JP2024089917A - Cell culture device and cell culture method - Google Patents

Abstract

The present invention provides a cell culture device that can promote cell proliferation by applying a mechanical stimulus with a simple configuration.
The 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. The culture substrate is disposed between a bottom plate of the box and the movable lid.
[Selected Figure] Figure 1

Description

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

Regenerative medicine aims to regenerate damaged organs and tissues using stem cells and other substances, restoring lost functions. There are various types of regenerative medicine, one of which 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 inventors believe that in order to form tissues outside the body in the same way as in the body, not only (1) cells, (2) three-dimensional culture substrate (corresponding to the extracellular matrix), (3) humoral regulatory factors, and (4) a nutrient supply system (corresponding to the vascular system), but also (5) mechanical stimuli are important. Based on this idea, the 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 therefor (see, for example, Patent Documents 1 and 2).

JP 2012-90584 A JP 2014-76026 A

As described above, the inventors (5) discovered that cell proliferation can be promoted by applying gravity in a direction different from normal as a mechanical stimulus, and (5) considered that cell proliferation and tissue formation efficiency could be further improved by applying an additional different mechanical stimulus to the cells.

The present invention was made in consideration of these points, and aims to provide a cell culture device with a simple configuration that can apply mechanical stimulation to promote cell proliferation, and a cell culture method using the same.

The present invention relates to the following cell culture device and cell culture method.
[1] A cell culture device comprising: a box body for containing a culture substrate on which cells have been fixed; and a movable lid body arranged within the box body so as to be movable in the depth direction of the box body, the culture substrate being arranged between a bottom plate of the box body and the movable lid body.
[2] The cell culture device described in [1], further comprising a fall prevention portion for preventing the movable lid body from falling off outside the box body.
[3] The cell culture device described in [1] or [2], further comprising an elastic member for pressing the movable lid body toward the culture substrate contained within the box body.
[4] The cell culture device described in any one of [1] to [3], further comprising a spacer disposed between the bottom plate and the movable lid.
[5] The cell culture device described in any one of [1] to [4], wherein at least one of the bottom plate of the box body and the movable lid body is a porous body.
[6] The cell culture device described in any one of [1] to [5], wherein the box body and the movable lid body are made of titanium.
[7] The cell culture device described in any one of [1] to [6], wherein the bottom plate of the box body and the movable cover body are made of titanium nonwoven fabric.
[8] A cell culture method comprising the steps of: accommodating a culture substrate having cells fixed thereon between a bottom plate of the box body and the movable lid body of the cell culture device described in any one of [1] to [7]; and culturing cells while rotating the cell culture device accommodating 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 a first embodiment 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 of 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 appearance of the cell culture device when the opening of the box is at the top, and FIG. 4B is a cross-sectional view showing the appearance of the cell culture device when the opening of the box is at the bottom. FIG. 5A is a plan view of a cell culture device according to a second embodiment of the present invention, and FIG. 5B is a cross-sectional view taken along line BB of FIG. 5A. FIG. 6 is a diagram showing an example of cell culture using the cell culture device according to the second embodiment. Figure 7A is a cross-sectional view showing the appearance of the cell culture device when the opening of the box is at the top, and Figure 7B is a cross-sectional view showing the appearance of the cell culture device when the opening of the box is at the bottom. 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.

The following describes an embodiment of the present invention with reference to the drawings.

[First embodiment]
(Configuration of cell culture device)
Fig. 1 is a perspective view of a cell culture device 100 according to a first embodiment of the present invention. Fig. 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 Figures 1 to 2B, the cell culture device 100 according to this embodiment has a box body 110, a movable lid body 120, and a fall prevention part 130. As shown in Figure 2B, the cell culture device 100 is used in a state where a culture substrate 140 on which cells have been fixed is placed between the bottom plate 111 of the box body 110 and the movable lid body 120.

The box 110 is a container with an opening for containing the culture substrate 140 on which cells have been fixed. In this embodiment, the box 110 has a bottom plate 111, side plates 112, and an opening 113. The shape and size of the box 110 are not particularly limited as long as it can perform the above-mentioned functions. For example, the shape of the box 110 in a plan view is approximately rectangular, with a length of about 8 to 10 cm and a width of about 3 to 5 cm. From the viewpoint of allowing the movable lid 120 to move freely in the depth direction of the box 110 within the box 110, it is preferable that the depth of the containing section (inside) of the box 110 is somewhat greater than the combined thickness of the culture substrate 140 and the movable lid 120.

The material constituting the box body 110 is not particularly limited as long as it does not adversely affect cell culture and has the necessary strength, but it is preferable that it can be sterilized by autoclave or the like. Examples of materials constituting the box body 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, the box body 110 and the 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 facilitating the movement of the movable lid 120 in the liquid medium, it is preferable that at least one of the box 110 and the movable lid 120 has a through hole, and more preferably, at least a part of the through hole is a porous body. For example, it is preferable that at least one of the bottom plate 111 of the box 110 and the movable lid 120 is a porous body, and more preferably, both the bottom plate 111 of the box 110 and the movable lid 120 are porous bodies. In this embodiment, the bottom plate 111 of the box 110 is made of a porous titanium nonwoven fabric (for example, Zellez by Hi-Lex Corporation or titanium fiber membrane by Osaka Yakin Kogyo Co., Ltd.), and the side plate 112 is made of a titanium plate. The thickness of the bottom plate 111 of the box 110 is not particularly limited, but is, for example, about 0.5 to 1 mm.

In addition, 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 material of the coating is not particularly limited and may be appropriately selected depending on the type of cells to be cultured. Examples of the coating material include proteins that constitute the 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 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 the cells have been fixed. The movable lid 120 is placed so that it can move freely in the depth direction of the box 110 when subjected to forces such as gravity. As will be described later, the movement of the movable lid 120 applies mechanical stimuli such as pressure and shear stress to the cells fixed to the culture substrate 140.

The shape and size of the movable lid 120 are not particularly limited as long as the above-mentioned functions can be performed. In this embodiment, the movable lid 120 is plate-shaped. From the viewpoint of allowing the movable lid 120 to move freely in the depth direction of the box 110 within the box 110, it is preferable that the horizontal size of the movable lid 120 (the vertical and horizontal sizes in FIG. 2A) is smaller than the horizontal size of the storage section of the box 110 with a certain margin. In addition, from the viewpoint of applying a mechanical stimulus to the cells settled on the culture substrate 140, it is preferable that the horizontal size of the movable lid 120 is larger than the horizontal size of the culture substrate 140 with a certain margin, and it is preferable that the movable lid 120 is configured to have a certain weight. The thickness of the movable lid 120 is appropriately adjusted according to the mechanical stimulus to be applied to the cells settled on the culture substrate 140, and may be thicker than the bottom plate 111 of the box 110.

The material constituting the movable lid 120 is not particularly limited as long as it does not adversely affect cell culture and has the necessary strength, but it is preferable that it can be sterilized by autoclave or the like. Examples of materials constituting the movable lid 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 110 and the movable lid 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 facilitating the movement of the movable lid 120 in the liquid medium, it is preferable that at least one of the box 110 and the movable lid 120 has a through hole, and more preferably, at least a part of the through hole is a porous body. For example, it is preferable that at least one of the bottom plate 111 of the box 110 and the movable lid 120 is a porous body, and more preferably, both the bottom plate 111 of the box 110 and the movable lid 120 are porous bodies. In this embodiment, the movable lid 120 is made of a porous titanium nonwoven fabric (for example, Zellez from Hi-Lex Corporation or a titanium fiber membrane from Osaka Yakin Kogyo Co., Ltd.).

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

The fall prevention part 130 is a member for preventing the movable lid body 120 from falling out of the box body 110. The configuration of the fall prevention part 130 is not particularly limited as long as it can perform the above-mentioned function. In the present embodiment, the fall prevention part 130 is two rods that are detachably attached near the opening 113 of the box body 110. In Figures 1 to 2B, four through holes are provided in the side plate 112 of the box body 110, but for example, four through holes may be provided at different heights. In this way, the height of the two rods that function as the fall prevention part 130 can be changed to adjust the movable range of the movable lid body 120.

The culture substrate 140 is a scaffold for culturing cells for proliferation or for forming tissues or organs. As described above, the culture substrate 140 is disposed between the bottom plate 111 of the box 110 and the movable lid 120. Like the movable lid 120, the culture substrate 140 is disposed so that it can freely move in the depth direction of the box 110 within the box 110 when subjected to a force such as gravity (for example, a change in the direction of gravity associated with the rotation of the lidded test tube 300 described below).

The type, shape and size of the culture substrate 140 are not particularly limited and can be appropriately selected from a known three-dimensional culture substrate according to the type of cells to be cultured. Examples of the culture substrate 140 include collagen fiber membrane (collagen gel), collagen sponge (e.g., collagen sponge and collagen sponge honeycomb from Koken Co., Ltd.), titanium nonwoven fabric (e.g., Zellez from Hi-Lex Corporation and titanium fiber membrane from Osaka Yakin Kogyo Co., Ltd.), and resin scaffolding materials (e.g., 3D Insert-PS and 3D Insert-PCL from 3D Biotek, NanoECM and NanoHep from Nanofiber solutions). In this embodiment, the culture substrate 140 is a plate-shaped collagen fiber membrane containing cells. In addition, one culture substrate 140 may be arranged between the bottom plate 111 and the movable cover 120 of the box body 110, or multiple culture substrates 140 may be arranged.

When culturing cells using the cell culture device 100 according to this embodiment, the cell culture device 100 is rotated so that the bottom plate 111 of the box body 110 and the movable cover body 120 alternate in the up-down position. The cell culture device 100 is rotated while immersed in a 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 accommodated in a lidded test tube 300. FIGS. 3A and 3B are diagrams showing the rotating lidded test tube 300 and the cell culture device 100 from the side of the lidded test tube 300, and FIG. 3C is a diagram showing a cross section taken along line CC in FIG. 3B. The cell culture device 100 is fixed in the lidded test tube 300, and the cell culture device 100 also rotates when the lidded test tube 300 is rotated. The method of installing the lidded test tube 300 is not particularly limited. For example, in a short-term culture of about 1 to 2 weeks, a test tube (BT-30, Nichiden Rika Glass Co., Ltd.) may be installed in a state in which an aluminum open-type cap (NE-30, Nichiden Rika Glass Co., Ltd.) is placed on the opening of the test tube, and the test tube may be installed in a state inclined at about 20 to 30 degrees and rotated. This allows the gas phase (5% CO ₂ ) required for the culture medium to be stably secured. On the other hand, in long-term culture, a tube 310 for supplying a culture medium previously equilibrated with a gas phase of 5% CO ₂ and a tube 310 for discharging the liquid culture medium in the capped test tube 300 may be connected to the capped test tube 300. This allows fresh liquid culture medium to be constantly supplied to the capped test tube 300, making it possible to continuously and automatically culture cells over a long period of time, such as several days to several months. From the viewpoint of efficiently replacing the liquid culture medium in the capped test tube 300 with a new one, it is preferable to place the tip of one of the two tubes 310 near the bottom of the test tube body of the capped test tube 300 and place the tip of the other tube 310 near the opening of the test tube body.

The type of the lidded test tube 300 is not particularly limited and is appropriately selected depending on the configuration of the cell culture device 100 and the type of cells to be cultured. The lidded test tube 300 may be made of glass, resin, or metal. The method of fixing the cell culture device 100 is also not particularly limited. For example, as shown in FIG. 3C, the size of the cell culture device 100 can be adjusted so that it can be accommodated in the lidded test tube 300 (so that it is in contact with the inside at multiple points when viewed in cross section), and the cell culture device 100 can be fixed to the lidded test tube 300 without using a separate fixing tool. The type of the tube 310 is also not particularly limited and is appropriately selected depending on the type of liquid medium. For example, the tube 310 is a silicone tube with an inner diameter of about 0.3 to 0.7 mm. The type of the 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 an appropriate temperature and in equilibrium with the gas phase (for example, 37° C., 5% carbon dioxide, 95% air). Liquid medium can be supplied manually, but when supplying a large amount of medium over a long period of time, it is preferable to use a pump or the like, and in that case, it is particularly preferable to rotate the lidded test tube 300 using the alternating rotation method described below.

The method of rotating the lidded test tube 300 containing the cell culture device 100 is not particularly limited. The angle of the lidded test tube 300 when rotating the lidded test tube 300 is also not particularly limited, and can be adjusted appropriately within a range of, for example, 0 to 45° with respect to the horizontal plane. The direction and speed of rotation are also not particularly limited, and can be adjusted appropriately. For example, the lidded test tube 300 containing the cell culture device 100 may be rotated continuously or intermittently in a clockwise or counterclockwise direction, or may be rotated alternately clockwise and counterclockwise (alternate rotation method). In addition, as long as the vertical positions of the bottom plate 111 of the box body 110 and the movable lid body 120 are alternately changed, the lidded test tube 300 containing the cell culture device 100 does not need to be rotated 360°, and may be rotated, for example, 180°. In addition, the covered test tube 300 containing the cell culture device 100 may be continuously rotated using the antigravity culture device described in Patent Document 1.

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

First, the culture substrate 140 on which cells have been fixed is placed in the cell culture device 100. More specifically, the culture substrate 140 on which cells have been fixed is placed between the bottom plate 111 and the movable lid 120 of the box body 110 of the cell culture device 100.

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, ES cells, Muse cells, DFAT cells, and other pluripotent stem cells, and various differentiated cells. The type of culture substrate 140 is appropriately selected depending on the type of cells, etc.

For example, a mixture of a suspension of pluripotent stem cells (e.g., DFAT cells) at a concentration of 1×10 ⁶ cells/mL and a collagen solution is fibrousized and left to stand for one day to stabilize. This results in a collagen fiber membrane (culture substrate 140) in which pluripotent stem cells are embedded. Also, the titanium nonwoven fabric as the bottom plate 111 of the box 110 and the titanium nonwoven fabric as the movable lid 120 are impregnated with a collagen solution that does not contain cells and covered. This results in a collagen-coated titanium nonwoven fabric as the bottom plate 111 and the movable lid 120. Then, the collagen fiber membrane (culture substrate 140) in which pluripotent stem cells are embedded is placed on the bottom plate 111, and the movable lid 120 is placed on top of it, and the anti-drop part 130 prevents the movable lid 120 from falling off (see FIGS. 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-C).

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

4A is a cross-sectional view showing the state of the cell culture device 100 when the opening 113 of the box body 110 is upward (vertically upward), and FIG. 4B is a cross-sectional view showing the state of the cell culture device 100 when the opening 113 of the box body 110 is downward (vertically downward). When the opening 113 is upward, as shown in FIG. 4A, the culture substrate 140 is placed on the bottom plate 111 and pressed by the movable lid 120. On the other hand, when the opening 113 is downward, as shown in FIG. 4B, the culture substrate 140 is placed on the movable lid 120 but is not pressed by the bottom plate 111. When the cell culture device 100 is rotated, the state of FIG. 4A and the state of FIG. 4B are alternately repeated, so that the movable lid 120 moves with the change in the direction of gravity, and the culture substrate 140 is intermittently pressed by the movable lid 120. Therefore, pressure or shear stress is applied intermittently to the cells in the culture substrate 140. The direction of gravity on the 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 as a mechanical stimulus to the cells, thereby promoting cell proliferation, cell growth, tissue formation, and organ formation.

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

As shown in Figures 5A and 5B, the cell culture device 200 according to this embodiment has a box body 110, a movable lid body 120, an elastic member 210, and a spacer 220. As shown in Figure 5B, the cell culture device 200 is used in a state in which a culture substrate 140 on which cells are fixed is placed between the bottom plate 111 of the box body 110 and the movable lid body 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 has an elastic member 210 and a spacer 220 instead of the fall-off prevention part 130. The same components as those in the cell culture device 100 according to the first embodiment are given the same reference numerals and the description thereof is omitted.

The elastic member 210 presses the movable lid 120 toward the culture substrate 140 contained in the box 110. When the opening 113 of the box 110 is upward (vertically upward), the elastic member 210 promotes the movable lid 120 to press the culture substrate 140, and when the opening 113 of the box 110 is downward (vertically downward), the elastic member 210 functions as a fall prevention part to prevent the movable lid 120 from coming off the box 110 and 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 FIG. 6). The elasticity of the elastic member 210 is not particularly limited as long as it can perform the above-mentioned functions, but it is preferable that it is adjusted so that the movable lid 120 does not press against the culture substrate 140 when the opening 113 of the box 110 is downward (vertically downward) (see FIG. 7B).

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

The spacer 220 is disposed between the bottom plate 111 of the box 110 and the movable lid 120, and prevents the movable lid 120 from tilting excessively when the elastic member 210 presses against the movable lid 120 due to the presence of a space between the bottom plate 111 and the movable lid 120 where the culture substrate 140 is not disposed. The thickness of the spacer 220 is not particularly limited as long as it does not prevent the movable lid 120 from pressing against the culture substrate 140 when the opening 113 of the box 110 is upward (vertically upward) and can perform the above function. The thickness of the spacer 220 is preferably slightly thinner than the thickness of the culture substrate 140, and is preferably, for example, 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-mentioned functions. The material of the spacer 220 is also not particularly limited, and may be, for example, metal, rubber, resin, ceramics, etc. 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-mentioned functions. In this embodiment, the cell culture device 200 has four spacers 220.

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

When culturing cells using the cell culture device 200 according to this embodiment, the cell culture device 200 is rotated so that the bottom plate 111 of the box body 110 and the movable cover body 120 alternate in the up-down direction. The cell culture device 200 is rotated while immersed in a liquid medium.

Figure 6 is a diagram showing an example of cell culture using a cell culture device 200. In this example, the cell culture device 200 is housed in a covered test tube 300 (e.g., a test tube (BT-30, Nichiden Rika Glass Co., Ltd.) covered with an aluminum open-type cap (NE-30, Nichiden Rika Glass Co., Ltd.)), and the covered test tube 300 is placed on a rotating culture table with a tilt of about 20 to 30 degrees. The cell culture device 200 is fixed in the covered test tube 300, and the cell culture device 200 rotates when the covered test tube 300 is rotated.

As in the first embodiment, the method of fixing the 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 just inside the lidded test tube 300 (so that it is inscribed at multiple points when viewed in cross section), the cell culture device 200 can be fixed to the lidded test tube 300 without using a separate fixing tool. Also, as in the first embodiment, the method of rotating the lidded test tube 300 containing the cell culture device 200 is not particularly limited.

(Cell Culture Method)
As in the first embodiment, the cell culture method according to the present embodiment includes (1) a step of accommodating the culture substrate 140 on which cells are fixed in the cell culture device 200, and (2) a step of culturing the cells while rotating the cell culture device 200. Each step will be described below.

First, the culture substrate 140 on which cells have been fixed is placed in the cell culture device 200. More specifically, the culture substrate 140 on which cells have been fixed is placed between the bottom plate 111 of the box body 110 and the movable lid body 120 of the cell culture device 200.

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

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

Figure 7A is a cross-sectional view showing the state of the cell culture device 200 when the opening 113 of the box body 110 is upward (vertically upward), and Figure 7B is a cross-sectional view showing the state of the cell culture device 200 when the opening 113 of the box body 110 is downward (vertically downward). When the opening 113 is upward, as shown in Figure 7A, the culture substrate 140 is pressed by the movable lid body 120 while being placed on the bottom plate 111. At this time, the culture substrate 140 is pressed not only by the weight of the movable lid body 120 but also by the pressing force of the elastic member 210. On the other hand, when the opening 113 is downward, as shown in Figure 7B, the culture substrate 140 is placed on the movable lid body 120 but is not pressed by the bottom plate 111. At this time, since the movable lid 120 is supported by the elastic member 210, not by the fall-off prevention part 130 (see embodiment 1) fixed to the box 110, the movable range of the movable lid 120 is not limited to an absolute range. For example, even if the thickness of the complex of the culture substrate 140 and the cells increases due to cell proliferation, the movable lid 120 can move to a position according to the thickness of the complex when in the state of FIG. 7B, and apply almost no pressure to the complex. When the cell culture device 200 is rotated, the state of FIG. 7A and the state of FIG. 7B are alternately repeated, so that the movable lid 120 moves with the change in the direction of gravity, and the culture substrate 140 is intermittently pressed by the movable lid 120. Therefore, pressure or shear stress is intermittently applied to the cells in the culture substrate 140. In addition, the direction of gravity with respect to the 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, cells can be cultured while applying pressure or shear stress to the cells intermittently as a mechanical stimulus, similar to the cell culture device 100 according to the first embodiment. 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, since the movable cover 120 is supported by the elastic member 210, pressure or shear stress can be applied to the cells intermittently, not continuously, even if the cells proliferate and the thickness of the complex of the culture substrate 140 and the cells increases.

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

[Example 1]
(1) Preparation of culture substrates A number of fibrous collagen membranes (FCMs) 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.

The method for preparing the collagen fiber membrane will be briefly explained. Collagen extracted from calf skin was treated with pepsin to obtain an acidic collagen solution. This acidic collagen solution was dialyzed against a weak alkaline solution to obtain collagen fibers. The suspension containing the collagen fibers was placed in a stainless steel mold measuring 10 cm long x 10 cm wide x 3 cm deep, freeze-dried, cross-linked with hexamethylene diisocyanate, washed, and freeze-dried to obtain a collagen fiber membrane. Finally, the collagen fiber membrane was pressed to adjust the thickness to 2 mm, and cut into a size of 5 mm long x 5 mm wide.

Each collagen fiber membrane containing a minimum amount of liquid medium was impregnated with a liquid medium containing 3×10 ⁵ DFAT cells and left to stand for 24 hours to allow the cells to settle in. The collagen fiber membrane swelled to more than twice its thickness.

(2) Preparation of cell culture device Two sets of cell culture device and lidded test tube according to the second embodiment were prepared (see Figs. 5A to 6). A titanium plate with a thickness of 0.1 mm was processed into an approximately rectangular tubular 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. A titanium nonwoven fabric with a length of 55 mm, a width of 26 mm, and a thickness of 1 mm was used as the bottom plate of the box, and a titanium nonwoven fabric with a length of 50 mm, a width of 23 mm, and a thickness of 1.5 mm was used as the movable lid. These two titanium nonwoven fabrics were impregnated with the acidic collagen solution and coated with collagen. Coil springs were used as the elastic members. These two coil springs have elasticity to the extent that they can support the movable lid without deformation if it is only the movable lid, and can support the movable lid while deforming when 10 collagen fiber membranes are added to the movable lid. A rectangular metal plate bent into a U-shape was used as the spacer. The thickness of the spacer was 3 mm. The test tube body of the lidded test tube was a glass culture tube having a depth of 120 mm, an outer diameter of 30 mm, and a thickness of 0.6 mm.

(3) Cell Culture In each of the two cell culture devices prepared in (2) above, ten collagen fiber membranes on which the DFAT cells prepared in (1) above were fixed were placed between the bottom plate of the box and the movable lid so as not to 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 cell culture device was placed in a lidded test tube tilted at about 20-30° and rotated alternately 240 times per hour (four round trips per minute), while the device was cultured for 7 days in a gas phase of 5% carbon dioxide and 95% air at 37°C (see Figure 6). The culture medium was not replaced. For the other cell culture device (Comparative Example), the cell culture device was placed in a lidded test tube tilted at about 20-30° and left stationary with the opening of the box positioned upward, while the device was cultured for 7 days in a gas phase of 5% carbon dioxide and 95% air at 37°C. This control experiment was used to create a comparative example that lacked all of the dynamic elements that are the core of the cell culture device. This comparative example should clearly show 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 the flow of the culture medium.

(4) Results After 7 days of culture, 10 collagen fiber membranes were removed from each cell culture device and placed 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 90 minutes of culture, the absorbance at 450 nm was measured for each well. The measured absorbance is proportional to the number of live cells.

Figure 8 is a graph showing the number of cells (absorbance) when the cells were cultured while rotating the lidded test tube (Example) and the number of cells (absorbance) when the cells were cultured without rotating the lidded test tube (Comparative Example). As shown in this graph, the cells proliferated six times more in the cell culture method according to the Example than in the cell culture method according to the Comparative Example.

[Example 2]
(1) Preparation of Culture Substrate Thirty collagen sponges (Honeycomb Disk 96, Koken Co., Ltd.) with a diameter of 6 mm and a thickness of 2 mm were prepared as culture substrates.

Each collagen sponge was soaked in a minimum amount of liquid medium, and then soaked in liquid medium containing 3×10 ⁵ DFAT cells, and left to stand for 24 hours to allow the cells to settle in. The collagen sponge swelled to more than twice its thickness.

(2) Preparation of cell culture device Three sets of cell culture devices and lidded test tubes according to the first embodiment were prepared (see Figs. 1 to 4B). As in the first embodiment, a box plate and a movable lid were prepared. Four through holes were formed in the side plate of the box plate, and two titanium rods were inserted as anti-drop parts. In one of the three sets, four through holes were formed at a position away from the bottom plate of the box plate so that the movable lid and culture substrate could be moved by rotating the cell culture device (so that the distance between the bottom plate and the movable lid would change) (Example: see Fig. 9A). In the remaining two sets, four through holes were formed at a position close to the bottom plate of the box plate so that the movable lid and culture substrate could not be moved even when the cell culture device was rotated (so that the distance between the bottom plate and the movable lid would not change) (Comparative example: see Figs. 9B and 9C). In addition, as in the cell culture device according to the second embodiment, a plurality of spacers were placed between the bottom plate of the box plate and the movable lid. As the spacers, rectangular metal plates bent into a U-shape were used. The thickness of the spacers was set to 3 mm. The test tube body of the lidded test tube was a glass culture tube having a depth of 120 mm, an outer diameter of 30 mm, and a thickness of 0.6 mm.

(3) Cell Culture In each of the three cell culture devices prepared in (2) above, ten collagen sponges with DFAT cells prepared in (1) above fixed thereon were placed between the bottom plate of the box and the movable lid so as not to 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.

Of the three cell culture devices, one cell culture device (Example) with a movable lid and culture substrate was cultured for 7 days at 37°C in a gas phase of 5% carbon dioxide and 95% air while rotating the lidded test tube in which the cell culture device was placed at an inclination of approximately 20 to 30° and alternating 240 times per hour (four round trips per minute) (see Figure 9A). The culture medium was not replaced.

As for another cell culture device (Comparative Example 1) in which the movable lid and culture substrate could not be moved, the lidded test tube in which the cell culture device was placed was tilted at an angle of about 20 to 30 degrees, and the cells were cultured for 7 days in a gas phase of 5% carbon dioxide and 95% air while rotating 240 times per hour (4 round trips per minute) at 37°C (see Figure 9B). The culture medium was not replaced.

For the last cell culture device (Comparative Example 2) in which the movable lid and culture substrate could not be moved, the lidded test tube in which the cell culture device was placed was tilted at an angle of about 20 to 30 degrees, and the cells were left stationary with the opening of the box positioned at the top, and cultured at 37°C in a gas phase of 5% carbon dioxide and 95% air for 7 days. The culture medium was not replaced.

(4) Results After 7 days of culture, 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 was proportional to the number of viable cells.

Figure 10 is a graph showing the number of cells (absorbance) when culturing while rotating a cell culture device in which the movable lid and culture substrate can be moved (Example), when culturing while rotating a cell culture device in which the movable lid and culture substrate cannot be moved (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). As shown in this graph, the cell culture method of the Example proliferated three times more cells than the cell culture method of Comparative Example 1 and 13 times more cells than the cell culture method of Comparative Example 2. This shows that cell proliferation can be further promoted by not only rotating the cell culture device but also adding mechanical stimulation by moving the movable lid and/or culture substrate.

The cell culture device and cell culture method according to the present invention are useful, for example, for forming organs or tissues in regenerative medicine. The cell culture device and cell culture method according to the present invention are also useful for producing artificial cultured meat in response to the upcoming global food crisis, particularly the difficulty in supplying edible meat.

REFERENCE SIGNS LIST 100, 200 Cell culture device 110 Box body 111 Bottom plate 112 Side plate 113 Opening 120 Movable lid 130 Fall-off prevention part 140 Culture substrate 210 Elastic member 220 Spacer 300 Lidded test tube 310 Tube

Claims (8)

A box for accommodating the culture substrate on which the cells are fixed;
A movable cover body disposed within the box body so as to be movable in a depth direction of the box body;
having
The culture substrate is disposed between the bottom plate of the box and the movable lid.
Cell culture device. The cell culture device according to claim 1, further comprising a fall prevention part for preventing the movable lid from falling off outside the box body. The cell culture device according to claim 1, further comprising an elastic member for pressing the movable lid against the culture substrate contained in the box. The cell culture device according to claim 3, further comprising a spacer disposed between the bottom plate and the movable lid. The cell culture device according to claim 1, wherein at least one of the bottom plate and the movable lid is a porous body. The cell culture device according to claim 1, wherein the box and the movable lid are made of titanium. The cell culture device according to claim 1, 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 and the movable cover of the box of the cell culture device according to any one of claims 1 to 7;
Culturing cells while rotating the cell culture device containing the culture substrate;
A cell culture method comprising the steps of:

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