JP7132640B2 - Cell culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell culture apparatus capable of agitating a culture medium, supplying nutrients to cells, and oxygenating cells with a simple operation, and culturing a large amount of cells.

Pluripotent stem cells (embryonic stem cells, induced pluripotent stem (iPS) cells) are recognized as an important cell source for regenerative medicine due to their unlimited proliferation potential and pluripotent potential. As regenerative medicine using stem cells, for example, treatments for liver cirrhosis, blood diseases, and myocardial infarction, construction of blood vessels, regeneration of bones and corneas, and securing of skin for transplantation are considered. In regenerative medicine, target cells or organs are grown from stem cells or the like in a culture dish and transplanted to humans. Recently, angiogenesis is performed from stem cells derived from bone marrow, and angina pectoris, myocardial infarction, etc. have been successfully treated.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, there is a demand for a cell culture apparatus that efficiently cultures large amounts of stem cells in an artificial environment.

FIG. 8 illustrates a conventional cell culture device. A conventional cell culture device comprises a first chamber 810, a porous scaffold 820, and a second chamber 830, as shown in FIG. The second chamber 830 has a bellows shape that can be compressed and decompressed. In FIG. 8, the second chamber 830 is in an uncompressed configuration, with the second chamber filled with culture solution and the scaffold 820 indirectly exposed to the gaseous environment to oxygenate the cells. In FIG. 9, the compressed shape of the second chamber 830 forces the culture solution upward into the first chamber 810, and the scaffold 820 is immersed in the culture solution to feed the cells. . Returning to FIG. 8 again, the second chamber 830 assumes an uncompressed shape, so that the culture solution moves downward and fills the second chamber 830 with the culture solution.

However, in the cell culture apparatus described above, since the second chamber 830 has a bellows shape, the cells that have migrated to the second chamber 830 enter the bellows-shaped convex portion to the outside, and thereafter enter the second chamber 830. Compression and decompression are less susceptible to up-and-down movement of the culture solution, which can make oxygenation difficult. In addition, even if the second chamber 830, especially the central part, has a bellows shape in a compressed state, the culture solution cannot be completely returned to the first chamber 810 due to its structure. Therefore, in order to seed cells onto the porous scaffold 820, the cell suspension is placed in the first chamber 810 and allowed to stand, or the bellows shape is compressed and uncompressed to allow cell attachment, but the resulting death Space and dead volume are inefficient as there is a cell suspension that cannot adhere.

Patent No. 4430317

The present invention has been made in view of such problems, and is a cell culture apparatus that enables sufficient nutrient supply and oxygenation to cells while having a simple structure, and enables appropriate mass culture of stem cells. intended to provide

The cell culture apparatus according to the present invention comprises a substantially cylindrical cell culture vessel having a flat bottom at the lower end and a hollow space filled with a medium for culturing cells, and a magnet or ferromagnetic material. A substantially disk having a smaller diameter than the diameter of the substantially cylindrical body of the cell culture vessel, provided with a plurality of first magnetized bodies at equal intervals around the periphery, and horizontally arranged in the hollow space of the cell culture vessel. and a plurality of second magnetized magnets each comprising a magnet corresponding to each of the first magnetized bodies of the dish-shaped bodies so as to attract the first magnetized body of each of the dish-shaped bodies by magnetic force. The plate-shaped body is positioned outside the cell culture vessel, the cell culture vessel is positioned inside the ring, and the cell culture vessel is moved upward to move the dish-shaped body upward in conjunction with magnetic force. The medium filled in the hollow space of the cell culture vessel is pushed up from below and moved downward to move the dish-shaped body downward in conjunction with the magnetic force, thereby filling the hollow space of the cell culture vessel. and a substantially ring-shaped annular body that moves in the vertical direction and causes the filled culture medium to drop by gravity.

INDUSTRIAL APPLICABILITY According to the present invention, although the structure is simple, it is possible to sufficiently supply nutrients and oxygen to cells, and to appropriately culture large amounts of stem cells.

It is a figure explaining the external appearance of the cell culture apparatus concerning this embodiment. It is a figure explaining in detail the vicinity of the plate-shaped body of the cell culture apparatus concerning this embodiment. It is a figure explaining in detail near the bottom part of the cell culture container of the cell culture apparatus concerning this embodiment. It is a figure explaining the storage part provided below the bottom part of the cell culture container concerning this embodiment. FIG. 2 is a plan view for explaining the ring-shaped body of the cell culture vessel according to the present embodiment, in which (a) shows a state in which the second magnetized body is sliding radially outward, and (b) shows the second magnet. In this state, the adherend is sliding radially inward. FIG. 10 is an enlarged view for explaining a state in which the second magnetized body slides radially inward; FIG. 10 is an enlarged view for explaining a state in which the second magnetized body slides radially outward; FIG. 2 is a diagram illustrating a conventional cell culture device in which a bellows-shaped chamber is in an uncompressible shape; FIG. 2 is a diagram illustrating a conventional cell culture device in which a bellows-shaped chamber is in a compressed shape;

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. The embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is as follows. The scope of the present invention is not limited to the embodiments, and other embodiments in which the configurations of the following embodiments are appropriately replaced by those skilled in the art are also included in the scope of the present invention.

(1) First Embodiment As shown in FIG. 1, a cell culture device 900 according to this embodiment includes a substantially cylindrical cell culture vessel 100, a substantially disk-shaped dish-shaped body 200, and a substantially ring-shaped and a storage section 400 in which the annular body 300 is stored. A substantially cylindrical body is a cylindrical body whose cross section on a horizontal plane includes not only a circle but also an ellipse, an oval shape, and the like. The term “substantially disk-shaped” means a shape including not only a circle but also an ellipse, an oval shape, and the like in plan view. The term "substantially ring-shaped" refers to a shape that includes not only an annular body but also an ellipsoidal annular body and an oval annular body in plan view.

The cell culture vessel 100 has an opening (not shown) at its upper end and a flat bottom 120 at its lower end. The cell culture vessel 100 has a hollow space 130 filled with a scaffold for culturing cells. A scaffold, a culture medium, and cells to be cultured can be put into the cell culture vessel 100 through an opening at the upper end thereof.

Cells to be cultured are not particularly limited, and can be either cells that are not susceptible to cytotoxicity or cells that are susceptible to cytotoxicity, but are preferably cells that are susceptible to cytotoxicity, and more preferably. , human embryonic stem (ES) cells, iPS cells, somatic stem cells and the like. Preferred somatic stem cells are, for example, adipose-derived or bone marrow-derived mesenchymal stem cells. Also, the medium used is a liquid medium containing, for example, glucose and the like.

The dimensions of the cell culture vessel 100 are not particularly limited, but for example, the volume is 250 ml (100 mm diameter, 50 mm height, 100 mm bottom), 500 ml (100 mm diameter, 100 mm height, 100 mm bottom) or 1000 ml. (diameter 100mm, height 200mm, bottom 100mm), etc.

The cell culture container 100 is a non-cell-adhesive container in which cells are less likely to adhere to the inner wall of the container. It is a plastic or glass container with a hydrophobic surface treatment such as

The cells cultured in the cell culture vessel 100 are vibrated in the vertical direction by the vibrating section 710 of the vibrating device 700 . The vibrating device 700 is, but not limited to, a vortex, for example.

The scaffold that fills the space 130 of the cell culture vessel 100 is, for example, a three-dimensional porous scaffold. A three-dimensional porous scaffold, for example, has an average porosity of 50-90%, preferably 80-90%, and an average pore size of 10-800 μm, preferably 200-400 μm. The scaffold may be a single scaffold or a scaffold consisting of an assembly of multiple cell-supporting pieces. The scaffold culture area is not particularly limited, but is, for example, 800 to 900,000 cm ² , preferably 800 to 45,000 cm ² . In the case of a scaffold consisting of an assembly of a plurality of cell-supporting pieces, for example, it may be a fiber assembly (nonwoven fabric, woven fabric, knitted fabric, etc.) made of thermoplastic resin fibers, or the thermoplastic resin may be kneaded in advance. It may be an assembly of sheets formed by forming after forming.

As shown in FIG. 2, the dish-shaped body 200 has a plurality of first magnetized bodies 210 equidistantly arranged on its periphery. The dish-shaped body 200 has, for example, a thickness of 15 mm, an outer diameter of 95 mm, and an inner diameter of 68 mm. The first magnetized body 210 is a ferromagnetic metal in consideration of the influence on cultured cells. For example, the first magnetized body 210 is a metal containing iron, cobalt, and nickel as main components. When the first magnetized body 210 is a magnet, it is preferable to use a magnet with a low magnetic force in order to suppress the influence on cultured cells.

Although the shape of the first magnetized body 210 is not particularly limited, it can be, for example, a cylindrical body with a diameter of 10 mm and a height of 10 mm. In this embodiment, six first magnetized bodies 210 are provided at equal intervals of 60 degrees around a substantially disk-shaped dish-shaped body 200 . The magnetic force of the first magnetized body 210 is 0 Gauss in the case of iron. The dish-shaped body 200 is arranged horizontally within the hollow space of the cell culture vessel 100 . The substantially disk-shaped dish-shaped body 200 has a smaller diameter than the diameter of the substantially cylindrical body of the cell culture vessel 100 . Specifically, if the inner diameter of the substantially cylindrical body of the cell culture vessel 100 is D ₁ , the diameter D ₂ of the substantially disk-shaped dish-shaped body 200 can be 0.90D ₁ to 0.998D ₁ . Therefore, the substantially disk-shaped dish-shaped body 200 does not contact the inner surface of the cell culture vessel 100 . In addition, it is possible to provide wheels arranged at regular intervals on the outer peripheral portion of the substantially disk-shaped dish-shaped body 200 and bring the wheels and the inner surface of the cell culture vessel 100 into contact with each other.

The substantially ring-shaped annular body 300 is located outside the cell culture vessel 100 and allows the cell culture vessel 100 to be located inside the ring. The thickness of the annular body 300 is, for example, the same as that of the dish-shaped body 200, which is 15 mm. The substantially ring-shaped annular body 300 includes a plurality of second magnetic bodies 310 made of magnets corresponding to the first magnetic bodies 210 of the dish-shaped body 200 . The shape of the second magnetized body 310 is not particularly limited, but may be, for example, a cylindrical body with a diameter of 10 mm and a height of 16 mm. The magnetic force of the second magnetized body 310 is, for example, 4500 to 4800 Gauss for a permanent magnet. The distance between one end of the first magnetized body 210 of the dish-shaped body 200 and the other end of the second magnetized body 310 of the annular body 300 is, for example, 0.1 mm to 10.0 mm. be. Since the second magnetized body 310 of each annular body 300 and the first magnetized body 210 of each dish-shaped body 200 correspond to each other, when the annular body 300 moves up and down along the cell culture vessel 100 , the height of the annular body 300 (that is, the position in the vertical direction) and the height of the dish-shaped body 200 match. It is also possible to express that the height of the ring-shaped body 300 and the height of the plate-shaped body 200 are the same as that the ring-shaped body 300 and the plate-shaped body 200 are aligned in the vertical direction.

A plurality of through holes 220 are provided in the central portion of the dish-shaped body 200 . The hole diameter of the through hole 220 is not particularly limited, but can be, for example, 1 mm to 5 mm.

The annular body 300 is supported by a lifting device 320 and moves up and down. Since the ring-shaped body 300 moves up and down, the dish-shaped body 200 interlocked with the ring-shaped body 300 by magnetic force also moves up and down. This allows agitation of the medium as well as oxygenation and nutrition to the cells. The vertical motion of the annular body 300 is, for example, simple harmonic motion or substantially simple harmonic motion. The cycle of vertical movement of the annular body 300 can be, for example, 0.5 times/hour to 12.0 times/hour. Approximately simple harmonic motion is, for example, a motion that moves from bottom to top, stops for a certain period of time, then moves from top to bottom, stops for a certain period of time, and then moves from bottom to top again. The stroke length of vertical movement of annular body 300 is greater than the height of cell culture vessel 100 . Specifically, the annular body 300 can move up and down in a range from near the top of the cell culture vessel 100 to below the bottom 120 of the cell culture vessel 100, and the stroke length of the vertical movement of the annular body 300 is, for example, 80mm to 260mm is possible. The upward or downward moving speed of the annular body 300 can be, for example, 0.5 mm/sec to 10.0 mm/sec.

As shown in FIG. 3, the cell culture vessel 100 has a bottom projection 125 protruding upward at the center of the bottom 120 and having a space inside. In other words, the cell culture vessel 100 has a bottom concave portion 126 having a shape corresponding to the bottom convex portion 125 in the central portion of the bottom portion 120 thereof. For example, when the diameter of the bottom 120 of the cell culture vessel 100 is 100 mm, the depth of the bottom recess 126 is 8 mm, and the inner diameter of the bottom recess 126 is 60 mm. The vibrating portion 710 of the vibrating device 700 is tightly fitted into the bottom recessed portion 126 .

The dish-shaped body 200 has a dish recess 230 in the center of its lower surface in which the bottom projection 125 of the cell culture vessel 100 is fitted. In FIG. 3, a gap is shown between the bottom protrusion 125 of the cell culture vessel 100 and the dish recess 230 of the dish-shaped body 200, but the bottom protrusion 125 is fitted into the dish recess 230 without any gap. It may be something that can be done.

As shown in FIG. 4 , a storage section 400 is provided below the bottom section 120 of the cell culture vessel 100 . The ring-shaped body 300 moves downward from the bottom portion 120 of the cell culture vessel 100 and is stored in the storage section 400 . The storage part 400 is substantially ring-shaped in plan view so that the substantially ring-shaped annular body 300 is stored therein.

The storage unit 400 is made of a ferromagnetic material, specifically a metal containing iron, cobalt, and nickel as main components. Since the storage part 400 is made of a ferromagnetic material, when the annular body 300 is stored in the storage part 400, the action of the magnetic force to the outside of the second magnetized body 310 inside the annular body 300 is caused by the storage part 400. Therefore, the influence of the magnetic force of the second magnetized body 310 on the cultured cells can be suppressed.

The storage unit 400 includes a bottom peripheral wall 401 that surrounds the bottom of the annular body 300, an outer peripheral wall 403 that surrounds the outer side of the annular body 400 and is provided outside the bottom peripheral wall 401, and a wall that surrounds the inner side of the annular body 400. and an inner peripheral wall 402 provided inside the bottom peripheral wall 401 of the enclosure. The thicknesses of the bottom peripheral wall 401, the inner peripheral wall 402, and the outer peripheral wall 403 are determined from the viewpoint of shielding the magnetic force of the second magnetized body 310 from the outside, and are not particularly limited, but are preferably 0.1 mm or more and 2 mm or less, preferably It is 0.5 mm or more and 1.5 mm or less, more preferably 0.8 mm or more and 1.2 mm or less.

The distance between the bottom peripheral wall 401 of the storage unit 400 and the bottom 120 of the cell culture vessel 100 is not particularly limited, but is, for example, 30 mm or more and 200 mm or less, preferably 40 mm or more and 150 mm or less, more preferably 50 mm or more and 100 mm or less.

The inner peripheral wall 402 and the outer peripheral wall 403 may be formed at the same height or may be formed at different heights. Both the height of the inner peripheral wall 402 and the outer peripheral wall 403 are preferably longer than the height of the second magnetized body 310 inside the annular body 300, and are not particularly limited, but are, for example, 12 mm or more and 50 mm or less, preferably 18 mm or more and 40 mm. Below, more preferably 24 mm or more and 30 mm or less.

Next, the mode of use of the cell culture apparatus according to this embodiment will be described.

First, the dish-shaped body 200 placed in the hollow space 130 of the cell culture vessel 100 is positioned near the bottom of the cell culture vessel 100. As shown in FIG. In this case, since the height of the ring-shaped body 300 and the height of the plate-shaped body 200 are the same, the ring-shaped body 300 is also positioned near the bottom of the cell culture vessel 100 . The height of the ring-shaped body 300 and the height of the dish-shaped body 200 are equal to each other when the height of the center of the second magnetized body 310 in the vertical direction and the center of the first magnetized body 210 in the vertical direction is means match. Then, the scaffold, medium, and cells to be cultured are put into the space 130 of the cell culture vessel 100 through an opening (not shown) at the upper end of the cell culture vessel 100 .

Next, the annular body 300 located near the bottom of the cell culture vessel 100 is moved upward. The second magnetized body 310 of each annular body 300 and the first magnetized body 210 of each dish-shaped body 200 correspond to each other, and the annular body 300 and the dish-shaped body 200 are interlocked by magnetic force to form the dish. The body 200 also moves upward. As the dish-shaped body 200 moves upward, the scaffold filled in the hollow space 130 of the cell culture vessel 100 and the cells adhering to the scaffold are pushed up by the dish-shaped body 200 from below. Move to Although the plate-shaped body 200 moves upward, since a plurality of through holes 220 are provided in the central portion of the plate-shaped body 200, the medium in the space 130 of the cell culture vessel 100 is not pushed upward and penetrates. The medium is agitated as it passes through hole 220 .

Next, the annular body 300 stops moving by positioning the dish-shaped body 200 near the top of the cell culture vessel 100 . When the dish-shaped body 200 is positioned near the top of the cell culture vessel 100, the height of the cell culture vessel 100 is L (the height L is the distance from the opening at the top of the cell culture vessel 100 to the bottom 120). ), the dish-shaped body 200 is positioned at a distance of, for example, 0.3L to 0.7L from the upper end of the cell culture vessel 100 downward. When the dish 200 is positioned near the top of the cell culture vessel 100, the medium is not forced upward, so the cells can be oxygenated, for example.

Next, the annular body 300 is moved downward. The annular body 300 and the dish-shaped body 200 are interlocked by the magnetic force, and the dish-shaped body 200 also moves downward. As the dish-shaped body 200 moves downward, the scaffold and the cells attached to the scaffold drop by gravity and are immersed in the medium to receive nutrients.

When the ring-shaped body 300 continues to move downward and is positioned near the bottom of the cell culture vessel 100, the dish-shaped body 200 comes into contact with the bottom projection 125 of the cell culture vessel 100, and the dish-shaped recess 230 of the dish-shaped body 200 It is fitted into the bottom projection 125 of the cell culture vessel 100 without any gap and stopped.

Annular body 300 is then moved further below bottom 120 of cell culture vessel 100 and stored in storage 400 shown in FIG. By storing the ring-shaped body 300 in the storage section 400, the magnetic force acting on the cultured cells of the second magnetized body 310 inside the ring-shaped body 300 is reduced.

After storing the annular body 300 in the storage part 400 for a certain period of time, the annular body 300 stored in the storage part 400 is moved upward. When the ring-shaped body 300 continues to move upward and the height of the ring-shaped body 300 and the height of the dish-shaped body 200 match near the bottom of the cell culture vessel 100, the second magnetized body of each ring-shaped body 300 Since 310 and the first magnetized bodies 210 of each dish-shaped body 200 correspond to each other, the annular body 300 and the dish-shaped body 200 are interlocked by the magnetic force, and the dish-shaped body 200 also moves upward.

After culturing, the dish concave portion 230 of the dish-shaped body 200 is tightly fitted into the bottom convex portion 125 of the cell culture vessel 100, and the vibrating portion 710 of the vibration device 700 is tightly fitted into the bottom concave portion 126 of the cell culture vessel 100. In this state, the vibrating device 700 is operated to transmit, for example, vertical vibration from the vibrating section 710 to the cultured cells in the cell culture vessel 100 . This makes it easier to collect cultured cells deeply embedded in the three-dimensional porous scaffold.

Thus, in the first embodiment, it is possible to oxygenate and supply nutrients to cells while suppressing the influence of magnetic force on cultured cells.

(2) Second Embodiment When a large amount of scaffolding such as a large number of non-woven fabric sheets is used, the weight of the scaffolding may hinder the upward movement of the dish-shaped body 200 . In order to properly raise the dish-shaped body 200 even when a large amount of scaffolding is used, it is necessary to ensure the action of the magnetic force between the first magnetized body 210 and the second magnetized body. For that purpose, it is necessary to reduce the distance between the first magnetized body 210 and the second magnetized body 310 . However, when the distance between the first magnetized body 210 and the second magnetized body 310 is shortened, if an unexpected external force is applied to the cell culture device 900, the annular body 300 stored in the storage part 400 will rise. In some cases, there is also a risk of colliding with the bottom portion 120 of the cell culture vessel 100 . For this reason, when the dish-shaped body 200 moves upward, it is necessary to pull the second magnetized body 310 in the annular body 300 toward the dish-shaped body 200, while the annular body 300 stored in the storage part 400 It is preferable that the second magnetized body 310 in the ring-shaped body 300 moves to the opposite side of the dish-shaped body 200 when it rises.

In order to solve the above-described problem, the cell culture device 900 according to this embodiment has the annular body 300 with a characteristic configuration in addition to the configuration of the above-described first embodiment. That is, in the cell culture device 900 according to this embodiment, as shown in FIGS. 5(a) and 5(b), the ring-shaped body 300 has a housing portion 350 in which the second magnetized body 310 is housed. The storage part 350 has a gap inside so that the second magnetized body 310 can slide in the radial direction of the annular body 300 . Thereby, the second magnetized body 310 is housed in the housing portion 350 so as to be slidable in the radial direction.

The housing portion 350 has a columnar hollow portion 351 extending in the radial direction of the annular body 300 and a widening portion 352 located radially outside the columnar hollow portion 351 . The columnar cross section of the columnar hollow portion 351 and the cross section of the second magnetized body 310 have the same shape, and the inner surface of the columnar hollow portion 351 and the outer surface of the second magnetized member 310 are in close contact. Movements other than radial sliding of the magnetized body 310 are restricted. That is, the second magnetized body 310 only slides in the radial direction of the annular body 300 and does not move in the direction perpendicular to the radial direction of the annular body 300, for example.

The spreading portion 352 is spatially continuous with the columnar hollow portion 351 . As shown in FIGS. 5(a) and 5(b), the cross-sectional area perpendicular to the radial direction of the annular body 300 is larger at the expanded portion 352 than at the columnar hollow portion 351. As shown in FIGS.

The second magnetized body 310 has a stopper portion 370 at the radially outer end of the annular body 300 . As shown in FIGS. 5(a) and 5(b), the cross-sectional area perpendicular to the radial direction of the annular body 300 is such that the stopper portion 370 is larger than the columnar hollow portion 351 and the spreading portion 352 is larger than the stopper portion 370. As shown in FIG. The stopper portion 370 is located within the flared portion 352 . The radial length of the annular body 300 of the stopper portion 370 (that is, the thickness of the stopper portion 370) is, for example, 1.5 mm.

The annular body 300 has a third magnetized body 360 on the radially outer side of the housing portion 350 . That is, as shown in FIGS. 5(a) and 5(b), the ring-shaped body 300 is provided with the third magnetized body 360 on the radially outer side of the spread portion 352. As shown in FIG. Like the first magnetized body 210, the third magnetized body 360 is a ferromagnetic metal. For example, the third magnetized body 360 is a metal containing iron, cobalt, and nickel as main components. The mass of the third magnetized body 360 is smaller than the mass of the first magnetized body 210 . When the third magnetized body 360 is a magnet, it is preferable to use a magnet with a low magnetic force in order to suppress the influence on cultured cells.

As described above, in the configuration according to the second embodiment, unlike the first embodiment, the ring-shaped body 300 includes the storage portion 350 that slidably stores the second magnetic body 310 and the third magnetic body 360. , and a stopper portion 370. This characteristic configuration is used as an attracting mechanism (as described later, when the height of the annular body 300 and the height of the dish-shaped body 200 are the same, the second magnetized body 310 has a mechanism that is attracted to the first magnetized body 210 instead of the third magnetized body 360).

Next, the mode of use of the cell culture apparatus according to this embodiment will be described.

First, the dish-shaped body 200 placed in the hollow space 130 of the cell culture vessel 100 is positioned near the bottom of the cell culture vessel 100. As shown in FIG. At this time, the height of the ring-shaped body 300 and the height of the dish-shaped body 200 are the same, and the ring-shaped body 300 is also positioned near the bottom of the cell culture vessel 100 .

The second magnetized body 310 is a magnet, but the first magnetized body 210 and the third magnetized body 360 are metal made of ferromagnetic material. The mass of the third magnetized body 360 is smaller than the mass of the first magnetized body 210 . As a result, when the height of the ring-shaped body 300 and the height of the plate-shaped body 200 match, the second magnetic body 310 is attracted to the first magnetic body 210 instead of the third magnetic body 360. As shown in 5(b) and FIG. 6, the second magnetized body 310 inside the ring-shaped body 300 slides in the housing portion 350 and moves radially inwardly of the ring-shaped body 300. As shown in FIG. The radially inner side is the side toward the center of the annular body 300 . Since the second magnetized body 310 is a magnet and the first magnetized body 210 is a ferromagnetic metal, the magnetic force acts on the first magnetized body 210 from the second magnetized body 310. , the first magnetized body 210 is fixed to the dish-shaped body 200, and the second magnetized body 310 is slidable in the radial direction of the annular body 300. The explanation is that it is attracted to the body 210 .

As shown in FIG. 5(b), the cross-sectional area perpendicular to the radial direction of the annular body 300 is larger in the order of the columnar hollow portion 351, the stopper portion 370, and the spread portion 352. When attracted to the first magnetized body 210, the stopper portion 370 abuts the radially inner end of the spread portion 352, thereby separating the radially inner end of the second magnetized body 310 and the cell culture vessel 100 from the outside of the cell culture vessel 100. A small gap of distance L2 is generated between the side surfaces. Although the distance L2 is not particularly limited, it is, for example, 0.05 mm to 0.15 mm, preferably 0.1 mm. As shown in FIG. 5(b), a gap is formed between the radially outer end of the stopper portion 370 and the radially inner end of the expanding portion 352, and the distance is, for example, 2.2 mm.

Next, the annular body 300 located near the bottom of the cell culture vessel 100 is moved upward. The second magnetized body 310 of each annular body 300 and the first magnetized body 210 of each dish-shaped body 200 correspond to each other, and the annular body 300 and the dish-shaped body 200 are interlocked by magnetic force to form the dish. The body 200 also moves upward. Since the first magnetized body 210 and the second magnetized body 310 are close to each other at a very small distance L2, even if a large amount of scaffolding such as a large number of non-woven fabric sheets is used, the scaffolding is There is no possibility that the upward movement of the dish-shaped body 200 will be hindered due to the weight of the plate.

Next, the annular body 300 stops moving by positioning the dish-shaped body 200 near the top of the cell culture vessel 100 . When the dish 200 is positioned near the top of the cell culture vessel 100, the medium is not forced upward, so the cells can be oxygenated, for example.

Next, the annular body 300 is moved downward. The annular body 300 and the dish-shaped body 200 are interlocked by the magnetic force, and the dish-shaped body 200 also moves downward. The scaffold and the cells attached to the scaffold are gravity-fed and immersed in the medium to feed the cells.

When the ring-shaped body 300 continues to move downward and is positioned near the bottom of the cell culture vessel 100, the dish-shaped body 200 comes into contact with the bottom projection 125 of the cell culture vessel 100, and the dish-shaped recess 230 of the dish-shaped body 200 It is fitted into the bottom protrusion 125 of the cell culture vessel 100 .

Next, the annular body 300 moves further downward than the bottom 120 of the cell culture vessel 100, while the dish 200 remains fitted in the bottom projection 125 of the cell culture vessel 100. FIG. As the annular body 300 moves downward, the distance between the second magnetized body 310 and the first magnetized body 210 increases, and the second magnetized body 310, which has been attracted to the first magnetized body 210, finally It is attracted to the third magnetized body 360 instead of the first magnetized body 210, and as shown in FIGS. It moves radially outward of the annular body 300 . The radially outer side is the side away from the center of the annular body 300 . Since the second magnetic body 310 is a magnet and the third magnetic body 360 is a ferromagnetic metal, the magnetic force acts on the third magnetic body 360 from the second magnetic body 310. , the third magnetized body 360 is fixed to the annular body 300, and the second magnetized body 310 is slidable in the radial direction of the annular body 300; It is explained that it is attracted to 360. The stopper portion 370 abuts on the radially outer end portion of the spread portion 352, thereby providing a distance L1 between the radially inner end portion of the second magnetized body 310 and the outer surface of the cell culture vessel 100. Become. Although the distance L1 is not particularly limited, it is, for example, 2.1 mm to 2.5 mm, preferably 2.3 mm.

Next, as shown in FIG. 4 , the annular body 300 is stored in the storage section 400 provided below the bottom 120 of the cell culture vessel 100 . When the ring-shaped body 300 is stored in the storage portion 400, the second magnetized body 210 is attracted to the third magnetized body 360, and the stopper portion 370 abuts on the radially outer end portion of the spread portion 352. ing. When the annular body 300 is stored in the storage unit 400, the effect of the magnetic force on the outside of the second magnetized body 310 is reduced by the storage unit 400, and the influence of the magnetic force on the cultured cells can be suppressed.

Next, the annular body 300 stored in the storage section 400 is moved upward. Since the radially inner end of the second magnetized body 310 and the outer surface of the cell culture vessel 100 are separated by a distance L1, the height between the upper end of the annular body 300 and the bottom 120 of the cell culture vessel 100 is When they are equal, even if an unexpected external force acts, the second magnetized body 310 of the annular body 300 is unlikely to come into contact with the cell culture vessel 100 .

Further, when the annular body 300 moves upward and rises to a height near which the height of the annular body 300 and the height of the dish-shaped body 200 coincide, the height shown in FIGS. In this way, the second magnetized body 310 is attracted to the first magnetized body 210 instead of the third magnetized body 360, and the second magnetized body 310 slides in the housing portion 350, causing the annular body 300 to move. , and the first magnetized body 210 and the second magnetized body 310 approach each other at a distance L2. In this state, the annular body 300 and the dish-shaped body 200 are interlocked, and the dish-shaped body 200 also moves upward.

In this way, in the second embodiment, even when a large amount of nonwoven fabric sheets are inserted, the precise up-and-down motion of the annular body 300 is ensured, and the influence of the magnetic force on the cultured cells is suppressed, while the cells are oxygenated. and nutrition.

(Example 1)
In this example, an example of cell culture using a cell culture device (the cell culture device of the first embodiment) having a storage section for storing an annular body will be described.

A dish-shaped body placed in the hollow space of the cell culture vessel was positioned near the bottom of the cell culture vessel. A scaffold, a medium, and cells to be cultured were put into the space of the cell culture vessel through the opening at the top of the cell culture vessel. The cells were 2.0×10 ⁷ adipose-derived mesenchymal stem cells (ADSC). 500 BioNOC II matrices from CESCO were used as scaffolds (3D culture). The stroke length of vertical movement of the annular body was 90 mm. That is, the height of the cell culture vessel was 60 mm, and the distance between the bottom of the cell culture vessel and the storage section was 30 mm. The upward or downward moving speed of the annular body was set to 1.0 mm/sec. The cycle of vertical movement of the annulus was 1.0 times/hour. The number of culture days was 7 days.

When the amount of sugar consumed by cells was measured using a GlucCell Handy Glucose Monitor (CESCO), the amount of sugar consumed by cells increased as the number of days increased. showed that the cells were properly cultured.

On the 7th day, a large amount of 3.0×10 ⁸ ADSCs were recovered from the cell culture vessel. Although it is difficult to efficiently recover the cultured cells buried deep in the three-dimensional porous scaffold without damaging them, after culturing, an enzyme solution containing trypsin-EDTA and caseinase is added to the cell culture vessel. A large amount of cultured cells can be recovered from the cell culture vessel by enzymatically treating the cultured cells by adding .

It was analyzed whether the recovered ADSCs retained a normal karyotype. After the collected ADSCs were fixed with Carnoy, simple karyotype analysis (Q-band) was performed (chromocenter Co., Ltd.). Simple karyotype analysis (Q-band analysis) identifies each chromosome based on the band pattern characteristic of the chromosome detected by the banding method, and analyzes the presence or absence of polyploidy, aneuploidy, translocation, etc. method. As a result, it was found that the collected ADSCs retained a normal karyotype, and the cells cultured using the cell culture apparatus according to this example maintained a normal karyotype.

That is, in Example 1, it was shown that the influence of the magnetic force on the cultured cells could be suppressed and the cells could be appropriately cultured.

(Example 2)
In this example, an example of cell culture using a cell culture device (the cell culture device of the second embodiment) that has a storage portion for storing an annular body and is provided with a so-called attracting mechanism will be described.

A dish-shaped body placed in the hollow space of the cell culture vessel was positioned near the bottom of the cell culture vessel. A scaffold, a medium, and cells to be cultured were put into the space of the cell culture vessel through the opening at the top of the cell culture vessel. The cells were 2.0×10 ⁸ adipose-derived mesenchymal stem cells (ADSC). As a scaffold, 5000 pieces of BioNOC II matrix from CESCO were used (3D culture). The stroke length of vertical movement of the annular body was 90 mm. That is, the height of the cell culture vessel was 60 mm, and the distance between the bottom of the cell culture vessel and the storage section was 30 mm. The upward or downward moving speed of the annular body was set to 1.0 mm/sec. The cycle of vertical movement of the annulus was 1.0 times/hour. The culture days were 10 days. During the 10-day culture period, the toroids moved up and down normally.

When the amount of sugar consumed by cells was measured using a GlucCell Handy Glucose Monitor (CESCO), the amount of sugar consumed by cells increased as the number of days increased. showed that the cells were properly cultured.

On the 10th day, a large amount of 3.0×10 ⁹ ADSCs were recovered from the cell culture vessel. It was analyzed whether the recovered ADSCs retained a normal karyotype. After the collected ADSCs were fixed with Carnoy, simple karyotype analysis (Q-band) was performed (chromocenter Co., Ltd.). As a result, it was found that the collected ADSCs retained a normal karyotype, and the cells cultured using the cell culture apparatus according to this example maintained a normal karyotype.

That is, in the second embodiment, even when a large amount of 5000 non-woven fabric sheets were inserted, cells could be cultured while ensuring accurate vertical movement of the annular body and suppressing the influence of the magnetic force on the cultured cells. shown.

(Example 3)
Differentiation into adipocytes, chondrocytes, and osteocytes was induced using ADSCs after large-scale culture and recovery according to Examples 1 and 2. Human Mesenchymal Stem Cell Functional IDentification Kit from R&D System was used for induction of differentiation. After adhering 10 ⁴ cells each to a 24-well culture plate, they were incubated for 14 days using a differentiation medium containing the ADipogenic Supplement and ITS Supplement supplied with the kit added to 50 ml of DMEM basal medium to induce differentiation into adipocytes. cultured. For induction of differentiation into chondrocytes, culture was performed for 21 days using a differentiation medium prepared by adding the ChonDrogenic Supplement and ITS Supplement attached to the kit to 50 ml of DMEM basal medium. To induce differentiation into osteocytes, culture was performed for 21 days using a differentiation medium prepared by adding the Osteogenic Supplement and ITS Supplement attached to the kit to 50 ml of DMEM basal medium.

In order to identify adipocytes, chondrocytes, and osteocytes after differentiation induction, OilReD-O, Alcian Blue, Alizarin ReD staining was performed after fixation with 4% paraformaldehyde. As a result, ADSCs after mass culture retained stem cell properties and pluripotency, and differentiated into adipocytes, chondrocytes, and osteocytes.

Can be used for cell culture.

100: Cell culture vessel
120: Bottom
125: bottom convex
126: bottom recess
130: Spatial part
200: Plate
220: through hole
230: Countersunk recess
210: 1st magnetized body
300: Toroid
310: Second magnetized body
320: Elevator
350: Storage part
351: Columnar hollow part
352: Spreading part
360: Third magnetized body
370: Stopper
400: Enclosure
401: Bottom peripheral wall
402: inner peripheral wall
403: Outer peripheral wall
700: Vibration device
710: Vibration part
900: Cell culture equipment

Claims (8)

a substantially cylindrical cell culture vessel having a flat bottom at the lower end and a hollow space filled with a scaffold for culturing cells;
A plurality of first magnetized bodies made of a ferromagnetic material are provided on the periphery at equal intervals, and the cells are horizontally arranged in a hollow space of the cell culture vessel without contact with the inner wall of the cell culture vessel. a substantially disk-shaped dish-shaped body having a diameter smaller than the diameter of the substantially cylindrical body of the culture vessel;
a plurality of second magnetic bodies composed of magnets corresponding to the first magnetic bodies of each of the dish-shaped bodies so as to attract the first magnetic bodies of each of the dish-shaped bodies by magnetic force; By positioning the cell culture vessel inside the ring outside the vessel and moving upward, the dish-shaped body is moved upward in conjunction with the magnetic force to enter the hollow space of the cell culture vessel. By pushing up the scaffold filled in the cell culture vessel from below and moving it downward, the scaffold filled in the hollow space of the cell culture vessel is moved downward by gravity in conjunction with the magnetic force. a substantially ring-shaped annular body that moves vertically,
and a storage section made of a ferromagnetic material, in which the ring-shaped body is stored by moving downward from the bottom of the cell culture container. The storage portion includes a bottom peripheral wall that surrounds the bottom of the annular body, an outer peripheral wall that surrounds the outer side of the annular body and is provided outside the bottom peripheral wall, and a peripheral wall that surrounds the inner side of the annular body. and an inner peripheral wall provided inside the bottom peripheral wall of the enclosure. The annular body includes a storage section in which the second magnetic bodies are accommodated with a gap therebetween so as to be slidable in the radial direction. a plurality of third magnetized bodies made of ferromagnetic material provided on the direction outside,
When the ring-shaped body moves downward from the bottom of the cell culture vessel and is stored in the storage section, the second magnetic body is attracted to the third magnetic body by magnetic force and moves through the storage section. slide radially outward,
When the ring-shaped body moves upward from the bottom of the cell culture vessel and aligns with the dish-shaped body, the second magnetized body is attracted to the first magnetized body by magnetic force and slides in the storage part. 3. The cell culture device according to claim 1, wherein the cell culture device moves radially inward by movement. 4. The cell culture apparatus according to claim 3, wherein the mass of said third magnetized body is smaller than the mass of said first magnetized body . In the storage part, the inner surface and the outer surface of the second magnetized body are in close contact with each other, so that movement of the second magnetized body other than sliding in the radial direction is restricted in the radial direction of the annular body. 5. The cell culture device according to claim 3, further comprising an extending columnar hollow portion and a widening portion positioned radially outward of the columnar hollow portion. The second magnetized body has a stopper portion located in the widening portion at the radially outer end of the annular body, and the cross-sectional area perpendicular to the radial direction of the annular body is the columnar hollow portion, 6. The cell culture device according to claim 5, wherein the size of the stopper portion is larger than that of the spread portion in this order. 7. The cell culture device according to any one of claims 1 to 6, wherein the scaffold filled in the cell culture container is an aggregate of a plurality of cell support pieces. 8. The cell culture apparatus according to any one of claims 1 to 7, wherein the cells to be cultured are mesenchymal stem cells.

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