JP7440853B1 - Cell culture device and cell culture method - Google Patents

Abstract

An object of the present invention is to provide a cell culture device that has a simple configuration and can promote cell proliferation by applying mechanical stimulation. A cell culture device includes a box for accommodating a culture substrate on which cells are fixed, and a movable lid disposed within the box so as to be movable in the depth direction of the box. have The culture substrate is arranged between the bottom plate of the box and the movable lid. [Selection diagram] Figure 1

Description

The present invention relates to a cell culture device and a cell culture method.

Regenerative medicine aims to use stem cells to regenerate damaged organs and tissues and restore lost functions. There are various types of regenerative medicine, but for example, there is a method in which organs and tissues artificially constructed from stem cells outside the patient's body are transplanted into the patient's body.

The present inventors have found that in order to form tissues in vitro similar to in vivo tissue, (1) cells, (2) three-dimensional culture substrate (corresponding to extracellular matrix), (3) humoral regulatory factors, We believe that not only (4) the nutritional supply system (corresponding to the vascular system) but also (5) mechanical stimulation is important. Based on this idea, the present inventors have proposed an anti-gravity culture method in which cells are cultured while applying gravity in a direction different from normal, and an apparatus used therein (see, for example, Patent Documents 1 and 2).

Japanese Patent Application Publication No. 2012-90584 Japanese Patent Application Publication No. 2014-76026

As mentioned above, the present inventors have found that (5) cell proliferation can be promoted by applying gravity in a direction different from normal as a mechanical stimulus; We thought that by applying stimulation to cells, we might be able to further improve cell proliferation and tissue formation efficiency.

The present invention has been made in view of the above, and aims to provide a cell culture device that has a simple configuration and can promote cell proliferation by applying mechanical stimulation, and a cell culture method using the same. .

The present invention relates to the following cell culture device and cell culture method.
[1] A box for accommodating a culture substrate on which cells are fixed, and a movable lid disposed within the box so as to be movable in the depth direction of the box, and the culture substrate A cell culture device, wherein the material is disposed between the bottom plate of the box and the movable lid.
[2] The cell culture device according to [1], further comprising a fall prevention part for preventing the movable lid from falling out of the box body.
[3] The cell culture device according to [1] or [2], further comprising an elastic member for pressing the movable lid toward the culture substrate housed in the box.
[4] The cell culture device according to any one of [1] to [3], further comprising a spacer disposed between the bottom plate and the movable lid.
[5] The cell culture device according to any one of [1] to [4], wherein at least one of the bottom plate of the box and the movable lid is a porous body.
[6] The cell culture device according to any one of [1] to [5], wherein the box body and the movable lid body are made of titanium.
[7] The cell culture device according to any one of [1] to [6], wherein the bottom plate of the box and the movable lid are made of titanium nonwoven fabric.
[8] A step of accommodating a culture substrate on which cells are fixed between the bottom plate of the box and the movable lid of the cell culture device according to any one of [1] to [7]; , a step of culturing cells while rotating the cell culture device containing the culture substrate.

According to the present invention, with a simple configuration, when culturing cells, pressure or shear stress can be applied to the cells as a mechanical stimulus to promote cell proliferation.

FIG. 1 is a perspective view of a cell culture device according to Embodiment 1 of the present invention. FIG. 2A is a plan view of the cell culture device according to Embodiment 1, and FIG. 2B is a cross-sectional view taken along line AA in FIG. 2A. 3A to 3C are diagrams showing an example of cell culture using the cell culture device according to the first embodiment. FIG. 4A is a cross-sectional view showing the state of the cell culture device when the opening of the box body is upward, and FIG. 4B is a cross-sectional view showing the state of the cell culture device when the opening of the box body is downward. It is a diagram. 5A is a plan view of a cell culture device according to Embodiment 2 of the present invention, and FIG. 5B is a cross-sectional view taken along line BB in FIG. 5A. FIG. 6 is a diagram showing an example of cell culture using the cell culture device according to the second embodiment. FIG. 7A is a cross-sectional view showing the state of the cell culture device when the opening of the box body is upward, and FIG. 7B is a cross-sectional view showing the state of the cell culture device when the opening of the box body is downward. It is a diagram. FIG. 8 is a graph showing the experimental results of Example 1. 9A to 9C are cross-sectional views for explaining the culture conditions of Example 2. FIG. 10 is a graph showing the experimental results of Example 2.

Embodiments of the present invention will be described below with reference to the drawings.

[Embodiment 1]
(Configuration of cell culture device)
FIG. 1 is a perspective view of a cell culture device 100 according to Embodiment 1 of the present invention. 2A is a plan view of the cell culture device 100, and FIG. 2B is a cross-sectional view taken along line AA in FIG. 2A.

As shown in FIGS. 1 to 2B, the cell culture device 100 according to the present embodiment includes a box body 110, a movable lid body 120, and a fall prevention part 130. As shown in FIG. 2B, the cell culture device 100 is used with a culture substrate 140 on which cells are fixed between the bottom plate 111 of the box 110 and the movable lid 120.

The box 110 is a container having an opening for accommodating a culture substrate 140 on which cells are fixed. In this embodiment, the box 110 has a bottom plate 111, a side plate 112, and an opening 113. The shape and size of the box 110 are not particularly limited as long as the above functions can be achieved. For example, the box 110 has a substantially rectangular shape in plan view, measuring approximately 8 to 10 cm in height and 3 to 5 cm in width. From the perspective of allowing the movable lid 120 to move freely in the depth direction of the box 110 within the box 110, the depth of the housing section (inside) of the box 110 is determined by the culture substrate 140 and the movable lid. It is preferable that the thickness be somewhat larger than the total thickness of the body 120.

The material constituting the box 110 is not particularly limited as long as it does not adversely affect cell culture and has the necessary strength, but it is preferably one that can be sterilized using an autoclave or the like. Examples of materials constituting the box 110 include metals such as titanium, titanium nitride, titanium alloys, and stainless steel, resins such as polypropylene, polystyrene, and polyethylene, and ceramics. In this embodiment, box body 110 and movable lid body 120 are made of titanium.

From the viewpoint of allowing oxygen and nutrients to reach the cells in the culture substrate 140 and from the viewpoint of making it easier to move the movable lid 120 in the liquid medium, at least one of the box 110 and the movable lid 120 has a through hole. It is preferable that the porous body has a porous body, and it is more preferable that at least a part thereof is a porous body. For example, it is preferable that at least one of the bottom plate 111 of the box body 110 and the movable lid body 120 is a porous body, and it is more preferable that both the bottom plate 111 of the box body 110 and the movable lid body 120 are porous bodies. In this embodiment, the bottom plate 111 of the box body 110 is made of a porous titanium nonwoven fabric (for example, Zellez of Hilex Corporation, titanium fiber membrane of Osaka Yakin Kogyo Co., Ltd.), and the side plate 112 is composed of a titanium plate. The thickness of the bottom plate 111 of the box body 110 is not particularly limited, but is, for example, about 0.5 to 1 mm.

Further, the inner surface of the box 110 that may come into contact with cells may be coated to promote cell proliferation and cell adhesion. The coating material is not particularly limited, and can be appropriately selected depending on the type of cells to be cultured. Examples of coating materials include proteins constituting extracellular matrix such as collagen, elastin, fibronectin, laminin, and vitronectin, and amino acid polymers such as polylysine and polyornithine.
In this embodiment, the bottom plate 111 of the box body 110 is coated with collagen.

The movable lid 120 is a lid placed inside the box 110 so as to cover the culture substrate 140 on which cells are fixed. The movable lid 120 is arranged so that it can freely move in the depth direction of the box 110 within the box 110 by receiving a force such as gravity. As will be explained later, the movement of the movable lid body 120 applies mechanical stimulation such as pressure and shear stress to the cells fixed on the culture substrate 140.

The shape and size of the movable lid 120 are not particularly limited as long as they can perform the above functions. In this embodiment, movable lid body 120 is plate-shaped. From the perspective of allowing the movable lid 120 to move freely in the depth direction of the box 110 within the box 110, the horizontal size of the movable lid 120 (the vertical and horizontal dimensions in FIG. 2A) (a) is preferably smaller than the horizontal size of the accommodating portion of the box body 110 by some margin. In addition, from the viewpoint of applying mechanical stimulation to cells fixed on the culture substrate 140, it is preferable that the horizontal size of the movable lid body 120 is larger than the horizontal size of the culture substrate 140 with some margin, It is preferable that the movable lid body 120 is configured to have a certain amount of weight. The thickness of the movable lid 120 is adjusted as appropriate depending on the mechanical stimulus to be applied to the cells fixed on the culture substrate 140, and may be thicker than the bottom plate 111 of the box 110.

The material constituting the movable lid body 120 is not particularly limited as long as it does not adversely affect cell culture and has the necessary strength, but it is preferably one that can be sterilized using an autoclave or the like. Examples of materials constituting the movable lid body 120 include metals such as titanium, titanium nitride, titanium alloys, and stainless steel, resins such as polypropylene, polystyrene, and polyethylene, and ceramics. As described above, in this embodiment, the box body 110 and the movable lid body 120 are made of titanium.

As described above, from the viewpoint of allowing oxygen and nutrients to reach the cells in the culture substrate 140 and from the viewpoint of making it easier to move the movable lid 120 in the liquid medium, at least one of the box 110 and the movable lid 120 is , preferably has a through hole, and more preferably at least a portion thereof is a porous body. For example, it is preferable that at least one of the bottom plate 111 of the box body 110 and the movable lid body 120 is a porous body, and it is more preferable that both the bottom plate 111 of the box body 110 and the movable lid body 120 are porous bodies. In the present embodiment, the movable lid body 120 is made of a porous titanium nonwoven fabric (for example, Zellez manufactured by Hilex Corporation, titanium fiber membrane manufactured by Osaka Yakin Kogyo Co., Ltd., etc.).

Furthermore, the movable lid body 120 that may come into contact with cells may be coated to promote cell proliferation and cell adhesion. The coating material is not particularly limited, and can be appropriately selected depending on the type of cells to be cultured. Examples of coating materials include proteins constituting extracellular matrix such as collagen, elastin, fibronectin, laminin, and vitronectin, and amino acid polymers such as polylysine and polyornithine.
In this embodiment, movable lid body 120 is coated with collagen.

The fall prevention part 130 is a member for preventing the movable lid 120 from falling off to the outside of the box body 110. The configuration of the drop-off prevention section 130 is not particularly limited as long as it can perform the above function. In this embodiment, the drop-off prevention part 130 is two rods detachably attached to the vicinity of the opening 113 of the box body 110. In FIGS. 1 to 2B, four through holes are provided in the side plate 112 of the box body 110, but four through holes may be provided at different heights, for example. By doing so, it becomes possible to adjust the movable range of the movable lid body 120 by changing the heights of the two rods that function as the falling-off prevention part 130.

The culture substrate 140 is a scaffold for culturing cells to proliferate or form tissues or organs. As described above, the culture substrate 140 is arranged between the bottom plate 111 of the box 110 and the movable lid 120. Similarly to the movable lid 120, the culture substrate 140 is also shaped like a box inside the box 110 by receiving a force such as gravity (for example, a change in the direction of gravity due to the rotation of the lidded test tube 300, which will be described later). 110 so as to be freely movable in the depth direction.

The type, shape, and size of the culture substrate 140 are not particularly limited, and can be appropriately selected depending on the type of cells to be cultured from a known three-dimensional culture substrate. Examples of the culture substrate 140 include a collagen fiber membrane (collagen gel), a collagen sponge (for example, Koken Co., Ltd.'s collagen sponge, collagen sponge honeycomb, etc.), a titanium nonwoven fabric (for example, Hilex Corporation's Zellez, Osaka These include titanium fiber membranes from Yakin Kogyo Co., Ltd.), resin scaffolds (e.g. 3D Insert-PS and 3D Insert-PCL from 3D Biotek, and NanoECM and NanoHep from Nanofiber solutions). In this embodiment, culture substrate 140 is a plate-shaped collagen fiber membrane containing cells. Moreover, one culture substrate 140 may be arranged between the bottom plate 111 of the box 110 and the movable lid 120, or a plurality of culture substrates 140 may be arranged.

When culturing cells using the cell culture device 100 according to the present embodiment, the cell culture device 100 is arranged such that the vertical position of the bottom plate 111 of the box body 110 and the movable lid body 120 is alternately changed. be rotated. The cell culture device 100 is rotated while being immersed in the liquid medium.

3A to 3C are diagrams showing an example of cell culture using the cell culture device 100. In this example, the cell culture device 100 is housed in a test tube 300 with a lid. 3A and 3B are views of the rotating lidded test tube 300 and the cell culture device 100 seen from the side of the lidded test tube 300, and FIG. 3C is a cross-sectional view taken along the line CC in FIG. 3B. FIG. The cell culture device 100 is fixed within a test tube 300 with a lid, and by rotating the test tube 300 with a lid, the cell culture device 100 also rotates. The method of installing the lidded test tube 300 is not particularly limited. For example, for short-term culture of about 1 to 2 weeks, cover the opening of the test tube (BT-30, Nichiden Rika Glass Co., Ltd.) with an open aluminum cap (NE-30, Nichiden Rika Glass Co., Ltd.). The test tube may be placed at an angle of about 20 to 30 degrees and rotated. This makes it possible to stably secure the gas phase (5% CO ₂ ) necessary for the culture medium. On the other hand, for long-term culture, the test tube 300 with a lid includes a tube 310 for supplying a medium that has been equilibrated with a gas phase of 5% CO ₂ in advance, and a tube 310 for discharging the liquid medium in the test tube 300 with a lid. The tube 310 may be connected. As a result, a fresh liquid medium is always supplied into the lidded test tube 300, and cells can be continuously and automatically cultured over a long period of time, such as from several days to several months. From the viewpoint of efficiently replacing the liquid medium in the lidded test tube 300 with a new one, the tip of one of the two tubes 310 is placed near the bottom of the test tube body of the lidded test tube 300. However, it is preferable to arrange the tip of the other tube 310 near the opening of the test tube body.

The type of lidded test tube 300 is not particularly limited, and is appropriately selected depending on the configuration of the cell culture device 100, the type of cells to be cultured, and the like. The test tube 300 with a lid may be made of glass, resin, or metal. Furthermore, the method of fixing the cell culture device 100 is not particularly limited. For example, as shown in FIG. 3C, by adjusting the size of the cell culture device 100 so that it can be accommodated within the lidded test tube 300 (so that it is inscribed at multiple locations when viewed in cross section), The cell culture device 100 can be fixed to the lidded test tube 300 without using a separate fixing device. The type of tube 310 is also not particularly limited, and is appropriately selected depending on the type of liquid medium and the like. For example, the tube 310 is a silicone tube with an inner diameter of about 0.3 to 0.7 mm. The type of liquid medium is also not particularly limited, and is appropriately selected depending on the type of cells to be cultured. The liquid medium is supplied at a suitable temperature, equilibrated with a gas phase (eg, 37° C., 5% carbon dioxide, 95% air). The liquid culture medium can be supplied manually, but if a large amount of culture medium is to be supplied over a long period of time, it is preferable to use a pump or the like. It is particularly preferred to rotate.

The method of rotating the lidded test tube 300 containing the cell culture device 100 is not particularly limited. Further, the angle of the lidded test tube 300 when rotating the lidded test tube 300 is not particularly limited, and may be adjusted as appropriate within the range of 0 to 45 degrees with respect to the horizontal plane, for example. Further, the rotation direction and rotation speed are not particularly limited, and may be adjusted as appropriate. For example, the lidded test tube 300 containing the cell culture device 100 may be rotated clockwise or counterclockwise continuously or intermittently, or may be rotated alternately clockwise and counterclockwise. (alternate rotation method). Furthermore, if the vertical positions of the bottom plate 111 of the box 110 and the movable lid 120 are alternately changed, the test tube 300 with a lid containing the cell culture device 100 does not need to be rotated 360 degrees. For example, it may be rotated by 180 degrees. Moreover, the test tube 300 with a lid containing the cell culture device 100 may be continuously rotated using the anti-gravity culture apparatus described in Patent Document 1.

(Cell culture method)
The cell culture method according to the present embodiment includes (1) a step of accommodating a culture substrate 140 on which cells are fixed in a cell culture device 100, and (2) a step of culturing cells while rotating the cell culture device 100. and has. Each step will be explained below.

First, the cell culture device 100 houses the culture substrate 140 on which cells are fixed. More specifically, a culture substrate 140 on which cells are fixed is housed between the bottom plate 111 of the box 110 of the cell culture device 100 and the movable lid 120.

The type of cells to be cultured is not particularly limited and can be appropriately selected depending on the purpose. Examples of cells to be cultured include iPS cells, pluripotent stem cells such as ES cells, Muse cells, and DFAT cells, and various differentiated cells. The type of culture substrate 140 is appropriately selected depending on the type of cells and the like.

For example, a mixture of a suspension of pluripotent stem cells (eg, DFAT cells) at a concentration of 1×10 ⁶ cells/mL and a collagen solution is fibroticized and stabilized by standing for one day. Thereby, a collagen fiber membrane (culture substrate 140) in which pluripotent stem cells are embedded can be obtained. Further, the titanium nonwoven fabric serving as the bottom plate 111 of the box body 110 and the titanium nonwoven fabric serving as the movable lid body 120 are impregnated and coated with a collagen solution that does not contain cells. Thereby, a titanium nonwoven fabric coated with collagen can be obtained as the bottom plate 111 and the movable lid 120. Then, a collagen fiber membrane (culture substrate 140) in which pluripotent stem cells are embedded is placed on the bottom plate 111, and a movable lid 120 is placed on top of the collagen fiber membrane, and the movable lid 120 is connected to the fall prevention part 130. (See Figures 2A and 2B). The assembled cell culture device 100 is placed in a lidded test tube 300 filled with a liquid medium (see FIGS. 3A to 3C).

Next, cells are cultured while rotating the cell culture device 100 containing the culture substrate 140 (see FIGS. 3A to 3C).

FIG. 4A is a sectional view showing the state of the cell culture device 100 when the opening 113 of the box 110 is facing upward (vertically upward), and FIG. FIG. When the opening 113 is upward, the culture substrate 140 is placed on the bottom plate 111 and is pressed by the movable lid 120, as shown in FIG. 4A. On the other hand, when the opening 113 is at the bottom, the culture substrate 140 is placed on the movable lid 120, but is not pressed by the bottom plate 111, as shown in FIG. 4B. When the cell culture device 100 is rotated, the state shown in FIG. 4A and the state shown in FIG. , will be intermittently pressed by the movable lid 120. Therefore, pressure or shear stress is intermittently applied to the cells in the culture substrate 140 as well. Furthermore, the direction of gravity relative to cells also changes periodically. As a result, cell proliferation, cell growth, tissue formation, and organ formation can be promoted.

(effect)
As described above, by using the cell culture device 100 according to the present embodiment, cells can be cultured while intermittently applying pressure or shear stress to the cells as mechanical stimulation. As a result, cell proliferation, cell growth, tissue formation, and organ formation can be promoted.

[Embodiment 2]
(Configuration of cell culture device)
5A is a plan view of a cell culture device 200 according to Embodiment 2 of the present invention, and FIG. 5B is a cross-sectional view taken along line BB in FIG. 5A.

As shown in FIGS. 5A and 5B, cell culture device 200 according to this embodiment includes a box 110, a movable lid 120, an elastic member 210, and a spacer 220. As shown in FIG. 5B, the cell culture device 200 is used with the culture substrate 140 on which cells are fixed placed between the bottom plate 111 of the box 110 and the movable lid 120.

The cell culture device 200 according to the second embodiment differs from the cell culture device 100 according to the first embodiment in that it includes an elastic member 210 and a spacer 220 instead of the falling-off prevention part 130. Components that are the same as those of the cell culture device 100 according to Embodiment 1 are designated by the same reference numerals, and the description thereof will be omitted.

The elastic member 210 presses the movable lid 120 toward the culture substrate 140 housed in the box 110. The elastic member 210 facilitates the movable lid 120 to press the culture substrate 140 when the opening 113 of the box 110 is located upward (vertically upward), and when the opening 113 of the box 110 is located downward. (vertically downward), it functions as a drop-off prevention part to prevent the movable lid 120 from coming off from the box body 110, and also controls the free fall of the movable lid 120. As will be described later, in this embodiment, the elastic member 210 presses the movable lid 120 while being disposed between the movable lid 120 and the side wall of the test tube body of the lidded test tube 300. (See Figure 6). The elasticity of the elastic member 210 is not particularly limited as long as it can perform the above function, but it is necessary to prevent the movable lid 120 from pressing the culture substrate 140 when the opening 113 of the box 110 is located downward (vertically downward). Preferably, it is adjusted (see FIG. 7B).

The configuration of the elastic member 210 is not particularly limited as long as it can perform the above function. Examples of the elastic member 210 include springs such as coil springs, leaf springs, and filigree springs. The material of the elastic member 210 is also not particularly limited, and includes, for example, metal, rubber, resin, ceramics, and the like. In this embodiment, elastic member 210 is a metal coil spring. The number of elastic members 210 is also not particularly limited as long as the above functions can be achieved. In this embodiment, cell culture device 200 has two elastic members 210.

The spacer 220 is arranged between the bottom plate 111 of the box body 110 and the movable lid 120, and when the elastic member 210 is pressing the movable lid 120, the spacer 220 is provided between the bottom plate 111 and the movable lid 120. This prevents the movable lid body 120 from tilting excessively due to the existence of a space where the base material 140 is not placed. The thickness of the spacer 220 is not particularly limited as long as it does not prevent the movable lid body 120 from pressing the culture substrate 140 when the opening 113 of the box body 110 is located upward (vertically upward) and can exhibit the above function. . The thickness of the spacer 220 is preferably slightly thinner than the thickness of the culture substrate 140, for example, preferably about 50 to 95% of the thickness of the culture substrate 140.

The configuration of the spacer 220 is not particularly limited as long as it can perform the above function. The material of the spacer 220 is also not particularly limited, and includes, for example, metal, rubber, resin, ceramics, and the like. In this embodiment, the spacer 220 is a rectangular metal plate bent into a U-shape. The number of spacers 220 is also not particularly limited as long as it can perform the above function. In this embodiment, cell culture device 200 has four spacers 220.

The culture substrate 140 has been described in Embodiment 1, so a description thereof will be omitted. As described above, one culture substrate 140 or a plurality of culture substrates 140 may be arranged between the bottom plate 111 of the box 110 and the movable lid 120. In this embodiment, a plurality of culture substrates 140 are arranged between the bottom plate 111 and the movable lid 120.

When culturing cells using the cell culture device 200 according to the present embodiment, the cell culture device 200 is arranged such that the vertical position of the bottom plate 111 of the box body 110 and the movable lid body 120 is alternately changed. be rotated. The cell culture device 200 is rotated while being immersed in the liquid medium.

FIG. 6 is a diagram showing an example of cell culture using the cell culture device 200. In this example, the cell culture device 200 is a test tube with a lid 300 (for example, a test tube covered with an open aluminum cap (NE-30, Nichiden Rika Glass Co., Ltd.) (BT-30, Nichiden Rika Glass Co., Ltd.) ), and the test tube 300 with a lid is installed on a rotating culture stand at an angle of about 20 to 30 degrees. The cell culture device 200 is fixed in a test tube 300 with a lid, and by rotating the test tube 300 with a lid, the cell culture device 200 also rotates.

Similar to Embodiment 1, the method for fixing cell culture device 200 is not particularly limited. For example, by adjusting the size of the cell culture device 200 so that it can be accommodated in the lidded test tube 300 (so that it is inscribed at multiple locations when viewed in cross section), cells can be grown without using a separate fixing device. The culture device 200 can be fixed to a test tube 300 with a lid. Moreover, similarly to Embodiment 1, the method of rotating the lidded test tube 300 containing the cell culture device 200 is not particularly limited.

(Cell culture method)
Similar to Embodiment 1, the cell culture method according to the present embodiment includes (1) storing the culture substrate 140 on which cells are fixed in the cell culture device 200; and (2) storing the cell culture device 200. and a step of culturing the cells while rotating. Each step will be explained below.

First, the cell culture device 200 houses the culture substrate 140 on which cells are fixed. More specifically, a culture substrate 140 on which cells are fixed is housed between the bottom plate 111 of the box 110 of the cell culture device 200 and the movable lid 120.

For example, a collagen solution is fibrillated and left to stand for one day to stabilize it. The resulting collagen gel is impregnated with a suspension of pluripotent stem cells (eg, DFAT cells) at a concentration of 3×10 ⁵ cells/mL, and left to stand for one day to allow the cells to settle. Thereby, a collagen fiber membrane (culture substrate 140) with pluripotent stem cells fixed thereon can be obtained. Further, the titanium nonwoven fabric as the bottom plate 111 of the box body 110 and the titanium nonwoven fabric as the movable lid body 120 are also impregnated with the collagen solution and coated. Thereby, a titanium nonwoven fabric coated with collagen can be obtained as the bottom plate 111 and the movable lid 120. Then, a collagen fiber membrane (culture substrate 140) on which pluripotent stem cells have settled is placed on the bottom plate 111, and a movable lid 120 is placed on top of it, and the elastic member 210 prevents the movable lid 120 from falling off. (See Figures 5A and 5B). The assembled cell culture device 200 is placed in a lidded test tube 300 filled with a liquid medium (see FIG. 6).

Next, cells are cultured while rotating the cell culture device 200 containing the culture substrate 140 (see FIG. 6).

FIG. 7A is a sectional view showing the state of the cell culture device 200 when the opening 113 of the box 110 is located upward (vertically upward), and FIG. FIG. When the opening 113 is upward, the culture substrate 140 is placed on the bottom plate 111 and is pressed by the movable lid 120, as shown in FIG. 7A. At this time, the culture substrate 140 is pressed not only by the weight of the movable lid 120 but also by the pressing force by the elastic member 210. On the other hand, when the opening 113 is at the bottom, the culture substrate 140 is placed on the movable lid 120, but is not pressed by the bottom plate 111, as shown in FIG. 7B. At this time, since the movable lid 120 is supported by the elastic member 210 instead of the fall prevention part 130 (see Embodiment 1) fixed to the box 110, the movable range of the movable lid 120 is absolutely limited. not limited to a certain range. For example, even if cells proliferate and the thickness of the complex between the culture substrate 140 and cells increases, the movable lid body 120 moves to a position corresponding to the thickness of the complex in the state shown in FIG. 7B. Little pressure can be applied to the composite. When the cell culture device 200 is rotated, the state shown in FIG. 7A and the state shown in FIG. , will be intermittently pressed by the movable lid 120. Therefore, pressure or shear stress is intermittently applied to the cells in the culture substrate 140 as well. Furthermore, the direction of gravity relative to cells also changes periodically. As a result, cell proliferation, cell growth, tissue formation, and organ formation can be promoted.

(effect)
As described above, by using the cell culture device 200 according to the present embodiment, similarly to the cell culture device 100 according to the first embodiment, it is possible to apply pressure or shear stress to cells as mechanical stimulation intermittently. Cells can be cultured. As a result, cell proliferation, cell growth, tissue formation, and organ formation can be promoted. Furthermore, in the cell culture device 200 according to the present embodiment, the movable lid body 120 is supported by the elastic member 210, so even if cells proliferate and the thickness of the culture substrate 140 and cell complex increases. , the pressure or shear stress applied to the cells can be applied intermittently rather than continuously.

The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited by these examples.

[Example 1]
(1) Preparation of culture substrate A plurality of fibrous collagen membranes (FCM) were prepared using collagen extracted from calf skin by the method described in Non-Patent Document 1. The size of each collagen fiber membrane was 5 mm long x 5 mm wide x 2 mm thick. In this example, these collagen fiber membranes were used as culture substrates.
[Non-Patent Document 1] Yoshinori Kuboki, et al., "Regeneration of periodontal ligament and cementum by BMP-applied tissue engineering", European Journal of Oral Sciences, Vol. 106, pp. 197-203.

A method for preparing a collagen fiber membrane will be briefly explained. Collagen extracted from calf skin was treated with pepsin to obtain an acidic collagen solution. Collagen fibers were obtained by dialyzing this acidic collagen solution using a weakly alkaline solution. This suspension containing collagen fibers was put into a stainless steel mold measuring 10 cm long x 10 cm wide x 3 cm deep and freeze-dried, cross-linked with hexamethylene diisocyanate, washed and freeze-dried to form a collagen fiber membrane. I got it. Finally, the collagen fiber membrane was pressed to a thickness of 2 mm, and cut into a size of 5 mm in length x 5 mm in width.

Each collagen fiber membrane containing a minimum amount of liquid medium was impregnated with a liquid medium containing 3 x 10 ⁵ DFAT cells, and the cells were allowed to settle for 24 hours. The collagen fiber membrane swelled to more than double its thickness.

(2) Preparation of cell culture device Two sets of the cell culture device and lidded test tube according to Embodiment 2 were prepared (see FIGS. 5A to 6). A titanium plate with a thickness of 0.1 mm was processed into a substantially rectangular tube shape with a length of 55 mm, a width of 26 mm, and a height of 10 mm to form the side plate of the box. Further, a titanium nonwoven fabric measuring 55 mm long, 26 mm wide, and 1 mm thick was used as the bottom plate of the box, and a titanium nonwoven fabric measuring 50 mm long, 23 mm wide, and 1.5 mm thick was used as the movable lid. These two titanium nonwoven fabrics were coated with collagen by impregnating them with the above acidic collagen solution. A coil spring was used as the elastic member. These two coil springs can support the movable lid without deforming if it is only the movable lid, and when 10 collagen fiber membranes are added to the movable lid, they can deform and support the movable lid. It has elasticity. As the spacer, a rectangular metal plate bent into a U-shape was used. The thickness of the spacer was 3 mm. A glass culture tube with a depth of 120 mm, an outer diameter of 30 mm, and a thickness of 0.6 mm was used as the test tube body of the lidded test tube.

(3) Cell culture In each of the two cell culture devices prepared in (2) above, between the bottom plate of the box and the movable lid body, 10 sheets of collagen on which the DFAT cells prepared in (1) above were fixed were placed. The fibrous membranes were arranged so that they did not overlap each other. The cell culture device containing the collagen fiber membrane was placed in a lidded test tube containing 40 mL of liquid medium, and two coil springs were further placed between the movable lid and the lidded test tube.

For one of the two cell culture devices (Example), the test tube with a lid in which the cell culture device was installed was tilted at approximately 20 to 30 degrees and rotated alternately 240 times per hour. The cells were cultured for 7 days at 37° C. in a gas phase of 5% carbon dioxide and 95% air while rotating the cells at a rate of 4 times per minute (see FIG. 6). No medium exchange was performed. Regarding the other cell culture device (comparative example), the test tube with a lid in which the cell culture device was installed was tilted at about 20 to 30 degrees and left standing with the opening of the box upwards. The cells were cultured for 7 days at ℃ in a gas phase of 5% carbon dioxide and 95% air. This control experiment provided a comparative example lacking all of the dynamic elements that are the core of a cell culture device. This comparative example should clearly demonstrate the effects of the dynamic elements of the cell culture device according to the present invention, namely the pressure fluctuation effect and the shear stress effect due to medium flow.

(4) Results After culturing for 7 days, 10 collagen fiber membranes were taken out from each cell culture device and placed one in each well of a 24-well plate. 0.4 mL of liquid medium and 0.04 mL of Cell Counting Kit-8 (CCK-8) reagent solution were added to each well, and after culturing for 90 minutes, the absorbance at 450 nm was measured for each well. The measured absorbance is proportional to the number of viable cells.

Figure 8 shows the number of cells (absorbance) when cultured while rotating a test tube with a lid (example) and the number of cells (absorbance) when cultured without rotating a test tube with a lid (comparative example). ). As shown in this graph, in the cell culture method according to the example, cells proliferated 6 times more than in the cell culture method according to the comparative example.

[Example 2]
(1) Preparation of culture substrate As a culture substrate, 30 collagen sponges (Honeycomb Disc 96, Koken Co., Ltd.) with a diameter of 6 mm and a thickness of 2 mm were prepared.

Each collagen sponge containing a minimum amount of liquid medium was impregnated with a liquid medium containing 3 x 10 ⁵ DFAT cells, and allowed to stand for 24 hours to allow the cells to settle. The collagen sponge swelled to more than double its thickness.

(2) Preparation of cell culture device Three sets of the cell culture device and lidded test tube according to Embodiment 1 were prepared (see FIGS. 1 to 4B). In the same manner as in Example 1, a box board and a movable lid were produced. Four through holes were formed in the side plate of the box, and two titanium rods were inserted as drop-off prevention parts. In one of the three sets, the movable lid and culture substrate were moved away from the bottom plate of the box plate so that they could be moved by rotating the cell culture device (so that the distance between the bottom plate and the movable lid changed). Four through holes were formed at the same positions (Example: see FIG. 9A). In the remaining two sets, the movable lid and culture substrate are placed close to the bottom plate of the box plate so that they do not move even when the cell culture device is rotated (so that the distance between the bottom plate and the movable lid does not change). Four through holes were formed (comparative example: see FIGS. 9B and 9C). Moreover, like the cell culture device according to Embodiment 2, a plurality of spacers were arranged between the bottom plate of the box and the movable lid. As the spacer, a rectangular metal plate bent into a U-shape was used. The thickness of the spacer was 3 mm. A glass culture tube with a depth of 120 mm, an outer diameter of 30 mm, and a thickness of 0.6 mm was used as the test tube body of the lidded test tube.

(3) Cell culture In each of the three cell culture devices prepared in (2) above, 10 sheets of collagen on which the DFAT cells prepared in (1) above were fixed were placed between the bottom plate of the box and the movable lid. The sponges were arranged so that they did not overlap each other, and two titanium rods were inserted to prevent the movable lid from falling off. The cell culture device containing the collagen sponge was placed in a lidded test tube containing 40 mL of liquid medium.

Among the three cell culture devices, for one cell culture device (example) in which the movable lid body and culture substrate can be moved, the test tube with the lid in which the cell culture device is installed is tilted approximately 20 to 30 degrees. The cells were cultured for 7 days at 37° C. in a gas phase of 5% carbon dioxide and 95% air while rotating alternately 240 times per hour (4 reciprocations per minute) (see FIG. 9A). No medium exchange was performed.

Regarding another cell culture device (Comparative Example 1) in which the movable lid and the culture substrate cannot be moved, the test tube with the lid in which the cell culture device is installed is tilted at approximately 20 to 30 degrees, and The cells were cultured for 7 days at 37° C. in a gas phase of 5% carbon dioxide and 95% air while rotating the cells alternately (four reciprocations per minute) (see FIG. 9B). No medium exchange was performed.

For the last cell culture device (Comparative Example 2) in which the movable lid and culture substrate cannot be moved, the test tube with the lid in which the cell culture device is installed is tilted at approximately 20 to 30 degrees, and the opening of the box is The cells were left standing in a state where the cell was placed above, and cultured for 7 days at 37° C. in a gas phase of 5% carbon dioxide and 95% air. No medium exchange was performed.

(4) Results After culturing for 7 days, 10 collagen sponges were taken out from each cell culture device, and the absorbance was measured in the same manner as in Example 1. The measured absorbance is proportional to the number of living cells.

Figure 10 shows the number of cells (absorbance) when cultured while rotating a cell culture device in which a movable lid and culture substrate can be moved (example) and a cell culture device in which a movable lid and culture substrate cannot be moved. Graph showing the number of cells (absorbance) when cultured while rotating (Comparative Example 1) and when culturing without rotating a cell culture device in which the movable lid and culture substrate cannot be moved (Comparative Example 2) It is. As shown in this graph, in the cell culture method according to the example, cells proliferated 3 times more than in the cell culture method according to Comparative Example 1, and 13 times more than in the cell culture method according to Comparative Example 2. This shows that cell proliferation can be further promoted by not only rotating the cell culture device but also applying mechanical stimulation by moving the movable lid and/or the culture substrate.

The cell culture device and cell culture method according to the present invention are useful, for example, in the formation of organs or tissues in regenerative medicine. Furthermore, the cell culture device and cell culture method according to the present invention are also useful for producing artificially cultured meat in response to the soon-to-be-seen global food crisis, particularly in response to difficulties in supplying edible meat.

100, 200 Cell culture device 110 Box 111 Bottom plate 112 Side plate 113 Opening 120 Movable lid 130 Falling prevention part 140 Culture substrate 210 Elastic member 220 Spacer 300 Test tube with lid 310 Tube

Claims (7)

A box body for accommodating a culture substrate on which cells are fixed;
a movable lid body disposed within the box body so as to be movable in the depth direction of the box body;
a fall prevention part for preventing the movable lid from falling out of the box;
has
The culture substrate is arranged between the bottom plate of the box and the movable lid,
Cell culture device. A box body for accommodating a culture substrate on which cells are fixed;
a movable lid body disposed within the box body so as to be movable in the depth direction of the box body;
an elastic member for pressing the movable lid toward the culture substrate housed in the box ;
has
The culture substrate is arranged between the bottom plate of the box and the movable lid,
Cell culture device. The cell culture device according to claim 2 , further comprising a spacer arranged between the bottom plate and the movable lid. The cell culture device according to claim 1 or 2 , wherein at least one of the bottom plate and the movable lid is a porous body. The cell culture device according to claim 1 or 2 , wherein the box body and the movable lid body are made of titanium. The cell culture device according to claim 1 or 2 , wherein the bottom plate and the movable lid are made of titanium nonwoven fabric. A step of accommodating a culture substrate on which cells are fixed between the bottom plate of the box and the movable lid of the cell culture device according to claim 1 or 2 ;
culturing cells while rotating the cell culture device containing the culture substrate;
A cell culture method comprising:

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