JP2004166603A - Method for seeding cell, method for culturing cell, and cell-cultured product obtained by the culture method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for seeding cells hardly causing reduction of the function of the cells under culture; to provide a method for culturing the cells by which the function of the cells is maintained for a long period; and to provide a cell-cultured product highly maintaining the function in a living body. <P>SOLUTION: The method for seeding the cells keeps the high contact state or high contact frequency of the cells by adding centrifugal force to the cells inserted into a container, and forms an aggregate comprising two or more cell layers on the inner wall of the container. The aggregate can be formed on a permeable membrane. The cells can be cultured while keeping the functions possessed by the cells good for a long period by culturing the aggregate. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cell seeding method, a cell culture method, and a cell culture used in the fields of cell culture, tissue culture, and the like. In particular, the present invention relates to a seeding method capable of culturing cells while maintaining the function of the cells, a cell culture obtained after seeding by this method, and a culturing method.
[0002]
[Prior art]
Conventionally, when culturing adherent animal cells, it is common to take the form of a so-called monolayer culture in which the cells spontaneously adhere to the bottom surface of the culture vessel and extend or adhere by flattening the cytoplasm. is there.
[0003]
However, cells in a so-called primary culture immediately after being taken out of a living body and beginning to culture often retain the characteristics of the tissue from which they are derived, but in most cases, the function maintained in the living body for several days or several days It is known to disappear on a weekly basis.
[0004]
Among the primary cultured cells, highly differentiated primary hepatocytes tend to lose their functions particularly during the monolayer culture period. For example, even if primary culture of rat hepatocytes is carried out in a monolayer culture in a flask, for example, ammonia metabolizing ability, which is one of the important functions of hepatocytes, is usually lost within about two weeks from the start of culture.
[0005]
In this regard, it has been reported that the formation of a highly functional cell tissue body can be induced by forcibly filling cells within or between hollow fibers using centrifugal force to increase the frequency of contact between cells. (For example, Patent Document 1).
[0006]
However, there is no known method capable of culturing cells while maintaining the function using a general-purpose culture vessel without using hollow fibers.
[0007]
[Patent Document 1]
JP-A-2001-128660 (column 3, line 49 to column 4, line 3)
[0008]
[Problems to be solved by the invention]
The present invention provides a method of seeding cells whose function is hardly reduced during culture, a method of culturing cells capable of maintaining cell functions for a long period of time, and a cell culture that highly maintains functions in vivo. The main purpose is to
[0009]
[Means for Solving the Problems]
In order to achieve the above object, the present inventor has conducted repeated studies, and by applying centrifugal force when seeding cells into a culture vessel, the cells are kept in a high contact state or a high contact frequency and the cells are layered. By forming aggregates in an overlapping state and culturing the cells in the state of the aggregates, it has been found that the cells can be cultured for a long period of time while maintaining the function of the cells at a high level.
[0010]
Based on the above findings, the present invention provides a cell seeding method, a cell culture method, and a cell culture described in each of the following items.
[0011]
Item 1. A method for seeding cells in which an aggregate composed of a plurality of cell layers is formed on the inner wall of a container by applying a centrifugal force to the cells placed in the container.
[0012]
Item 2. Item 2. The cell seeding method according to Item 1, wherein the container has a plurality of wells, and a centrifugal force is applied to the cells in a state where the cells are placed in one or more wells.
[0013]
Item 3. Item 3. The method for seeding cells according to Item 1 or 2, wherein the cells are hepatocytes.
[0014]
Item 4. A method for seeding cells in which a cell is placed on a permeable membrane of a container having a permeable membrane therein and centrifugal force is applied to the cells to form an aggregate composed of a plurality of cell layers on the permeable membrane.
[0015]
Item 5. The container according to item 4, wherein the container has a plurality of wells provided with a permeable membrane inside, and applies a centrifugal force to the cells while the cells are placed on the permeable membrane in one or more wells. Seeding method.
[0016]
Item 6. Item 6. The method for seeding cells according to Item 4 or 5, wherein the cells are hepatocytes.
[0017]
Item 7. Item 7. A cell culture obtained by culturing cells seeded by the method according to any one of Items 1 to 6.
[0018]
Item 8. Aggregates consisting of multiple cell layers on the permeable membrane by applying centrifugal force to the cells while placing the cells on the permeable membrane in a permeable container in which all or part of the bottom surface is made of a permeable membrane Forming a;
Culturing the aggregate in a state in which the permeable container is fitted in a culture container having a size that allows the permeable container to be fitted therein.
[0019]
Item 9. Item 9. The cell culture method according to Item 8, wherein in the aggregate forming step, a centrifugal force is applied to the cells while the permeable container is fitted in the culture container.
[0020]
Item 10. Item 10. The method for culturing a cell according to Item 8 or 9, wherein the cell is a hepatocyte.
[0021]
Item 11. On the permeable membrane by applying centrifugal force to the cells while placing the cells on one or more permeable membranes of a container having a plurality of permeable wells, all or a part of the bottom surface of which is formed of a permeable membrane Forming an aggregate consisting of a plurality of cell layers at the same time;
Culturing the aggregate in a state where the permeable well is internally fitted in the well of the culture vessel having a plurality of wells sized to allow the permeable well to be embedded therein.
[0022]
Item 12. Item 12. The method for culturing cells according to Item 11, wherein in the aggregate forming step, a centrifugal force is applied to the cells while the permeable well is fitted in the well of the culture container.
[0023]
Item 13. Item 13. The method for culturing a cell according to Item 11 or 12, wherein the cell is a hepatocyte.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
(1) Cell seeding method
Method for seeding cells on inner wall of container In the first method for seeding cells on the inner wall of the present invention, centrifugal force is applied to the cells in a state where the cells are placed in the inner wall of the container, whereby the inner wall of the container is formed of a plurality of cells. This is a method of forming an aggregate.
[0025]
Generally, seeding of cells refers to seeding cells on the bottom surface (culture surface) of the culture medium. In the present invention, the cells are seeded on the inner wall of the container in an aggregate state. In the present invention, the container inner wall includes a container bottom surface, a side surface, and the like.
[0026]
In the present invention, an aggregate of cells refers to a group of cells in a state where cells are highly adhered to each other or in a state where they are frequently adhered to such an extent that spontaneous aggregation of dispersed cells cannot be achieved. In the aggregate of the present invention, the cells are not a monolayer, but form a plurality of cell layers by overlapping regularly or irregularly.
[0027]
Such aggregates can be obtained by suspending the cells in an appropriate liquid medium or buffer and applying a centrifugal force within a range that does not damage the cells according to the type of the cells using a usual centrifuge. Can be formed. The centrifugal force varies depending on the type of cells, but is preferably about 2 to 2000 × G, particularly preferably about 4 to 500 × G. For example, in the case of primary rat hepatocytes, it is preferable to apply a centrifugal force of usually 1500 × G or less, particularly 400 × G or less. In the case of primary rat hepatocytes, the lower limit of the centrifugal force is not particularly limited as long as cell aggregates are formed, and is usually about 5 × G. When a layered aggregate is formed on the inner wall of the culture vessel (including the bottom surface and the side wall) or a permeable membrane described later, if the centrifugal force is too high, the original form and function of the cells are lost. Conversely, if the centrifugal force is too low, the contact between cells is weak, and it does not lead to the formation of cell aggregates capable of expressing high functions. Such a problem does not occur within the scope of the present invention. The centrifugation time may be appropriately set within a range where a cell aggregate can be formed.
[0028]
Aggregates are formed on the inner wall of the container by applying a centrifugal force. Depending on the type of centrifuge, the aggregate may be formed on the bottom surface of the container, or may be formed on the bottom surface and side surfaces of the container.
[0029]
In placing the cells in the container, the number of cells to be placed in the container is adjusted by applying a centrifugal force in the above range so that aggregates in which the cells are generally overlapped by about 2 to 20 layers, especially about 5 to 10 layers are obtained. It is desirable to adjust. If there are too many cells in the aggregates, the cells in the central layer of the aggregates will be insufficiently nutrient and gas exchange will be insufficient, and if there are too few cells, the number of cells will be small and they will perform well. Agglomerates are difficult to form. Such a problem does not occur within the scope of the present invention.
[0030]
The number of cell layers of the aggregates can be confirmed by, for example, cutting out a vessel wall on which the layered aggregates are placed or a permeable membrane described below, fixing them in formalin, preparing paraffin-embedded sections, and observing them with a microscope. .
[0031]
The container in which cells are seeded may have a plurality of wells. When a container having a plurality of wells is used, a plurality of cell aggregates can be formed at once by applying a centrifugal force to the cells while the cells are placed in the plurality of wells. Forming a plurality of aggregates in a plurality of wells is advantageous when performing a plurality of cell cultures at once.
[0032]
After the cell aggregate is formed on the inner wall of the container or the inner wall of the well of the container, the cell aggregate may be cultured as it is or by adding a cell culture solution. Alternatively, cells may be cultured in a state where the supernatant is removed and a cell culture solution is further placed in a container or well. Thereby, culture can be performed while satisfactorily expressing or maintaining the functions originally possessed by the cells.
Method for seeding cells on permeable membrane In the second method for seeding cells of the present invention, the cells are centrifuged while the cells are placed on the permeable membrane in a container having a permeable membrane inside. This is a method of forming an aggregate composed of a plurality of cell layers on a permeable membrane by applying force.
[0033]
The permeable membrane is a membrane having a large number of pores that do not allow cells to pass therethrough but allow the passage of culture solution components such as water, salts, and proteins, and usually have a pore size of about 0.1 to 5 μm, particularly about 0.2 to 3 μm. It is preferable that it has.
[0034]
The thickness of the permeable membrane is not particularly limited as long as the membrane has appropriate strength and good substance permeability is maintained, but is not particularly limited, but usually about 10 to 200 μm, particularly preferably about 20 to 100 μm is preferable. Adopted.
[0035]
The material of the permeable membrane is not particularly limited, as long as it has no cytotoxicity and does not deteriorate or decompose due to sterilization, washing, contact with a culture solution and the like. For example, a permeable membrane made of cellulose resin, polyolefin such as polyethylene and polypropylene, polysulfone, polyethersulfone, fluororesin, polycarbonate, acrylic resin and the like can be employed.
[0036]
The magnitude of the centrifugal force, the time for applying the centrifugal force, and the number of cells placed on the permeable membrane are the same as those described for the first cell seeding method of the present invention.
[0037]
A container provided with a permeable membrane, a container in which all or a part of the bottom surface of the container is formed of a permeable membrane, a container in which all or part of the side surface in addition to the bottom surface of the container is also formed of a permeable membrane, or Alternatively, the container may be a container or the like in which the entire surface of the container is formed of a permeable membrane. In these cases, after forming the cell aggregate on the permeable membrane of the container, the permeable container is fitted into another culture container, and in this state, the cell culture solution is put into the culture container to remove the cells. It can be cultured. Further, as described later, an aggregate can be formed in a state where the permeable container is fitted in the culture container.
[0038]
Further, in this permeable container, a permeable membrane may be provided horizontally or substantially horizontally at an intermediate position in the depth direction of the container. In this case, the cell suspension is placed on the permeable membrane, and the liquid that has passed through the permeable membrane is accumulated in the container by applying a centrifugal force. After the formation of the aggregate, the cell culture solution may be put in the container and cultured.
[0039]
The container for seeding cells may have a plurality of wells with a permeable membrane. When a container having such a plurality of wells is used, a plurality of cell aggregates can be formed at once by applying a centrifugal force to the cells while the cells are placed on the permeable membranes of the plurality of wells. Forming a plurality of aggregates on a plurality of permeable membranes is advantageous when performing a plurality of types of cell culture at once.
[0040]
The position where the permeable membrane is provided in the well is the same as the case where the permeable membrane is provided in a normal container.
[0041]
When an aggregate is formed on the permeable membrane, nutrients can be supplied through the permeable membrane, that is, from the lower surface side of the aggregate in the subsequent culture. As a result, cells can be cultured more effectively while maintaining the functions inherent to the cells.
(3) Cell culture The cell culture of the present invention is a cell culture obtained by culturing cells seeded by the first or second cell seeding method of the present invention.
[0042]
The liquid culture medium for cell culture used in culturing the aggregates is not particularly limited, and for example, Dulbecco's modified Eagle medium, Williams E medium, Ham's F-10 medium, F-12 medium, RPMI-1640 medium, MCDB153 medium, 199 medium A conventionally known basal medium for cell culture, such as a culture medium, to which a conventionally known growth factor or an antioxidant suitable for culturing each cell can be added, if necessary, can be used.
[0043]
Furthermore, cells can be cultured in a state where the aggregates are covered with a collagen gel or an agarose gel containing a liquid culture medium for cell culture. Thereby, the cells can be protected from physical damage to the cells when the medium is replaced.
[0044]
The temperature at the time of culturing varies depending on the type of cells, but may be generally about 36 to 37 ° C. The culture time varies depending on the use of the obtained cell culture, but can be about 12 hours or more, particularly about 24 to 96 hours.
[0045]
By this level of culturing, a cell culture that satisfactorily expresses the function that the cell has maintained in vivo can be obtained.
(4) Cell culturing method The cell culturing method of the present invention relates to a method in which cells are placed on a permeable membrane in a permeable container in which all or part of the bottom surface is formed of a permeable membrane. Forming a plurality of cell layer aggregates on the permeable membrane by applying centrifugal force;
And culturing the aggregate in a state where the permeable container is fitted in a culture container having a size that allows the permeable container to be fitted therein.
[0046]
In the aggregate formation step, the number of cells placed on the permeable membrane, the magnitude of the centrifugal force, and the time for applying the centrifugal force are as described above for the first cell seeding method of the present invention.
[0047]
In the permeable container, all or a part of the bottom surface is made of a permeable membrane, but all or a part of the side surface may be made of a permeable membrane. Further, the entire surface may be formed of a permeable film.
[0048]
The material, pore size, thickness and the like of the permeable membrane are as described above for the second cell seeding method of the present invention.
[0049]
The permeable container may have a plurality of wells in which all or a part of the bottom surface is formed of a permeable membrane. In this case, a plurality of cell aggregates can be formed at once by applying a centrifugal force to the cells with the cells placed on the permeable membranes of the plurality of wells. Forming a plurality of aggregates on a plurality of permeable membranes is convenient for culturing a plurality of types of cells.
[0050]
The position where the permeable membrane is provided in the well is the same as the case where the permeable membrane is provided in a normal container.
[0051]
In the cell culturing step, a cell culture solution is filled in the culture container in a state where the permeable container formed with cell aggregates is fitted in a culture container having a size capable of fitting the permeable container therein. Incubate.
[0052]
Further, in a state where the permeable container is fitted in such a culture container in advance, cells can be placed in the permeable container and centrifugal force can be applied to the cells. In this case, after the cell aggregates are formed on the permeable membrane, the culture solution can be filled and cultured in the culture vessel without moving the permeable membrane on which the aggregates are formed, which is convenient. .
[0053]
Specifically, for example, a permeable container (a so-called culture insert) having a plurality of wells and having a well bottom surface formed of a permeable membrane is provided with a plurality of sized containers capable of fitting the wells of the permeable container therein. Attach the wells to a culture vessel (so-called multi-well plate) so that the wells engage each other, then put the cell suspension into each well of the culture insert, and centrifuge the whole multi-well plate fitted with the culture insert. By applying a centrifugal force to the cells, cell aggregates can be formed on the permeable membrane of the culture insert. Next, after an appropriate amount of liquid culture medium for cell culture is put into each well of the multi-well plate, the cells can be cultured as they are.
[0054]
FIG. 1 shows a cross section of an example in which a cell aggregate is formed on a permeable membrane while a culture insert is fitted in a multiwell plate. In this example, a culture insert 2 having a bottom surface made of a permeable membrane 21 is attached to a multi-well plate 1 such that wells are fitted to each other. The cell aggregate 3 is formed on the permeable membrane 21.
[0055]
A culture insert having such a permeable membrane is commercially available under the trade name of, for example, a membrane culture insert (manufactured by Iwaki Glass Co., Ltd.) and having a plurality of wells such as 6 wells, 12 wells, and 24 wells.
Cells The method of seeding the first and second cells of the present invention, the method of culturing the cells of the present invention, and the cells to be subjected to the cell culture of the present invention are not limited to those described above. Sexual animal cells are preferred. The origin of the cells is not particularly limited, and cells derived from any animal such as human, mouse and rat can be used. In addition, the adherent animal cells can be used for both primary cultured cells and established cells. The method of the present invention is particularly suitable for preserving primary cultured cells in which it is difficult to maintain cell functions. Primary culture cells may be derived from any tissue such as liver, kidney, nerve, and lung.In particular, human hepatocytes that rapidly lose their differentiation function by monolayer culture are used as target cells in the method of the present invention. It is suitable.
[0056]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited to these Examples.
[0057]
Example 1
A permeable container having 24 wells and having a polycarbonate permeable membrane on the bottom surface of each well (trade name: Transwell / 24 well plate, pore size: 3.0 μm, culture area: 0. 100 μl of 1.8 × 10 ⁵ cell suspensions of rat hepatocytes / 100 μl prepared by collagenase digestion were placed in each well of 33 cm ² (Corning Coaster). Next, the cells were spontaneously sedimented for 5 minutes, and then centrifuged at 60 × G for 90 seconds to form cell aggregates on the permeable membrane. The number of cell layers of the aggregates was determined by cutting out a permeable membrane on which the layered aggregates were mounted, fixing them with formalin, and then preparing paraffin-embedded sections and observing them with a microscope. Was.
[0058]
Transwells in which hepatocyte aggregates were formed on each of the permeable membranes were placed on a 24-well cell culture plate (manufactured by Falcon) as they were, and the transwells were placed in the wells of the cell culture plate. It was placed so as to fit.
[0059]
Next, in a well of the cell culture plate, 13.5 g / L of Dulbecco's modified eagle medium (DMEM; manufactured by Gibco), 60 mg / L proline, 50 ng / ml EGF (manufactured by Toyobo Co., Ltd.), 10 mg / L insulin (Manufactured by Sigma), 7.5 mg / L hydrocortisone (manufactured by Sigma), 50 μg / L linoleic acid (manufactured by Sigma), 100,000 U / L potassium penicillin G (manufactured by Nacalai Tesque), 100 mg / L streptomycin sulfate ( 650 μl of a serum-free medium (HSFM) containing 1.05 g / L sodium bicarbonate (manufactured by Nacalai Tesque) and 1.19 g / L HEPES (manufactured by Nacalai Tesque), 5% carbon dioxide gas And incubated at 37 ° C. for 1 month in an atmosphere of 95% air.
[0060]
Comparative Example 1
Cells were cultured under the same conditions as in Example 1 except that no centrifugal force was applied.
[0061]
Comparative Example 2
2.0 × 10 ⁶ primary rat hepatocytes prepared in the same manner as in Example 1 were seeded in a flask (manufactured by Falcon) having a culture area of 25 cm ² , and the same medium (HSFM) as in Example 1 was used. Under the same culture conditions as in Example 1 (in an atmosphere of 5% carbon dioxide and 95% air), monolayer culture was performed at 37 ° C. for one month.
[0062]
Cell function evaluation Hepatocytes have a function of metabolizing ammonia and drugs in addition to the ability to synthesize proteins such as albumin. In particular, it is known that the ammonia metabolizing ability usually disappears promptly with the lapse of days of culture. Here, albumin production and ammonia metabolism were measured over time.
[0063]
For each sample of Example 1, Comparative Example 1 and Comparative Example 2, the amount of albumin produced and the amount of ammonia metabolized were measured at 1, 3, 7, 14, 21, and 28 days after the start of culture.
[0064]
The amount of albumin produced was determined by measuring the amount of albumin in the medium by the EIA method using anti-rat albumin, and the amount of ammonia metabolism was determined by measuring the amount of ammonia consumed in the medium by an automatic analyzer.
[0065]
The measurement results of the albumin production amount are shown in FIG. 2, and the measurement results of the ammonia metabolism amount are shown in FIG. As is clear from FIGS. 2 and 3, the cells of Example 1 in which hepatocyte aggregates were formed on the permeable membrane using centrifugal force and then cultured, showed high albumin producing ability and ammonia metabolism. It has been shown to express and maintain the ability.
[0066]
On the other hand, in the cells of Comparative Example 2 in which the culture was performed without centrifugation and in Comparative Example 3 in which the monolayer culture was performed, no difference was observed from the cells of Example 1 in the initial stage of the culture. After that, its function quickly disappeared.
[0067]
【The invention's effect】
According to the present invention, a method of seeding cells whose function is hardly reduced during culture, a method of culturing cells capable of maintaining the function of cells for a long period of time, and a cell culture having a high level of function in vivo Provided.
[0068]
Furthermore, by applying centrifugal force, aggregates forming multiple cell layers are formed, and by creating a state in which the frequency of contact between cells is extremely increased, cell aggregates that express the functions inherent to cells satisfactorily Is obtained. When this cell aggregate is cultured, even if it is cultured for a long period of time, the function of the cell is less likely to decrease than in the case of ordinary monolayer culture or the like.
[0069]
In the present invention, the aggregate is formed in a layer on the inner wall of the container or on the permeable membrane. According to this method, a large number of aggregates can be easily obtained using a general-purpose container (such as a multiwell plate), an instrument (such as a culture insert), and a general-purpose device (a centrifuge). In addition, since the centrifugal force can be applied widely and uniformly, an aggregate composed of a large number of cells can be obtained by increasing the area of the container or the permeable membrane.
[0070]
In addition, such aggregates are easily induced by the tissue, and the tissue formed from the cell aggregates satisfactorily expresses the functions inherent in the cells.
[0071]
Further, in the case of forming an aggregate on the permeable membrane of the permeable container by applying centrifugal force to the cells in a state where the permeable container and the culture container are combined in the cell culture method of the present invention, The culture can be performed by filling the culture vessel with the cell culture solution, and the culture of the aggregate can be easily performed.
[0072]
In particular, by using a culture insert that is a permeable container having a plurality of wells and a multi-well plate that is a culture container having a plurality of wells, a plurality of cell aggregates can be easily obtained at the same time, and various types of cells can be obtained. A cell culture can be easily obtained at once without using a special device or the like.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of a state in which a culture insert is mounted on a multi-well plate and a cell aggregate is formed on a permeable membrane in a well of the culture insert.
FIG. 2 is a graph showing the amount of albumin produced per unit number of cells.
FIG. 3 is a graph showing ammonia metabolism per unit cell number of cells.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Multi-well plate 2 Culture insert 21 Permeable membrane 3 Cell aggregate

Claims (13)

A method for seeding cells in which an aggregate composed of a plurality of cell layers is formed on the inner wall of the container by applying a centrifugal force to the cells placed in the container. The method according to claim 1, wherein the container has a plurality of wells, and a centrifugal force is applied to the cells in a state where the cells are placed in one or more wells. 3. The method according to claim 1, wherein the cells are hepatocytes. A method for seeding cells in which a cell is placed on a permeable membrane of a container having a permeable membrane therein and centrifugal force is applied to the cells to form an aggregate composed of a plurality of cell layers on the permeable membrane. The cell according to claim 4, wherein the container has a plurality of wells provided with a permeable membrane therein, and the cell is subjected to centrifugal force with the cells placed on the permeable membrane in one or more wells. Seeding method. The method according to claim 4 or 5, wherein the cells are hepatocytes. A cell culture obtained by culturing cells seeded by the method according to claim 1. Aggregates consisting of multiple cell layers on the permeable membrane by applying centrifugal force to the cells while placing the cells on the permeable membrane in a permeable container in which all or part of the bottom surface is made of a permeable membrane Forming a;
Culturing the aggregates in a state in which the permeable container is fitted in a culture container having a size that allows the permeable container to be fitted therein. The method for culturing cells according to claim 8, wherein in the aggregate forming step, a centrifugal force is applied to the cells while the permeable container is fitted in the culture container. The method for culturing cells according to claim 8 or 9, wherein the cells are hepatocytes. On the permeable membrane by applying centrifugal force to the cells while placing the cells on one or more permeable membranes of a container having a plurality of permeable wells, all or a part of the bottom surface of which is formed of a permeable membrane Forming an aggregate consisting of a plurality of cell layers at the same time;
Culturing the aggregate in a state where the permeable well is internally fitted in the well of the culture vessel having a plurality of wells sized to allow the permeable well to be embedded therein. The method for culturing cells according to claim 11, wherein in the aggregate forming step, a centrifugal force is applied to the cells while the permeable well is fitted in the well of the culture vessel. The method for culturing cells according to claim 11 or 12, wherein the cells are hepatocytes.

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