JP6566549B2 - Cultivation method using culture apparatus - Google Patents

Description

The present invention relates to a culture method using a culture apparatus.

Mass culture of microorganisms, insect cells, plant cells, animal cells and the like is widely used for the production of various substances useful as pharmaceuticals, foods, cosmetics, and raw materials thereof.
Patent Document 1 describes a culture bag in which a stirring device having a stirring blade is stored in an airtight manner. In this culture apparatus, a high shear (shear stress) was generated in the system by the stirring blade during stirring. In addition, particles were generated due to sliding of the bearing portion of the stirring blade. Furthermore, there were problems in terms of price and a method for joining the stirring blade and the culture bag.
Patent Document 2 describes a culture device in which a culture bag made of a flat bag is fixed to a tray-like support device. In the case of this culture apparatus, since the flat bag is placed horizontally, there has been a problem of increasing the occupied area in increasing the scale.

Japanese Patent No. 4986659 Japanese Patent No. 5214714

This invention is made | formed in view of the said situation, and makes it a subject to provide the culture method using the culture apparatus which can culture from a small scale to a large scale, without replacing | exchanging a container.

In order to solve the above-mentioned problem, a culture bag made of a soft packaging material and an outer shell container for storing the culture bag, and a side surface for storing the contents of the culture bag and the culture bag for the outer shell container are stored. The side walls are inclined so that the width of the upper part is wide and the width of the lower part is narrow, and when the contents are stored in the culture bag, the culture bag is deformed by the weight of the contents. The outer shell container is replaced by using a culture apparatus that stirs and mixes the contents in the culture bag while the culture bag is supported by the outer shell container by closely contacting the inner surface of the outer shell container. A culture method characterized by adding a culture medium to the same culture bag and performing multi-stage culture while scaling up in stages .

It is preferable that the contents can be stirred and mixed by shaking the outer shell container containing the culture bag.
It is preferable that the side wall portion of the outer shell container has an inverted conical shape, and the angle θ formed by the opposing buses of the inverted cone is 25 to 90 °.
It has a flat bottom wall at the lower end of the side wall of the outer shell container, and the diameter R1 at the upper end of the side wall is 1.5 to 5 times the diameter R2 at the lower end of the side wall. Is preferred.
It is preferable that the culture bag is a gusset bag and has a spout.

In the culture method, when the content contains cells and a medium, the cells can be cultured.

  According to the present invention, it is easy to scale up from a small volume to a large capacity without replacing the container. Moreover, when shaking a container, it can stir from a small quantity to a large volume within one container with a low shear (shear stress), and an efficient cell culture can be performed. Moreover, since there is no stirring blade, generation | occurrence | production of the particle by the bearing part of a stirring blade can be suppressed. Even if the apparatus is contaminated by culturing, it is only necessary to replace the culturing bag, and it is not necessary to discard the stirring blade and the stirring magnet. Therefore, it is possible to provide a culturing apparatus that is less expensive than the conventional one.

It is (a) front view and (b) sectional drawing which show an example of a culture bag. It is (a) front view and (b) top view which show an example of an outer shell container. It is a perspective view which shows an example of the outer shell container which accommodated the culture bag. It is a perspective view which shows an example of the shaking apparatus used for a culture apparatus. (A) is a top view which shows an example of an outer shell container, (b) is a top view which shows an example of the shaking operation | movement of an outer shell container. It is a perspective view which shows typically an example of a motion of the contents accommodated in the culture bag.

  Hereinafter, based on a preferred embodiment, the present invention will be described with reference to the drawings.

  FIG. 1 shows an example of the culture bag 10. FIG.1 (b) is sectional drawing which follows the SS line | wire of Fig.1 (a). The culture bag 10 is composed of a soft packaging material such as a film 12. In the example shown in FIG. 1 (b), a pair of gusseted films 12b is disposed between a pair of gussetless films 12a, and a sealing portion 11 is provided at the peripheral edge of the films 12a and 12b, thereby sealing the gusset. Show the bag. The shape of the culture bag 10 is inclined on the side so that the upper part is wide and the lower part is narrow. The side surface of the culture bag 10 is a surface that surrounds the content in a state in which the content is accommodated, and is configured by, for example, the inner end of the seal portion 11 or the inner surface of the film 12. In the case of FIG. 1, the side surface can be tilted in a state in which the contents are accommodated by tilting the seal portion 11 serving as a side edge of the culture bag 10. The seal part 11 may be widened or the film 12 may be left outside the seal part 11. For example, the periphery 14 of the culture bag may have a rectangular shape as indicated by a two-dot chain line. It is more preferable that the culture bag 10 is a multiple packaging bag such as a double bag or a triple bag because the liquid is difficult to leak.

Although the material of the film 12 which comprises the culture bag 10 is not specifically limited, Thermoplastic resins, such as a polystyrene, polyamide, polyester, polyolefin, and these laminated bodies are mentioned. The film 12 used for the culture bag 10 is preferably the same material as the film used in the purification process. By using the same film in the culturing process and the purification process, it is possible to easily ensure the product quality of the pharmaceutical product to be produced. The material, thickness, etc. of each film 12a, 12b used as the film 12 may be the same or different.
Although the capacity | capacitance (size) of the culture bag 10 is not specifically limited, For example, it can be set as the magnitude | size of 0.1L-5000L, Preferably it is 1-2000L, More preferably, it is 20-2000L. The culture bag 10 may be single use (disposable).

A plurality of tubes (not shown) can be connected to the culture bag 10 for the purpose of adding or removing contents such as a culture medium or atmospheric gas. In addition, the culture bag 10 can include a spout 13. The spout 13 can be provided with a cock, a cap, a tapered spout and the like. A tube may be connected to the spout 13. In the case of this embodiment, the spout 13 is fixed to the culture bag 10 by sealing the base of the spout 13 to the back side of the film 12. Examples of the sealing method include heat sealing (heat sealing), ultrasonic sealing, high frequency sealing, cooling sealing, and the like. The base of the spout 13 may cover the entire lower part (bottom) of the culture bag 10, and the side of the base of the spout 13 may be sealed to the side of the culture bag 10.
The material of the tube is not particularly limited, but a material excellent in chemical resistance, weather resistance, etc., such as silicone and thermoplastic elastomer, is preferable. The tube may be provided with a filter, a flow monitor, a flow meter, a valve, a pump, and the like. The culture bag 10 and the tube are preferably sterilized before the culture. The sterilization means can be appropriately selected according to the purpose of culturing, and specific examples include radiation such as γ rays, gas such as ethylene oxide, heating with water vapor, and the like.

FIG. 2 shows an example of the outer shell container 20. 2A is a front view, and FIG. 2B is a top view. FIG. 3 is a perspective view of the state in which the culture bag 10 of FIG. 1 is accommodated in the outer shell container 20 of FIG. The outer shell container 20 can accommodate the entire culture bag 10 as shown in FIG. 3 in order to protect the culture bag 10 made of a soft packaging material. The culture container 40 used in the culture apparatus of the present embodiment includes the culture bag 10 and the outer shell container 20.
The configuration of the outer shell container is not particularly limited, but the outer shell container 20 of FIG. 2 includes a side wall portion 21 and a bottom wall portion 22. The bottom wall portion 22 is provided at the lower end portion of the side wall portion 21. The upper part of the outer shell container 20 may be opened, or may be configured to be closed by providing a lid. When a lid is provided, internal temperature fluctuations can be suppressed. The lid may be detachable from the outer shell container 20, or may be connected to the outer shell container 20 by a hinge or the like so as to be opened and closed.

The material of the outer shell container 20 is not particularly limited, but preferably has a hardness (rigidity) capable of protecting the culture bag 10, for example, a composite such as metal such as stainless steel, resin, wood, laminated material, fiber reinforced plastic, etc. Materials.
As shown in FIG. 2A, the inner space of the outer shell container 20 has a shape in which the side wall portion 21 is inclined so that the upper portion is wide and the lower portion is narrow. The width is a dimension of a horizontal cross-sectional shape. For example, in the case of a circle, the width corresponds to a diameter. Examples of the shape of the side wall portion 21 of the outer shell container 20 include a conical shape (inverted conical shape) reduced downward, for example, an inverted conical shape (conical shape having a bottom surface on the upper side). When the outer shell container 20 has an inverted cone shape, the bottom area of the outer shell container 20 is reduced, and the culture bag 10 is accommodated and supported in the outer shell container 20 even when the capacity of the culture bag 10 is small. Can do. Moreover, since the cross section of the upper part of the outer shell container 20 becomes wide, even when the capacity of the culture bag 10 is large, the culture bag 10 can be housed and supported in the outer shell container 20. Therefore, it is easy to scale up from a small amount to a large capacity without replacing the outer shell container 20. In order to stabilize the support of the culture bag 10, the bottom wall portion 22 of the outer shell container 20 is preferably flat.

From the viewpoint of accommodating the culture bag 10 having a small capacity to a large capacity, the angle θ formed by the opposing buses of the inverted cone of the outer shell container 20 is preferably 25 to 90 °. Specific examples of the angle θ include 25 °, 30 °, 45 °, 60 °, and 90 °, but are not particularly limited thereto. In FIG. 2A, θ = 45 °. The cone bus line is an intersection line between the side surface of the cone and a plane including the axis of the cone, and appears on both sides around the axis. In FIG. 2A, straight lines located at both ends of the angle θ correspond to these buses. When the angle θ is too small, it is difficult to make a difference between the upper width and the lower width of the outer shell container 20, and it is difficult to widen the range of capacities that can be accommodated. If the angle θ is too large, the side wall portion 21 may become horizontal and the support of the culture bag 10 may become unstable.
In consideration of shapes other than the cone, θ is an angle formed by opposing side surfaces in the polygonal pyramid, or two sides appearing on both sides of the vertical axis when the side surface is cut by a cross section including the vertical axis (vertical direction). It can be defined as an angle formed. When the axis of the cone or other cone shape (vertical direction) is perpendicular to the bottom surface (horizontal direction), the angle formed between the side surface (side wall portion) and the bottom surface (bottom wall portion) of the outer shell container is inside the outer shell container. If φ, φ = 90 ° + (θ / 2). φ is preferably within the range of 102.5 to 135 °. When the container does not have a bottom surface (bottom wall portion), the horizontal plane can be regarded as the bottom surface. When the angle formed by the side surface (side wall portion) of the outer shell container and the vertical axis is χ, χ = θ / 2. χ is preferably within a range of 12.5 to 45 °. In the case of a cone, the angles θ, φ, and χ are constant values, but in the case of other shapes, the angles θ, φ, and χ may take different values for each side position within a predetermined angle range. In that case, it is preferable that θ, φ, or χ at each position on the side surface is within the above-mentioned range over the entire circumference of the side surface.
Regarding the ratio of the width of the upper part of the outer shell container 20 to the width of the lower part, for example, the upper part is preferably 1.5 to 5 times the lower part. For example, the diameter R1 at the upper end of the side wall 21 is preferably 1.5 to 5 times the diameter R2 at the lower end of the side wall 21.

  It is preferable that the contents of the culture bag 10 accommodated in the outer shell container 20 include a liquid (medium) and a gas. Since the outer shell container 20 has an inverted cone shape, the culture bag 10 that is in close contact with the outer shell container 20 also has an inverted cone shape as shown in FIG. When the contents are shaken, the contact area of the contents 15 with the gas increases, and the movement of the substance at the gas-liquid interface is promoted. The volume ratio of gas to liquid (gas / liquid volume ratio) is not particularly limited, and the liquid may be more than the gas, the gas may be more than the liquid, or about half. The gas-liquid volume ratio is preferably in the range of, for example, 1: 9 to 9: 1. It is preferable that the culture bag 10 is in close contact with the outer shell container 20 and the outer shell container 20 supports the side surface of the culture bag 10 in the whole or most of the range in which the culture bag 10 contains at least the liquid (medium). In the portion where the gas is stored in the culture bag 10, the upper part (part) of the culture bag 10 may be exposed on the outer shell container 20 (outside).

  The medium accommodated in the culture bag 10 is not particularly limited as long as it can be stirred by shaking, and may be a solution, emulsion, suspension, dispersion, gel, or the like. The medium may be a uniform single-phase composition or may be composed of two or more phases, such as a liquid containing solids. The medium is preferably sterilized before culturing. Furthermore, the culture medium can contain living organisms (culture objects) such as microorganisms, insect cells, plant cells, animal cells, tissues, cell sheets, and cell clusters. In culturing that requires a scaffold, solids such as granules, lumps, foams, and fibers may be mixed into the medium as a culture substrate.

The gas accommodated in the culture bag 10 may be an aerobic atmosphere containing air (or an O ₂ -containing gas) or an anaerobic atmosphere having a low O ₂ concentration. The composition of the gas is not particularly limited, but two or more mixed gases are common. Examples of gaseous components include oxidizing gases such as oxygen (O ₂ ), inert gases such as nitrogen (N ₂ ) and argon (Ar), acidic gases such as carbon dioxide (CO ₂ ), ammonia (NH ₃ ), and the like. And a reducing gas such as hydrogen sulfide (H ₂ S). The gas supplied to the culture bag 10 is preferably passed through a filter. Gases such as air, oxygen and carbon dioxide are preferably introduced from one or both of the lower side (liquid side) and the upper side (gas side).

  The side wall 21 of the outer shell container 20 has a window 23 for visually confirming the amount (liquid level) of the contents of the culture bag 10 accommodated in the outer shell container 20. The window 23 is provided as a slit-like cut in the vertical direction of the side wall portion 21. The window 23 may be opened or may be closed with a transparent material. Moreover, as shown in FIG.2 (b), the bottom wall part 22 has the hole 24 for letting a tube pass. As a structure for connecting the outer shell container 20 to the shaking device 30 (see FIG. 5), a connecting portion 25 is provided.

  The arrangement and number of tubes provided in the culture bag 10 are not particularly limited, and a desired number can be provided at an appropriate position. The arrangement of the tubes can be, for example, one or more of the upper, lower and side portions of the culture bag. Through these tubes, sampling of contents, introduction and discharge of fluids (gas, liquid, powder, etc.), supply of nutrients, removal of waste products, etc. can be performed. A configuration is also possible in which a circulation path including a culture bag and an external device is provided via a tube, and a part of the culture solution is processed by the external device and then returned to the culture bag.

  As shown in FIG. 3, when the culture bag 10 is accommodated in the outer shell container 20, the culture bag 10 is deformed according to the weight of the contents and is in close contact with the inner surface of the outer shell container 20. By contacting the culture bag 10 and the outer shell container 20 over a wide area, they adhere to each other without mechanical fitting, engagement, chemical adhesion, and adsorption, and an adhesive force that can withstand shaking is generated. The inner surface of the outer shell container 20 is smooth and is preferably polished by, for example, a buff. Although the inner surface may have gentle irregularities, the thickness of the film 12 constituting the culture bag 10 has minute irregularities and shape changes (acute angle portions, etc.) that cause damage or deterioration of the film 12. Preferably not.

  The outer shell container 20 preferably has a temperature adjustment mechanism (means) so that the temperature of the culture bag 10 and its contents can be adjusted. Examples of the temperature adjusting mechanism include, but are not limited to, a jacket structure that allows a heat medium such as circulating water to pass through, a heating device such as a rubber heater, and the like.

  FIG. 4 shows an example of a shaking device 30 that shakes the outer shell container 20. The shaking device 30 includes a base portion 31 and a frame portion 32 that surround the outer shell container 20, and a power source 33 that shakes the outer shell container 20. As shown in FIG. 3, the shaking device 30 can shake the outer shell container 20 while containing the culture bag 10. The culture apparatus (shaking culture apparatus) of the present embodiment includes an outer shell container 20 (the culture container 40 described above) that contains the culture bag 10 and a shaking apparatus 30, and the outer shell container 20 that contains the culture bag 10. The contents in the culture bag 10 can be cultured while being stirred and mixed.

  FIG. 5B shows an example of the shaking operation of the outer shell container. In the present embodiment, the planar shape of the outer shell container 20 is circular, and the three-dimensional shape is an inverted conical shape with the central axis oriented substantially up and down. FIG. 5A shows the center 20c and the radius 20r of the outer shell container 20. FIG. In the shaking operation of FIG. 5B, the center 20c of the outer shell container 20 rotates (revolves) on the track C having the radius R. In this case, the amplitude of shaking is twice the radius R. Further, the outer shell container 20 may be rotated (rotated) around the center 20c. Although the radius R in the illustrated example is smaller than the radius 20r, the illustrated ratio does not particularly accurately represent the actual ratio.

  At the time of shaking, it is preferable that the outer shell container 20 containing the culture bag 10 rotates in the horizontal direction. The rotation direction may include both a horizontal component and a vertical component. The vertical component is preferably smaller than the horizontal component (including the case where the vertical component is 0). Efficient stirring can be achieved by incorporating rotation instead of shaking in one direction. The shear (shear stress) during stirring is less likely to be applied to the culture bag, and efficient culture can be achieved. The rotation speed and the direction of rotation can be appropriately controlled. By changing the rotation speed and the rotation direction, not only the horizontal liquid stirring but also the vertical stirring can be realized.

  It is preferable to set the number of rotations, amplitude, etc. according to the culture system, and to control by a program. For example, in the case of E. coli culture, it is preferable to increase the dissolved oxygen concentration (DO) in the solution, and in order to increase the DO, it is preferable to vigorously shake and mix the gas and liquid. . On the other hand, when culturing animal cells, it is preferable to suppress bubbling of the culture solution and gently agitate. The shaking culture apparatus of this embodiment can perform culture by controlling one or more of the following control factors, for example.

・ Rotation speed and shaking direction of the apparatus ・ pH of the medium
・ Air flow rate of medium ・ Temperature of medium ・ Level of liquid level (wave height)

  As for the shaking direction, it is preferable to repeat right rotation and left rotation or forward rotation and reverse rotation in an arbitrary pattern. It is preferable to build a system so that the rotation speed and rotation speed of the shaking device can be controlled by a control panel or the like. It is preferable that the shaking device can arbitrarily switch the rotation direction. Therefore, it is preferable that the power source and the rotation device include a mechanism capable of rotating in both forward and reverse directions. Moreover, it is preferable to control a rotation pattern by a program, for example. The rotation pattern is not particularly limited, but several examples will be given below.

(1) Forward rotation → Stop → Reverse rotation → Stop → Forward rotation → Stop → Reverse rotation → (Repeat)
(2) Forward rotation->Stop-> Forward rotation->Stop-> Reverse rotation->Stop-> Reverse rotation-> (Repeat)
(3) Forward rotation → Stop → Forward rotation → Stop → Forward rotation → Stop → Forward rotation → (Repeat)

  The rotation speed and amplitude of shaking of the outer shell container can be set arbitrarily, but when rotated, the wave height of the contents in the culture bag (for example, the average height, the maximum height, the height of the liquid surface) It is preferable to detect the height of the wave and adjust the rotational speed from the height of the wave by feedback control or the like in order to keep the increase width of the difference) within a certain range. The rotation speed is preferably 0.1 to 2000 rpm (times / min), more preferably 0.1 to 200 rpm, and 0.1 to 100 rpm, and still more preferably 10 to 80 rpm. The amplitude is preferably 0.1 to 100 mm, more preferably 2 to 100 mm, and still more preferably 10 to 30 mm. When the rotational direction changes, the rotational speed is the rotational speed (non-negative value) regardless of the rotational direction, such as the sum of the right rotational speed and the left rotational speed, or the sum of the positive rotational speed and the reverse rotational speed. The sum of

The culture bag preferably includes a sensor for measuring the pH or DO of the contents and a feedback control mechanism based on data obtained by the sensor.
As for the hydrogen ion index (pH), the pH in the system can be known by inserting a pH electrode into the culture bag or attaching a chip whose color changes with pH in the system. When it is desired to make the system acidic, the pH can be changed to the acidic side by introducing CO ₂ gas or dropping an acid solution. When CO ₂ gas is introduced, CO ₂ gas may be introduced from the top surface of the culture bag or intermittently from the bottom of the culture bag in order to prevent the CO ₂ gas from being unnecessarily dissolved in the solution. preferable. When it is desired to make the system basic, the pH can be made basic by adding an alkaline solution. The acid or alkali inlet for making the pH constant may be a tube connected to the culture bag 10 or a spout, or may be provided elsewhere.
Regarding DO (dissolved oxygen concentration), it is preferable to measure the air flow rate, apply feedback control, and introduce oxygen. As a method for supplying oxygen (O ₂ ), it is preferable to introduce oxygen from a tube having an outlet in the solution (under the liquid level) so that oxygen is distributed in the solution.

  The temperature of the contents (medium) can be adjusted using the temperature adjustment function (described above) of the outer shell container 20. Although the culture temperature depends on the type of organism to be cultured, for example, in the case of microorganisms, it is preferably 4 to 40 ° C, particularly preferably 25 to 37 ° C. When it is used as a stirrer for a medium or the like without culturing, it may be cooled (eg, 4 to 20 ° C.). The temperature control can be feedback controlled by incorporating a thermometer in the culture bag 10 and using the measured value. The thermometer may be a contact-type thermometer, but a stationary non-contact thermometer is preferable.

  The culture apparatus of the present embodiment can be applied to both fed-batch culture (fed batch system) and perfusion culture (perfusion system). Organisms and cells to be cultured are not particularly limited, but include fungi including E. coli, yeast, microorganisms, insect cells, plant cells, animal cells, CHO (Chinese Hamster Ovary) cells for production of biopharmaceuticals, HeLa cells, It can be used for culturing COS cells, iPS cells for regenerative medicine, stem cells such as mesenchymal stem cells, and animal cells such as differentiated tissue cells. Among these, it is suitable for CHO cell culture.

  The culture apparatus and culture method using the same according to the present embodiment are particularly suitable for large-scale culture. The shaking device is preferably equipped with a counter balance, particularly in the case of a large scale. When the counter balance is installed, the apparatus can be operated stably even if a culture bag containing a large volume of medium is used. By mounting two or more culture bags on one shaking device, they may function as a counter balance with each other.

  For example, an example of the process of culturing cells is given. First, the contents including the cells and the medium are accommodated in the culture bag 10. The culture bag 10 is accommodated in the outer shell container 20, and the first stage culture is performed while stirring and mixing by shaking or the like. Next, a part or all of the old medium is removed from the culture bag, or cells are left in the culture bag while the old medium remains in the culture bag, and a new medium is added to the culture bag. Further, the second stage culture is performed while stirring and mixing the culture bag 10 containing the cells and the medium by shaking or the like. Thus, when the culture is scaled up, since the culture bag 10 and the outer shell container 20 are inverted cones, even if the same culture bag 10 is used, the outer shell container 20 can be stably supported. Multi-stage culture can be performed while adding a medium to the culture bag 10 and scaling up in stages. Since the culture bag has an inverted pyramid shape, it is easy to settle the cells at the bottom and drain the supernatant from the culture bag. Since it is not necessary to transfer the cells from the culture bag, damage to the cells can be suppressed. Here, the culture target is not limited to cells, but can be scaled up in the case of an individual or tissue.

As mentioned above, although this invention has been demonstrated based on suitable embodiment, this invention is not limited to the above-mentioned embodiment, A various change is possible in the range which does not deviate from the summary of this invention.
The apparatus for stirring the contents in a state where the culture bag is accommodated in the outer shell container is not limited to the shaking apparatus, and a stirring blade, a magnet, a stirring bar, or the like may be used.
The above shaking apparatus can be used not only for culturing but also for stirring, preparation, chemical reaction, fermentation and the like of the composition (medium etc.).

DESCRIPTION OF SYMBOLS 10 ... Culture bag, 11 ... Seal part, 12, 12a, 12b ... Film, 13 ... Outlet, 14 ... Periphery of culture bag, 15 ... Contents, 20 ... Outer shell container, 21 ... Side wall part, 22 ... Bottom wall Part, 23 ... window, 24 ... hole, 25 ... coupling part, 30 ... shaking device, 31 ... base part, 32 ... frame part, 33 ... power source, 40 ... culture vessel.

Claims (6)

Including a culture bag made of flexible packaging material and an outer shell container for containing the culture bag, and a side surface for storing the contents of the culture bag and a side wall portion for storing the culture bag of the outer shell container, respectively. The width of the lower portion is inclined so that the width of the lower portion is narrowed, and when the contents are accommodated in the culture bag, the culture bag is deformed by the weight of the contents and is formed on the inner surface of the outer shell container. By using the culture apparatus that stirs and mixes the contents in the culture bag while the culture bag is supported by the outer shell container, the same culture is performed without replacing the outer shell container. by adding medium to the bag, stepwise while scaling up the culture method characterized by carrying out the cultivation of multiple stages. By shaking the shell vessel containing the culture bag, a method of culturing according to claim 1, characterized by mixing and stirring the contents. The culture method according to claim 1 or 2, wherein a side wall portion of the outer shell container has an inverted conical shape, and an angle θ formed by opposing buses of the inverted cone is 25 to 90 °. It has a flat bottom wall portion at the lower end portion of the side wall portion of the outer shell container, and the diameter at the upper end portion of the side wall portion is 1.5 to 5 times the diameter at the lower end portion of the side wall portion. The culture method according to any one of claims 1 to 3. The culture method according to any one of claims 1 to 4, wherein the culture bag is a gusset bag and includes a spout. The culture method according to any one of claims 1 to 5, wherein the contents include cells and a medium, and the cells are cultured.

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