Patent Description:
Large-scale culture of microorganisms, insect cells, plant cells, animal cells, and the like is widely used for producing medicines, foods, and cosmetics, as well as various substances useful as raw materials thereof and the like.

Patent Document <NUM> describes a culture bag airtightly housing a stirring device provided with a rotating shaft having stirring blades. In this culture apparatus, the considerably high shear (shearing stress) due to the stirring blades was generated in the system upon stirring. Additionally, there was generation of particles due to gliding of a bearing portion of the stirring blades. Furthermore, there were the problem of the cost, and the problem of a method of connecting the stirring blades and the culture bag.

Patent Document <NUM> describes a culture apparatus in which a culture bag composed of a flat bag is fixed in a tray-like supporting device. In the case of this culture apparatus, since the flat bag is arranged horizontally, there was the problem that an occupation area is increased upon scale up.

<CIT> discloses a cell culture apparatus comprising a culture bag placed in a containment outer vessel. The containment vessel is positioned onto an orbital shaken platform. Orbital shaking induces motion to liquid contained in the bag ("shake mixing"). The culture bag is placed in direct contact with heating elements, and a neoprene insulation sheet is used to insulate the container wall from the outside environment.

The present invention has been made in view of the above-mentioned circumstances, and an object thereof is to provide a shake-type culture apparatus which can perform large scale culture without using stirring blades and a culture method using the same.

In order to solve the above-mentioned problems, the present invention provides a shaking culture apparatus as defined in claim <NUM>, and a culture method as defined in claim <NUM>.

It is preferable that the outer shell container housing the culture bag rotates in a horizontal direction.

It is preferable that the outer shell container housing the culture bag has a rotation number of shaking of <NUM> to <NUM>,<NUM> rpm and an amplitude of <NUM> to <NUM>.

The culture apparatus comprises a device for detecting a height of a wave of the contents in the culture bag, and is provided with an adjustment mechanism for adjusting the rotation number of shaking of the outer shell container housing the culture bag so that the height of the wave of the contents becomes constant.

It is preferable that the outer shell container comprises a jacket structure through which circulating water can be passed or a rubber heater.

The culture bag is a gusset bag, and is provided with a spout as defined in claim <NUM>.

It is preferable that the culture apparatus is provided with a sensor for measuring a pH or a dissolved oxygen concentration (DO) of the contents in the culture bag, and a feedback control mechanism based on data obtained by the sensor.

It is preferable that the culture bag is provided with an injection port for pouring an acid or an alkali to make a pH of the contents constant.

It is preferable that the culture bag can be connected to each tube for adding and taking out the contents.

It is preferable that the culture bag is provided with a mechanism for supplying air or oxygen to the contents.

It is preferable that an internal space of the outer shell container has a cylindrical shape, a circle of a cylinder is positioned on a bottom face of an outer shell, and a ratio of a diameter of the circle and a height of the cylinder is <NUM>:<NUM> to <NUM>:<NUM>.

It is preferable that the contents are housed in the culture bag, and the culture bag is deformed due to the weight of the contents, to be attached firmly to an internal surface of the outer shell container, so that the culture bag is supported by the outer shell container.

Also, the present invention provides a culture method using the shake-type culture apparatus.

According to the present invention, by using the outer shell container and not using the stirring blades, scale up from a small volume to a large volume is easy. Additionally, by shaking the container, a small volume to a large volume can be stirred in one container with the low shear (shearing stress), and effective cell culture can be performed. Additionally, since the culture apparatus has no stirring blades, generation of particles due to a bearing portion of the stirring blades can be suppressed. Even when the apparatus is contaminated by culture, since it is enough to exchange the culture bag, and it is not necessary to dispose the stirring blades and the stirring magnet, the culture apparatus which is less expensive than before can be provided.

Referring to the drawings, the present invention will be illustrated below based on preferable embodiments.

<FIG> shows one example of a culture bag <NUM>. This culture bag <NUM> is composed of a soft packaging material such as films <NUM>, <NUM>. <FIG> shows a gusset bag which is sealed by arranging one pair of films <NUM> with a gusset between one pair of films <NUM>, and providing a sealing portion <NUM> at the peripheral edge of the films <NUM>, <NUM>. Examples of a shape of the bag include a cylindrical type, a square cylindrical type, and the like. When the culture bag <NUM> is a multiple packaging bag such as a double bag and a triple bag, since the liquid is hardly leaked, this is more preferable.

A material of the films constituting the culture bag <NUM> is a thermoplastic resin such as a polystyrene, a polyamide, a polyester, and a polyolefin, or a laminate thereof. It is preferable that the film used in the culture bag <NUM> is the same material as that of the film used in a purification step. By using the same material in a culturing step and a purification step, it becomes easy to secure product quality of the prepared medicines.

The volume (size) of the culture bag <NUM> is not particularly limited, but can be, for example, the size of <NUM> to <NUM>,<NUM> and preferably <NUM> to <NUM>,<NUM>. The culture bag <NUM> may be single use (disposable).

For the purpose of addition, taking out, or the like of the contents such as a culture medium and the atmospheric gas, a plurality of tubes <NUM>, <NUM> can be connected to the culture bag <NUM>. Additionally, the culture bag <NUM> can be provided with a spout <NUM>. A cock, a cap, a taper-shaped pouring port, or the like can also be provided in the spout <NUM>. Tubes <NUM>, <NUM> may also be connected to the spout <NUM>. In the case of the present embodiment, by sealing a base <NUM> of the spout <NUM> on a back side of the film <NUM>, the spout <NUM> is fixed to the culture bag <NUM>.

A material of the tube is not particularly limited, but a material excellent in chemical resistance, weather resistance, and the like such as silicone and a thermoplastic elastomer is preferable. In the tube, a filter, a flow monitor, a flowmeter, a valve, a pump, and the like may be provided. It is preferable that the culture bag <NUM> and the tubes <NUM>, <NUM> have been sterilized before culturing. A sterilizing means can be appropriately selected depending on the culturing purpose or the like, and specific examples thereof include radiation such as γ-ray; the gas such as ethylene oxide; heating with the water vapor or the like; and the like.

<FIG> shows one example of an outer shell container <NUM>. Additionally, <FIG> shows a cross-sectional view of the state where the culture bag <NUM> of <FIG> is housed in the outer shell container <NUM> of <FIG>. To protect the culture bag <NUM> composed of a soft packaging material, the outer shell container <NUM> can house the entirety of the culture bag <NUM> as shown in <FIG>.

A configuration of the outer shell container is not particularly limited, and the outer shell container <NUM> as shown in <FIG> is provided with a container body <NUM> having a side wall portion 21a and a bottom wall portion 21b, and a lid portion <NUM> for closing an upper opening of the side wall portion 21a. When the entirety or the majority can be housed by the side wall portion 21a in a vertical direction of the culture bag <NUM>, the lid portion <NUM> may be omitted. A lower portion of a side face of the culture bag <NUM> can also be covered with the container body <NUM>, and an upper portion of a side face of the culture bag <NUM> can also be covered with the lid portion <NUM>.

In order to suppress temperature variation of the interior, it is preferable that an opening of the outer shell container <NUM> for taking in and out the culture bag 10is closed with the lid portion <NUM>. The lid portion <NUM> may be of the detaching type so that it can be separated from the container body <NUM>, or may be openably/closably connected to the container body <NUM> with a hinge or the like.

It is preferable that a material of the outer shell container <NUM> (for example, a container body <NUM> and a lid portion <NUM>) has such hardness (rigidity) that the culture bag <NUM> can be protected, and examples thereof include a metal such as stainless, a resin, a timber, a glued laminated timber, a composite material such as a fiber-reinforced plastic.

It is preferable from a view point of the stirring efficiency that an internal space of the outer shell container has a cylindrical shape, and a circular portion of a cylinder is positioned on a bottom face of the outer shell container. The ratio of a diameter (internal diameter) of a circle positioned on a bottom face of a cylinder of an internal space and the height of a cylinder is, for example, <NUM>:<NUM> to <NUM>:<NUM>. The internal diameter and the height may be equal (ratio is <NUM>:<NUM>).

It is preferable that the contents of the culture bag <NUM> housed in the outer shell container <NUM> contain the liquid (culture medium) <NUM> and the gas <NUM>. The volumetric ratio of the gas and the liquid (gas-liquid volumetric ratio) is not particularly limited, and the liquid may be more than the gas, or the gas may be more than the liquid, or both may be in approximately equal amounts. The gas-liquid volumetric ratio is preferably, for example, in the range of <NUM>:<NUM> to <NUM>:<NUM>. It is preferable that in the entirety or the majority of the range that the culture bag <NUM> houses at least the liquid (culture medium) <NUM>, the culture bag <NUM> is firmly attached to the outer shell container <NUM>, and the outer shell container <NUM> supports a side face of the culture bag <NUM>. At a portion at which the gas <NUM> is housed in the culture bag <NUM>, an upper portion (a part) of the culture bag <NUM> may be exposed above (outside) the outer shell container <NUM>.

The culture medium <NUM> is not particularly limited as far as it can be stirred by shaking, and may be a solution, an emulsion, a suspension, a dispersion, a gel or the like. The culture medium <NUM> may be a uniform monophasic composition, or may be composed of two or more phases, for example, the liquid containing the solid matter, and the like. It is preferable that the culture medium <NUM> has been sterilized before culturing. Furthermore, the culture medium <NUM> can contain living bodies (culturing subject) such as microorganisms, insect cells, plant cells, animal cells, tissues, cell sheets, and cell masses. In culture requiring a scaffold, the solid matter such as a particle, a mass, an expanded body, and a fiber may also be mixed into a culture medium as a culture substrate.

The gas <NUM> may be an aerobic atmosphere containing the air (or O<NUM>-containing gas), or may be an anaerobic atmosphere having the low O<NUM> concentration. The composition of the gas <NUM> is not particularly limited, but is generally a mixed gas of two or more gases. Examples of a component of the gas <NUM> include an oxidative gas such as oxygen (O<NUM>); an inert gas such as nitrogen (N<NUM>) and argon (Ar); an acidic gas such as carbon dioxide (CO<NUM>); a basic gas such as ammonia (NH<NUM>); a reducing gas such as hydrogen sulfide (H<NUM>S); and the like. As the gas to be supplied to the culture bag <NUM>, the gas which has been passed through a filter is preferable. It is preferable that the gas such as the air, oxygen, and carbon dioxide is introduced from any one or both of a lower side (liquid side) or upper side (gas side).

The side wall portion 21a has a window <NUM> for visually confirming an amount of the contents (liquid surface) of the culture bag <NUM> housed in the outer shell container <NUM>. The window <NUM> is provided as a slit-like notch in a vertical direction of the side wall portion 21a. The window <NUM> may be opened, or may be closed with a transparent material. Additionally, the bottom wall portion 21b and the lid portion <NUM> have holes <NUM>, <NUM> through which tubes <NUM>, <NUM> are passed.

In the present embodiment, there were two tubes <NUM> on an upper surface of the culture bag <NUM> and two tubes <NUM> on a lower surface of the culture bag <NUM>, but arrangement and the number of tubes <NUM>, <NUM> are not particularly limited, and the desired number of tubes can be provided at appropriate positions. Tubes can be arranged, for example, at any one or two or more of an upper portion, a lower portion, and a side portion of the culture bag. Through these tubes, sampling of the contents, introduction and discharge of the fluid (gas, liquid, powder, and the like), supply of nutrients, removal of waste products, and the like can be performed. A configuration is also possible that, through tubes, a circulation pathway including the culture bag and an external device is provided, and after a part of the culture liquid is treated with the external device, it is returned to the culture bag.

When the culture bag <NUM> is housed in the outer shell container <NUM> as shown in <FIG>, the culture bag <NUM> is deformed according to the weight of the contents, and is firmly attached to an internal surface of the outer shell container <NUM>. By contact between the culture bag <NUM> and the outer shell container <NUM> over a wide area, they are firmly attached even without mechanical fitting or engaging, or chemical adhesion or adsorption, and a fixing force enduring shaking is generated. It is preferable that an internal surface of the outer shell container <NUM> is smooth, and has been polished, for example, by puffing or the like. There may be mild irregularity on an internal surface, but it is preferable that the culture bag does not have such fine irregularity and shape change (acute angle or the like) that damage and deterioration of films <NUM>, <NUM> are caused to the thickness of films <NUM>, <NUM> constituting the culture bag <NUM> (see <FIG>).

The outer shell container <NUM> has a temperature adjusting mechanism (means) so that temperature adjustment of the culture bag <NUM> and the contents thereof can be performed. Examples of the temperature adjusting mechanism include, but are not limited to, a jacket structure through which a heat medium such as circulating water can be passed; a heating device such as a rubber heater; and the like.

<FIG> shows one example of a shaking device <NUM> which shakes the outer shell container <NUM>. The shaking device <NUM> comprises a base portion <NUM> and a frame portion <NUM> surrounding the outer shell container <NUM>, and a power source <NUM> which shakes the outer shell container <NUM>. The shaking device <NUM> can shake the outer shell container <NUM> while the culture bag <NUM> is housed, as shown in <FIG>. The shake-type culture apparatus of the present embodiment is provided with the outer shell container <NUM> housing the culture bag <NUM>, and the shaking device <NUM>, and can perform culture while the contents in the culture bag <NUM> are stirred and mixed by shaking the outer shell container <NUM> housing the culture bag <NUM>.

<FIG> shows one example of a shaking action of the outer shell container. In the present embodiment, a planar shape of the outer shell container <NUM> is a circular shape, and a three-dimensional shape thereof is a cylinder with a central axis being toward approximately vertical. <FIG> shows a center 20c and a radius 20r of the outer shell container <NUM>. In the shaking action of <FIG>, the center 20c of the outer shell container <NUM> rotates (revolves in orbit) on an orbit C of a radius R. In this case, the amplitude of shaking is two times of the radius R. Further, the outer shell container <NUM> may rotate (turn on its axis) around the center 20c. The radius R of an illustrated example is smaller than the radius 20r, but the illustrated ratio does not particularly represent the actual ratio precisely.

It is preferable that upon shaking, the outer shell container <NUM> housing the culture bag <NUM> rotates in a horizontal direction. A rotation direction may include both a horizontal component and a vertical component. It is preferable that the magnitude of the vertical component is smaller than the magnitude of the horizontal component (including the case where the vertical component is <NUM>). Not by shaking in one direction, but by incorporating rotation, effective stirring can be performed. The shear (shearing stress) at stirring is hardly applied to the culture bag, and effective culture can be attained. A speed and a direction of rotation can be appropriately controlled. By changing the rotation speed and the rotation direction, not only stirring of the liquid in a horizontal direction, but also stirring in a vertical direction can be realized.

It is preferable that the rotation number, the amplitude, and the like are set depending on the system of culture, and are controlled by a program. For example, in culture of Escherichia coli, it is preferable that culture is performed by increasing the dissolved oxygen concentration (DO) in a solution. To increase the DO, it is preferable to perform vigorous shaking to intensify mixing of the gas and the liquid. Meanwhile, in culture of animal cells, it is preferable to suppress foaming of the culture liquid and perform mild stirring. The shake-type culture apparatus of the present embodiment can perform culture, for example, by controlling one or two or more of the following control factors.

Regarding a shaking direction, it is preferable that right rotation and left rotation, or forward rotation and reverse rotation are repeated at an arbitrary pattern. It is preferable to construct a system so that the rotational speed and the rotation number of the shaking device can be controlled with a control panel or the like. It is preferable that, in the shaking device, a rotation direction can be arbitrarily switched. For this reason, it is preferable that a power source and a rotating device include a mechanism which can perform rotation in both forward and reverse directions. Additionally, it is preferable to control a rotation pattern, for example, by a program. The rotation pattern is not particularly limited, but examples include the following several examples.

The rotation number and the amplitude of shaking of the outer shell container can be arbitrarily set, and it is according to the present invention to detect the height of a wave, and adjust the rotation number from the height of a wave by feedback control or the like, in order that, when rotated, an increase width of the height of a wave of the contents in the culture bag (for example, average height of liquid level, maximum height, height difference, and the like) is within a certain range. The rotation number is preferably <NUM> to <NUM>,<NUM> rpm (rotation per minute), more preferably <NUM> to <NUM> rpm, further preferably <NUM> to <NUM> rpm, and most preferably <NUM> to <NUM> rpm. The amplitude is preferably <NUM> to <NUM>, more preferably <NUM> to <NUM>, and further preferably <NUM> to <NUM>. In addition, when a rotation direction is changed, the rotation number is the total of the rotation numbers (non-negative value) not depending on a rotation direction, such as a sum of the right rotation number and the left rotation number, or a sum of the forward rotation number and the reverse rotation number.

It is preferable that the culture bag is provided with a sensor for measuring a pH or DO of the contents, and a feedback control mechanism by data obtained by the sensor.

Concerning the hydrogen ion exponent (pH), a pH in the system can be known by inserting a pH electrode into the culture bag, or sticking a chip of which the color changes by a pH, in the system. When the system is desired to be acidic, a pH can be changed to an acidic side by introducing a CO<NUM> gas, or adding an acid solution dropwise. When a CO<NUM> gas is introduced, it is preferable to introduce a CO<NUM> gas from an upper surface of the culture bag, or intermittently introduce the gas from a bottom portion of the culture bag, in order to prevent a CO<NUM> gas from being dissolved in a solution more than necessary. When the system is desired to be basic, a pH can be made to be basic by adding an alkali solution. A port for placing an acid or an alkali for making a pH constant may be tubes <NUM>, <NUM> or a spout <NUM>, or may be provided at another place.

Concerning DO (dissolved oxygen concentration), it is preferable to measure an airflow quantity, perform feedback control, and introduce oxygen. As a method of supplying oxygen (O<NUM>), it is preferable to introduce oxygen through a tube having an outlet in a solution (under liquid level) to diffuse oxygen in the solution.

Concerning a temperature of the contents (culture medium), temperature adjustment can be performed using the temperature adjusting mechanism (as described above) of the outer shell container <NUM>. A culture temperature depends on a kind of an organism to be cultured and, for example, in the case of microorganisms, is preferably <NUM> to <NUM> and particularly preferably <NUM> to <NUM>. When used as a device of stirring a culture medium or the like without culture, the contents may be cooled (for example, <NUM> to <NUM>). For managing a temperature, feedback control can be performed by incorporating a thermometer into the culture bag <NUM> and utilizing a measured value thereof. The thermometer may be a contact thermometer, but a stationary non-contact thermometer is preferable.

The shake-type culture apparatus of the present embodiment can be applied to both of feeding culture (fed-batch system) and perfusion culture (perfusion system). An organism, a cell or the like to be cultured is not particularly limited, and the culture apparatus can be used for culturing bacteria including Escherichia coli, yeast, microorganisms, insect cells, plant cells, animal cells, CHO (Chinese Hamster Ovary) cells for producing biomedicines, HeLa cells, COS cells, iPS cells for use in regenerative therapy, stem cells including mesenchymal stem cells, animal cells such as differentiated tissue cells , and the like. Among them, the culture apparatus is suitable for culturing CHO cells.

The shake-type culture apparatus of the present embodiment and a culture method using the same are particularly suitable for a large-scale culture. It is preferable that the shaking device is loaded with a counter balance, particularly in the case of large scale. When the counter balance is loaded, the apparatus can be stably operated even using the culture bag containing a large volume of a culture medium. By loading one shaking device with two or more culture bags, they may mutually function as the counter balance.

The present invention has been illustrated above based on suitable embodiments, but the present invention is not limited to the above-mentioned embodiments, and can be variously modified in a range not departing from the gist of the present invention.

Claim 1:
A shaking culture apparatus (<NUM>) comprising:
a culture bag (<NUM>) made from a thermoplastic resin film or a laminate of thermoplastic resin film;
an outer shell container (<NUM>) being arranged to house the entirety of said culture bag (<NUM>) to be able to perform adjustment of a temperature thereof;
a power source (<NUM>) for shaking said outer shell container (<NUM>) housing said culture bag (<NUM>),
a device for detecting a height of liquid level of contents in said culture bag (<NUM>); and
an adjustment mechanism for adjusting a rotation number of shaking of said outer shell container (<NUM>) housing said culture bag (<NUM>) so that the height of the liquid level of said contents becomes constant,
wherein
said culture bag (<NUM>) is provided with a spout (<NUM>), and said spout (<NUM>) is fixed to said culture bag (<NUM>) by sealing a base (<NUM>) of said spout (<NUM>) on a back side of a film (<NUM>) of said culture bag (<NUM>), and
said contents in said culture bag (<NUM>) can be stirred and mixed by shaking said outer shell container (<NUM>) housing said culture bag (<NUM>) so that a center (20c) of said outer shell container (<NUM>) revolves in orbit on an orbit C of radius R;
wherein the culture bag (<NUM>) is a gusset bag.