WO2017090760A1

WO2017090760A1 - Culture device

Info

Publication number: WO2017090760A1
Application number: PCT/JP2016/085072
Authority: WO
Inventors: 樋口　朗; 浩幸内藤; 慶司本庄; 透安孫子; 林部　和弥; 工藤　泰之; 広和小田桐
Original assignee: 株式会社京都製作所; デクセリアルズ株式会社
Priority date: 2015-11-27
Filing date: 2016-11-25
Publication date: 2017-06-01
Also published as: JPWO2017090760A1; JP6600364B2

Abstract

A culture device comprising: a culture bag provided with a culture part in which a culture space storing a culture solution therein for performing culture is formed and which is formed of a deformable material; a plurality of bag-pressing parts which are to be pressed from outside so as to deform different sections of the culture part of the culture bag; and a control part which controls the bag-pressing parts so that different sections of the culture part are deformed at different timings by the bag-pressing parts to thereby generate a flow of the culture solution within the culture space.

Description

Incubator

The present invention relates to a culture apparatus used for culturing microorganisms and animal and plant cells.

Conventionally, disposable culture bags have been used for culturing microorganisms and animal and plant cells. For example, as described in Patent Document 1, the culture bag is a bag body in which a culture solution in which a culture target (for example, cells) is suspended at a constant concentration (number) is accommodated. The culture bag also includes a port for supplying a mixed gas whose concentration is controlled, such as oxygen and carbon dioxide, a port for supplying or collecting a culture solution, a port for acquiring a sample, and the like. . Such a culture bag is made of an elastomer material, and is maintained in a prescribed shape by the pressure of the mixed gas during use.

Also, the position and orientation of such a culture bag is periodically changed in order to promote culture, for example, to promote cell proliferation. For example, the culture bag described in Patent Document 1 is fixed on a stage that swings about a swing axis. The amount of mixed gas taken into the culture solution, for example, the amount of dissolved oxygen, is determined by the rocking stroke, the rocking angle, and the rocking speed, which are the rocking conditions of the culture bag. The rocking condition is determined by the nature of the culture object (for example, a cell).

This causes the culture fluid to flow and generate waves on the liquid surface, increasing the area of the liquid surface (gas-liquid interface). Further, the generated wave of the culture wall is broken by colliding with the inner wall surface of the culture bag. As a result, the mixed gas is actively taken into the culture solution. In addition, the culture solution is agitated, and the incorporated gas reaches the entire culture solution. As a result, the proliferation of cells in the culture medium is promoted.

Japanese translation of PCT publication No. 2010-540228

By the way, although depending on the type of culture target and the conditions of rocking of the culture bag, the gas involved in the collision between the wave of the culture solution and the inner wall surface of the culture bag may become bubbles. In particular, bubbles are generated when the culture wave is large.

When the bubble bursts, the cells around the bubble are damaged by the impact caused by the bubble, and in some cases, cell death may be caused. In addition, bubbles may aggregate to form large bubbles (foam), and dissolution of the mixed gas into the culture solution may be inhibited by the large bubbles.

Furthermore, if the flow of the culture solution collides with the inner wall surface of the culture bag and the flow direction of the culture solution changes suddenly, shear stress occurs, and if the generated shear stress is large, the cells may be damaged. The larger the wave of the culture solution, the greater the shear stress and the more damaged the cells.

Therefore, the larger the wave of the culture solution, the more the culture of the culture target (for example, cell proliferation) can be inhibited.

In order to suppress the occurrence of waves in the culture medium to such an extent that the culture can be inhibited, the periodic change in the position and orientation of the culture bag should be suppressed, that is, the change amount should be reduced and the period should be lengthened. Can be considered. For example, in the case of the culture apparatus described in Patent Document 1, it is conceivable to reduce the swing stroke amount of the stage on which the culture bag is placed and to reduce the swing speed thereof.

However, in this case, it is possible to suppress the generation of waves of a culture solution that is large enough to inhibit the culture, but as a result, the culture solution does not flow sufficiently, such as oxygen taken into the culture solution. There is a possibility that the amount of gas is insufficient, and the culture efficiency (for example, cell growth rate) of the culture target is reduced.

Therefore, an object of the present invention is to suppress the generation of bubbles in a culture solution that causes damage and damage to a culture target and the generation of waves in the culture solution that cause shear stress in culture performed while flowing the culture solution in a culture bag.

In order to solve the above technical problem, according to one aspect of the present invention,
A culture bag having a culture part in which a culture space for containing a culture solution and performing culture is formed and made from a deformable material;
First and second bag pressing portions that are deformed by pressing different parts of the culture portion of the culture bag;
By controlling the first and second bag pressing portions, the first and second bag pressing portions cause different portions of the culture portion to be deformed at different timings, thereby generating a culture fluid flow in the culture space. And a culture device having a portion.

According to the present invention, in the culture performed while flowing the culture solution in the culture bag, it is possible to suppress the generation of bubbles in the culture solution that cause damage to the culture target and shear stress.

1 is a schematic perspective view of a culture bag used in a culture apparatus according to an embodiment of the present invention. Top view of culture bag A longitudinal sectional view along the Yb axis of FIG. Longitudinal sectional view along the Xb axis in FIG. The figure which shows the some expansion | swelling bag part of a culture bag The block diagram which shows the structure of the culture apparatus which concerns on one embodiment of this invention The figure for demonstrating the generation | occurrence | production method of the flow of the culture solution by several culture bag parts The figure for demonstrating the generation | occurrence | production method of the flow of the culture solution by the some culture bag part following FIG. 7A. The figure for demonstrating the generation | occurrence | production method of the flow of the culture solution by the some culture bag part following FIG. 7B. The figure for demonstrating the generation | occurrence | production method of the flow of the culture solution by several culture bag parts following FIG. 7C. The figure which shows the some expansion | swelling bag part of the culture bag used for the culture apparatus which concerns on another embodiment of this invention. The top view of the culture | cultivation bag used for the culture apparatus which concerns on another embodiment of this invention. Longitudinal sectional view along the Xb axis in FIG. The figure which shows schematically the culture apparatus which concerns on different embodiment of this invention.

A culture apparatus according to one embodiment of the present invention includes a culture bag that includes a culture part in which a culture space in which culture medium is stored and culture is formed, and is made of a deformable material, and is pressed from the outside. By controlling the 1st and 2nd bag press part which changes the different part of the culture part of a culture bag, and the 1st and 2nd bag press part, the 1st and 2nd bag press part of a culture part A control unit that deforms different portions at different timings and generates a flow of the culture solution in the culture space.

According to this aspect, the culture solution can be flowed in the culture space without changing the position and orientation of the culture bag. Therefore, the generation | occurrence | production of the wave of the culture solution which produces the bubble which can damage a culture | cultivation object and the share stress which arise by changing the position and orientation of a culture bag can be suppressed.

The first and second bag pressing portions are a plurality of inflatable bags that are inflated by receiving supply of fluid, and the culture device supplies the fluid to the first and second inflatable bags, respectively. And the control unit controls the first and second fluid supply units so that different portions of the culture unit are deformed at different timings due to the pressures caused by the expansion of the first and second expansion bags. May be.

The culture bag includes an inner bag portion and an outer bag portion that accommodates the inner bag portion, the inner bag is the culture portion, and the inflatable bag has an outer surface of the inner bag portion and an inner surface of the outer bag portion at a plurality of locations. You may comprise by the some division | segmentation space formed by joining and dividing | segmenting the space between this inner bag part and an outer bag part into plurality.

The culture space of the culture part of the culture bag may be endless, and the control part may control the first and second bag pressing parts so that the culture medium circulates in the endless culture space. Thereby, the culture solution can circulate. As a result, it is possible to further suppress the generation of waves of the culture solution CF that can cause bubbles and shear stress that can damage the culture target.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

FIG. 1 schematically shows a culture bag used in a culture apparatus according to an embodiment of the present invention. FIG. 2 is a top view of the culture bag 100. FIG. 3 is a cross-sectional view taken along the Yb axis of FIG. FIG. 4 is a cross-sectional view along the Xb axis of FIG.

As shown in FIG. 2, the culture bag 100 is a bag body in which microorganisms and cells are cultured using a culture solution. The culture bag 100 is also made from a deformable material, such as polyethylene, so that it can be compressed when discarded for single use.

The culture bag 100 includes a main body 102 for culturing microorganisms and cells by containing a culture solution in which a culture target (for example, cells) is suspended at a certain concentration (number), and a sheet for holding the main body 102. Shaped bracket portion 104.

As shown in FIGS. 3 and 4, the main body 102 of the culture bag 100 is a double-structured bag, and the inner bag 106 is a culture unit for accommodating the culture solution CF and performing culture. . The main body 102 includes a cover part (outer bag part) 108 that accommodates the culture part 106 so as to cover the culture part 106 over the whole.

Although the reason will be described later, the culture part 106 is made of a deformable material, preferably an elastic material such as an elastomer material. The cover part 108 that accommodates the culture part 106 is made of a material that is harder (or less likely to deform) than the material of the culture part 106.

As shown in FIG. 2, the culture part (inner bag part of the main body part 102) 106 includes a culture space 110 in which the culture solution CF is accommodated and cultured. In the case of the present embodiment, the culture space 110 is an endless circumferential space in which the culture solution can circulate, and is annular, and more specifically, an annular (doughnut-shaped) space having a circular longitudinal section. is there.

Here, some terms are defined for the annular culture space 110. First, the rotation direction of the annular culture space 110 that is the rotation space is defined as R1. An axis orthogonal to the plane including the circumferential direction R1 is defined as a third bag axis Zb. The axes that are included in the plane including the rotation direction R1, are orthogonal to the third bag axis Zb, and are orthogonal to each other are defined as first and second bag axes Xb and Yb. Moreover, the circumferential direction of the longitudinal section of the annular culture space 110 orthogonal to the circumferential direction R1 is defined as the longitudinal section circumferential direction R2.

Furthermore, in the case of the present embodiment, since the culture space 110 has an annular shape, the third bag axis Zb is set as a central axis that passes through the center of the annular shape. Further, the sheet-like bracket portion 104 is developed along the first and second bag axes Xb and Yb.

The bracket portion 104 that holds the main body portion 102 of the culture bag 100 functions as a bracket for attaching the culture bag 100 to a culture device described later.

In the case of the present embodiment, the main body portion 102 is provided in the bracket portion 104 so as to penetrate the bracket portion 104. That is, the main body portion 102 is divided into an upper half 102a (a portion located on the upper side when attached to a culture apparatus described later) and a lower half 102b by the bracket portion 104. However, the culture space 110 of the main body portion 102 passes through the bracket portion 104.

In the case of the present embodiment, the main body 102 of the culture bag 100 is provided with a plurality of ports (hoses) 112, 114, 116, 118, and 120.

Each of the plurality of

ports

112, 114, 116, 118, and 120 communicates with the culture space 110 of the culture unit 106.

The culture medium port 112 is a port used when supplying the culture medium CF to the culture space 110 of the culture unit 106 and collecting the culture medium CF from the culture space 110. The culture medium port 112 is provided in the upper half 102 a of the main body 102.

The sampling port 114 is used for acquiring a sample of microorganisms or cells cultured in the culture space 110 of the culture unit 106. A specified amount of a culture solution (for example, a cell suspension) can be collected from the culture bag 100 via the port 114. The progress of the culture can be known by observing the collected suspension with a microscope or the like. For example, the degree of cell growth can be measured by counting the number of cells through a microscope. The sampling port 110 is a port including a luer lock connector with a valve, for example. The sampling port 114 extends from the lower half 102 b of the main body portion 102 and opens at the bracket portion 104.

The first gas supply port 116 is a port used for supplying a mixed gas such as oxygen and carbon dioxide necessary for culture into the culture space 110 of the culture unit 106. The gas supply port 116 extends from the lower half 102 b of the main body 102.

The exhaust port 118 is a port used for exhausting the culture space 110 of the culture unit 106 or adjusting the pressure in the culture space 110 by the exhaust. The exhaust port 118 extends from the upper half 102 a of the main body 102.

The second gas supply port 120 is used to supply a mixed gas such as oxygen and carbon dioxide necessary for culture into the culture space 106 of the culture unit 102, similarly to the first gas supply port 116. Port. The second gas supply port 120 extends from the upper half 102 a of the main body 102. Although details will be described later, in the case of the present embodiment, the second gas supply port 120 is mainly used, and the first gas supply port 116 is used in an auxiliary manner.

The position and longitudinal section of the annular circumferential direction R1 on the main body 102 where the culture medium port 112, the sampling port 114, the first gas supply port 116, the exhaust port 118, and the second gas supply port 120 are provided. The position in the circumferential direction R <b> 2 may be changed depending on the use (culture type) of the culture bag 100. In addition, the first and second

gas supply ports

116 and 120 and the exhaust port 118 are provided with filters for suppressing entry of foreign matter into the culture space 110 of the culture bag 100.

Further, as shown in FIG. 2, in addition to the plurality of

ports

112, 114, 116, 118, and 120, the body portion 102 of the culture bag 100 further includes a plurality of air supply ports 122 </ b> A, 122 </ b> B, 122 </ b> C, And 122D are provided. These air supply ports will be described.

FIG. 5 is a cross-sectional view of the main body 102 along the circular direction R1 of the annular culture space 110 and perpendicular to the radial direction D (see FIG. 2).

As shown in FIG. 5, these

air supply ports

122A, 122B, 122C, and 122D do not communicate with the culture space 110, but communicate with the

expansion bag portions

124A, 124B, 124C, and 124D.

Specifically, the

expansion bag portions

124A, 124B, 124C, and 124D are configured by a plurality of spaces formed between the culture portion 106 and the cover portion 108. For example, the outer surface of the culture unit 106 and the inner surface of the cover unit 108 are joined (for example, welded) at a plurality of joints 126, so that a plurality of independent divided spaces, that is, the

expansion bag portions

124A, 124B, 124C, and 124D is formed. In the case of the present embodiment, each of these

inflatable bag portions

124A, 124B, 124C, and 124D is an annular space that circulates the culture portion 106 in the longitudinal section circumferential direction R2.

As shown in FIG. 2, the culture medium port 112, the sampling port 114, the first gas supply port 116, the exhaust port 118, and the second gas supply port 120 are provided in the culture part (inner bag part) 106. In order to communicate with the culture space 110, the culture portion 106 and the cover portion (outer bag portion) 108 are provided at a joint 126 (cross-hatched portion).

The

air supply ports

122A, 122B, 122C, and 122D communicate with the plurality of

inflatable bag portions

124A, 124B, 124C, and 124D, respectively. Although details will be described later, each of the plurality of

expansion bag portions

124A, 124B, 124C, and 124D receives supply of compressed air intermittently and repeats expansion and contraction. Thereby, each of the

expansion bag parts

124A, 124B, 124C, and 124D intermittently presses different parts of the culture part 106 from the outside, thereby deforming the different parts of the culture part 106 at different timings. . As a result, a flow of the culture solution CF in the circulation direction R1 is generated in the culture space 110.

FIG. 6 shows a configuration of the culture apparatus 10 for performing culture using the culture solution CF in a state where the culture solution CF circulates in the circulation direction R1 in the annular culture space 110 of the culture bag 100. It is a block diagram.

As shown in FIG. 6, the culture apparatus 10 includes a vent valve 50 connected to the exhaust port 118 of the culture bag 100, a flow rate adjustment valve 52 connected to the first gas supply port 116, and a second gas supply port. A flow control valve 54 connected to 120 is included.

The vent valve 50 is a valve for adjusting the pressure in the culture space 110 by exhausting the culture bag 110 from the culture space 110 to the outside. Therefore, the vent valve 50 is disposed between the exhaust port 118 of the culture bag 100 and the outside air. By adjusting the opening degree of the vent valve 50, the pressure of the culture space 110 is adjusted.

The flow

rate adjusting valves

52 and 54 are valves for adjusting the amount of mixed gas of oxygen and carbon dioxide supplied to the culture space 110 of the culture bag 100. The flow control valve 52 is connected to the first gas supply port 116 of the culture bag 100, and the flow control valve 54 is connected to the second gas supply port 120.

The

flow control valves

52 and 54 are connected to an oxygen source (for example, an oxygen cylinder) 61 and a carbon dioxide source (for example, a carbon dioxide cylinder) 62 through on-off

valves

57 and 58 and

flow control valves

59 and 60.

Specifically, the flow rate adjustment valve 52 is connected to a compressed air source (for example, an air cylinder) 63 via an on-off valve 57. Further, the flow rate adjustment valve 54 is connected to the compressed air source 63 via the on-off valve 58. Further, an oxygen source 61 is connected between the on-off

valves

57 and 58 and the compressed air source 63 via a flow rate adjustment valve 59. A carbon dioxide source 62 is connected between the on-off

valves

57 and 58 and the compressed air source 63 via the flow rate control valve 60.

Oxygen from the oxygen source 61 and carbon dioxide from the carbon dioxide source 62 are mixed together with the compressed air from the compressed air source 63. The mixed gas accompanying the compressed air is supplied to only the flow control valve 54 or both of the

flow control valves

52 and 54, that is, only the second gas supply port 120 or both the first and second

gas supply ports

116 and 120. Sent to. The amount of oxygen and the amount of carbon dioxide in the mixed gas are adjusted by changing the opening degree of the

flow control valves

59 and 60. Further, by selectively opening and closing the on-off

valves

57 and 58, only the second gas supply port 120 through only the flow control valve 54 or both of the first and second through the flow control valves 54 and 56 are provided. The mixed gas is supplied to both of the

gas supply ports

116 and 120. Furthermore, the supply amount of the mixed gas to the first and second

gas supply ports

116 and 120 is adjusted by changing the opening degree of each of the flow

rate adjustment valves

52 and 54.

Thereby, oxygen and carbon dioxide are supplied to the culture space 110 of the culture bag 100 through the second gas supply port 120, and the amount of oxygen supplied to the culture space 110 by the flow

rate control valves

54, 59, and 60, that is, The oxygen concentration in the culture solution CF is adjusted, and the amount of carbon dioxide supplied to the culture space, that is, the pH value of the culture solution CF is adjusted. Moreover, the shape of the culture part 106 (culture space 110) of the culture bag 100 is maintained in a substantially constant shape by the compressed air.

When the oxygen concentration or pH value in the culture solution CF is lower than the set value, the on-off valve 57 is opened, and oxygen and carbon dioxide are mixed into the culture space 110 of the culture bag 100 via the first gas supply port 116. Gas is additionally supplied. Thus, by providing a plurality of ports (in the case of the present embodiment, the first and second gas supply ports 116 and 120) for supplying the mixed gas to the culture space 110 of the culture bag 100, the culture solution CF It is possible to finely control the oxygen concentration and pH value.

Note that when the culture space 110 of the culture bag 100 is filled with the culture solution CF, the second gas supply port 120 and the exhaust port 118 are not used.

The culture apparatus 10 also has

flow control valves

66A, 66B, 66C, and 66D and a switching valve as an air supply unit that supplies compressed air to the plurality of

expansion bag portions

124A, 124B, 124C, and 124D of the culture bag 100. 68A, 68B, 68C, and 68D.

The

flow control valves

66A, 66B, 66C, and 66D are connected to the

expansion bag portions

124A, 124B, 124C, and 124D of the culture bag 100 through the

air supply ports

122A, 122B, 122C, and 122D. Further, the flow

rate adjusting valves

66A, 66B, 66C, and 66D are connected to the outside air and the compressed air source 70 via the switching

valves

68A, 68B, 68C, and 68D. By switching the switching

valves

68A, 68B, 68C, and 68D, the switching

valves

68A, 68B, 68C, and 68D, the

flow control valves

66A, 66B, 66C, and 66D, the

air supply ports

122A, 122B, 122C, and Compressed air is supplied from the compressed air source 70 to each of the

expansion bag portions

124A, 124B, 124C, and 124D of the culture bag 100 via 122D. Alternatively, the air in the

expansion bag portions

124A, 124B, 124C, and 124D of the culture bag 100 includes the

air supply ports

122A, 122B, 122C, and 122D, the

flow control valves

66A, 66B, 66C, and 66D, and the switching valve. 68A, 68B, 68C, and 68D are discharged to the outside air. The compressed air source 63 and the compressed air source 70 may be the same compressed air source.

The culture apparatus 10 further includes a pH sensor 72, a temperature sensor 74, and a dissolved oxygen sensor 76 in order to monitor the state of the culture solution CF during culture. The pH sensor 72 detects the pH value of the solvent liquid CF in the culture space 110, the temperature sensor 74 detects the temperature of the solvent liquid CF, and the dissolved oxygen sensor 76 detects the oxygen concentration of the solvent liquid CF.

Based on the state of the culture fluid CF during culture, that is, the detection results of the pH sensor 72, the temperature sensor 74, and the dissolved oxygen sensor 76, the vent valve 50, the

flow control valves

52, 54, 59, and 60, and the on-off valve The culture apparatus 10 has a control box 80 for controlling 57 and 58 and the heater 78. The heater 78 is for adjusting the temperature of the culture solution CF in the culture space 110 of the culture bag 100.

The control box 80 includes a valve control unit 82 that controls the plurality of

valves

50, 52, 54, and 57 to 60, and a sensor management unit 84 that acquires detection values of the pH sensor 72, the temperature sensor 74, and the dissolved oxygen sensor 76. A temperature control unit 86 that controls the heater 78, and a flow generation unit 88.

First, the sensor management unit 84 of the control box 80 is connected to the pH sensor 72, the temperature sensor 74, and the dissolved oxygen sensor 76, and is detected by the pH value of the culture solution CF detected by the pH sensor 72 and the temperature sensor 74. The temperature of the culture solution CF and the oxygen concentration of the solvent solution CF detected by the dissolved oxygen sensor 76 are periodically acquired.

The valve control unit 82 controls the plurality of

valves

50, 52, 54, and 57 to 60 so that the pH value and the oxygen concentration of the solvent liquid CF acquired by the sensor management unit 84 are maintained at the set values. . The temperature control unit 86 controls the heater 78 so that the temperature of the solvent liquid CF acquired by the sensor management unit 84 is maintained at the set value.

The flow generator 88 is connected to the

flow control valves

66A, 66B, 66C, and 66D and the switching

valves

68A, 68B, 68C, and 68D, and based on the flow rate of the culture fluid CF set by the user, To control. By controlling these valves, the flow generator 88 controls the expansion and contraction of the

expansion bag portions

124A, 124B, 124C, and 124D of the culture bag 100, respectively. Thereby, a flow of the culture solution CF is generated in the culture space 110 of the culture bag 100. It should be noted that the control for the

flow control valves

66A, 66B, 66C, and 66D and the switching

valves

68A, 68B, 68C, and 68D, that is, the expansion patterns of the

expansion bag portions

124A, 124B, 124C, and 124D that generate the flow of the culture fluid CF. Will be described later.

The culture environment (pH value, temperature, and oxygen concentration of the culture medium CF) set by the user is maintained by the valve control unit 82, the sensor management unit 84, and the temperature control unit 86. The valve control unit 82, the sensor management unit 84, the temperature control unit 86, and the flow generation unit 88 can output a control signal (current) to each of the plurality of

valves

50, 52, 54, and 57 to 60. , A detection signal (current) from the pH sensor 72, the temperature sensor 74, and the dissolved oxygen sensor 76 can be received, and driving power can be supplied to the heater 78. Further, the

flow control valves

66A, 66B, 66C and 66D and switching

valves

68A, 68B, 68C and 68D can output control signals (currents) to, for example, circuit boards.

Moreover, the culture apparatus 10 has a control unit 90 for the user to set culture conditions. The control unit 90 is, for example, a computer, such as an input device 92 such as a mouse or a keyboard for inputting culture conditions desired by the user, and a display for the user to check the culture conditions and the state during culture. And an output device 94. The control unit 90 commands the control box 80 to maintain the culture conditions (pH value, temperature, oxygen concentration, and flow rate of the culture medium CF) set by the user via the input device 92.

Hereafter, the expansion patterns of the

expansion bag portions

124A, 124B, 124C, and 124D that generate the flow of the culture solution CF will be described with reference to FIGS. 7A to 7D. Here, the expansion pattern in which the flow of the culture solution CF flowing in the direction of arrow A is generated will be described.

First, as shown in FIG. 7A, after the inflation bag portion 124A is inflated, the inflation bag portion 124B is inflated. Most of the culture fluid CF pressed by the expansion of the expansion bag portion 124B flows in the direction of arrow A. That is, most of the culture fluid CF does not flow to the expansion bag portion 124A side where the flow path cross-sectional area is small due to the expansion of the expansion bag portion 124A, but the flow path cross-sectional area is increased because the expansion bag portion 124C is contracted. It flows toward the large inflation bag portion 124C.

Next, as shown in FIG. 7B, in the state where the inflatable bag portion 124B is inflated, the inflatable bag portion 124C on the downstream side in the arrow A direction is inflated with respect to the inflatable bag portion 124B. Most of the culture solution CF pressed by the expansion of the expansion bag portion 124C flows in the arrow A direction. Note that the expansion bag portion 124A is contracted substantially simultaneously with the expansion of the expansion bag portion 124C.

Subsequently, as shown in FIG. 7C, in the state where the inflatable bag portion 124C is inflated, the inflatable bag portion 124D on the downstream side in the arrow A direction is inflated with respect to the inflatable bag portion 124C. Most of the culture solution CF pressed by the expansion of the expansion bag 124D flows in the direction of arrow A. The inflation bag portion 124B is contracted substantially simultaneously with the inflation of the inflation bag portion 124D.

Then, as shown in FIG. 7D, in the state where the inflatable bag portion 124D is inflated, the inflatable bag portion 124A on the downstream side in the arrow A direction is inflated with respect to the inflatable bag portion 124C. Most of the culture solution CF pressed by the expansion of the expansion bag portion A flows in the direction of arrow A. The inflation bag portion 124C is contracted substantially simultaneously with the inflation of the inflation bag portion 124A.

As shown in FIGS. 7A to 7D, the culture solution CF flows in the direction of arrow A by sequentially inflating the plurality of

inflatable bag portions

124A, 124B, 124C, and 124D. In addition, the culture solution CF is intermittently accelerated by the expansion of the

expansion bag portions

124A, 124B, 124C, and 124D. Therefore, the flow of the expansion liquid CF is not a constant flow, but a pulsating flow having a different speed (pressure) in the circulation direction R1. As a result, the culture solution CF flows in the circulation direction R1 while being stirred.

In addition, while adjusting the time between the timing which each expansion | swelling

bag part

124A, 124B, 124C and 124D expand | swells, adjusting the expansion | swelling speed of each of these expansion | swelling bag parts, adjusting the speed | rate of the culture solution CF. Is possible.

According to the present embodiment as described above, in the culture performed while flowing the culture solution CF in the culture bag 100, the generation of bubbles of the culture solution that can cause damage to the culture target and shear stress is suppressed. be able to.

Specifically, in the case of the present embodiment, the culture solution CF can be flowed in the culture space 110 without changing the position and orientation of the culture bag 100. Therefore, generation | occurrence | production of the wave of the culture solution which produces the bubble which can damage a culture | cultivation object and the share stress which generate | occur | produce by changing the position and orientation of the culture bag 100 can be suppressed.

Further, in the case of the present embodiment, as shown in FIG. 2, since the culture space 110 in which the culture solution CF is contained and cultured is an annular space, the culture solution CF can circulate. Therefore, the generation | occurrence | production of the wave of the culture solution CF which produces the bubble which can damage a culture | cultivation object, and a shear stress is further suppressed.

More specifically, the inner wall surface of the culture space 110 and the culture solution are compared with the case where the flow direction changes in a disordered manner due to the circulation of the culture solution CF (by restricting the flow direction to the circulation direction R1). Collisions with CF waves are suppressed. Specifically, the occurrence of a collision in which the flow direction of the culture fluid CF changes rapidly (for example, the flow direction is reversed) is suppressed. Thereby, generation | occurrence | production of the bubble and the shear stress of the grade which can damage a culture target (for example, cell) are suppressed.

In addition, as the culture solution CF circulates (by restricting the flow direction to the circulation direction R1), the generated waves of the culture solution CF are also smaller than when the flow direction changes randomly. That is, the generation | occurrence | production of the wave of a culture solution of the magnitude | size which is the extent which produces the bubble which can damage a culture | cultivation object (for example, cell), and a shear stress is suppressed.

Furthermore, since the annular culture space 110 through which the culture solution CF flows is an endless peripheral space in which the culture solution CF can circulate, the occurrence of a region where the flow rate is substantially zero (so-called stagnation) in the culture solution CF is suppressed. Is done. Therefore, the culture target is prevented from aggregating in a region where the flow rate is substantially zero. As a result, damage to the culture target is suppressed.

In the present application, the “endless circular space in which the culture medium can circulate” refers to an inner surface (for example, in FIG. 2) in which the culture liquid can circulate and restricts the movement of the culture liquid to the center of rotation. It means a space provided with an annular culture space 110 shown in FIG. Further, instead of the annular space of the present embodiment, the culture space may be a three-dimensional shape space such as an “8” shape that intersects three-dimensionally.

Furthermore, as shown in FIG. 7A, the convex portions generated in the culture space 110 when the inflated bag portions 124A to 124D of the present embodiment press the culture portion 106 of the culture bag 100 from the outside are necessarily rounded. The shape becomes distorted. Therefore, since it is not as sharp as an impeller that rotates and stirs the culture solution, the shear stress generated by this convex portion is smaller than the shear stress caused by the impeller. Further, in the case of the impeller, a large shear stress is locally generated, but in the case of this convex portion, a small shear stress is generated in a distributed manner. Therefore, the damage to the culture target is also suppressed by the shape feature of the convex portion.

As mentioned above, although the present invention has been described with reference to the above-described embodiment, the embodiment of the present invention is not limited to this.

For example, in the case of the above-described embodiment, as shown in FIG. 6, the

expansion bag portions

124A, 124B, 124C, and 124D of the culture bag 100 communicate with the outside air via the switching

valves

68A, 68B, 68C, and 68D. It shrinks by being done. Alternatively, the

expansion bag portions

124A, 124B, 124C, and 124D may be contracted by sucking the internal air by a vacuum pump (not shown). In this case, the expansion bag portion is contracted at high speed. Therefore, if the expansion of the expansion bag portion is also performed at a high speed, a high-speed flow of the culture solution CF can be generated.

In the case of the above-described embodiment, as shown in FIG. 5, the

expansion bag portions

124A, 124B, 124C, and 124D are annular spaces that circulate around the culture portion 106 in the circumferential direction R2 in the longitudinal section. The embodiment of the present invention is not limited to this.

For example, the culture bag 200 used in the culture device according to another embodiment shown in FIG. 8 has a plurality of

inflatable bag portions

208A, 208B, 208C, and 208D, similarly to the culture bag 100 of the above-described embodiment. Is provided. However, the

expansion bag portions

208A, 208B, 208C, and 208D shown in FIG. 8 are not annular spaces that circulate in the circumferential direction of the longitudinal section of the culture space 212.

For example, a sheet 204 made of a deformable material is attached to a part of an inner peripheral surface of an annular cover portion 206 having a circular vertical cross section through a plurality of joints. Thereby, a plurality of

inflatable bag portions

208A, 208B, 208C, and 208D that are independent spaces are formed between the sheet 204 and the inner peripheral surface portion of the cover portion 206 to which the sheet 204 is attached. Yes. On the other hand, a culture part is constituted by the sheet 204 and the part of the cover part 206 to which the sheet 204 is not attached, and a culture space 212 is formed therein.

Air supply ports

210A, 210B, 210C, and 210D are communicated with the

expansion bag portions

208A, 208B, 208C, and 208D.

As with the culture bag 100 shown in FIGS. 7A to 7D, the flow of the culture solution can be formed by inflating the

expansion bag portions

208A, 208B, 208C, and 208D in order.

Furthermore, in the case of the above-mentioned embodiment, as shown in FIG. 2, the culture space 110 of the culture bag 100 is annular, but the embodiment of the present invention is not limited to this.

For example, FIG. 9 is a top view of a culture bag having a substantially rectangular culture space. FIG. 10 is a cross-sectional view along the Xb axis of FIG.

The culture bag 300 shown in FIGS. 9 and 10 has a substantially rectangular shape, and is a double bag having an inner bag portion (culture portion) 302 and an outer bag portion (cover portion) 304 that accommodates the inner bag portion 302. It is a bag of structure. In addition, a substantially rectangular culture space 312 is formed in the culture unit 302.

The outer surface of the culture part (inner bag part) 302 and the inner surface of the cover part (outer bag part) 304 are joined by a plurality of joints 308, whereby a plurality of

inflatable bag parts

306A, 306B, 306C and 306D are formed. Each of these

inflatable bag portions

306A, 306B, 306C, and 306D is an annular space and communicates with the

air supply ports

310A, 310B, 310C, and 310D.

As shown in FIG. 9, the plurality of

inflatable bag portions

306A, 306B, 306C, and 306D are arranged in the longitudinal direction of the culture bag 300 (first bag axis Xb direction). Therefore, when the plurality of

inflatable bag portions

306A, 306B, 306C, and 306D are inflated in order, a culture solution flow in the longitudinal direction of the culture bag 300 is generated.

In the case of the culture bag 300, the culture solution cannot circulate like the above-described culture bag 100 having the annular culture space 110. Therefore, in order to suppress the collision of the flow caused by the expansion of each of the plurality of expansion bag portions, the following expansion pattern of the expansion bag portion is executed.

For example, each of the plurality of

inflatable bag portions

306A, 306B, 306C, and 306D is inflated one by one in order. When the flow of the culture solution generated by the expansion of one expansion bag portion is almost subsided, the next expansion bag portion expands to newly generate a flow of the culture solution. Thereby, the flow of the culture solution can be generated while suppressing the generation of bubbles.

In the case of the above-described embodiment, as shown in FIGS. 7A to 7D, the plurality of

inflatable bag portions

124A, 124B, 124C, and 124D, which are means for pressing and deforming the culture portion 106 of the culture bag 100, Although it is provided in the culture bag 100, the embodiment of the present invention is not limited to this. For example, the expansion bag may be provided not on the culture bag side but on the culture device side. In this case, the culture bag has a simple structure.

Furthermore, in the case of the above-described embodiment, as shown in FIGS. 7A to 7D, the means for pressing and deforming the culture unit 106 of the culture bag 100 includes a plurality of inflated parts supplied with fluid (compressed air). Although the

expansion bag portions

124A, 124B, 124C, and 124D, the embodiment of the present invention is not limited to this.

For example, the culture apparatus 500 according to another embodiment shown in FIG. 11 includes a plurality of bag pressing portions 502 that press the annular culture portion 402 of the culture bag 400 from the outside to partially deform the culture portion 402. , 504, 506, and 508. As shown in FIG. 11, the plurality of

bag pressing portions

502, 504, 506, and 508 are arranged at equal intervals in the circumferential direction R1. Since the

bag pressing portions

502, 504, 506, and 508 have the same configuration, the bag pressing portion 502 will be described. The bag pressing portion 502 includes a pair of

bag pressing bars

502A and 502B that sandwich the culture unit 402.

For example, when the pair of

bag pressing bars

506A and 506B of the bag pressing unit 504 sandwich the culture unit 402 and the pair of

bag pressing bars

506A and 506B of the bag pressing unit 506 sandwich the culture unit 402, the culture unit 402 is sandwiched. A flow of the culture solution CF in the direction of arrow A toward the bag pressing portion 508 occurs.

Finally, in the case of the above-described embodiment, as shown in FIG. 5, different portions of the culture unit 106 of the culture bag 100 are deformed at different timings to generate a flow of the culture solution CF in the culture space 110. There are four bag pressing portions (inflatable bag portions) 124A, 124B, 124C, and 124D, but the embodiment of the present invention is not limited to this. There may be at least two bag pressing portions.

As described above, the embodiment has been described as an example of the technique in the present invention. For this purpose, the accompanying drawings and detailed description are provided. Accordingly, among the components described in the accompanying drawings and the detailed description, not only the components essential for solving the problem, but also the components not essential for solving the problem in order to illustrate the technology. May also be included. Therefore, it should not be immediately recognized that these non-essential components are essential as those non-essential components are described in the accompanying drawings and detailed description.

In addition, since the above-described embodiment is for illustrating the technique in the present invention, various modifications, replacements, additions, omissions, and the like can be made within the scope of the claims and the equivalents thereof.

The disclosures of the specification, drawings, and claims of Japanese Patent Application No. 2015-232255 filed on November 27, 2015 are incorporated herein by reference in their entirety. .

Claims

A culture bag having a culture part in which a culture space for containing a culture solution and performing culture is formed and made from a deformable material;
First and second bag pressing portions that are deformed by pressing different parts of the culture portion of the culture bag;
By controlling the first and second bag pressing portions, the first and second bag pressing portions cause different portions of the culture portion to be deformed at different timings, thereby generating a culture fluid flow in the culture space. And a culture device.
A plurality of inflatable bags in which the first and second bag pressing portions are inflated by receiving a supply of fluid;
Having first and second fluid supply parts for supplying fluid to the first and second inflatable bags, respectively;
2. The control unit according to claim 1, wherein the control unit controls the first and second fluid supply units such that different portions of the culture unit are deformed at different timings by pressing due to expansion of the first and second expansion bags. Culture device.
The culture bag includes an inner bag portion and an outer bag portion that houses the inner bag portion,
The inner bag is the culture part,
The inflatable bag has a plurality of divided spaces formed by joining the outer surface of the inner bag portion and the inner surface of the outer bag portion at a plurality of locations and dividing the space between the inner bag portion and the outer bag portion into a plurality of spaces. The culture apparatus according to claim 2, comprising:
The culture space of the culture part of the culture bag is endless,
The culture apparatus according to any one of claims 1 to 3, wherein the control unit controls the first and second bag pressing units so that the culture solution circulates in the endless culture space.