JP2021065118A - Suspension culture device and suspension culture method - Google Patents

Abstract

To provide a culture device and culture method that allow the culture of three-dimensional tissue having sufficient thickness.SOLUTION: A suspension culture device 1 has a culture tank 11 that stores an aqueous two-phase culture medium composed of an outer phase 101 and an internal phase 102. The internal phase 102 is used as a culture phase and is suspended in the outer phase 101. The suspension culture device 1 has suspension means that maintains the suspended state of the internal phase 102. The suspension means maintains the suspended state of the internal phase 102 by, for example, fluidizing the outer phase 101 in the culture phase 11.SELECTED DRAWING: Figure 1

Description

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

In recent years, in the medical field, regenerative medicine that artificially creates tissues and organs using cells to restore the functions of injured tissues and organs has attracted attention. Tissues and organs of multicellular organisms including humans have a three-dimensional structure composed of a plurality of cells. Therefore, a technique for culturing a cell tissue having a three-dimensional structure in vitro has been proposed.

U.S. Patent Application Publication No. 2016/0091487

The technique disclosed in Patent Document 1 uses an aqueous two-phase culture solution consisting of an immersion polymer phase and a droplet polymer phase, and the droplet polymer is used. This is a technique for culturing three-dimensional aggregates of cells in a phase. According to such a technique, cells can be cultured without being adversely affected by medium exchange and preventing evaporation of droplets. However, since the cells are cultured in the droplet polymer phase settled at the bottom of the water-immersed polymer phase, the cells spread in a plane during the culture, and there is a problem that sufficiently thick cells cannot be cultured.

The present invention has been made in view of the above, and an object of the present invention is to provide a culturing apparatus and a culturing method capable of culturing a three-dimensional tissue having a sufficient thickness.

In the present invention, an aqueous two-phase culture solution containing an outer phase and an inner phase as a culture phase floating in the outer phase, a culture tank containing the aqueous two-phase culture solution, and a floating state of the inner phase are provided. The present invention relates to a suspension culture apparatus comprising a suspension means for maintaining.

The floating means may maintain the floating state of the inner phase by flowing the outer phase in the culture tank.

The flow velocity at which the outer phase flows in the culture tank may be controlled according to the floating state of the inner phase in the culture tank.

The suspension culture device may include a circulation mechanism that collects the external phase that has flowed out of the culture tank and allows it to flow back into the culture tank for circulation.

The specific gravity of the inner phase is larger than the specific gravity of the outer phase, and the floating means causes the outer phase to flow in from below the culture tank and flows out from above the culture tank to cause the outer phase to flow out. May be maintained in a floating state.

The floating means may maintain the floating state of the internal phase by rotating the culture tank.

The aqueous two-phase culture solution may be composed of a dextran phase and a polyethylene glycol phase.

The outer phase may be a polyethylene glycol phase and the inner phase may be a dextran phase.

Further, the present invention is a suspension culture method for culturing cells in a culture solution, wherein the culture solution is an aqueous two-phase culture solution containing an outer phase and an inner phase as a culture phase floating in the outer phase. The present invention relates to a suspension culture method in which the culture is performed while maintaining the suspension state of the internal phase.

In the floating culture method, the floating state of the inner phase may be maintained by flowing the outer phase in the culture tank.

The flow velocity at which the outer phase flows in the culture tank may be controlled according to the floating state of the inner phase in the culture tank.

In the suspension culture method, the external phase that has flowed out of the culture tank may be recovered and flowed back into the culture tank for circulation.

The specific gravity of the inner phase is larger than the specific gravity of the outer phase, and the outer phase is allowed to flow in from below the culture tank and the outer phase is allowed to flow out from above the culture tank to maintain the floating state of the inner phase. You may.

In the suspension culture method, the suspension state of the internal phase may be maintained by rotating the culture tank.

The aqueous two-phase culture solution may be composed of a dextran phase and a polyethylene glycol phase.

The outer phase may be a polyethylene glycol phase and the inner phase may be a dextran phase.

According to the present invention, it is possible to provide a culturing apparatus and a culturing method capable of culturing a three-dimensional tissue having a sufficient thickness.

It is a schematic diagram which shows the suspension culture apparatus which concerns on 1st Embodiment. It is a schematic diagram which shows the suspension culture apparatus which concerns on 2nd Embodiment. It is a figure which shows the fluorescence image of the cell cultured by the suspension culture method which concerns on Example. It is a figure which shows the fluorescence image of the cell cultured by the culture method which concerns on a comparative example. It is a figure which shows the fluorescence image of the cell cultured by the suspension culture method which concerns on Example.

Hereinafter, the suspension culture apparatus and the suspension culture method according to the embodiment of the present invention will be described. The present invention is not limited to the following embodiments.

[First Embodiment]
As shown in FIG. 1, the suspension culture device 1 according to the present embodiment includes a culture solution 10, a culture tank 11, a pump 20, an incubator 30, a flow path 40, and a position detection device 50.

The culture solution 10 is an aqueous two-phase culture solution containing an outer phase 101 and an inner phase 102 as a culture phase. By using the aqueous two-phase culture solution for culturing, the adverse effect on the object to be cultured can be reduced. The aqueous two-phase culture solution is, for example, a culture solution separated into two phases, which is produced by combining two aqueous polymers having different chemical structures. The combination of such an aqueous polymer is not particularly limited, and is, for example, polyethylene glycol, propylene glycol, methoxypolyethylene glycol, dextran, dextran sulfate, polyether, polyaspartate, polyamine, polylysine, sodium carboxymethyl cellulose, polyvinyl alcohol. , Ethylene / propylene copolymer, and a combination of two polymers selected from the group consisting of polyvinylpyrrolidone.
When the culture solution 10 according to the present embodiment is described as a polyethylene glycol phase (hereinafter, may be referred to as “PEG phase”) and a dextran phase (hereinafter, referred to as “DEX phase”) obtained by combining polyethylene glycol and dextran. It is preferable that the aqueous two-phase culture solution is composed of (there is). Further, it is preferable that the outer phase 101 is the PEG phase and the inner phase 102 as the culture phase is the DEX phase.

The aqueous biphasic culture medium contains a medium suitable for culturing cells to be cultured. The medium is not particularly limited, and conventionally known media are used. For example, a medium obtained by adding serum or serum substitute to a basal medium is used.

The aqueous two-phase culture solution is prepared by dissolving the above two types of polymers in a medium, mixing them, and separating them naturally or by applying centrifugal force.
The method for preparing the aqueous two-phase culture solution will be described below by taking an aqueous two-phase culture solution composed of a PEG phase and a DEX phase as an example. First, a PEG solution is prepared by dissolving polyethylene glycol in a medium. The molecular weight and concentration of polyethylene glycol are not particularly limited, but for example, polyethylene glycol having a molecular weight of 8000 to 20000 and a concentration of 2.5 to 14% by weight can be used. Similarly, a DEX solution is prepared by dissolving dextran in a medium. The molecular weight and concentration of dextran are not particularly limited, but for example, those having a molecular weight of 20000 to 500,000 and a concentration of 3.2 to 14% by weight can be used. After autoclave sterilization of the prepared PEG solution and DEX solution, the PEG solution and DEX solution are mixed and separated into two phases by centrifugation or the like. As a result, a solution separated into two phases, a PEG phase containing a large amount of polyethylene glycol and a DEX phase containing a large amount of dextran, can be obtained. Then, for example, by dropping the DEX phase into the prepared container filled with the PEG phase, an aqueous two-phase culture solution having the PEG phase as the outer phase and the DEX phase as the inner phase can be obtained.

The outer phase 101 exists around the inner phase 102 in the culture tank 11. Further, the foreign phase 101 is stored in the reservoir 32. As shown in FIG. 1, the outer phase 101 in the culture tank 11 and the outer phase 101 in the reservoir 32 circulate through the flow path 40 by the pump 20. As a result, the environment of the internal phase 102 is maintained in the same environment as that in the incubator 30 through the external phase 101. Further, the floating state of the inner phase 102 is maintained by the flow of the outer phase 101 in the culture tank 11.

The internal phase 102 is used as a culture phase for culturing cells and is prepared as a suspension of cells. For example, when the DEX phase is used as the internal phase 102, the internal phase 102 is prepared by suspending cells in the DEX phase.
The inner phase 102 floats in the outer phase 101 in the culture tank 11. As shown in FIG. 1, the internal phase 102 floats by forming spherical or long spherical droplets. The size and number of the internal phases 102 are not particularly limited. The inner phase 102 has a higher specific gravity than the outer phase 101, for example, and the gravity acting on the inner phase 102 is larger than the buoyancy. In this case, the internal phase 102 gradually settles in the culture tank 11. However, when the outer phase 101 flows in the culture tank 11 by a floating means such as a pump 20, an upward drag acts against the inner phase 102, and the floating state of the inner phase 102 is maintained.

Examples of the culture to be cultured in the internal phase 102 include mammalian cells including humans. The type of cell is not particularly limited, and examples thereof include somatic cells, progenitor cells thereof, and mixed cells thereof. Specifically, stem cells such as embryonic stem cells, artificial pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells, nerve stem cells, and skin stem cells, myocardial cells, heart wall cells, hepatocytes, fibroblasts, and osteoblasts. , Vascular endothelial cells, hepatic stem cells, mesenchymal cells, pancreatic islet cells, cartilage cells, epithelial cells and the like, cancer cells, cancer-derived cell lines and the like. The cells to be cultured are preferably adhesive cells. When the adherent cells are cultured in a plane, they spread on a plane to form a two-dimensional tissue, but by suspending the adherent cells, a three-dimensional agglomerate of cells can be formed.

The culture tank 11 is a container for containing the culture solution 10. The culture tank 11 is provided with an inflow port 111 into which the outer phase 101 flows in and an outflow port 112 in which the outer phase 101 flows out. The inflow port 111 is provided below the culture tank 11, and the outflow port 112 is provided above the culture tank 11. The shape of the culture tank 11 is not particularly limited, and for example, a cylindrical one or a square tubular one is used. The material of the culture tank 11 is preferably a light-transmitting member. By forming the culture tank 11 with a light-transmitting member, the position of the internal phase 102 in the culture tank 11 can be easily grasped from the outside. The light transmissive member is not particularly limited, but for example, a transparent resin such as glass or vinyl chloride is used.

The pump 20 is a device capable of transporting a liquid at a constant flow velocity. The pump 20 is a floating means for maintaining the floating state of the inner phase 102 in the culture tank 11 by supplying the outer phase 101 from below the culture tank 11 and collecting it from above. Further, the pump 20 constitutes a circulation mechanism for circulating the external phase 101 together with the flow path 40 described later. Specifically, the pump 20 and the flow path 40 are configured so that the external phase 101 flowing out of the culture tank 11 is collected in the reservoir 32 and supplied to the culture tank 11 again for circulation. The pump 20 is preferably located outside the incubator 30.
As the pump 20, for example, a roller pump is used. The roller pump has a roller inside, a rotating body to which the roller is attached, a motor for rotating the rotating body, and a housing (not shown). When the rotating body rotates, the roller pump squeezes the tube through which the liquid flows by the roller and presses it against the housing to send the liquid in the tube. The roller pump is not particularly limited, and conventionally known ones are used.

The incubator 30 is a device that accommodates an object to be cultured such as cells and maintains and controls an environment suitable for culturing. The incubator 30 includes a chamber 31 and a reservoir 32.

The chamber 31 has a door that can be opened and closed. Inside the chamber 31, for example, temperature, humidity, CO ₂ concentration and the like are adjusted to predetermined conditions. Inside the chamber 31, a hot water heater, an electric heater, a humidifier, a carbon dioxide gas cylinder, a temperature / humidity sensor, a CO ₂ concentration sensor, and the like are provided to adjust the above conditions (not shown). The above-mentioned predetermined conditions differ depending on the type of tissue to be cultured, but for example, when culturing mammalian cells including humans, the conditions of temperature 37 ° C., humidity 100%, and CO ₂ concentration 5% are applied. ..

The reservoir 32 is a container arranged in the chamber 31 and in which the foreign phase 101 is stored. The reservoir 32 is a container whose part is opened toward the inside of the chamber 31, and is configured to be gas exchangeable with the atmosphere in the chamber 31. The reservoir 32 is connected to the culture tank 11 via the second flow path 43, and is connected to the pump 20 via the third flow path 44. In addition, it is connected to the first flow path 41 via the bubble removing portion 42.

The flow path 40 is a flow path through which the foreign phase 101 as a culture solution flows. The flow path 40 includes a first flow path 41, a bubble removing portion 42, a second flow path 43, and a third flow path 44. These flow paths are made of a flexible resin such as a silicone resin.

The first flow path 41 is arranged between the pump 20 and the culture tank 11. The upstream end of the first flow path 41 is connected to the pump 20, and the downstream end is connected to the inflow port 111 of the culture tank 11.
The bubble removing portion 42 is a vent pipe arranged between the first flow path 41 and the reservoir 32. The upstream end of the bubble removing portion 42 is connected to an arbitrary portion in the middle of the first flow path 41, and the downstream end is connected to the reservoir 32. The inner diameter of the bubble removing portion 42 is preferably larger than the inner diameter of the first flow path 41. As a result, the flow velocity in the bubble removing section 42 is reduced, and the bubbles in the first flow path 41 can be removed more reliably.
The second flow path 43 is arranged between the culture tank 11 and the reservoir 32. The upstream end of the second flow path 43 is connected to the outlet 112 of the culture tank 11, and the downstream end is connected to the reservoir 32.
The third flow path 44 is arranged between the reservoir 32 and the pump 20. The upstream end of the third flow path 44 is connected to the reservoir 32 and the downstream end is connected to the pump 20.

The position detection device 50 detects a floating state such as the position of the internal phase 102 in the culture tank 11. Further, the flow velocity of the outer phase 101 flowing into and out of the culture tank 11 is automatically controlled according to the floating state of the inner phase 102. The position detection device 50 includes an image sensor 51, a control unit 52, and a communication unit 53.

The image sensor 51 is a camera device including an image sensor that reads light through an optical component such as a lens, and has a function of converting an image of a photographed object into a digital signal. The shape and color of the internal phase 102 in the culture tank 11 are registered in advance in the image sensor 51, and the position coordinate data of the internal phase 102 in the captured image can be acquired.

The control unit 52 controls the pump 20 through the communication unit 53 based on the position coordinate data of the internal phase 102 acquired by the image sensor 51. For example, when the central position of the internal phase 102 is lower than the intermediate position in the vertical direction of the culture tank 11, control is performed so as to increase the flow velocity of the pump 20. Further, when the central position of the internal phase 102 is higher than the intermediate position in the vertical direction of the culture tank 11, control is performed so as to reduce the flow velocity of the pump 20. By performing the above control, the control unit 52 can stably suspend the internal phase 102 so as to be in a fixed position in the culture tank 11.

The method of circulating the outer phase 101 in the apparatus and suspending the culture in the inner phase 102 using the suspension culture apparatus 1 described above is as follows.
When the outer phase 101 is pumped by using the pump 20, the outer phase 101 flows in the direction of the arrow 132 and flows into the culture tank 11 through the first flow path 41. At this time, the bubbles mixed in the outer phase 101 flow into the bubble removing section 42 and are removed before flowing into the culture tank 11. The bubble removing section 42 communicates with the reservoir 32 in the incubator 30, and the foreign phase 101 collected together with the bubbles flows into the reservoir 32. The outer phase 101 flowing into the culture tank 11 from the inflow port 111 provided below the culture tank 11 flows from the lower side to the upper side against the direction of gravity 121, and the inner phase 102 in the culture tank 11 is suspended. .. The outer phase 101 flows out from the outflow port 112 provided above the culture tank 11. The foreign phase 101 flowing out of the culture tank 11 flows in the direction of the arrow 131 through the second flow path 43, and flows into the reservoir 32 in the incubator 30. The foreign phase 101 that has flowed into the reservoir 32 is kept under a predetermined condition and stored in the reservoir 32. The foreign phase 101 stored in the reservoir 32 is supplied to the pump 20 through the third flow path 44.
The flow velocity of the outer phase 101 flowing from the pump 20 into the culture tank 11 is controlled according to the floating state of the inner phase 102 in the culture tank 11. As a result, the floating state of the internal phase 102 can be maintained. At this time, the flow velocity of the outer phase 101 may be controlled by using the position detection device 50.

According to the suspension culture device 1 and the culture method according to the first embodiment described above, the following effects are achieved.
The suspension culture device 1 was configured to include an outer phase 101, an aqueous two-phase culture solution 10 containing an inner phase 102 as a culture phase suspended in the outer phase 101, a culture tank 11, and a suspension means. As a result, the floating state of the internal phase 102 can be maintained, and a three-dimensional tissue having a sufficient thickness can be cultured.

The floating means of the inner phase 102 was a pump 20 for flowing the outer phase 101 in the culture tank 11. As a result, the floating state of the internal phase 102 can be maintained.

The flow velocity of the outer phase 101 flowing into and out of the culture tank 11 is controlled according to the floating state of the inner phase 102 in the culture tank 11. As a result, the floating state of the internal phase 102 in the culture tank 11 can be stably maintained, and the object to be cultured can be cultured for a long period of time.

A flow path 40 through which the outer phase 101 flows is provided, and the outer phase 101 flowing out of the culture tank 11 is configured to circulate so as to flow into the culture tank 11 again via the reservoir 32 arranged in the incubator 30. did. A pump 20 and a flow path 40 were used as the circulation mechanism. As a result, the foreign phase 101 that has flowed out of the culture tank 11 can be reused. Further, since the outer phase 101 is supplied to the culture tank 11 via the reservoir 32, the environment in the culture tank 11 can be maintained in the same environment as in the incubator 30. Therefore, the culture environment can be maintained together with the floating state of the internal phase 102.

The floating means of the inner phase 102 is a pump 20 that allows the outer phase 101 to flow in from below the culture tank 11 and the outer phase 101 to flow out from above the culture tank 11. As a result, when the specific gravity of the inner phase 102 is larger than that of the outer phase 101 and the inner phase 102 gradually settles in the culture tank 11, the floating state of the inner phase 102 can be maintained.

[Second Embodiment]
As shown in FIG. 2, the suspension culture device 1A according to the second embodiment includes a culture solution 10, a culture tank 11A, a rotation mechanism 12, and an incubator 30.
Hereinafter, each configuration of the present embodiment will be described, but the description may be omitted for the parts common to the first embodiment.

The culture tank 11A is a container for accommodating the culture solution 10, and is arranged in the incubator 30. The culture tank 11A is a closed container in which the culture solution 10 does not enter and exit from the outside. The shape of the culture tank 11A is not particularly limited, and is, for example, a cylindrical shape or a square tubular shape as in the first embodiment. The material of the culture tank 11A has a permeability to gases such as O ₂ and CO _2, which is impermeable to liquid such as culture fluid 10, it is preferable to use a gas permeable resin. Thereby, the environment of the culture solution 10 can be made similar to that in the chamber 31.
The gas permeable resin is not particularly limited, and conventionally known ones can be used. For example, low-density polyethylene, low-density polystyrene, silicone resin, polyisoprene, polybutadiene, ethylene-vinyl acetate copolymer and the like can be mentioned. The gas permeability of these gas-permeable resins decreases as the thickness increases. Therefore, it is preferable that at least a part of the culture tank 11A is made of a thin gas permeable resin. Further, a part thereof may be composed of an arbitrary material for ensuring strength. Alternatively, a support such as a frame for ensuring strength may be separately provided.

The rotation mechanism 12 rotatably supports the culture tank 11A in the incubator 30. The configuration of such a rotation mechanism 12 is not particularly limited. For example, the rotating mechanism 12 may be provided with self-supporting legs to rotatably support both end portions of the culture tank 11A, or the culture tank 11A may be suspended and rotatably supported in the incubator 30. Good.
The rotation mechanism 12 further includes a drive device such as a motor. The rotation mechanism 12 can rotate the culture tank 11A by the driving device.

The method of rotating the culture tank 11A and suspend-culturing the object to be cultured in the internal phase 102 using the suspension culture device 1A described above is as follows.
The inner phase 102 gradually settles in, for example, the outer phase 101 due to the difference in specific gravity from the outer phase 101. Before the inner phase 102 reaches the bottom surface of the culture tank 11A, the rotation mechanism 12 is used to rotate the culture tank 11A. As a result, the floating state of the inner phase 102 can be maintained without the inner phase 102 reaching the wall surface of the culture tank 11A. The rotation speed of the rotation mechanism 12 is controlled so that the floating state of the internal phase 102 is maintained.

According to the suspension culture device 1A according to the second embodiment, the following effects are exhibited.
The suspension culture device 1A was configured to include a rotation mechanism 12 for rotating the culture tank 11A. As a result, the culture tank 11A is rotated, so that the floating state of the internal phase 102 in the culture tank 11A can be maintained. In the above configuration, since stirring in the internal phase 102 is unlikely to occur, adhesion between cells is promoted. Therefore, it is preferable because a three-dimensional tissue having a sufficient thickness can be cultured.

The present invention is not limited to the above embodiment, and can be appropriately modified.

The culture tank 11 according to the first embodiment has been described as having an inlet 111 of the outer phase 101 formed below and an outlet 112 formed above, but the present invention is not limited to this configuration. When the specific gravity of the inner phase 102 is smaller than that of the outer phase 101 and the buoyancy acting on the inner phase 102 is larger than that of gravity, the inner phase 102 gradually rises in the culture tank 11. In this case, the inlet of the outer phase 101 may be formed above the culture tank 11, and the outlet may be formed below the culture tank 11. As a result, the outer phase 101 flows downward in the culture tank 11, and the inner phase 102 has a downward effect. Even with such a configuration, the floating state of the internal phase 102 in the culture tank 11 can be maintained.

In the above embodiment, the PEG phase is used as the outer phase 101, and the DEX phase is used as the inner phase 102, which is the culture phase, but the present invention is not limited to this configuration. A DEX phase can be used as the outer phase 101, and a PEG phase can be used as the inner phase 102. Moreover, the PEG phase can also be used as a culture phase.

In the above embodiment, the object to be cultured in the internal phase 102 has been described as mammalian cells including humans, but the composition is not limited to this. Cells other than mammals and bacteria such as Escherichia coli and Pseudomonas aeruginosa can also be cultured in the internal phase 102.

Although the suspension culture device 1 according to the first embodiment has been described as having the position detection device 50, the present invention is not limited to this configuration. The flow rate of the outer phase 101 flowing in the culture tank 11 may be controlled by other configurations.

The suspension culture device 1A according to the second embodiment has been described as having a culture solution 10, a culture tank 11A, a rotation mechanism 12, and an incubator 30, but is not limited to this configuration. The suspension culture device 1A may be configured to include the position detection device 50 as in the suspension culture device 1 according to the first embodiment.
In this case, the position detection device 50 can be configured to be communicable with the rotation mechanism 12, and the rotation speed of the rotation mechanism 12 can be controlled according to the floating state of the internal phase 102. As a result, the floating state of the internal phase 102 can be stably maintained.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.

<Preparation of culture solution (Examples and Comparative Examples)>
For PEG (molecular weight 35,000) and DEX (molecular weight 500000) as solutes, solutions (each concentration 5% by weight and concentration 10% by weight) were prepared using a culture solution as a medium, and these were mixed and separated into two phases. A two-phase culture solution consisting of a PEG phase and a DEX phase was prepared.

<Culture conditions>
(Example)
The relative fabricated DEX phase were suspended ^{2.0 × 10 6 (cells / ml} ) and made as murine fibroblasts (NIH-3T3). Next, 10 μl of the suspended DEX phase was added dropwise to the cylindrical culture tank (Tigon tube, inner diameter 4 mm) filled with the prepared PEG phase. Further, the DEX phase was suspended in the PEG phase for 3 hours by imparting a flow from downward to upward to the PEG phase. The PEG phase to be circulated was adjusted to 37 ° C. and 5% CO _{2 using an incubator.}

(Comparison example)
The relative fabricated DEX phase were suspended ^{2.0 × 10 6 (cells / ml} ) and made as murine fibroblasts (NIH-3T3). Next, 10 μl of the suspended DEX phase was added dropwise to the dish filled with the PEG phase prepared above, and the mixture was allowed to stand in an _{incubator (37 ° C., 5% CO 2) for 3 hours.}

[Thickness evaluation by confocal microscope imaging]
From the cell clusters contained in the DEX phase of Examples and Comparative Examples cultured under the above conditions, five cell clusters existing near the central part of the DEX phase were arbitrarily selected and imaged using a confocal microscope. ..
FIG. 3 is a Z-stack image of a cell mass cultured by the culture method of the example (128Slice, 1 μm / Slice), and FIG. 4 is a Z-stack image of a cell mass of a comparative example (30Slice, 1 μm). / Slice). Specifically, FIGS. 3 (A) and 4 (A) are images obtained by capturing the cell mass from the Z-axis direction, respectively, and FIGS. 3 (B) and 4 (B) show the X-axis direction and FIG. 3 (C). ) And FIG. 4C are Z-stack images obtained by photographing the thickness of the cell mass in the Z-axis direction from the Y-axis direction.
As a result of analyzing the captured image, the average value of the size of the cell mass in the Z-axis direction of the example was 78.8 μm (N = 5). The maximum value of the size of the cell mass in the Z-axis direction of the comparative example was 12 μm (N = 5).

As shown in FIGS. 3 and 4, the cell mass cultured by the culture method according to the example has a larger size in the Z-axis direction than the cell mass cultured by the culture method according to the comparative example, and has a three-dimensional tissue. It was confirmed that it was formed.

[Staining of dead cells with Propidium Iodide (PI)]
The cell mass cultured by the culture method according to the example was stained with dead cells using propidium iodide (PI) and imaged using a microscope. FIG. 5 (A) is a bright field image, and FIG. 5 (B) is a fluorescence image obtained by staining dead cells with PI. The dotted line portion in FIG. 5B indicates a portion corresponding to the position of the cell mass in the bright field image.

As shown in FIG. 5, when the cell mass cultured by the culture method according to the example was stained with PI, no fluorescent staining was confirmed in most of the cell clusters, and most of the cell mass cultured by the culture method according to the example was live cells. It was confirmed that.

1, 1A suspension culture device, 10 aqueous two-phase culture solution (culture solution), 101 outer phase (polyethylene glycol phase), 102 inner phase (dextran phase), 11, 11A culture tank, 12 rotation mechanism (floating means), 20 pumps (Floating means)

Claims (16)

An aqueous two-phase culture solution containing an outer phase and an inner phase as a culture phase suspended in the outer phase.
A culture tank containing the aqueous two-phase culture solution and
A suspension culture apparatus comprising a suspension means for maintaining the suspension state of the internal phase. The floating culture apparatus according to claim 1, wherein the floating means maintains the floating state of the inner phase by flowing the outer phase in the culture tank. The suspension culture apparatus according to claim 2, wherein the flow velocity at which the outer phase flows in the culture tank is controlled according to the floating state of the inner phase in the culture tank. The suspension culture apparatus according to any one of claims 1 to 3, further comprising a circulation mechanism for collecting the outer phase flowing out of the culture tank and allowing it to flow into the culture tank again for circulation. The specific gravity of the inner phase is larger than the specific gravity of the outer phase.
The floating means allows the foreign phase to flow in from below the culture tank and at the same time.
The suspension culture apparatus according to any one of claims 1 to 4, wherein the floating state of the inner phase is maintained by flowing out the outer phase from above the culture tank. The suspension culture apparatus according to claim 1, wherein the suspension means maintains the suspension state of the internal phase by rotating the culture tank. The suspension culture apparatus according to any one of claims 1 to 6, wherein the aqueous two-phase culture solution comprises a dextran phase and a polyethylene glycol phase. The suspension culture apparatus according to claim 7, wherein the outer phase is a polyethylene glycol phase and the inner phase is a dextran phase. A suspension culture method in which cells are cultured in a culture medium.
The culture solution is an aqueous two-phase culture solution containing an outer phase and an inner phase as a culture phase floating in the outer phase.
A suspension culture method in which culture is performed while maintaining the floating state of the internal phase. The suspension culture method according to claim 9, wherein the floating state of the inner phase is maintained by flowing the outer phase in the culture tank. The suspension culture method according to claim 10, wherein the flow velocity at which the outer phase flows in the culture tank is controlled according to the floating state of the inner phase in the culture tank. The suspension culture method according to any one of claims 9 to 11, wherein the foreign phase that has flowed out of the culture tank is recovered, and the foreign phase flows into the culture tank again to be circulated. The specific gravity of the inner phase is larger than the specific gravity of the outer phase.
The foreign phase is allowed to flow in from below the culture tank, and at the same time,
The suspension culture method according to any one of claims 9 to 12, wherein the floating state of the inner phase is maintained by flowing out the outer phase from above the culture tank. The suspension culture method according to claim 9, wherein the floating state of the internal phase is maintained by rotating the culture tank. The suspension culture method according to any one of claims 9 to 14, wherein the aqueous two-phase culture solution comprises a dextran phase and a polyethylene glycol phase. The suspension culture method according to claim 15, wherein the outer phase is a polyethylene glycol phase and the inner phase is a dextran phase.

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