JP2012090584A - Method and apparatus for antigravity culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new culture method for enhancing growth of cells by giving new physical stimulation to cells.SOLUTION: There is provided an antigravity culture method for culturing cells by giving antigravity stimulation reacting with gravity from a scaffold on which cells are fixed to the direction of the cells while the cells are fixed on a culture substrate.

Description

  The present invention relates to a method for culturing cells.

  In a living body, cells repeat normal growth and differentiation in a three-dimensional environment, and grow into normal tissues. However, conventionally, cell culture research has traditionally been performed in a two-dimensional flat container for the convenience of experiments. As a result, recently, the results of research on culture using only a two-dimensional plate are considerably different from those of a three-dimensional environment, and a number of inconveniences have been revealed. In particular, when stem cells were cultured on a conventional plate, it became clear that they formed cancer (teratomas). Accordingly, a cell culture platform having an appropriate three-dimensional structure has been rapidly demanded (Non-patent Document 1). However, at present, there is no definitive version of a three-dimensional cell growth base that allows cells to grow normally into tissues without becoming cancerous.

Nature, 423: 21 August, (2003) p869-872

  An object of this invention is to provide the novel culture method which accelerates | stimulates the proliferation of a cell by giving a new physical stimulus with respect to a cell.

  The present inventor has found that cell culture is promoted by applying a new physical stimulus called antigravity stimulation in cell culture, and has completed the present invention.

  The present invention (1) is an antigravity culture method for culturing cells by applying antigravity stimulation that causes gravity to act on cells fixed on a culture substrate from the anchored scaffold to the cell direction.

  The present invention (2) is an antigravity culture method according to the invention (1) in which cells are fixed on a culture substrate and the culture substrate is rotated to alternately apply gravity stimulation and antigravity stimulation to the cells. It is.

  The present invention (3) is the antigravity culture method of the invention (2), wherein the culture substrate is a porous three-dimensional culture substrate.

  The present invention (4) is the antigravity culture method of the invention (2), wherein the culture substrate is a two-dimensional flat plate culture dish.

  According to the present invention (5), the culture substrate is placed and fixed in a container, and a culture solution is introduced to rotate the container. Gravity culture method.

  The present invention (6) is the antigravity culture method of the invention (5), wherein the angle of the culture substrate is adjusted.

  This invention (7) is the antigravity culture method of the said invention (5) or (6) whose said culture solution is 1-1000 ml.

  The present invention (8) is the antigravity culture method according to any one of the inventions (2) to (7), wherein the rotation speed is 1 to 100 rpm.

  The present invention (9) is the antigravity culture method of the invention (3), wherein the culture substrate is a foamed polyurethane coated with calcium phosphate.

The present invention (10) uses a container and a device for rotating the container,
Put the cells in the container,
The anti-gravity culture method according to any one of the inventions (1) to (9), wherein the container is rotated and cultured.

  In the present invention (11), cells are fixed on a culture substrate, the culture substrate is fixed above the gravity direction, and the cells are fixed below the gravity direction to give an antigravity stimulus to the cells. The antigravity culture method of the invention (1).

  The present invention (12) is the antigravity culture method of the invention (11), wherein the culture substrate is a two-dimensional flat plate culture dish.

  The present invention (13) is the antigravity culture method of the invention (11) or (12), wherein the culture is carried out in a culture solution.

The present invention (14) uses a container and a device for rotating the container,
Put the cells in the container,
The anti-gravity culture according to any one of the inventions (11) to (13), wherein a flat plate culture dish is fixed in the vessel, the vessel is rotated, and the plate is stationary and applied with anti-gravity stimulation. Is the method.

  In the present invention (15), the flat plate culture dish is fixed by an inner wall fixing means so that the angle of the plate is adjusted to be horizontal, the cells are fixed on the flat plate culture dish, and the culture base is The anti-gravity culture method according to the invention (14), wherein the cells are cultured upward in the direction and directed downward in the direction of gravity.

  The present invention (16) is the antigravity culture method according to any one of the inventions (1) to (15), wherein the cells are osteoblasts and the proliferation is promoted by the culture.

The present invention (17) includes a cylindrical container rotating means for tilting and rotating the cylindrical container;
A motor connected to the cylindrical container rotating means via a rotation speed reducing means;
It is an antigravity culture apparatus characterized by having.

  The present invention (18) is the antigravity culture apparatus according to the invention (17), characterized by having a drive power source for driving the motor.

  The present invention (19) is the antigravity culture apparatus according to the invention (17) or (18), wherein the rotation speed reducing means is a planetary gear.

As for this invention (20), the said cylindrical container rotation means is:
A frame that fixes a plurality of rolls in parallel via a rotation axis;
Fixing means for fixing the frame body obliquely,
The antigravity culture apparatus according to any one of the inventions (17) to (19), wherein at least one rotating shaft of the plurality of rolls and the motor are rotatably connected.

  The present invention (21) is the antigravity culture device of the invention (20), wherein the rotating shaft and the motor are rotatably connected by a flexible joint.

  According to the present invention, since the cells are activated by the antigravity stimulation that causes gravity to act from the scaffold in which the cells are fixed in the cell direction, there is an effect of promoting the proliferation of the cells. In addition, by rotating and culturing, even when a three-dimensional culture substrate is used, cells can be trapped on the culture substrate with high efficiency that cannot be obtained by conventional methods. In addition, by using the culture solution, the culture solution circulates in the three-dimensional culture substrate, and new culture solution is supplied one after another to the cell surface. Done. Cells that have been grown and differentiated can be easily released from the culture substrate by a conventional method using triprin or collagenase.

FIG. 1 is a schematic perspective view of an antigravity culture apparatus according to the present invention. FIG. 2 (a) is a schematic plan view of the antigravity culture apparatus according to the present invention, and FIG. 2 (b) is a schematic side view. FIG. 3 shows parts used in the anti-gravity culture apparatus according to the present invention, FIG. 3 (a) is a schematic view of parts used when a three-dimensional culture base is used, and FIG. It is the schematic of the components used when using the cell which grew in the monolayer or the multilayer, without using a three-dimensional culture base. FIG. 4 is a graph showing experimental results when cells were cultured by the culture methods according to Examples and Comparative Examples. FIG. 5 is a graph showing experimental results when cells were cultured by the culture methods according to Examples and Comparative Examples. FIG. 6 is a graph showing experimental results when cells were cultured by the culture methods according to Examples and Comparative Examples. FIG. 7 is a graph showing experimental results when cells were cultured by the culture methods according to Examples and Comparative Examples.

  The cell culture method according to the present invention is an antigravity culture method in which cells are cultured while applying antigravity stimulation that causes gravity to act in the cell direction from the scaffold on which the cells are fixed. The present inventor has found that cell proliferation is promoted by applying antigravity stimulation to the cells.

  Here, the “anti-gravity stimulation” means a physical stimulation applied to the cells by applying gravity from the scaffold where the cells are fixed to the cells. In addition, “gravity stimulation” means physical stimulation applied to cells by applying gravity in the direction of the scaffold where the cells are fixed. The “gravity change stimulus” means a physical stimulus given to a cell by changing the direction of action of gravity applied to the cell fixed to the culture substrate.

  The culture method according to the present invention has been completed by finding that cell activity is activated by applying antigravity stimulation to cells. That is, by culturing cells suspended from a fixed culture base, antigravity stimulation is given to the cells, and cell proliferation is activated. By being arranged so as to be suspended from the ceiling in this way, the cells are cultured while clinging to the adhesive surface to the culture substrate. Furthermore, since the shape of the cell itself swells downward, the cell tries to maintain its shape. These factors are thought to be stimulating stimuli such as cell proliferation.

  In the culturing method according to the present invention, it is preferable to culture cells by applying a gravity change stimulus such as giving an antigravity stimulus to a cell or alternately giving a gravity stimulus and an antigravity stimulus. . Examples of a method for culturing cells while applying antigravity stimulation include a method in which cells are fixed on a culture substrate, the culture substrate is fixed above the gravity direction, and the cells are fixed below the gravity direction. . That is, a method of culturing in a state where cells are inverted by inverting the substrate on which the cells are fixed can be mentioned. It has been found that culturing of cells is promoted by culturing in such a state. Since the arrangement of the skeleton or the like that forms the cells is changed by gravity by placing the cells in an antigravity state, it is presumed that this is a stimulus that promotes cell culture.

  As a method of culturing cells while alternately applying weight stimulation and antigravity stimulation, for example, it is preferable to rotate the cells while being fixed to a substrate such as a culture substrate. By culturing by such a method, for example, unlike the case of rotating and floating in the medium, the cell rotates from the viewpoint of the cells fixed on it by rotating the base. It seems that the gravity field of the fixed base is reversed. This is considered to cause rearrangement in the cell and the like and anti-gravity stimulation, which promotes cell culture and differentiation.

  The rotation includes intermittent rotation and continuous rotation. In intermittent rotation, the cell is once fixed with the bottom face down, and after a certain time, it is rotated 180 degrees to turn the bottom surface up, and then the culture is continued after a certain time to rearrange the skeleton in the cell. In this way, the cytoskeleton rearrangement is repeated as necessary. Cytoskeletal rearrangement stimuli are critical stimuli for cells, which affect cell differentiation and proliferation.

  Here, the culture base may be a porous three-dimensional culture base or a two-dimensional (planar) culture base such as a dish as shown in FIG. For example, when a dish is used, a special effect can be expected by rotation. That is, when rotated by 180 °, gravity acts on the cell from the free surface opposite to the culture base, and can give the cell an effect of promoting specific differentiation simultaneously with proliferation.

The culture substrate used in the present invention is preferably a porous three-dimensional culture substrate. Here, the three-dimensional culture substrate means a substrate having a three-dimensional space such as bubbles and gaps inside the substrate.
According to the conventional method, when cells are seeded by dropping a cell suspension onto a porous 3D culture substrate, the cells pass through the substrate and almost all fall to the bottom. Had become a major technical obstacle. On the other hand, by rotating the base as in the method according to the present invention, the chance that the cells that fall through and come into contact with the base again and settle on the base increases. Therefore, by rotating the cell base and the cells, the cells are trapped on the culture base with high efficiency that was not obtained by the conventional method, and thereby, the anchorage-dependent cells such as osteoblasts are made into the three-dimensional culture base. It has the effect of sowing and fixing.

  In the culture method according to the present invention, the culture substrate is placed in a container, and further cultured by introducing a culture medium and rotating the container, while applying antigravity stimulation to the cells. Is preferred. By introducing the culture solution in this way, the culture solution circulates in the three-dimensional culture substrate, and new culture solution is supplied one after another to the cell surface. To be done. By being arranged in such an environment, an environment suitable for cell culture can be provided. When using a culture solution, it is preferable to introduce the cell suspension in which the cells are suspended into the container together with the culture substrate.

  Here, the culture substrate is not particularly limited, but it is preferable to use a porous three-dimensional culture substrate such as a porous body such as a polymer or ceramics or a fiber assembly material such as titanium mesh. As the porous substrate of the polymer, a foam of a biocompatible material such as urethane or those coated with collagen on the surface thereof can be used. Moreover, as a titanium mesh, the nonwoven fabric etc. of a titanium fiber are mentioned. The ceramic porous substrate may be a porous substrate made of a single ceramic, or a substrate in which a ceramic is coated on the surface of a porous material such as a polymer. Further, it is preferable to use a porous cell culture substrate in which the surface of foamed polyurethane is coated with calcium phosphate. Even in the state where calcium phosphate is coated, it is preferable that the cells have cells (bubbles, compartments) inside and the cells are in communication with each other because the cells can easily grow. The calcium phosphate is not particularly limited, but it is preferable to use hydroxyapatite or β-tricalcium phosphate (βTCP). As the polyurethane foam, it is preferable to use a product having a geometric shape and chemical properties particularly suitable for cell culture among known polyurethanes. Here, it is preferable that the foam cell has a front-rear and left-right spread of 200 to 600 μm. The foamed polyurethane is preferably hydrophilic, and exhibits hydrophilicity and water absorption that absorbs 100 μL of Dulbecco's phosphate buffered saline without leaving the surface within 3 minutes. More preferred.

  The method for producing the porous culture substrate is not particularly limited. For example, an apatite precipitation step of mixing a foam having open cells and a treatment solution, bubbling carbon dioxide, and adding calcium chloride and dibasic potassium phosphate. Can be manufactured. Here, the treatment solution preferably contains sodium chloride. By obtaining a porous cell culture substrate by such a production method, a coated surface in which spherical crystals are aggregated is formed on the surface of the polyurethane foam. By thus obtaining spherical crystals, cell proliferation is further promoted.

  Here, the rotation speed according to the present invention is preferably 1 to 100 rpm, and more preferably 3 to 20 rpm. By culturing at such a rotation speed, cell proliferation is particularly promoted.

  In the culture method according to the present invention, when only anti-gravity stimulation is applied, the culture base is preferably a two-dimensional flat plate culture dish. For example, after culturing and fixing cells on a flat plate culture dish, it is preferable to invert the culture dish and culture the cells hanging on the ceiling. By culturing in this way, antigravity stimulation can be applied to the cells. Further, the base may be reversed again after a predetermined time has elapsed.

  Further, in the case of the above method using a plate culture dish, it is preferable that the culture is performed in a culture solution. By reversing in the culture solution, it is possible to prevent the fixed cells from falling. Furthermore, since it is reversed in the liquid, the antigravity stimulus becomes a milder stimulus than in the air.

  The cells that can be cultured in the present invention are not particularly limited. For example, osteoblasts, odontoblasts, periodontal ligament cells, chondrocytes, fibroblasts, cardiomyocytes, hepatocytes, pancreatic cells, ES cells, and Examples include anchorage-dependent cells such as iPS cells.

  The culture solution used in the present invention can be appropriately selected according to the cells to be cultured, and is not particularly limited. For example, a liquid containing nutrients such as a carbon source, vitamins, and inorganic salts is preferable. Although the quantity of a culture solution is not specifically limited, 1-1000 ml is suitable and 1-10 ml is more suitable.

  The culture period of the antigravity culture method according to the present invention can be appropriately set according to the cells to be cultured and is not particularly limited, but is preferably 1 day to 5 weeks, more preferably 2 to 4 weeks, 3 weeks is particularly preferred.

  In addition, since the cell culture environment can be applied by appropriately changing a known environment, it is not particularly limited, but it is preferable to culture under a constant temperature condition such as an incubator.

  Hereinafter, an antigravity culture apparatus suitably used in the present invention will be described, and further, a culture method using the apparatus will be described in detail.

《Anti-gravity culture device》
FIG. 1 is a schematic perspective view of an antigravity culture apparatus 1 according to the present invention, FIG. 2 (a) is a schematic plan view of the antigravity culture apparatus, and FIG. 2 (b) is the antigravity culture apparatus. It is a schematic side view of an apparatus.

  The antigravity culture apparatus 1 includes a cylindrical container rotating unit 11 that tilts and rotates a cylindrical container, a motor 13 that is connected to the cylindrical container rotating unit 11 via a rotation speed reducing unit 12, and the motor. Drive power source 14 for driving. By using the apparatus, the cells arranged in the cylindrical container can be subjected to antigravity stimulation by the rotation of the container. Each of the above-described components is preferably installed on the substrate 15. In this way, it is easy to carry by installing on a single substrate. The rotation speed reduction means 12 and the motor 13 may be installed on a rubber cushion base 17. The positional relationship of the flexible joint can be adjusted by adjusting the height of the cushion base 17. Further, since the apparatus according to the present invention is provided with a driving power source (for example, battery) 14, it is not necessary to separately introduce a power source from outside, and the apparatus is directly placed in the culture apparatus and the door is closed. Therefore, it is preferable because it can be cultured. Further, if necessary, an external power source can be introduced from the gap in the door of the culture apparatus using a flat lead wire as usual.

  The cylindrical container rotating means 11 includes a frame body 113 that fixes a plurality of rolls 111a to 111c in parallel via rotating shafts 112a to 112c (FIG. 2A). The plurality of rolls 111 a to 111 c are rotatably fixed inside the frame body 113. In addition, the roll has a configuration in which, for example, a core bar 1111 such as a round bar is used at the center, and the periphery thereof is covered with a cushioning material 1112 having moderate frictional properties while being soft such as urethane foam or rubber. It is preferable to have (FIG. 2A). Further, the plurality of rolls 111 are arranged so that the rotation axes thereof are parallel to each other, and an interval d that allows the cylindrical container to be placed between adjacent rolls (the interval d between the rolls is smaller than the diameter of the cylindrical container). It is preferable to have (Fig. 2 (a)).

  It is preferable that a holding portion 1131 for preventing the cylindrical container from slipping down is provided on either one of the sides that fix the rotation shaft of the frame body 113. In addition, it is preferable to have fixing means 114 for fixing the frame body 113 obliquely. By having the fixing means, the cylindrical container can be tilted and rotated, so that an antigravity stimulus can be applied to the cells arranged in the cylindrical container. Furthermore, it is preferable that the fixing means is configured so that the inclination angle of the frame body can be adjusted. More specifically, for example, a plate-like body 1141 having a fixing hole 1142 in the upper portion is arranged vertically on the left and right of the frame body 113, and a screw 1143 for fixing the frame body 113 to the fixing hole 1142 is provided. Furthermore, it is preferable to provide washers 1144 on both sides of the fixing hole 1142. Here, the shape of the fixing hole 1142 is preferably formed to be elongated in the vertical direction, and the frame can be moved up and down by forming the fixing hole 1142 in this manner. Even when the body interferes with the substrate 15, the interference can be prevented. Moreover, since it can move up and down, distance adjustment of either of the rotating shafts 112a-c and the flexible joint for transmitting rotation of a motor becomes easy.

  At least one of the plurality of rolls is rotatably connected to a rotating shaft and a motor. Since at least one rotating shaft is connected to the motor, the cylindrical container can be rotated by placing the cylindrical container between the rolls, and is further adjacent to the cylindrical container. Since the roll also rotates, the cylindrical container can be rotated stably.

  The connection between the rotating shaft and the motor is preferably connected by a flexible joint 16 via the rotation speed reducing means 12. As the flexible joint 16, for example, a resin tube such as rubber or plastic can be used. By using the flexible joint in this way, it is possible to freely set the inclination of the frame body 113. It is preferable to adjust the positional relationship between the flexible joint so that the center of the flexible joint is the intersection of the two axes of the rotation shaft 112b and the rotation speed reduction means rotation shaft.

  The rotation speed reduction means 12 is not particularly limited, but it is preferable to use a planetary gear. Since the speed reduction mechanism can be made more compact by using the planetary gear, the entire apparatus can be made less bulky. For example, the rotational speed of the motor can be reduced to 1/400 by the planetary gear.

  The drive power supply 14 is not particularly limited as long as power can be supplied independently, but a primary battery or a secondary battery such as a battery can be used. For example, a battery box is provided as shown in the figure. Also good. By having a drive power supply that can supply power independently as described above, the motor can be driven even if it is placed in a closed system such as an incubator.

  FIG. 3A is a schematic configuration diagram of a container and a culture base used when a three-dimensional culture base is used, and FIG. 3B is a container and inner wall fixing means used when a flat plate culture dish is used. FIG.

  When using a three-dimensional culture substrate as shown in FIG. 3A, the container is preferably a glass cylindrical container 21, and the opening of the cylindrical container is covered with a breathable aluminum cap 23. It is preferable that A circular three-dimensional culture base 25 is provided in the glass container, and the three-dimensional culture base 25 is fixed to the inner wall of the cylindrical container 21. Here, it is preferable that the angle of the three-dimensional culture substrate 25 is adjusted. The culture solution l is introduced into the cylindrical container 21.

  As in the case of using a three-dimensional culture base in a flat plate culture dish as shown in FIG. 3 (b), the container is preferably a glass cylindrical container 21, and further, an opening of the cylindrical container. Is preferably covered with a breathable aluminum cap 23. Here, it is preferable that the plate culture dish 28 has a circular shape having a diameter smaller than the inner diameter of the cylindrical container. The flat plate culture dish is not particularly limited, but is preferably made of titanium or plastic. Furthermore, it is preferable that an inner wall fixing means 29 such as an O-ring (for example, made of silicon) is provided on the periphery of the plate culture dish. It is preferable to adjust the angle by the inner wall fixing means. For example, when the cylindrical container 21 is tilted at an inclination when the cylindrical container 21 is rotated by the antigravity culture apparatus, it is preferably fixed so as to be in the horizontal direction. It is.

<Antigravity culture method>
An example of an antigravity culture method using the above apparatus will be described. As a specific example of the antigravity culture method according to the present invention, there is a method in which cells are seeded in the container using an apparatus for rotating the container, and the container is rotated and cultured. In this way, the cells are cultured while applying anti-gravity stimulation to the cells by rotating the container. It is preferable that the culture base is fixed and rotated on the inner wall of the cylindrical container to be rotated, and the culture is performed while inverting the direction of gravity with respect to the cells.

  Although it does not specifically limit as a container, The above-mentioned cylindrical container is suitable and a test tube is mentioned more suitably. That is, by placing the cylindrical container on the cylindrical container rotating means 11, the container rotates, and an antigravity stimulus can be given to the cells. In addition, although the inclination of a cylindrical container is not specifically limited, 0-80 degrees is suitable with respect to the horizontal, 5-60 degrees is more suitable, and 10-40 degrees is still more suitable.

  When using a three-dimensional culture substrate, the disk-shaped three-dimensional culture substrate 25 to which various cells are fixed is fixed to the inner wall of the cylindrical container 21 and then cultured while being rotated on an antigravity device. . The opening of the cell is covered with a breathable aluminum cap 23.

  When using a plate culture base, various cells are first cultured as a monolayer cell layer in the plate culture dish 26. The plate culture dish 26 is fixed to the inside of the cylindrical container 21 through an O-ring 29 provided at the periphery. Due to the presence of this O-ring, it can be fixed to the inner wall of the glass cell at any angle so that the bottom of the plate culture dish is perpendicular to the direction of gravity. As a result, the plate culture dish can be fixed perpendicularly to the direction of gravity and upside down by 180 °, and as a result, the dynamic effects of gravity and antigravity can be given to the cells, respectively. .

Example 1: Antigravity culture method Using the antigravity culture apparatus 1, cells were cultured. 50,000 cells of the osteoblast MC3T3-E1 are seeded on a culture base coated with collagen on a three-dimensional culture base (hydroxyapatite-coated foamed polyurethane: porous culture base). Eagle's minimum essential medium) was placed in a test tube, placed on a roll inclined at 25 ° to the horizontal, rotated at a speed of 4 rpm, and a culture test was performed in an incubator. Two days later, two weeks later, and four weeks later, the number of cells was observed by performing a color reaction (MTS measurement method) for measuring mitochondrial enzyme activity and measuring the absorbance at 450 nm. The cell number increased significantly after 2 weeks. The result is shown by the solid line in FIG.

Comparative Example 1: Static culture method The results of conventional static culture, which is a control experiment, are shown by dotted lines in FIG. A cell culture test was conducted under the same conditions as in Example 1 except that the three-dimensional culture substrate seeded with the prepared osteoblasts was placed on a 96-well dish and statically cultured. Two days later, two weeks later, and four weeks later, the number of cells was subjected to a color reaction and the absorbance was measured at 450 nm. As a result, the number of cells in the conventional stationary state grew with time, but cultured in an antigravity apparatus. Compared with the case, it is about a half in 2-4 weeks.

Example 2 and Comparative Example 2
The culture according to Example 2 was performed in the same manner as in Example 1 except that polyurethane coated only with collagen was used as the three-dimensional culture substrate. Moreover, the culture which concerns on the comparative example 2 was performed on the conditions similar to Example 2 except not having rotated. The results of culturing according to Example 2 and Comparative Example 2 were observed in the number of cells after 1 week, 2 weeks, 3 weeks, and 6 weeks, and are shown in FIG. At any point in time, the antigravity culture example showed a higher cell number than the comparative example cultured by the conventional method.

Example 3 and Comparative Example 3
The culture according to Example 3 was performed in the same manner as in Example 1 except that a commercially available titanium mesh was used as the three-dimensional culture base. Moreover, the culture which concerns on the comparative example 3 was performed on the conditions similar to Example 3 except not having rotated. The results of culturing according to Example 3 and Comparative Example 3 were observed by the number of cells after 3 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 7 weeks, and are shown in FIG. At any point in time, the antigravity culture example showed a higher cell number than the comparative example cultured by the conventional method. In particular, from the second week to the fourth week, the value was 1.4 to 1.6 times higher.

Example 4 and Comparative Example 4
Cells were cultured using the antigravity culture apparatus 1. 125,000 osteoblasts MC3T3-E1 were seeded on a two-dimensional plate culture base (titanium culture dish having an inner diameter of 13 mm and a depth of 2 mm) and cultured for 4 hours. Thereafter, in Example 4, the culture medium (Eagle's minimum essential medium) is placed in a test tube, the dish is inverted, and the culture dish is directed upward with respect to the direction of gravity and the cells are directed downward in the direction of gravity. And cultured for 48 hours while applying antigravity stimulation. In Comparative Example 4, the culture solution (Eagle's minimum essential medium) is placed in a test tube and placed perpendicular to the direction of gravity for 48 hours without inverting the dish (in the same direction as the 4-hour culture). Cultured. The number of cells after 48 hours was observed by performing a color reaction (MTS measurement method) for measuring mitochondrial enzyme activity and measuring the absorbance at 450 nm. As a result, in Example 4 to which antigravity stimulation was applied, the number of cells significantly increased. The result is shown in FIG.

  The antigravity culture method and the antigravity culture apparatus according to the present invention have been completed based on a new discovery that cells are activated by antigravity stimulation. According to the conventional culturing method, the cells are simply fixed on the culture substrate and cultured, but it is shown that cell proliferation can be promoted by turning over the substrate after cell fixation. This means that the cells are activated by a very simple operation and is expected to be applicable to the entire cell culture.

  The antigravity culture method and the antigravity culture apparatus according to the present invention can be applied universally to a three-dimensional culture base without using a complicated and expensive apparatus. As an example of application, iPS cells are currently difficult to cancerize in conventional plate culture, but with this device, the natural kinetics of gravity at the same time as the three-dimensional cell house. New developments can be expected with stimulation.

  According to the antigravity culture apparatus of the present invention, the effect of gravity on cells can be examined on a desk without going to space. In short, in conventional cell culture devices, cells were gently laid on the floor, but the anti-gravity culture device according to the present invention was attached to the lower surface of the ceiling like a gecko or a ninja. Make it live. It becomes a stimulus (antigravity stimulus), and the cells will behave differently from on the floor, which is presumed to have a different effect on how the cancer drugs work. There are 60 trillion cells (10 12) in one body. In the body, instead of living quietly on a flat plate, it receives power from the top, bottom, left and right and moves. By alternately applying gravity from the top to the bottom alternately by the antigravity culture apparatus, the culture can be performed outside the body by bringing it closer to a state close to a living body.

1: Antigravity culture device 11: cylindrical container rotating means 12: rotational speed reducing means 13: motor 14: driving power source

Claims (21)

  An antigravity culture method of culturing cells by applying an antigravity stimulus that causes gravity to act on cells fixed on the culture substrate from the scaffold in which the cells are fixed in the cell direction.   The antigravity culture method according to claim 1, wherein the cells are fixed on the culture substrate, and the culture substrate is rotated to alternately apply gravity stimulation and antigravity stimulation to the cells.   The antigravity culture method according to claim 2, wherein the culture base is a porous three-dimensional culture base.   The antigravity culture method according to claim 2, wherein the culture substrate is a two-dimensional flat plate culture dish.   The antigravity culture method according to any one of claims 2 to 4, wherein the culture substrate is placed and fixed in a container, a culture solution is introduced, and the container is rotated.   The antigravity culture method according to claim 5, wherein an angle of the culture base is adjusted.   The antigravity culture method according to claim 5 or 6, wherein the culture solution is 1-1000 ml.   The antigravity culture method according to any one of claims 2 to 7, wherein the rotation speed is 1 to 100 rpm.   The antigravity culture method according to claim 3, wherein the culture substrate is foamed polyurethane coated with calcium phosphate. Using a container and a device for rotating the container,
Put the cells in the container,
The antigravity culture method according to any one of claims 1 to 9, wherein the culture is performed by rotating the container.   The anti-gravity stimulus is applied to the cells by fixing the cells on the culture substrate, fixing the culture substrate on the upper side with respect to the direction of gravity, and fixing the cells on the lower side in the direction of gravity. Gravity culture method.   The antigravity culture method according to claim 11, wherein the culture substrate is a two-dimensional flat plate culture dish.   The antigravity culture method according to claim 11 or 12, wherein the culture is performed in a culture solution. Using a container and a device for rotating the container,
Put the cells in the container,
The antigravity culture method according to any one of claims 11 to 13, wherein a flat plate culture dish is fixed in the container, the container is rotated, and the culture is performed by applying a static antigravity stimulus. .   The flat plate culture dish is fixed by an inner wall fixing means, and the angle of the flat plate culture dish is adjusted to be horizontal, the cells are fixed on the flat plate culture dish, and the culture base is moved in the direction of gravity. The antigravity culture method according to claim 14, wherein the cells are cultured on the upper side toward the lower side in the gravity direction.   The antigravity culture method according to any one of claims 1 to 15, wherein the cells are osteoblasts and promote proliferation by the culture. A cylindrical container rotating means for tilting and rotating the cylindrical container;
A motor connected to the cylindrical container rotating means via a rotation speed reducing means;
An antigravity culture apparatus characterized by comprising:   The antigravity culture apparatus according to claim 17, further comprising a driving power source for driving the motor.   The antigravity culture apparatus according to claim 17 or 18, wherein the rotation speed reduction means is a planetary gear. The cylindrical container rotating means is
A frame that fixes a plurality of rolls in parallel via a rotation axis;
Fixing means for fixing the frame body obliquely,
The antigravity culture apparatus according to any one of claims 17 to 19, wherein at least one rotating shaft of the plurality of rolls and the motor are rotatably connected.   The antigravity culture apparatus according to claim 20, wherein the rotating shaft and the motor are rotatably connected by a flexible joint.

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