WO2010131715A1

WO2010131715A1 - Culture apparatus

Info

Publication number: WO2010131715A1
Application number: PCT/JP2010/058124
Authority: WO
Inventors: 幸二西田; 義則大家; 伸吾大竹; 悟前田; Tadashi OTAKE (大竹直)
Original assignee: 株式会社大竹; 国立大学法人東北大学; 大竹千恵子
Priority date: 2009-05-14
Filing date: 2010-05-13
Publication date: 2010-11-18
Also published as: JP2012147678A

Abstract

A culture apparatus provided with a culture container (1), wherein the culture container comprises: a container body (11) enclosing a closed space (110) therein; a scaffold (123) for holding cells in the closed space; a scaffold fixation device (12) for fixing the scaffold; an injection port (13), which is provided at the center of the bottom face of the container body, for injecting a medium into the closed space; a discharge port (14), which is provided in the upper part of the container body, for discharging the medium from the closed space; a diaphram (15), which is provided on the top face of the container body, for increasing and reducing pressure in the closed space through vertical vibration; and a rectifying member (16), which is provided above the injection port, for flowing the injected medium toward the circumferential section of the closed space.

Description

Incubator

The present invention relates to a culture apparatus for culturing cells efficiently, and particularly to a culture apparatus for one specimen for culturing cells for regenerative medicine.

As a method of inducing regeneration of living tissue for regenerative medicine, there is a method of promoting cell proliferation and differentiation in vitro using a culture apparatus. Techniques for realizing such a method are disclosed in Patent Documents 1 to 8, for example.
JP-A-4-356184 JP 2004-89138 A JP 2004-350557 A JP 2006-264647 A JP 2007-20493 A JP 2007-110932 A JP 2002-316136 A JP 2007-167002 A

Incidentally, a medium is used for culturing cells. The medium is preferably always fresh because it supplies nutrients to the cells and removes wastes from the cells. Therefore, the replacement of the medium is a very important operation in the culture apparatus. However, when an operator manually changes the medium, a skillful technique is usually required, and the cells may be infected with bacteria. Therefore, there is a demand for an apparatus that automatically changes the culture medium.

Furthermore, such an apparatus is required to have excellent culture efficiency, low cost, and small size.

An object of the present invention is to provide a culture apparatus capable of automatically exchanging a medium.

The present invention provides a culture apparatus including a culture container for culturing cells using a culture medium, wherein the culture container includes a container body having a sealed space, a scaffold for holding cells in the sealed space, and a scaffold. Scaffolding fixture for fixing, provided at the center of the bottom of the container body, an inlet for injecting the medium into the sealed space, and provided at the top of the container body. Is provided on the top surface of the container body, and is provided above the diaphragm and the injection port to pressurize and depressurize the sealed space by vibrating up and down. A rectifying member for allowing the culture medium to flow toward the peripheral edge in the sealed space, and the culture vessel is depressurized when the diaphragm is moved upward, thereby reducing the pressure from the inlet. Medium injection Possible and then, when the diaphragm moves down, the closed space is pressurized, thereby, it adapted to allow the discharge of the medium from the outlet, and characterized in that.

According to the present invention, by oscillating the diaphragm up and down, the medium can be injected into the sealed space from the inlet and discharged from the outlet. Therefore, the medium in the sealed space can be replaced little by little by vibrating the diaphragm. Thereby, automatic exchange of a culture medium is attained.

It is a front view which shows the culture apparatus of 1st Embodiment of this invention. It is a front view of a culture container. It is a top view of a culture container. It is a side view of a culture container. FIG. 5 is a VV cross-sectional view of FIG. 3. It is a disassembled perspective view of a scaffold, a scaffold fixing tool, and both horizontal arms. It is a top view of a scaffold, a scaffold fixture, and both horizontal arms. FIG. 8 is a sectional view taken along line VIII-VIII in FIG. It is a front view of a 1st check valve. It is a longitudinal cross-sectional view of a 1st check valve. It is a XI arrow partial view of FIG. It is a perspective view of a culture container. FIG. 13 is a sectional view taken along line XIII-XIII in FIG. 12. It is a longitudinal cross-sectional view which shows sowing work. It is a longitudinal cross-sectional view which shows the movement state of a culture medium. It is a front view which shows the culture container of an airtight state independently. It is a front view which shows the culture part of an airtight state independently. It is a front view which shows the culture part of a double sealing state. It is a longitudinal cross-sectional view which shows the modification of a rotational drive mechanism. It is a perspective view of the modification of a scaffold fixing tool. It is a XXI arrow line view of FIG. FIG. 21 is a sectional view taken along line XXII-XXII in FIG. 20. It is a longitudinal cross-sectional view of the culture container which has three through-holes for needles. It is a front view of another modification of a scaffold fixture. It is a XXV arrow line view of FIG. It is a longitudinal cross-sectional view of the culture container which has a deformation | transformation structure replaced with the through-hole for needles. It is a longitudinal cross-sectional view which shows the sowing jig and culture container used for another sowing operation | work. FIG. 28 is a longitudinal sectional view showing a step subsequent to FIG. 27. It is a front view which shows the modification of a culture apparatus. It is a front view which shows the culture apparatus of 2nd Embodiment of this invention. It is a longitudinal cross-sectional view of an exposed part. It is a longitudinal cross-sectional view of another exposed part.

DESCRIPTION OF SYMBOLS 1 Culture container 21 Injection medium tank 22 Medium injection pipe 23 Discharge medium tank 24 Medium discharge pipe 25 First check valve 26 Second check valve 27 First clamp member 28 Second clamp member 3 Container drive mechanism 31 1st motor 311 Horizontal rotating shaft 32 Rotating member 33 Arm 34 1st connecting member 35 Rod 36 Rod guide 4 Rotation drive mechanism 41 2nd motor 42 2nd connection member 5 pH measuring device 6 Imaging mechanism 105 Container drive control unit 106 Rotation drive Control unit 107 pH measurement control unit 108 Imaging control unit

[First Embodiment]
FIG. 1 is a front view showing a culture apparatus according to a first embodiment of the present invention. The culture apparatus 100 includes an apparatus main body 101, a thermostatic chamber 103 in which the apparatus main body 101 is accommodated, and a control unit 109.

The control unit 109 includes a container drive control unit 105, a rotation drive control unit 106, a pH measurement control unit 107, and an imaging control unit 108. These

control units

105, 106, 107, and 108 can be operated by an operator.

The apparatus main body 101 includes a culture unit 102, a container drive mechanism 3, and a rotation drive mechanism 4 on a substrate 111. A container drive control unit 105 is connected to the container drive mechanism 3. A rotation drive control unit 106 is connected to the rotation drive mechanism 4.

The culture unit 102 includes a culture vessel 1, an infusion medium tank 21, a medium infusion pipe 22, a discharge medium tank 23, and a medium discharge pipe 24. The culture part 102 has established the airtight structure as a whole.

A first check valve 25 and a second check valve 26 are provided in the medium injection pipe 22 and the medium discharge pipe 24, respectively. In addition, a first clamp member 27 and a second clamp member 28 are provided in the medium injection pipe 22 and the medium discharge pipe 24, respectively.

A pH measuring device 5 is provided in the culture medium discharge pipe 24. A pH measurement control unit 107 is connected to the pH measuring device 5. The culture vessel 1 is provided with a photographing mechanism 6. A photographing control unit 108 is connected to the photographing mechanism 6.

In the culture unit 102, members that come into contact with the culture medium, specifically, the culture medium tank 21 for injection, the culture medium injection pipe 22, the culture vessel 1, the culture medium tank 23 for discharge, the culture medium discharge pipe 24, and the first check valve 25. The second check valve 26 is made of a material that does not adversely affect the living body, that is, a biocompatible material. The biocompatible material is appropriately selected from, for example, resin, metal, glass, and the like. Also, these members can be discarded after use.

The culture apparatus 100 is configured such that the medium flows from the infusion medium tank 21 to the discharge medium tank 23 via the medium infusion pipe 22, the culture container 1, and the medium discharge pipe 24. . That is, the culture apparatus 100 of this embodiment is a perfusion type.

(Culture container 1)
2, 3, and 4 are a front view, a plan view, and a side view of the culture vessel 1. 5 is a cross-sectional view taken along the line VV of FIG. The culture vessel 1 is a vessel for culturing cells with a medium. The culture container 1 includes a container body 11 having a sealed space 110, a scaffold 123 for holding cells in the sealed space 110, a scaffold fixture 12 for fixing the scaffold 123, and a medium in the sealed space 110. From the inlet 13 for injecting the medium, the outlet 14 for discharging the culture medium from the sealed space 110, the diaphragm 15 that pressurizes and depressurizes the sealed space by vibrating up and down, and the inlet 13 And a rectifying member 16 for flowing the injected culture medium toward the peripheral edge in the sealed space 110.

The container body 11 has a rectangular parallelepiped shape, and includes a bottom surface portion 111, first to fourth

side surface portions

112, 113, 114, 115, and a top surface portion 116. The sealed space 110 is surrounded by these six surface portions.

The injection port 13 extends from the lower portion of the first side surface portion 112 of the container body 11 through the bottom surface portion 111 and opens upward at the center of the bottom surface portion 111. The rectifying member 16 is a plate material, and is arranged horizontally via the legs 161 above the injection port 13 with a slight gap 163 therebetween. The center of the rectifying member 16 and the center of the injection port 13 coincide with each other in a plan view, and the rectifying member 16 secures a uniform gap 162 at the periphery in the sealed space 110. The discharge port 14 is formed through the upper portion of the first side surface portion 112.

The diaphragm 15 is made of, for example, a stretchable synthetic rubber. The diaphragm 15 constitutes an upper surface portion 116 of the container main body 11. The peripheral edge portion 151 of the diaphragm 15 is fixed to the upper end surfaces of the four side surface portions of the container main body 11 by the ring plate 192 with screws 193, whereby the diaphragm 15 is fixed at the center portion thereof. 151 can vibrate up and down.

In the culture container 1, when the diaphragm 15 moves upward, the inside of the sealed space 110 is depressurized, so that the medium can be injected into the sealed space 110 from the inlet 13, and when the diaphragm 15 moves downward, the sealed space 110. Since the inside is pressurized, the medium in the sealed space 110 can be discharged from the discharge port 14. Therefore, when the diaphragm 15 vibrates, the medium can be injected and discharged, that is, the medium can be exchanged in the sealed space 110.

As shown in FIG. 5, the scaffold fixture 12 is supported in the sealed space 110 by the first horizontal arm 181 and the second horizontal arm 182. The first horizontal arm 181 extends from the scaffold fixture 12, and a distal end 1811 is slidably inserted into a recess 1121 on the inner surface of the first side surface portion 112. The second horizontal arm 182 extends from the scaffold fixture 12 through the third side surface 114. The third side surface portion 114 is provided with a sealing member 1142 that seals the through hole 1141 via the O-ring 1143. The second horizontal arm 182 penetrates the sealing member 1142 in an airtight manner and is rotatable via a bearing 1144.

6 and 7 are an exploded perspective view and a plan view of the scaffold 123, the scaffold fixture 12, and both

horizontal arms

181 and 182, respectively. 8 is a cross-sectional view taken along the line VIII-VIII in FIG. The scaffold fixture 12 includes a scaffold fixture body 122 in which a through hole 121 in the thickness direction is formed in a disc, and an O-ring 124. The through hole 121 is configured by communicating a large diameter portion 1211 and a small diameter portion 1212, and has a stepped portion 1213. The seeded sheet-like scaffold 123 is fixed to the scaffold fixture 12 by sandwiching the peripheral edge between the stepped part 1213 and the O-ring 124, and constitutes a cell holding surface (culture surface).

Furthermore, a needle through hole 185 is formed in the first side surface portion 112. The needle through-hole 185 can be sealed with a stopper 186 so that an injection needle for seeding cells can be inserted toward the seeding sheet-like scaffold 123 of the scaffold fixture 12 in the sealed space 110. Is formed.

(Injection medium tank 21 and discharge medium tank 23)
The infusion medium tank 21 and the discharge medium tank 23 are made of, for example, deformable synthetic rubber. The infusion medium tank 21 is installed on the substrate 111, and is therefore installed on the same plane as the culture vessel 1.

(Medium injection pipe 22 and culture medium discharge pipe 24)
The culture medium injection pipe 22 and the culture medium discharge pipe 24 are made of, for example, deformable synthetic rubber. The medium injection pipe 22 connects the injection medium tank 21 and the injection port 13 of the culture vessel 1. The medium discharge pipe 24 communicates the discharge medium tank 23 with the discharge port 14 of the culture vessel 1.

9 and 10 are a front view and a longitudinal sectional view of the first check valve 25, respectively. The first check valve 25 has a spherical valve structure. That is, the first check valve 25 has a spherical valve 252 in the valve space 251. The valve space 251 has an inlet 253 and an outlet 254. The inlet 253 has a mortar shape with a gradually increasing diameter. The outlet 254 has a double structure having a main port 2541 and a sub port 2542. The spherical valve 252 has a size that can completely block the inlet 253 and the main port 2541, but cannot block the auxiliary port 2542.

In the first check valve 25, when the medium flows in from the inlet 253, the spherical valve 252 is pushed by the medium and moves to the outlet 254 side. However, since the spherical valve 252 blocks the main port 2541 but does not block the sub port 2542, the culture medium flows through the sub port 2542. Therefore, in the first check valve 25, the flow of the medium from the inlet 253 toward the outlet 254 is ensured. On the other hand, when the culture medium flows in from the outlet 254, the spherical valve 252 is pushed by the culture medium and closes the inlet 253. Therefore, in the first check valve 25, the flow of the medium from the outlet 254 toward the inlet 253 is blocked. Thereby, the 1st check valve 25 exhibits a backflow prevention function.

The second check valve 26 also has the same structure and function as the first check valve 25.

In the first check valve 25, when the weight of the spherical valve 252 is large, a large pressure is required to flow the culture medium from the inlet 253 to the outlet 254. Conversely, when the weight of the spherical valve 252 is small, a small pressure is sufficient. That is, according to the first check valve 25, the medium injection pressure can be changed by changing the weight of the spherical valve 252. Similarly, the discharge pressure of the culture medium can be changed by the second check valve 26. Therefore, according to the

check valves

25 and 26, it is possible to adjust the pressure necessary for exchanging the culture medium.

Furthermore, in the first check valve 25, when the diameter of the auxiliary port 2542 is small, a large pressure is required to flow the medium from the inlet 253 to the outlet 254. Conversely, when the diameter of the auxiliary port 2542 is large, a small pressure is sufficient. That is, according to the first check valve 25, the medium injection pressure can be changed by changing the diameter of the sub-port 2542. Similarly, the discharge pressure of the culture medium can be changed by the second check valve 26. Therefore, according to the

check valves

The first clamp member 27 is provided between the first check valve 25 and the injection port 13 in the medium injection pipe 22. The second clamp member 28 is provided between the second check valve 26 and the discharge port 14 in the culture medium discharge pipe 24. The first clamp member 27 is provided so that the pipe 22 can be closed. The second clamp member 28 is provided so that the pipe 24 can be closed.

(Container drive mechanism 3)
The container drive mechanism 3 is connected to the diaphragm 15. As shown in FIG. 1, the container drive mechanism 3 includes a first motor 31, a rotating member 32, an arm 33, a first connecting member 34, a rod 35, and a rod guide 36.

The first motor 31 is fixed to a vertical plate 112 erected on the substrate 111. The first motor 31 has a horizontal rotation shaft 311 and is capable of on / off pulse control. The rotating member 32 is a disc and is provided so as to rotate in a vertical plane by a horizontal rotating shaft 311. FIG. 11 is a partial view taken along arrow XI in FIG. A plurality (three in this case) of screw holes 321 are formed in the rotating member 32. Each screw hole 321 is separated from the rotation center 322 by a distance P. The distance P is different for each screw hole 321. The arm 33 extends downward from the rotating member 32. The upper end 331 of the arm 33 is detachably connected to any one screw hole 321 by a screw 323. The rod 35 extends vertically downward from the lower end 332 of the arm 33. The upper end 351 of the rod 35 is detachably connected to the lower end 332 of the arm 33 via the first connecting member 34. A lower end 352 of the rod 35 is fixed to the center of the diaphragm 15. The rod guide 36 is a horizontal plate having a through hole 361 in the center, and is supported above the diaphragm 15 by screws 193. The rod guide 36 is disposed so that the rod 35 penetrates the through hole 361 (FIG. 5) in the vertical direction, and thereby guides the movement of the rod 35 in the vertical direction.

In the container drive mechanism 3, when the first motor 31 is operated, the rotating member 32 is rotated together with the horizontal rotating shaft 311, and the arm 33 and the rod 35 are moved up and down. Thereby, the diaphragm 15 vibrates up and down. When the horizontal rotation shaft 311 makes one revolution, the rod 35 reciprocates up and down once, so that the diaphragm 15 vibrates once. When the horizontal rotation shaft 311 rotates continuously, the diaphragm 15 vibrates continuously. When the diaphragm 15 vibrates, the medium is injected and discharged in the sealed space 110, and the medium moves, that is, the medium is replaced.

According to the container drive mechanism 3, the amplitude of the diaphragm 15 can be set arbitrarily. That is, the amplitude of the diaphragm 15 can be adjusted by the position of the screw hole 321 to which the upper end 331 of the arm 33 is connected. That is, when the upper end 331 of the arm 33 is connected to the screw hole 321 having the maximum distance P, the amplitude of the diaphragm 15 becomes maximum, and the upper end 331 of the arm 33 is set to the screw hole 321 having the minimum distance P. Are coupled, the amplitude of the diaphragm 15 is minimized. For example, when the distance P is 4 mm, the amplitude is 8 mm. By the way, when the diaphragm 15 moves downward, the volume of the sealed space 110 decreases correspondingly, and an amount of the medium corresponding to the decreased amount is discharged, and when the diaphragm 15 moves upward, the corresponding amount. The volume of the sealed space 110 increases, and an amount of medium corresponding to the increase is injected. Therefore, the magnitude of the amplitude of the diaphragm 15 corresponds to the amount of medium injected and discharged (movement amount). If the amplitude of the diaphragm 15 is large, the amount of movement of the medium per vibration of the diaphragm 15 is large.

Further, according to the container driving mechanism 3, the rotation speed of the first motor 31 can be arbitrarily set by controlling the on / off pulse of the first motor 31. Therefore, the vibration speed of the diaphragm 15 can be arbitrarily set. That is, the time T1 required for one rotation of the horizontal rotating shaft 311 is the time T1 required for the diaphragm 15 to vibrate once. The time T1 can be adjusted by on / off pulse control of the first motor 31. That is, when the on-pulse is set to be long, the time T1 is shortened, and when the off-pulse is set to be long, the time T1 is lengthened. Time T1 corresponds to the moving speed of the culture medium. That is, when the time T1 is short, the moving speed of the medium is high, and when the time T1 is long, the moving speed of the medium is low.

Furthermore, according to the container drive mechanism 3, the operation time T2 of the first motor 31 can be arbitrarily set by using a timer. Therefore, the driving time T2 of the diaphragm 15 can be arbitrarily set.

(Container drive control unit 105)
The container drive control unit 105 is connected to the first motor 31 of the container drive mechanism 3 so that the rotation speed and the drive time T2 of the first motor 31 can be arbitrarily set.

(Rotation drive mechanism 4)
A rotation driving mechanism 4 is connected to the scaffold fixture 12. The rotational drive mechanism 4 includes a second motor 41, a second connecting member 42, and a second horizontal arm 182.

The second motor 41 is fixed to the vertical plate 112. As shown in FIG. 5, the second motor 41 has a horizontal rotation shaft 411 and is capable of on / off pulse control. The horizontal rotating shaft 411 is detachably connected to the second horizontal arm 182 via the second connecting member 42.

In the rotation drive mechanism 4, when the second motor 41 is operated, the second horizontal arm 182 is rotated together with the horizontal rotation shaft 411, and the scaffold fixture 12 is rotated. When the horizontal rotation shaft 411 makes one rotation, the scaffold fixture 12 also makes one rotation. When the horizontal rotation shaft 411 rotates continuously, the scaffold fixture 12 also rotates continuously. The rotation of the scaffold fixture 12 means that the scaffold 123 rotates.

According to the rotation drive mechanism 4, the rotation speed of the second motor 41 can be arbitrarily set by performing on / off pulse control of the second motor 41. Therefore, the rotation speed of the scaffold fixture 12 can be set arbitrarily. That is, the time S1 required for one rotation of the horizontal rotating shaft 411 is the time S1 required for one rotation of the scaffold fixture 12. The time S1 can be adjusted by on / off pulse control of the second motor 41. That is, when the on-pulse is set longer, the time S1 becomes shorter, and when the off-pulse is set longer, the time S1 becomes longer. Time S1 corresponds to the rotational speed of the scaffold fixture 12. That is, when the time S1 is short, the rotation speed of the scaffold fixture 12 is high, and when the time S1 is long, the rotation speed of the scaffold fixture 12 is low.

Further, according to the rotation drive mechanism 4, the operation time S2 of the second motor 41 can be arbitrarily set by using a timer. Therefore, the driving time S2 of the scaffold fixture 12 can be arbitrarily set.

(Rotation drive control unit 106)
The rotation drive control unit 106 is connected to the second motor 41 of the rotation drive mechanism 4 so that the rotation speed and drive time S2 of the second motor 41 can be arbitrarily set.

(PH measuring device 5)
The pH measuring device 5 is provided between the second check valve 26 and the discharge medium tank 23 in the medium discharge pipe 24. The pH measuring device 5 is provided so that the pH of the medium flowing through the medium discharge pipe 24 can be measured.

(PH measurement control unit 107)
The pH measurement control unit 107 operates the pH measuring device 5, acquires and analyzes measurement data, and can detect the culture state. Usually, when pH is 6.5 or less or 8.5 or more, it is judged that culture | cultivation is in an insufficiency state.

(Photographing mechanism 6)
The culture container 1 is provided with an imaging mechanism 6 for observing the cells of the scaffold fixture 12. FIG. 12 is a perspective view of the culture vessel 1. 13 is a cross-sectional view taken along the line XIII-XIII in FIG. In addition, in FIG. 13, the scaffold fixture 12 rotates and is in a vertical state. The photographing mechanism 6 has a camera 61 and a light source 62. The camera 61 has a function of taking an image of a cell and detecting a light transmission amount of the light source 62. The camera 61 is rotatably provided via a bearing 1134 in a recess 1133 of a sealing member 1132 fitted in a through hole 1131 formed in the center of the second side surface portion 113. Since the sealing member 1132 is colorless and transparent, the camera 61 can photograph the inside of the container body 11. The light source 62 is provided on the fourth side surface 115 and at a position facing the camera 61. A through hole 1151 is formed at the center of the fourth side surface portion 115. The through hole 1151 is airtightly closed from the outside by a colorless and transparent plate member 1152. The plate member 1152 is fixed to the fourth side surface portion 115 by a plate body 1153. The light source 62 is held in the through hole 1154 of the plate body 1153. Since the plate member 1152 is colorless and transparent, the light source 62 can illuminate the inside of the container body 11.

(Shooting control unit 108)
The imaging control unit 108 can operate the camera 61 and the light source 62 to acquire and analyze imaging data and detect the culture state. As the photographing data, for example, a photographed image of a cell, a light transmission amount of the light source 62, and the like are used.

(Operation and effect of culture apparatus 100)
First, an initial setting operation is performed manually. That is, as shown in FIG. 1, since the infusion medium tank 21 is installed on the same plane as the culture container 1, when the weight 219 is placed on the infusion medium tank 21, the infusion medium tank 21 is removed. The medium is pushed out. As a result, the medium is injected through the medium injection pipe 22 to the lower surface of the seeding sheet-like scaffold 123 fixed to the scaffold fixture 12 in the culture vessel 1. Then, the medium injection is temporarily stopped by removing the weight 219.

Next, the cell suspension is seeded on the scaffold 123 by manual work. That is, as shown in FIG. 14, the needle 202 of the syringe 201 filled with the cell suspension to be cultured is inserted into the sealed space 110 from the needle through hole 185, and the cell suspension is placed on the scaffold 123. Eject. As a result, the cell suspension is seeded on the surface (cell holding surface) of the scaffold 123. After seeding the cell suspension, the needle 202 is extracted, and the needle through hole 185 is sealed with a stopper 186 as shown in FIG.

Then, after the cells in the cell suspension have settled, the weight 219 is placed on the infusion medium tank 21 again, and the medium is spread until just before the second check valve 26 of the medium discharge pipe 24. .

Then, the container drive control unit 105 and the rotation drive control unit 106 are operated to operate the container drive mechanism 3 and the rotation drive mechanism 4, and, if necessary, the pH measurement control unit 107 and / or the imaging control unit 108. To operate the photographing mechanism 6 and / or the pH measuring device 5. Thereby, the culture apparatus 100 operates as follows and exhibits a remarkable effect.

(1) When the container driving mechanism 3 operates, the diaphragm 15 vibrates up and down. When the diaphragm 15 moves upward, the culture medium that has spread from the culture medium tank 21 for injection to the culture medium injection pipe 22 is injected into the sealed space 110 from the injection port 13. When the diaphragm 15 moves downward, the medium in the sealed space 110 is discharged from the discharge port 14 to the medium discharge pipe 24. Therefore, when the diaphragm 15 vibrates, the culture medium in the sealed space 110 is gradually replaced. That is, the medium is automatically changed.

(2) Since the culture unit 102 has established a sealed structure, automatic medium exchange is performed without being contaminated by bacteria.

(3) Since the culture unit 102 is provided in the thermostatic chamber, the culture is performed at an optimum temperature.

(4) FIG. 15 shows the moving state of the medium in the culture vessel 1. Since the medium injected from the injection port 13 collides with the rectifying member 16 from below, the momentum is weakened and the medium is evenly distributed in the lateral direction, and moves upward through the gap 162 and stagnates. Further, the amount of the medium injected from the injection port 13 is a constant amount corresponding to the amplitude of the diaphragm 15. Therefore, the injected medium is layered in the sealed space 110. Then, the medium injected one after another gently moves upward while maintaining the layered state. When the medium reaches the height position of the discharge port 14, the discharge port 14 is moved when the diaphragm 15 moves downward. Discharged from. Thereby, the cells of the scaffold 123 are successively exposed to new medium that has moved gently. Thus, the cells can be gently touched with fresh media and cultured without unnecessary stress. Therefore, the culture efficiency is improved.

(5) When the amplitude of the diaphragm 15 is large, the amount of the medium injected from the injection port 13 is large. When the amount of the medium to be injected is large, the contact efficiency between the cells of the scaffold 123 and the fresh medium is improved. Therefore, the culture efficiency is also improved from this point.

(6) When the vibration speed of the diaphragm 15 is high, the moving speed of the medium in the sealed space 110 is high. When the moving speed of the medium is high, the contact efficiency between the cells of the scaffold 123 and the fresh medium is improved. Therefore, the culture efficiency is also improved from this point.

(7) When the driving time of the diaphragm 15 is long, the contact time between the cells of the scaffold 123 and the fresh medium is long. Therefore, the culture efficiency is also improved from this point.

(8) When the rotation drive mechanism 4 is operated, the scaffold fixture 12, that is, the scaffold 123 rotates. When the scaffold 123 rotates, the cell holding surface is positively brought into contact with a fresh medium, and waste products attached to the cell holding surface are positively removed. Therefore, the culture efficiency is also improved from this point.

(9) When the rotation speed of the scaffold fixture 12 is high, the effect of the above (8) is improved.

(10) When the driving time of the scaffold fixture 12 is long, the effect of the above (8) is improved.

(11) Since the first check valve 25 is provided in the medium injection pipe 22 and the second check valve 26 is provided in the medium discharge pipe 24, the injection medium tank 21 and the discharge medium tank 23, no pressure difference is generated. Therefore, the medium is exchanged smoothly.

(12) Since both the first check valve 25 and the second check valve 26 have a spherical valve structure, the medium flows lightly in both the

pipes

22 and 24. Therefore, also from this point, the medium is exchanged smoothly.

(13) When the pH measuring device 5 is activated, the measurement data is acquired and analyzed by the pH measurement control unit 107, and the culture state is detected. Therefore, when the detection result indicates an inadequate culture state, the operator operates the container drive control unit 105 and the rotation drive control unit 106 so that the container drive mechanism 3 and the rotation drive mechanism 4 are cultured. Can be set to conditions that can promote

(14) When the imaging mechanism 6 operates, the imaging control unit 108 acquires and analyzes the imaging data, and detects the culture state. Therefore, when the detection result indicates an inadequate culture state, the operator operates the container drive control unit 105 and the rotation drive control unit 106 so that the container drive mechanism 3 and the rotation drive mechanism 4 are cultured. Can be set to conditions that can promote

(Another usage mode of the culture apparatus 100)
The culture apparatus 100 can be used in the following manner.

(A) As shown in FIG. 16, the culture vessel 1 can be transported alone in a sealed state. 1, the rod 35 and the arm 33 are disconnected by releasing the first connecting member 34, and the horizontal arm 182 and the horizontal rotating shaft 411 are disconnected by releasing the second connecting member 42. When the culture medium injection pipe 22 is cut and closed using the first clamp member 27 and the culture medium discharge pipe 24 is cut and closed using the second clamp member 28, the culture vessel 1 is in the state shown in FIG. It becomes. According to this, the culture vessel 1 can be moved to another place without contaminating the cells being cultured.

(B) As shown in FIG. 17, the culture unit 102 can be transported alone in a sealed state. 1, the rod 35 and the arm 33 are disconnected by releasing the first connecting member 34, and the horizontal arm 182 and the horizontal rotating shaft 411 are disconnected by releasing the second connecting member 42. Then, the culture part 102 will be in the state of FIG. According to this, the culture unit 102 can be moved to another place without contaminating the cells and culture medium being cultured, and the culture can be easily resumed at that place.

(C) As shown in FIG. 18, the culture unit 102 can be transported in a double sealed state by being housed in the thermostatic chamber 103. In FIG. 18, the first motor 31 of the container drive mechanism 3 and the second motor 41 of the rotation drive mechanism 4 are arranged outside the thermostatic chamber 103. In the container drive mechanism 3, the horizontal rotation shaft 311 of the first motor 31 and the rotation member 32 are releasably connected by

magnets

391 and 392 with the wall 1031 of the thermostatic chamber 103 interposed therebetween, and The second connecting member 42 is composed of

magnets

491 and 492, and the horizontal rotating shaft 411 and the horizontal arm 182 of the second motor 41 sandwich the wall 1031 of the thermostatic chamber 103 between them, and the

magnets

491 and 492 , Releasably connected. According to this, the culture unit 102 can be moved to another place without contaminating the cultured cells and medium, and the culture can be easily resumed with little preparation work at that place.

(Modified configuration of the culture apparatus 100)
The culture device 100 having the above configuration can employ the following modified configuration.

(A) FIG. 19 is a longitudinal sectional view showing a modification of the rotation drive mechanism 4. In this rotation drive mechanism 4, the horizontal arm 182 and the horizontal rotation shaft 411 are releasably connected by

magnets

493 and 494. The through hole 1141 of the third side surface portion 114 is closed with a resin plate 1149, and the magnet 493 and the magnet 494 are connected with the resin plate 1149 interposed therebetween. According to this configuration, the second motor 41 can be easily detached from the culture vessel 1.

(B) FIGS. 20 to 22 show modified examples of the scaffold fixture 12. 20 is a perspective view of the scaffold fixing tool 12A, FIG. 21 is a view taken along the arrow XXI in FIG. 20, and FIG. 22 is a sectional view taken along the line XXII-XXII in FIG. The scaffold fixture 12A is configured by stacking the three scaffold fixtures 12 of FIG. 8 through spacers 129 and fixing them with screws 128 that pass through the scaffold fixture body 122. . As a result, the scaffold fixture 12 </ b> A stacks and fixes the three scaffolds 123 at intervals 127. According to this configuration, a large amount of cells can be cultured at the same time at the same time.

When the scaffold fixture 12A is used, the fourth side surface 115 of the culture vessel 1 has three needles corresponding to the three scaffolds (cell holding surfaces) 123 as shown in FIG. A through hole 185 is formed.

(C) FIGS. 24 and 25 show another modification of the scaffold fixture 12. The scaffold fixture 12B is formed of a cylindrical body. FIG. 24 is a front view of the scaffold fixture 12B, and FIG. 25 is a view taken along arrow XXV in FIG. The cylindrical surface 1251 of the cylindrical body 125 is a net plate. Both ends of the cylindrical body 125 are closed with a porous plate 1252 through which the medium can pass. According to this scaffold fixture 12B, it is possible to culture the adherent cells well. That is, the adherent cells are seeded on atelocollagen or the like, filled in the cylindrical body 125 as a culture, and exposed to the culture medium without being adhered to the cylindrical surface 1251 by the rotation of the scaffold fixture 12B. The

(D) FIG. 26 is a longitudinal sectional view showing a modified example in place of the needle through-hole 185. In this example, the culture vessel 1 has a rubber wall portion 1129 in a part of the first side surface portion 112. The rubber wall 1129 allows the injection needle 202 for seeding the cell to penetrate toward the scaffold 123 in the sealed space 110, and closes the penetrated hole after the injection needle is removed. ,It is configured. According to this, since the operation | work of plugging the hole penetrated after extracting the injection needle 202 is unnecessary, workability | operativity can be improved.

(E) FIG. 27 and FIG. 28 are longitudinal sectional views showing modifications of the culture vessel 1. This culture vessel 1 does not have a special configuration (cell through-hole 185, rubber wall 1129, etc.) for seeding cells. In this example, sowing is performed using a sowing jig 80. The seeding jig 80 includes a cylindrical main body 81 and an O-ring 82 provided on the outer peripheral surface of the cylindrical main body 81. The seeding operation using the seeding jig 80 is performed before attaching the upper surface part 116 in the assembly process of the culture vessel 1 in the clean bench. That is, first, as shown in FIG. 27, the seeding jig 80 is positioned above the sealed space 110. Next, as shown in FIG. 28, the seeding jig 80 is inserted into the sealed space 110 until it comes into contact with the scaffold fixture 12. At this time, the airtightness of the sealed space 110 is ensured by the O-ring 82. Next, the solution 99 containing cells is injected into the internal space of the cylindrical body 81 and left to stand. As a result, the cells settle and are seeded on the surface of the seeding sheet-like scaffold 123 of the scaffold fixture 12. Thereafter, the solution is removed, and the seeding jig 80 is taken out from the sealed space 110. The culture vessel 1 has a simple configuration.

(F) FIG. 29 is a front view showing a modification of the culture apparatus 100. In the culture apparatus 100, the first check valve 25 has a general structure, not a spherical valve structure. According to this configuration, since the culture medium injection pipe 22 can be arranged in the lateral direction toward the injection port 13 of the culture vessel 1, the length of the culture medium injection pipe 22 can be reduced. Can be small.

[Second Embodiment]
FIG. 30 is a front view showing a culture device 100A according to the second embodiment of the present invention. This culture apparatus 100A is different from the culture apparatus 100 of the first embodiment in the following points, and is otherwise the same.

(I) Only the dual-purpose medium tank 29 is used as the medium tank. That is, the culture medium injection pipe 22 and the culture medium discharge pipe 24 communicate with the culture vessel 1 and the combined culture medium tank 29.

(Ii) The combined medium tank 29, the medium injection pipe 22, the culture vessel 1, and the medium discharge pipe 24 constitute a culture part 102A, and the culture part 102A has a gas-permeable structure. Specifically, the lid 291 of the combined medium tank 29 and a part or all of the medium injection pipe 22 and the medium discharge pipe 24 are made of a gas permeable material.

(Iii) The culture unit 102A is provided in the carbon dioxide incubator 91A. The inside of the carbon dioxide incubator 91A is maintained in a sterilized state. In the carbon dioxide incubator 91A, the temperature, humidity, oxygen partial pressure, carbon dioxide partial pressure, and nitrogen partial pressure are controlled.

(Iv) An exposure unit 28 is provided in the combined medium tank 29. Specifically, as shown in FIG. 31, the exposure unit 28 includes an umbrella unit 281 connected to the outlet end of the medium discharge pipe 24 in the combined medium tank 29, and is used for the medium. The medium discharged from the discharge pipe 24 is sprayed by the umbrella portion 281.

(V) In the culture apparatus 100A, the culture medium flows from the dual-purpose medium tank 29 to the dual-purpose medium tank 29 via the medium injection pipe 22, the culture container 1, and the medium discharge pipe 24. Yes. That is, the culture apparatus 100A of the present embodiment is a reflux type.

In the culture unit 102A, members that come into contact with the culture medium, specifically, the combined culture medium tank 29, the culture medium injection pipe 22, the culture container 1, the culture medium discharge pipe 24, the first check valve 25, and the second reverse valve. The stop valve 26 is made of a material that does not adversely affect the living body, that is, a biocompatible material. The biocompatible material is appropriately selected from, for example, resin, metal, glass, and the like. Also, these members can be discarded after use.

Further, in the culture apparatus 100A, the mechanism 3A using the linear actuator is used as the container driving mechanism, but the same mechanism 3 as in the first embodiment may be used. The container drive mechanism 3 </ b> A includes a linear actuator 37, a first connecting member 38, and a rod 39. The linear actuator 37 has a vertical telescopic shaft 371 and is capable of on / off pulse control. The rod 39 is detachably connected to the lower end portion of the vertical telescopic shaft 371 via the first connecting member 38, and extends vertically downward from the lower end portion of the vertical telescopic shaft 371 to the diaphragm 15. It is fixed at the center of In the container driving mechanism 3A, when the linear actuator 37 is operated, the vertical telescopic shaft 371 and the rod 39 move up and down, thereby vibrating the diaphragm 15 up and down.

(Operation and effect of culture apparatus 100A)
The operation of the culture apparatus 100A is the same as the operation of the culture apparatus 100 of the first embodiment except for the following points.

That is, part or all of the culture medium injection pipe 22 and the culture medium discharge pipe 24 are made of a gas permeable material, so that when the culture medium flows through both the

pipes

22 and 24, the culture medium is in the carbon dioxide incubator 91A. Be exposed to gas. Further, when the medium is discharged from the medium discharge pipe 24 to the combined medium tank 29, the medium is exposed to the gas entering the combined medium tank 29 from the carbon dioxide incubator 91 </ b> A by the exposure unit 28. Thereby, carbon dioxide gas, oxygen, etc. are taken in into a culture medium. Therefore, the medium can be reused.

In the initial setting operation, the medium is pushed out from the combined medium tank 29 by the pump 85.

(Another usage mode of the culture apparatus 100A)
The culture apparatus 100A can be used in a mode in which the culture unit 102A is accommodated in the carbon dioxide incubator 91A, similarly to the mode shown in FIG.

(Modified configuration of culture apparatus 100A)
The culture device 100A can employ the following modified configuration.

(A) As shown in FIG. 32, the umbrella portion 282 of the exposed portion 28 is supported by the support rod 283 directly below the outlet end portion of the medium discharge pipe 24 in the combined medium tank 29.

(B) The exposure unit 28 is provided outside the combined medium tank 29.

Example 1
The culture apparatus 100 of the first embodiment was used.

(1) Condition (a) The amplitude of the diaphragm 15 was set to 8 mm. As a result, the amount of the medium moved by one vibration of the diaphragm 15 was 2.5 cc.
(B) As the first motor 31, a motor having the ability to rotate 5 times per minute was used.
(C) As the on / off pulse control by the container drive control unit 105, the on pulse time was set to 0.5 seconds and the off pulse time was set to 0.5 seconds. The driving time of the first motor 31 is 2 minutes.

(2) Results Diaphragm 15 was vibrated five times, and 12.5 cc of the medium was replaced.

(Example 2)
The culture apparatus 100 of the first embodiment was used.

(1) Condition (a) Same as Example 1.
(B) Same as Example 1.
(C) As the on / off pulse control by the container drive control unit 105, the on pulse time was set to 0.5 seconds and the off pulse time was set to 20 seconds. The driving time of the first motor 31 was 10 minutes.

(2) Results Diaphragm 15 vibrated about 1.2 times, and about 3 cc of medium was changed.

Since the culture apparatus of the present invention can perform culture while automatically exchanging the medium, it has great industrial utility value.

Claims

In a culture apparatus equipped with a culture vessel for culturing cells with a medium,
The culture container
A container body having a sealed space;
A scaffold for holding the cells in an enclosed space;
A scaffold fixture for securing the scaffold,
Provided in the center of the bottom surface of the container body, and an inlet for injecting the medium into the sealed space;
Provided in the upper part of the container body, and a discharge port for discharging the medium from the sealed space;
A diaphragm that is provided on the upper surface of the container body and pressurizes and depressurizes the sealed space by vibrating up and down; and
A rectifying member that is provided above the inlet and for flowing the injected medium toward the peripheral edge in the sealed space;
With
When the diaphragm moves upward, the sealed space depressurizes the sealed space, thereby allowing the medium to be injected from the inlet, and when the diaphragm moves downward, the sealed space is pressurized and thereby discharged. The medium can be discharged from the
A culture apparatus.
An infusion medium tank for containing a medium used for culture;
An infusion pipe for the medium connecting the infusion medium tank and the inlet of the culture container;
A discharge medium tank for containing the medium discharged from the culture container;
A medium discharge pipe that communicates the medium tank for discharge and the discharge port of the culture container;
Is further provided,
The medium injection pipe is provided with a first check valve,
The medium discharge pipe is provided with a second check valve,
The infusion medium tank, the medium infusion pipe, the culture container, the medium discharge pipe, and the discharge medium tank constitute the culture part, and the culture part has established a sealed structure,
When the diaphragm vibrates up and down, the culture unit is configured so that the culture medium flows from the culture medium tank for injection to the culture medium tank for discharge via the medium injection pipe, the culture container, and the medium discharge pipe.
The culture apparatus according to claim 1.
A combined medium tank for containing a medium used for culture;
An infusion pipe for medium that communicates between the combined medium tank and the inlet of the culture container;
A culture medium discharge pipe connecting the combined medium tank and the discharge port of the culture container;
Is further provided,
The medium injection pipe is provided with a first check valve,
The medium discharge pipe is provided with a second check valve,
The combined medium tank, the medium injection pipe, the culture vessel, and the medium discharge pipe constitute a culture part, and the culture part has a gas-permeable structure and is provided in the carbon dioxide incubator. And
An exposure unit that exposes the medium that has passed through the medium discharge pipe to the gas in the carbon dioxide incubator;
When the diaphragm vibrates up and down in the culture part, the culture medium flows from the dual-purpose medium tank to the dual-purpose medium tank via the medium injection pipe, the culture container, the medium discharge pipe, and the exposure part.
The culture apparatus according to claim 1.
A container drive mechanism that vibrates the diaphragm up and down at an arbitrary amplitude, at an arbitrary vibration speed, and for an arbitrary drive time;
A container drive control unit for controlling a vibration speed and a drive time in the container drive mechanism;
Further comprising
The culture apparatus according to claim 2 or 3.
A rotation drive mechanism for rotating the scaffold fixture around the horizontal axis at an arbitrary rotational speed and for an arbitrary drive time;
A rotation drive control unit for controlling a rotation speed and a drive time in the rotation drive mechanism;
Further comprising
The culture apparatus according to claim 2 or 3.
A pH measuring device for measuring the pH of the medium flowing through the medium discharge pipe;
A pH measurement control unit that operates a pH measuring device, acquires and analyzes measurement data, and detects a culture state;
Further comprising
The culture apparatus according to claim 2 or 3.
An imaging mechanism for observing the cells of the scaffold in the culture vessel;
An imaging control unit that activates the imaging mechanism, acquires and analyzes the imaging data, and detects the state of the culture;
Further comprising
The culture apparatus according to claim 2 or 3.
The infusion medium tank is installed on the same plane as the culture container,
The culture apparatus according to claim 2.
One or both of the first check valve and the second check valve have a spherical valve structure.
The culture apparatus according to claim 2 or 3.
A first clamping member capable of closing the pipe is provided in the medium injection pipe,
The culture medium discharge pipe is provided with a second clamp member capable of closing the pipe.
The culture apparatus according to claim 2 or 3.
The container drive mechanism has a first connection member that can release the connection with the culture container.
The culture apparatus according to claim 4.
The rotation drive mechanism has a second connecting member that can release the connection with the culture vessel.
The culture apparatus according to claim 5.
The container drive mechanism includes a first motor, a rotating member, an arm, a first connecting member, a rod, and a rod guide.
The first motor has a horizontal rotation axis and is capable of on / off pulse control.
The rotating member is provided so as to rotate in a vertical plane by a horizontal rotating shaft,
The arm is detachably connected to a position separated by an arbitrary distance from the rotation center of the rotating member, and extends downward.
The rod is detachably connected to the lower end of the arm via the first connecting member, and extends vertically downward from the lower end of the arm and is fixed to the center of the diaphragm.
The rod guide is provided to guide the vertical movement of the rod,
When the first motor is actuated, the container driving mechanism rotates the rotating member together with the horizontal rotation shaft, and the arm and the rod move up and down, thereby vibrating the diaphragm up and down.
The culture apparatus according to claim 4.
The horizontal rotation shaft of the first motor and the rotation member are detachably connected.
The culture apparatus according to claim 13.
The container drive mechanism includes a linear actuator, a first connecting member, and a rod;
The linear actuator has a vertical telescopic axis and is capable of on / off pulse control,
The rod is detachably connected to the lower end of the vertical telescopic shaft via the first connecting member, and extends vertically downward from the lower end of the vertical telescopic shaft and is fixed to the center of the diaphragm. And
When the linear actuator is actuated, the container drive mechanism moves the vertical telescopic shaft and rod up and down, thereby vibrating the diaphragm up and down.
The culture apparatus according to claim 4.
The rotational drive mechanism includes a second motor, a second connecting member, and a horizontal arm,
The second motor has a horizontal rotation axis and is capable of on / off pulse control.
The horizontal arm extends through the side surface of the container body,
One end of the horizontal arm is fixed to the scaffold fixture, and the other end of the horizontal arm is detachably connected to the horizontal rotation shaft of the second motor via the second connecting member,
When the second motor is actuated, the rotation drive mechanism rotates the horizontal rotation shaft and the horizontal arm, thereby rotating the scaffold fixture.
The culture apparatus according to claim 5.
The second connecting member has a magnet connecting structure;
The culture apparatus according to claim 16.
The imaging mechanism has a camera and a light source that illuminates the cells of the scaffold,
The camera is installed inside the culture container, or installed outside the culture container so that it can be photographed through the observation window of the culture container.
The culture apparatus according to claim 7.
Scaffolding fixtures are fixed by stacking multiple scaffolds at intervals.
The culture apparatus according to claim 1.
The scaffold fixture is composed of a cylindrical body that can be filled with a culture seeded with cells, and the cylindrical surface of the cylindrical body is composed of a mesh plate.
The culture apparatus according to claim 1.
The culture container has a needle through-hole on the side surface,
The needle through-hole is formed so that an injection needle for seeding cells can be inserted toward the scaffold in the sealed space and can be sealed with a stopper.
The culture apparatus according to claim 1.
The culture container has a rubber wall part in a part of the side part,
The rubber wall portion is configured so that the injection needle for seeding cells can be penetrated toward the scaffold in the sealed space, and the through-hole is closed after the injection needle is removed. Yes,
The culture apparatus according to claim 1.