagnet material. 5, when the three-dimensional cell culture apparatus of the present invention includes an electromagnet protrusion, the culture plate 110 includes a plurality of wells 130 included in the culture plate, a cover for covering the culture plate, (120) and a protrusion of an electromagnet material extending from a surface of the cover portion in contact with the culture plate toward the inside of each well of the culture plate, and connected to each tip for controlling the magnetism of the protrusion of the electromagnet material An external power supply unit 160, and a control unit 170. In the present invention, the control unit 170 adjusts the power applied to the external power supply unit 160, the intensity of the power applied to the external power supply unit 160, and the application time thereof, thereby adjusting the magnetic force generated in the protrusions of the electromagnet material . Thus, various types of cell cultures are possible. That is, it is possible to cultivate the cells in various ways such as periodically applying magnetism in the course of culturing the cells, applying magnetism only until the cells coagulate, and then not applying magnetism.

The overall shape of the culture plate 10 and the shape of the well are not particularly limited and concretely include not only a round bottom flask but also a 6 well plate and a 96 well plate plate used for culturing cells generally in the art And may be appropriately selected depending on the purpose of culture. Fig. 6 shows a case where the culture plate is in the form of a multi-well, and Fig. 7 shows a case in which the culture plate is in the form of a circular plate as a preferred embodiment of the present invention.

In the three-dimensional cell culture apparatus of the present invention, the shape and the number of the tips can be appropriately selected depending on the capacity of the well for culturing the cells, and are not particularly limited, and the culture plate 10 ) Comprises a plurality of wells 30 for cell culture, it is possible that the protrusions are formed to include one per well 30 for culturing the cells.

It is also possible to include a plurality of protrusions in the well for culturing the cells as shown in FIG. When a plurality of tips are provided in a well for culturing one cell as described above, spleoids of the same type can be formed simultaneously in each well where the protrusions are located in the well for culturing the cells, When the cultured cells are further grown, they are also effective when the culture medium is replaced or separated at once.

In the three-dimensional cell culture equipment of the present invention, the length of the projecting portion 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 relative to the depth of the well, the number of cells contacting 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 efficiency of the battery is reduced.

In the present invention, the tip can be cultured three-dimensionally even when the tip is not in contact with the cell culture fluid in the well, and the cells can be cultured three-dimensionally even in the contacted state. When cells are cultured in contact with each other, it is preferable that the magnetism acts more directly. However, since the cells stick to the surface of the protrusions, it is preferable to perform appropriate surface treatment on the protrusions. At this time, as the surface treatment of protrusions, a method of coating a polymer material (a highly water repellent substance such as PEG) that interferes with cell attachment or covering a material that prevents direct contact with a cell can be used.

FIG. 8 shows a three-dimensional cell culture method using a three-dimensional cell culture apparatus according to the present invention. A three-dimensional cell culture method using the three-dimensional cell culture apparatus of the present invention comprises i) culturing the cells to be cultured in a spoloid form in a culture solution containing nanoparticles; ii) receiving a culture solution containing the cultured cells in a well of a culture plate portion; iii) covering the surface of the culture plate portion with a cover portion including the protrusions so that the protrusions are positioned inside the wells; iv) culturing the cells three-dimensionally while forming the spheroids by magnetism of the protrusions; It is configured to include.

In the three-dimensional cell culture method of the present invention, cells that are desired to be cultured in a spoloid form in a culture solution containing nanoparticles are basically cultured.

As described above, the magnetic nanoparticles can be introduced into cells by basal culture of the cells in a culture solution containing the nanoparticles, or the surface of the nanoparticles can be modified to adhere to cell membranes.

Methods for introducing magnetic nanoparticles into cells can be performed by a method commonly used by those skilled in the art. The magnetic nanoparticles are preferably contained in a cell culture medium at a concentration of 10 pg to 1000 pg / cell, The cells are cultured in the contained medium so that the magnetic nanoparticles are introduced into the cells.

In the present invention, super magnetic or ferromagnetic iron oxide nanoparticles are used as the magnetic nanoparticles. Preferably, the oxides contained in the nanoparticles are selected from the group consisting of 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 super-magnetic 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.

When the cells are cultured in this manner, the cells containing the magnetic nanoparticles gather near the tips due to the magnetism of the protruding portions of the cover portion, and as a result, three-dimensional cell cultures in which the cells are mutually agglomerated are made possible.

In the subsequent step, it is possible to further grow the size of the spheroids by increasing the number of cells in the three-dimensional cell cultured spleen by continuously culturing the aggregated cells while maintaining the state.

In the present invention, it is possible to regulate the cell type during the basal culture or to cultivate the fusion spolide in various forms in the spoloid formation step after the basal culture.

In the present invention, a method for producing a fused spheroid as possible is schematically shown in Fig.

Specifically, there are provided: A) a method of randomly mixing a plurality of types of cells in a randomly mixed speroid form, that is, a base culture,

B) forming a core-shell type spheroid formed by sequential introduction of cells, that is, forming a spheroid from the first cell, and then forming a shell part with the first cell spheroid as a core part How to,

It is possible to form various forms of spoiloids, such as the formation of spoeloids based on C) spoloids, i.e., first and second cell spoids.

In the method A, a plurality of kinds of cells to be cultured are cultured in a basal culturing step, and the cells to be cultured in the plurality of kinds of spoil are mixed in a culture solution containing the nanoparticles and simultaneously cultured The plurality of types of cells coagulate and randomly mix to form spheroids.

In the above B method, a plurality of cells, which are desired to be spoold cultured, are selected from a plurality of first cells and a plurality of second cells, and the respective cells are separately cultured in respective cultures containing nanoparticles, Is contained in the well of the culture plate portion.

The surface of the culture plate portion is covered with a cover portion including the protrusions so that the protrusions are positioned inside the wells and the first cells are cultured while forming the spheroids by the magnetism of the protrusions. Thereafter, the culture medium containing the cultured second cells is supplied to the wells of the first cell spleen-formed culture plate portion, and the second cells are cultured by the magnetism of the protrusions into the core portions of the first cell spoloids , And forming a spheroid to form a shell part.

In the above C method, a plurality of types of cells desired to be cultured in the form of spoil are separately cultured in respective cultures containing the nanoparticles, and the culture solution containing the cultured plural kinds of cells is placed in a well of a separate culture plate Accept.

The cover portion including the protrusions covers the surface of the culture plate portion so that the protrusions are positioned inside the wells and the plural kinds of cells are cultured while forming the primary spoloids by the magnetism of the protrusions.

The first spheroids of the cultured plural kinds of cells thus formed are mixed and the primary spheroids of the mixed plural kinds of cells are cultured while forming secondary spheroids by the magnetic properties of the protrusions.

In the three-dimensional cell culture apparatus and the three-dimensional cell culture method using the same, the magnetic nanoparticles are introduced into the cells, and the cells are cultured while applying magnetism using the magnetic protrusions to inoculate cells in the cover , It is possible to form spleoids in the suspended state in the culture medium in which the cells are basically cultured.

Figure 1 shows the shape of a plate bottom used for three-dimensional culture of conventional cells.
FIGS. 2 and 3 show a process and apparatus of a current culture method used for three-dimensional culture of conventional cells.
4 is a cross-sectional view of a three-dimensional cell culture apparatus according to an embodiment of the present invention.
FIGS. 5 and 6 show a three-dimensional cell culture apparatus using an electromagnet according to another embodiment of the present invention.
7 is a cross-sectional view of a three-dimensional cell culture apparatus according to another embodiment of the present invention.
FIG. 8 shows a method of three-dimensionally culturing cells by the three-dimensional cell culture method of the present invention.
9 is a photograph of a cover part of a three-dimensional cell culture apparatus manufactured according to an embodiment of the present invention.
FIGS. 10 to 12 show the result of culturing cells using the three-dimensional cell culture apparatus of the present invention, introducing a fluorescent substance into the cells, and observing them with a fluorescence microscope
Fig. 13 shows the viability of cells cultured using the three-dimensional cell culture apparatus of the present invention.
FIG. 14 shows the state of each cell by randomly extracting three-dimensionally cultured cells formed in a cell culture apparatus when the cell culture well includes a plurality of protrusions according to an embodiment of the present invention.
Fig. 15 shows the results of measurement of the distribution of proteins involved in the internal skeleton in the case of culturing cells using the three-dimensional culture apparatus of one embodiment of the present invention and the two-dimensional cultured comparative example.
FIG. 16 shows the response of each cell to a toxic substance in the case of culturing the cells using the three-dimensional culture apparatus of one embodiment of the present invention and in the two-dimensional cultured comparative example.
FIGS. 17 to 19 show the measurement results of the process of forming a fusion spoloid using a three-dimensional culture apparatus of an embodiment of the present invention.
Fig. 20 schematically shows a method for producing a fused spheroid, in the present invention.

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

< Example  1>

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

A 96-well plate was used as the culture plate, and a tip-shaped protrusion was made of NdFeB, which is a permanent magnet material, and attached to the cover of the 96-well plate to prepare a three-dimensional cell culture apparatus. The cover portion of the manufactured cell culture apparatus is shown in Fig.

< Example  1-2> Foundation culture

As a cell line, HeLa cells were used as magnetic nanoparticles and Fe ₃ O ₄ end Cells were cultured in the contained medium and cultured to introduce 800 pg of particles into the cells.

< Example  1-3> Three-dimensional cell culture

800 pg of the cells cultured in Example 1-2 were distributed to each of the three-dimensional cell culture apparatuses prepared in Example 1 to contain 500 cells per well. Cells were loaded with a material that stained Hoechst cells to facilitate observation.

The cover plate shown in FIG. 9 was covered with the culture plate, followed by incubation for 5 minutes. Three-dimensional aggregation process of the cells with time was measured with a fluorescence microscope. The results are shown in FIG.

As shown in FIG. 10, it can be confirmed that the cells are coagulated and three-dimensionally cultured five minutes after covering with a cover including a tip-shaped protrusion.

< Example  1-4> Three-dimensional cell culture according to culture concentration

The cells cultured on the basal culture in the above Example 1-2 per well of the culture plate prepared in Example 1-1 were each distributed so as to contain the above 125 to 1000 cells and after 5 minutes, -axis scanning, and reconstructed into a three-dimensional image. The results of confirming whether or not the cells were cultured in three dimensions and the results of three-dimensional images were shown in Fig.

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

< Example  1-5> Three-dimensional cell culture according to the concentration of magnetic nanoparticles used for the basic culture

The HeLa cells were basically cultured while varying the concentration of the magnetic nanoparticles introduced into cells at the time of the basal culture in the range of 200-800 pg / cell in Example 1-2, and the culture plate prepared in Example 1-1 The cells were moved to a well of 1000 cells / well, and a cover was attached, and the cells were three-dimensionally cultured for 5 minutes.

In FIG. 12, the degree of cell culture for each concentration can be confirmed. It can be confirmed that the difference in size of the three-dimensional cell, sphere, generated depending on the concentration of the magnetic nanoparticles per well in the culture plate is not large.

< Experimental Example  1 > 3-dimensionally cultured cells viability  Confirm

The cells cultured three-dimensionally in Example 1-5 were continuously cultured for 5 days, and the viability was confirmed by fluorescence observation using the Live / DEAD viability kit. The results are shown in FIG.

When Live / DEAD viability kit is used, when cells are mixed with Calcein AM (2 μM) and EthD-1 soultion (4 μM) after the incubation, living cells are green and dead cells are red fluorescence. As shown in FIG. 13, no cell death was observed until day 5, and it was confirmed that the cells were cultured three-dimensionally as the cells proliferated.

< Example  2> cell culture Well Multiple  When the protrusion is included

< Example  2-1> Three-dimensional cell culture device manufacturing

When a plurality of protrusions are contained in a well for culturing the cells, a circular plate is used as a culture plate to confirm whether or not the three-dimensional cell culture is carried out, and a plurality of tip-shaped magnets are attached to the cover of the circular plate at regular intervals Thereby producing a three-dimensional cell culture apparatus as shown in Fig.

< Example  2-2> Three-dimensional cell culture

800 pg cells cultured on the basis of the above Example 1-2 were distributed to contain 500 cells per well of the culture plate prepared in Example 2-1.

After the cells having the magnetic particles prepared in Example 2-1 were introduced into the prepared three-dimensional cell culture apparatus, the cells were cultured in three dimensions by attaching a cover, and the cultured three-dimensional cultured cells were randomly And the state of each cell is shown in Fig.

When a plurality of protrusions are included in one well, it can be seen that a plurality of spoloids having the same biological property as the three-dimensionally formed state can be obtained at a position where the protrusions are formed.

< Comparative example > Two-dimensional culture

HeLa cells and mesenchymal stem cells were suspended in a 96-well plate, respectively, with the culture medium. After a certain period of time, the cells were submerged in the bottom of the plate and cells were cultured in an incubator by a general culture method.

< Experimental Example  2> Actin  Dyeing and Confocal  Microscopy comparison

Two different types of HeLa and mesenchymal stem cells were cultured with 3-dimensional spheroids according to the method of Example 1, and the protein distribution in the cultured spoloid was compared with the protein distribution in the 2-dimensional cultured cells of the comparative example Actin was stained with Alexa 488-conjugated phalloidin and compared with a confocal microscope. The results are shown in Fig.

As shown in FIG. 15, the three-dimensional cell cultured spleroid of Example 1 and the two-dimensional cultured cell of Comparative Example exhibited different protein distributions. In the three-dimensional cell cultured spleroid of Example 1, The organic influences, along with the organic binding of actin, tend to outward bias the distribution of proteins.

< Experimental Example  3> Staurosporin ( staurosporine ) To cytotoxicity

After 500 μM of staurosporine, which is a toxic substance, was treated with the three-dimensional cell cultured spleen of Example 1 and the two-dimensional cultured cells of the comparative example for 6 hours, viability Was confirmed by fluorescence observation, and the results are shown in Fig.

In Fig. 16, red fluorescence means dead cells. As shown in FIG. 16, in the case of the three-dimensional cell cultured spoloid of Example 1 of the present invention, the reaction to the toxic drug appears from the outside of the spleoid, Show.

< Example  3-1> Speroid  Fusion between fusion ) Speroid  formation

Two mesenchymal stem cells were cultured in primary spleen, and the two cultured stem cell spleoids were mixed and cultured in a second culture medium in the cell culture apparatus prepared in Example 1 above.

FIG. 17 shows the process of forming secondary spheroids according to the incubation time. Cells forming one of the two primary spoloids were reddish by lipid membrane staining, and the cells forming the other spoloid were blue stained with nuclear staining. In FIG. 17, it can be seen that as the time passes, the two primary spoloids are fused to form a new secondary fusion spoloid.

< Example  3-2> Core-shell type Speroid  formation

HEK-293 cells with fluorescent proteins were cultured on a basal plate, followed by primary spore culture, and the cultured spoloids were subjected to secondary spore culture in a medium containing HeLa cells with GFP green to obtain core- And the results are shown in Fig. It can be confirmed from FIG. 18 that the core-shell structure spoil is formed.

< Example  3-3> Random mixing type Speroid  formation

HeLa cells with GFP fluorescence and HEK-293 cells with Red fluorescence protein were respectively incubated and then separated and mixed to form spheroids. The results are shown in FIG. In Fig. 19, it can be confirmed that the mixed cells form spleoid.

Claims (16)

A culture plate portion including a well for culturing the cells;
A cover portion covering the culture plate portion; 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,
3D cell culture apparatus, characterized in that the protrusions show a magnetic
The method of claim 2,
Wherein the protruding portion comprises a permanent magnet.
The method of claim 2,
Wherein the protruding portion includes an electromagnet.
The method of claim 4, wherein
A controller for controlling a magnitude and a supply time of a current supplied to the electromagnet, and a power unit for supplying power to the electromagnet for a predetermined period of time,
The method of claim 1,
Wherein the length of the protrusion is 1/20 to 9/10 of the depth of the well.
The method of claim 1,
Wherein the protrusions are formed so as to include one per well for culturing the cells.
The method of claim 1,
Wherein the protrusions are formed to include a plurality of cells per well for culturing the cells.
The method of claim 1,
Wherein the protruding portion has a pointed end.
Culturing the cells to be cultured in the form of spoil in a culture solution containing nanoparticles;
Receiving a culture medium containing the cultured cells in a well of a culture plate portion;
Covering a surface of the culture plate portion with a cover portion including a protrusion so that the protrusion is positioned inside the well; And
The method of three-dimensional culture of 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 protrusion.
11. The method of claim 10,
Wherein the step of accommodating the culture solution containing the cultured cells in the well of the culture plate part accommodates the end of the projected part so as not to contact the cultured cells with the culture solution, Dimensional culture method.
11. The method of claim 10,
Wherein the step of accommodating the culture solution containing the cultured cells in the well of the culture plate part accommodates the end of the projected part so as to be in contact with the cultured cell and the culture solution, Lt; / RTI &gt;
11. The method of claim 10,
In the step of basically culturing the cells to be cultured in the form of spoil in the culture solution containing the nanoparticles
Characterized in that a plurality of types of cells to be cultured in the spoloid form are present and the cells to be cultured in the plurality of kinds of spoloid types are mixed and simultaneously cultured in the culture medium containing the nanoparticles A three-dimensional culture method of cells using a culture apparatus. 11. The method of claim 10,
Separately culturing the first cells and the second cells, which are desired to be cultured in the form of spheroids, in respective cultures containing nanoparticles;
Receiving a culture medium containing the cultured first cells in a well of a culture plate portion;
Covering the surface of the culture plate portion with a cover portion including the protrusion so that the protrusion is positioned inside the well;
Culturing the first cells to form spheroids by magnetization of the protrusions;
Supplying a culture medium containing the cultured second cells to a well of the first cell spleen-formed culture plate portion; And
Wherein the second cells are cultured with the first cell spheroids as a core part and forming spheroids forming a shell part by the magnetic properties of the protrusions, wherein the three-dimensional culture using the three- Way.
11. The method of claim 10,
Separately culturing a plurality of kinds of cells desired to be cultured in a spoveroid form in respective cultures containing nanoparticles;
Receiving a culture solution containing the cultured plural kinds of cells in a well of another culture plate portion;
Covering the surface of the culture plate portion with a cover portion including the protrusion so that the protrusion is positioned inside the well;
Culturing the plurality of types of cells while forming primary spheredoids by the magnetism of the protrusions;
Mixing spheroids of each of the cultured plural kinds of cells; And
And a step of culturing the mixed spheroploids of the plurality of kinds of cells while forming secondary spherooids by the magnetism of the protrusions, and culturing the spheroplasts in a three-dimensional culture apparatus using the three-dimensional culture apparatus.
11. The method of claim 10,
Wherein 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. Three - dimensional culture method.

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