JP2006141325A - Culturing device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culturing device without requiring the selection of an installing place on culturing over several days to several months, without having problems such as dust-forming, vibration, noise, etc., and capable of operating working parts such as an air cylinder, etc., even in the time of power failure. <P>SOLUTION: The gas concentration of the gas fed from a gas cylinder such as a carbon dioxide gas cylinder in a temperature-keeping box means arranged with a culturing container means, is adjusted by a gas concentration-adjusting means. In such the culturing, the use of carbon dioxide gas, etc., is essential. Therefore, the gas pressure of the gas cylinder is used as a power source so as to drive the working parts such as an air cylinder, etc., used for a medicine-feeding means or waste liquid-discharging means. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a culture apparatus for culturing cells, and more particularly, to a culture apparatus capable of automatically performing operations associated with culture for several days to several months.

Cultivation is performed manually by complicated passage processes such as replacement of the medium in the incubator and re-seeding to make the cell density appropriate. Normally, these operations are carefully performed in a relatively clean atmosphere in which the concentration of suspended particulates in the atmosphere is suppressed by a clean environment generation technology cultivated in the semiconductor manufacturing field in order to avoid the occurrence of contamination and the like. However, even in this clean atmosphere, it is not enough to avoid contamination. When culturing cells in a circular petri dish usually used as an incubator, hold the petri dish lid upward to change the medium. While paying attention not to mix bacteria, it was necessary to perform complicated and difficult operations such as quickly inserting the pipetter into the gap between the petri dish and its lid and not touching the surroundings such as the edge of the petri dish. Such work is frequently and routinely performed and is usually performed by a very skilled worker.
USP 5,985,653

  Conventionally, a culture apparatus has been provided with many air cylinders, and the pressure for driving the air cylinder is performed by taking compressed air from a compression cylinder provided outside the culture apparatus, or The compressed air was acquired by operating an air compressor built in the culture apparatus.

  Thus, when taking in compressed air from an external compression cylinder, piping for letting compressed air pass from a compressed air generation source (compression cylinder) to a culture device is required. Usually, such a pipe is fixed to a wall, a ceiling or the like, so that the installation location of the culture apparatus is limited or sometimes cannot be installed. In addition, when an air compressor is installed in the culture apparatus, there is no restriction on the installation place as described above, but there are problems such as dust generation, vibration, and noise caused by installing the air compressor in the apparatus. there were. Further, since the air compressor does not operate at the time of a power failure, there is a problem that it is impossible to generate pressure.

  The object of the present invention is to operate an operating part such as an air cylinder without a problem of dust generation, vibration, noise, etc., even in the event of a power failure, without selecting an installation location during culture for several days to several months. It is in providing the culture apparatus which can be performed.

The first feature of the culturing apparatus of the present invention is the incubator means for culturing cells, the incubator means for culturing inside, the outer box means for setting the incubator means in a state suitable for culturing, Chemical supply means for supplying unused chemicals from outside the outer box means to the incubator means in the outer box means, and unnecessary waste liquid etc. from the incubator means in the outer box means. A culture apparatus comprising: a waste liquid discharging means for discharging to the outside; and a gas concentration adjusting means for adjusting a gas concentration in the outer box means by supplying a gas from a gas cylinder into the outer box means. And opening / closing means for opening and closing at least one of the discharge or supply paths of the chemical supply means and the waste liquid discharge means.
The second feature of the culture apparatus of the present invention is that the path is a tube and the opening / closing means is a pinch valve.
In the outer box means in which the incubator means is arranged, the gas concentration supplied from a gas cylinder such as a carbon dioxide cylinder is adjusted by the gas concentration adjusting means. Thus, in the culture, use of carbon dioxide gas or the like is essential. In view of this, the gas cylinder gas pressure is used as a power source to drive an operating portion such as an air cylinder used in the chemical supply means and the waste liquid discharge means.

  According to the present invention, it is possible to operate an operating part such as an air cylinder without a problem of dust generation, vibration, noise, etc. without selecting a place for installation during culture for several days to several months, even during a power failure. it can.

  Hereinafter, embodiments of a culture apparatus to which the present invention is applied will be described with reference to the drawings. FIG. 1 is a block diagram showing a basic configuration of a culture apparatus to which the present invention is applied.

  The incubator 1 is a container for culturing cells, and is connected to a reserve tank 4 into which unused chemicals are injected via a pump 3 and a flexible tube member 2. The waste liquid tank 7 stores used chemicals, and is connected to the incubator 1 through the pump 6 and the flexible tube member 5. The driving means 8 rotates the incubator 1. The camera 9 observes the cultured cells inside by the light emitted from the light source 10 that has passed through the incubator 1. The system controller 11 is connected to the pump 3, the pump 6, the driving unit 8, the camera 9, and the light source 10, and controls the pump 3, the pump 6, the driving unit 8, the camera 9, and the light source 10.

  FIG. 2 is a detailed view of the mechanism part of the culture apparatus to which the present invention is applied, and shows an actual configuration in which the system controller 11 in FIG. 1 is omitted. The incubator 38 is preferably made of a transparent non-toxic material, preferably polystyrene or polyethylene terephthalate. A gas permeable membrane 16 is attached to the surface of the main body 15 of the incubator 38. The surface of the incubator 38 is preferably modified so as to have hydrophilicity so that cells can be easily attached. A tube connecting member 19 for injecting chemicals is provided in the approximate center of the incubator 38 and functions to flow a chemical such as the medium 17 into the incubator 38. At this time, the inclined portion 381 softens the impact of various liquids falling to prevent the cultured cells from being damaged.

  Cells adhere to the bottom of the incubator 38 and culture is performed there. The tube connecting member 18 is a discharge port for discharging the old culture medium in which the waste products of the cells are eluted and the nutrients in the culture medium are reduced. The incubator 38 is fixed on the rotor 22, and the rotor 22 is freely supported at, for example, three places in the circumferential direction so that the cam follower 27 can rotate in the direction of arrow E below. Further, an internal gear (not shown) is formed below the rotor 22, and this gear meshes with a pinion 28 fitted to an output shaft of an incubator drive motor 29 fixed to a heat insulating box (frame) 30. The cable drum 25 winds the wiring of the pinch valve 24 provided on the rotor 22. The winding drum 26 winds up the wiring of the pinch valve 24 even when the rotor 22 rotates, and the wiring is automatically wound up so as not to get entangled with other protrusions by loosening the wiring. For example, it can be realized by constantly applying tension to the cable using a spring.

  The supply tube 21 is connected to a tube connection member 19 provided in the approximate center of the incubator 38. The guide member 35 guides the supply tube 21. The supply tube 21 is fixed to the frame 30 by a tube fixing member 36 provided above the guide member 35, and the tube from the tube fixing member 36 to the tube connecting member 19 passes through the inside of the guide member 35. It is designed to move freely.

  The culture medium tank 67 stores an unused culture medium, the buffer solution tank 68 stores a buffer solution, and the cell release agent tanks 69, 70, and 71 store cell release agents. Each tank 67, 68, 69, 70, 71 is provided in the heat insulation box 80. The pinch valves 72, 105, 73, 74, and 75 control liquid feeding from the tanks 67, 68, 69, 70, and 71. The pinch valve 66 controls injection of pre-culture cells to be described later. The air inlets 78 and 79 are for introducing air in the atmosphere to prevent liquid accumulation in the tube, and are filters for removing impurities in the atmosphere (the eye size is preferably 0.2 μm or less. ). The tubes taken out from the tanks 67, 68, 69, 70, 71 are connected to the supply tube 21 described above and can be fed by the ironing pump 37. The squeeze pump 37 is a pump that feeds the liquid in the tube by sandwiching the tube with a roller and rotating the roller.

  The waste liquid tube 23 is connected to a tube connecting member 18 provided on the bottom surface of the incubator 38 and guided outside the frame 30 by a guide member 99. A tube fixing member 100 is provided below the guide member 99. The waste liquid tube 23 is fixed by the tube fixing member 100, and the waste liquid tube 23 is interposed between the tube fixing member 100 and the tube connecting member 18. Is designed to move freely. The old culture medium produced by elution of cell waste products and a decrease in nutrients in the culture medium is stored in the waste liquid tank 102 in the waste liquid collection box 98 through the waste liquid tube 23 by the peristaltic pump 101. The pinch valve 103 controls the liquid feeding to the waste liquid tank 102, and the pinch valve 104 controls the liquid feeding state when the waste liquid is fed to the waste liquid tank 102 by the ironing pump 101.

  The shutter motor 50 opens and closes an opening provided on the right side surface of the frame 30 with a shutter 51, and a wire connected to the shutter 51 is wound around a rotating shaft thereof. By controlling the rotation of the shutter motor 50, the shutter 51 can be moved in the direction of arrow A (vertical direction in the drawing). A container 52 that stores cells before culture is supported by the holder 62. The holder 62 can be moved in the arrow B direction (left and right direction in the drawing) by a motor 63 having a feed screw. A rubber material is provided on the upper surface of the container 52 and is covered with outside air (not shown). The needle 53 is connected to the cell injection tube 56 and is fixed to the pipettor arm 55. The pipetter arm 55 is supported by a shaft 54 and can be rotated by a pipetter rotation motor 57 in the direction of arrow D1. The rotating member 58 is a member that rotates together with the shaft 54, and includes a pipettor vertical movement motor 59 and a pulley 60. A pulley fixed to the output shaft of the pipettor vertical movement motor 59 and the pulley 60 are connected by a belt 61, and a part of the belt 61 is fixed to the shaft 54.

  The shaft 54 moves up and down by driving the pipettor vertical movement motor 59. The pinch valve 66 controls the liquid feeding state when feeding the pre-culture cells with the ironing pump 37. A needle 39 is fixed to the pipettor arm 55, and an air filter 40 is provided at one end thereof. The function of the needle 39 is to prevent the inside from becoming a printing pressure and difficult to suck cells when the container 52 is formed of a hard plastic material. In addition, although the cell before culture | cultivation demonstrated that it was attracted | sucked by the ironing pump 37, you may make it liquid-feed by pumping air from the needle | hook 39 to the container 52. FIG.

  The shutter motor 81 opens and closes an opening provided on the left side surface of the frame 30 with a shutter 82, and a wire connected to the shutter 82 is wound around a rotating shaft thereof. By controlling the rotation of the shutter motor 81, the shutter 82 can be moved in the direction of arrow F (the vertical direction in the drawing). A container 84 for storing cells after culture is supported by the holder portion 93. The holder 93 can be moved in the arrow G direction (left and right direction in the drawing) by a motor 94 having a feed screw 95. A rubber material is provided on the upper surface of the container 84 and is covered from the outside (not shown). The needle 83 is connected to the cell injection tube 84 and is fixed to the pipetter arm 85. The pipettor arm 85 is supported by a shaft 87 and can be rotated in the direction of the arrow D2 by a pipetter rotation motor 88. The rotating member 89 is a member that rotates together with the shaft 87, and includes a pipettor vertical movement motor 90 and a pulley 91. A pulley fixed to the output shaft of the pipetter vertical movement motor 90 and the pulley 91 are connected by a belt 92, and a part of the belt 92 is fixed to the shaft 87.

  The shaft 87 is moved up and down by driving the pipettor up-and-down motor 90. The pinch valve 103 controls the liquid feeding state when feeding cells after culturing with the ironing pump 101. A needle 41 is fixed to the pipettor arm 55, and an air filter 42 is provided at one end. The function of this needle 41 is to prevent the inside from becoming a positive pressure and making it difficult for cells to be discharged when the container 84 is formed of a hard plastic material. In addition, although the cell after culture | cultivation demonstrated that it liquid-fed with the ironing pump 101, you may make it liquid-feed by pumping air to the incubator 38. FIG.

  The light source 34 supplies light into the frame 30 from the lower side of the frame 30 and includes a filter 33 on the light emission side. The CCD camera 31 includes a lens and is used for observing cells cultured in the incubator 38 from an observation window 32 provided on the upper side of the frame 30 and determining timing at the time of passage. It is what is done. The light source 34 is preferably of a type in which a plurality of LEDs are arranged flat in order to prevent unevenness in the brightness of the image, but may be composed of a single LED or lamp if the amount of light is sufficient. The filter 33 includes an ND filter for reducing the amount of light incident on the CCD camera 31 and an appropriate bandpass filter for obtaining a contrast suitable for cell observation. This filter may be provided in front of the CCD camera 31. The ND filter is preferably on the front surface of the CCD camera 31, and the band-pass filter is preferably on the front surface of the light source 34 when it cuts short-wavelength light that is harmful to cells. The heater 108 keeps the inside of the frame 30 at a constant temperature based on the detected temperature from the temperature sensor 106. The fan 65 agitates the air in the frame 30. The stands 96 and 97 stand the entire culture apparatus upright on the floor. The joint 107 includes a filter for removing impurities when supplying a mixed gas in which the ratio of carbon dioxide, nitrogen, and oxygen is controlled.

  Although the gas permeable membrane 16 attached to the upper surface of the incubator 38 is illustrated so as to cover the entire surface, it may be partially provided. Needless to say, it is better to increase the humidity inside the frame 30 in order to prevent evaporation of the culture medium. In this case, it is easy and effective to arrange a tray containing water therein. When the front surface of the incubator 38 is not covered with the gas permeable membrane 16, the mixed gas supplied from the joint 107 may be directly supplied to the inside of the incubator 38, or may be dissolved in a medium or the like. good.

  In addition, the frame 30 has a shape that substantially covers the whole, but may have a structure that covers only the peripheral portion of the incubator 38. That is, the case where the two sets of pipettors are provided on the left and right as a part of the frame 30 has been described, but the two sets of pipetters may be arranged outside the frame 30 by being configured separately from the frame 30. .

  FIG. 3 is a diagram showing a detailed configuration of the incubator 38 of FIG. 3A is a plan view of the incubator 38 of FIG. 2 as viewed from above, and FIG. 3B is a side sectional view thereof. In FIG. 3A, the tube connecting member 19 is provided in the vicinity of the rotation center at a position separated from the circle center of the incubator 38 by a distance L1. The positions and shapes of the tube connecting member 18 and the weir 20 for discharging the old culture medium may be as in the modified examples 112, 113, and 114. In the modified example 112, the weir 20 is omitted, in the modified example 113, the tube connecting member 18 is provided on the side surface of the incubator 38, and in the modified example 114, the opening of the tube connecting member 18 is provided. Is provided in contact with the bottom surface of the incubator 38.

  In the modified examples 112 and 114, the cells may aggregate in a hollow portion other than the opening of the tube connecting member 18 due to the centrifugal force accompanying the rotation of the incubator 38. Since the cells can be discharged out of the incubator 38, it is more preferable. The position of the tube connecting member 18 may be arranged anywhere in the incubator 38 and is not particularly limited. Further, the distance L1 is not particularly limited. However, it is preferable that the circle center and the rotation center of the incubator 38 are shifted from the viewpoint of uniform seeding of cells. Cell aggregation can also be avoided by placing the tube connecting member 18 near the center of the circle of the incubator 38, for example.

  Here, the rotation includes at least one of rotation, eccentric rotation, parallel movement, and reciprocal parallel movement and a combination thereof, and is an operation particularly useful for stirring and homogenizing cells and liquids. For example, the culture medium or the neutralized cell release agent may be discharged from the incubator by tilting the incubator. In addition, for uniform seeding of cells in the incubator, the incubator may be vibrated. In manual culture, uniform seeding can be achieved by operating the incubator to draw an 8-shaped locus. As described above, the rotation operation is the simplest and easy to configure, but it is not necessary to limit the rotation operation, and various translation operations or a combination of rotation and translation operations may be used.

  FIG. 4 is a block diagram showing the details of the control block diagram of the culture apparatus of FIG. 2 and connecting a plurality of culture apparatuses and planting them. The culture apparatus of FIG. 2 is indicated by a large block 127 in FIG. Each pinch valve 24, 66, 72, 73, 74, 75, 76, 77, 103, 104, temperature sensor 106, heater 108, fan 65, squeezing pumps 37, 101, container movement motors 94, 63, incubator drive motor 29, the pipetter vertical movement motor 59, the pipetter rotation motor 57, the pipetter vertical movement motor 90, the pipetter rotation motor 88, the shutter motors 50 and 81, etc. are connected to the bus 121 via the I / O 120, respectively. The CCD camera 31 is connected to the bus 121 via the image capturing board 250 and the I / O 120. A CPU 122, an operation console 123, an operation device 126, a memory 124, and a computer network driver 125 are connected to the bus 121.

  In FIG. 4, a computer network is provided outside, and a culture device 127 and a plurality of other culture devices 128 and 129 are connected to this computer network, and a control and monitoring device connected to this computer network. The plurality of culture apparatuses 127, 128, and 129 are monitored and controlled by 130 from each remote place. The control monitoring device 130 can be supported by a general-purpose personal computer. In the case of a computer network, there is no particular limitation as long as it is a bidirectional data communication means. For the purpose of simply recognizing the state of the culture apparatus from a remote location, one-way data communication means may be used. The number of culture apparatuses connected to this computer network is not particularly limited. When using multiple culture devices, it is always possible to remotely monitor the state of each culture device during a culture period that normally takes a long time of several weeks by connecting with data communication means. Is preferred.

  The function of the control monitoring device 130 is a function that sequentially monitors the operation of the culture device described in FIG. 5 and emits a signal to the outside in the event of an abnormality. Although the monitoring function is omitted, each culture device may be responsible for the monitoring function, or the control monitoring device 130 may undertake the monitoring function. For example, the control and monitoring device 130 can confirm the operation of each culture device in a time-sharing manner, and the method of informing the culture device to the outside when each cell culture device malfunctions is the easiest.

  FIG. 5 is a flowchart for explaining the operation of the culture apparatus. Hereinafter, the operation of the culture apparatus will be described with reference to FIGS. Since the CPU 122, the memory 124, and the bus 121 shown in FIG. 4 are techniques used in general-purpose computers, detailed operations of the CPU 122, the memory 124, and the bus 121 are omitted, and only the operation of each actuator will be described below.

Step S51: “Start”
This is a process of starting the operation of the culture apparatus 127 and starting when the operator presses the start switch of the operation device 126 of the console 123. In addition, as shown in FIG. 4 described above, when a plurality of culture apparatuses and a control monitoring apparatus are connected to a computer network, the start switch may be pushed on the control monitoring apparatus side. At this time, the chemical is already set in the culture apparatus 127 in the incubator 38 and each of the tanks 67, 68, 69, 70, 71.

Step S52: “Medium injection”
The pinch valve 72 is opened, the squeeze pump 37 is operated, and the medium in the medium tank 67 is fed through the tube 21. The medium to be fed flows through the path indicated by arrows J1 and J into the incubator 38, and becomes the medium 17 in the incubator 38. When a preset amount of medium is injected, the operation of the squeeze pump 37 is stopped and the pinch valve 72 is closed. The set value of the medium amount here is stored in the memory 124 in advance. In this embodiment, the pump 101 which will be described later is also the same, but the liquid charge is determined in the operation time of the pump without providing a means for liquid charge.

Step S53: “Injecting the container 52”
When the operator operates the corresponding switch of the operating device, the shutter motor 50 operates and the shutter 51 slides in the arrow A direction (upward direction). After the shutter 51 is raised by a predetermined amount, the container moving motor 63 operates and the holder moves in the arrow B direction (right direction). After this movement, the operator places the container 52 containing the pre-culture cells in the holder 62. Thereafter, the container moving motor 63 rotates in the opposite direction to the above, and the holder 62 moves in the direction of arrow B (leftward). The shutter motor 50 rotates, and the shutter 51 moves in the arrow A direction (downward direction) and closes.

Step S54: “Driving the pipetter and transferring the cells to the incubator 38”
The container moving motor 63 rotates forward and backward in small increments to suspend the cells in the container 52. Although details will not be described, an actuator for vibrating the container 52 may be provided inside the holder 62. Before and after this operation, the pipetter rotation motor 57 operates and the pipetter arm 55 rotates. Next, the pipettor vertical movement motor 59 is operated, the pipetter arm 55 is lowered, and the needle 53 is inserted into the container 52. The pinch valve 66 is opened and the ironing pump 37 is operated. Thereby, the pre-culture cells in the container 52 are sucked out, and the cells are fed through the tube 56 in the direction of the arrow J2 → J, passed through the tube 21, and injected into the incubator 38. After this injection is completed, the pinch valve 66 is closed and the ironing pump 37 is stopped.

Step S55: “Shuffling the incubator, homogenizing and sowing”
The motor 28 rotates and the cells injected into the incubator 38 are suspended for homogenization and seeding. The homogenization and seeding of cells is a treatment necessary for efficient culture because excessive cell density may lead to cell alteration. In addition, when the injection of cells before culture is confirmed, the motor 28 does not start rotating, but the cultured cells are adhesion-dependent cells (cells that are cultured by recognizing and adhering to a solid) It is better to rotate the motor 28 while the pre-culture cells are being injected into the incubator 38. Note that step S55 may be followed by step S56 or jump to step S58. Here, the case of jumping to step S58 will be described.

Step S58: “Culture”
In this step, the pre-culture cells enter the culture. During the culture, the temperature sensor 106 and the heater 108 control the inside of the frame 30 to a temperature suitable for the culture (around 37 ° C.). As a result, the atmosphere inside the frame 30 is also agitated so that there is no temperature unevenness.

Step S56: “Drain medium”
This step is a step that can be appropriately executed before step S58 is executed. The pinch valve 24 and the pinch valve 104 are opened, the squeeze pump 101 is operated, and the medium 17 in the incubator 38 moves the tube 23. Then, the medium is sent (discharged) to the waste liquid tank 102. After the liquid feeding (discharge) is completed, the ironing pump 101 is stopped, and the pinch valve 24 and the pinch valve 104 are closed.

Step S57: “Injecting new medium”
This step is also a step that can be appropriately executed before executing Step 6. The pinch valve 72 is opened, the peristaltic pump 37 is operated, and a new medium is injected into the incubator 38. After the injection of this medium, the pinch valve 72 is closed and the squeezing pump 37 is stopped.

Step S59: “Passing timing?”
During the above culture, the time may be determined in advance, or a switch may be provided on the console to instruct the operator to operate, but cell quality can be stabilized by using images as follows. Contributes to That is, the light source 34 appropriately emits light, and the CCD camera 31 acquires an image of cells cultured in the incubator 38. Cells in the initial stage of culture are very low in density at many locations and often form a partially dense state (colony). The colony is captured and measured by the CCD camera 31 by the operation of the incubator drive motor 28. If the cells of this colony part do not reach confluence, they are subsequently cultured. The determination of whether or not the cell is confluent may be the same as the cell count in step S60 described later. If necessary at that time, the process proceeds to step S58 after performing steps S56 and 57. Moreover, if it has reached confluence, it will be a subculture timing, and it progresses to the following step S60.

Step S60: “Is the target number of cells?”
The number of cells is counted or calculated based on information from the CCD camera 31. As a result, if the number of cells has reached a value preset by the operator, the process proceeds to step S68, and if not, the process proceeds to step S61.

Step S61: “Drain medium”
The process of step S61 to step S67 is a process executed when the number of cells has not reached the value set by the operator in advance. First, in this step, the pinch valve 24 and the pinch valve 104 are opened, the squeeze pump 101 operates, the medium 17 in the incubator 38 passes through the tube 23, and the medium is sent (discharged) to the waste liquid tank 102. Is done. After the liquid feeding (discharge) is completed, the ironing pump 101 is stopped, and the pinch valve 24 and the pinch valve 104 are closed.

Step S62: “Wash incubator with buffer”
The pinch valve 105 is opened, the squeezing pump 37 is operated, and the buffer solution is injected from the buffer solution tank 68 into the incubator 38. After the injection, the pinch valve 105 is closed and the ironing pump 37 is stopped. The incubator drive motor 28 rotates to rotate the incubator 38 to spread the buffer solution to the bottom of the incubator. Thereafter, the pinch valve 24 is opened and the squeeze pump 101 is operated to send the buffer solution in the incubator 38 to the waste liquid tank 102.

Step S63: “Injecting cell peeling agent”
The pinch valve 73 is opened, the squeezing pump 37 is operated, and the cell release agent is injected from the cell release agent tank 69 into the incubator 38. After the injection, the pinch valve 73 is closed and the ironing pump 37 is stopped. The incubator drive motor 28 rotates to spread the cell detachment agent to the bottom of the incubator.

Step S64: “Injecting neutralizing agent”
Here, the neutralizing agent is used as a medium. Although various things are utilized as the above-mentioned cell detachment agent, the cell detachment agent neutralized by this serum is assumed here, assuming that the medium contains serum. Therefore, the operation is the same as in step S52 described above, and the pinch valve 74 is opened and the neutralizing agent is injected from the tank 70.

Step S65: “Shuffling the incubator, homogenizing and sowing”
The same process as step S55 is performed. That is, the motor 28 rotates and the cells injected into the incubator 38 are suspended for homogenization and seeding. Then, after a predetermined time has elapsed, that is, after the cells adhere, the process proceeds to the next step S66.

Step S66: “Discharging the neutralized cell detaching agent”
The pinch valve 24 and the pinch valve 104 are opened, and the squeeze pump 101 feeds (discharges) the culture medium to the movement 102. After the liquid feeding (discharge) is completed, the ironing pump 101 is stopped, and the pinch valve 24 and the pinch valve 104 are closed.

Step S67: “New medium injection”
The same process as step S52 is performed. That is, the pinch valve 72 is opened, the ironing pump 37 is operated, and the medium in the medium tank 67 is fed through the tube 21. The medium to be fed flows through the path indicated by arrows J1 and J into the incubator 38, and becomes the medium 17 in the incubator 38. When a preset amount of medium is injected, the operation of the squeeze pump 37 is stopped and the pinch valve 72 is closed.

  The processes in steps S68 to S71 are executed when the number of cells has reached a value set in advance by the operator, and is the same as the processes in steps S61 to S64 described above.

Step S68: “Drain medium”
The same process as step S61 is performed. That is, the pinch valve 24 and the pinch valve 104 are opened, the squeezing pump 101 operates, the medium 17 in the incubator 38 passes through the tube 23, and the medium is sent (discharged) to the waste liquid tank 102. After the liquid feeding (discharge) is completed, the ironing pump 101 is stopped, and the pinch valve 24 and the pinch valve 104 are closed.

Step S69: “Wash the incubator with buffer”
The same process as step S62 is performed. That is, the pinch valve 105 is opened, the squeeze pump 37 is operated, and the buffer solution is injected from the buffer solution tank 68 into the incubator 38. After the injection, the pinch valve 105 is closed and the ironing pump 37 is stopped. The incubator drive motor 28 rotates to rotate the incubator 38 to spread the buffer solution to the bottom of the incubator. Thereafter, the pinch valve 24 is opened and the squeeze pump 101 is operated to send the buffer solution in the incubator 38 to the waste liquid tank 102.

Step S70: “Injecting cell peeling agent”
The same process as step S63 is performed. That is, the pinch valve 73 is opened, the squeeze pump 37 is operated, and the cell release agent is injected from the cell release agent tank 69 into the incubator 38. After the injection, the pinch valve 73 is closed and the ironing pump 37 is stopped. The incubator drive motor 28 rotates to spread the cell detachment agent to the bottom of the incubator.

Step S71: “Injecting neutralizing agent”
The same process as step S64 is performed. That is, the pinch valve 74 is opened and the neutralizing agent is injected from the tank 70. Then, after a predetermined time has elapsed, that is, after the cells adhere, the process proceeds to the next step S72.

Step S72: “Draining neutralized cell detachment agent”
The same processing as in step 66 is performed. That is, the pinch valve 24 and the pinch valve 104 are opened, and the medium is fed (discharged) to the motion 102 by the ironing pump 101. After the liquid feeding (discharge) is completed, the ironing pump 101 is stopped, and the pinch valve 24 and the pinch valve 104 are closed.

Step S73: “Drive the pipetter to transfer the cells to the container”
The pipetter rotation motor 88 operates to rotate the pipetter arm 85. Next, the pipettor vertical movement motor 90 is operated, the pipetter arm 85 is lowered, and the needle 83 is inserted into the container 84. The pinch valves 24 and 103 are opened, and the ironing pump 101 operates. Thereby, the cultured cells in the incubator 38 are sucked out, and the cells are fed (transferred) through the tube 23 in the direction of the arrow P1. Then, it passes through the tube 86 and is injected into the cell storage container 84.

Step S74: “Transporting the cell storage container out of the apparatus”
The shutter motor 81 operates and the shutter 82 rises. After raising the predetermined amount, the container moving motor 94 operates and the holder moves in the direction of arrow G. As a result, the operator can obtain the cell storage container 84 containing the cultured cells.

Step S75: “End”
The operator can obtain the container 84 containing pure cultured cells without contamination as compared with the state before the culture. When the pre-culture cells placed in the container 52 are cells contained in the bone marrow fluid in step S53, in order to remove unnecessary cells (blood-related cells) other than the target cells, step S62, Step S63, step S64, and step S66 may be inserted between step S56 and step S57.

  FIG. 6 is a diagram showing an example of the operation of “shuffling the incubator and homogenizing / seeding” in step S55 of FIG. The incubator 38 repeats forward and reverse rotation as shown in FIG. For example, when the stop is once in the forward direction, once in the reverse direction, and once in the last forward direction, the stop is performed slowly. That is, by shortening the initial acceleration time and deceleration time (t1, t2, t3, t4, t5), the culture medium 17 of the incubator 38 is undulated and becomes suspended. Furthermore, by taking a longer deceleration time (t6) of the last operation, the culture medium decreases its speed while continuing to flow in the circumferential direction due to its inertia and eventually stops. This ensures that the cells are evenly seeded. The last deceleration time (t6) may be an S curve.

  FIG. 6B is a schematic sketch diagram of the simulation result showing the seeded state of the cells. In the dark color near the center (inner periphery S2), in the last operation (deceleration time t6), the flow tangential speed is low, so that the cells are relatively aggregated. The cell is shown thinly seeded. The number of forward / reverse rotations, rotational speed, and angular acceleration (t1, t2, t3, t4, t5, t6) are not particularly limited, and cells may be evenly spread over the front surface of the incubator 38 depending on conditions. it can. However, for example, by intentionally realizing the operation of aggregating cells in the vicinity of the center as described above, it may be convenient for determining the timing of confluence by observing an image of only that portion. That is, it is not necessary to observe the entire surface of the incubator 38, and the quality of the cells is not deteriorated by culturing excessively. Further, such density control is suitable when culturing cell types that easily grow according to the density.

  FIG. 7 is a diagram showing a first modification of the incubator 38 of the culture apparatus according to the above-described embodiment. FIG. 7 (A) is a view as seen from above, and FIG. A side view is shown. For example, four incubators 170a to 170d are placed on the rotor 22 so that the center of rotation is the center of the four incubators 170a to 170d. The incubators 170a to 170d in this figure are formed in substantially the same cylindrical shape, and the tube connecting members 174a to 174d of the incubators 170a to 170d are connected by a tube 171. In FIG. 7B, the incubators 170a and 170c are omitted. This tube 171 performs the same function as the tube 21 of FIG. The tube connecting members 175a to 175d are for discharging the old culture medium. Here, the number of the incubators 170a to 174d is four, but the number is not particularly limited to four, and two, three, or six may be freely selected. Moreover, you may arrange | position an incubator in piles. The culture area can be freely changed by dividing the incubator into a plurality of pieces as shown in FIG. As a result, when the cells are adhesive cells (for example, mesenchymal stem cells), the number of cells that can be cultured is often proportional to the area, and the number of cells in culture can be adjusted. The operation is the same as that in the processing flow of FIG. 5. By shuffling as indicated by the arrow M, the medium in the incubators 170a to 174d finally flows in the direction of the arrow Q, and the incubator 170a. Cells within ˜174d will be seeded uniformly.

  FIG. 8 is a diagram showing a second modification of the incubator 38 of the culture apparatus according to the above-described embodiment. When cultured, cells vary depending on the cell type, but generally increase in a colony shape. Therefore, when it becomes confluent, it is necessary to sow, that is, passaging, on a relatively clean surface of a larger area. Incubators 167, 168, and 169 shown in FIG. 8A are examples of an incubator that is suitable for application to culture when such characteristics are remarkable. The incubator 167 has a generally circular petri dish structure, and a supply tube 182 having the same function as the supply tube 21 of FIG. The incubator 168 has substantially the same envelope structure as that of the incubator 167, but is provided with a single culture auxiliary plate 189 therein. The incubator 169 has the same envelope structure as that of the incubator 167, but two culture auxiliary plates 191 and 192 are provided inside. A waste liquid tube 185 is connected to the bottom surface of the incubator 169. Each of the incubators 167, 168, and 169 are connected by connecting tubes 183 and 184, respectively, and the liquid feeding is controlled by pinch valves 186, 187, and 188 provided in the middle thereof. These incubators 167, 168, 169 and pinch valves 186, 187, 188 are fixed to the rotor 22 shown in FIG. FIG. 8B is a diagram showing a positional relationship with the rotation center axis of each of the incubators 167, 168, and 169. As shown in FIG. By shuffling in the direction shown, the cells in the incubators 167, 168, and 169 are uniformly seeded.

  The operation of the culture apparatus using the incubators 167, 168, and 169 in FIG. 8 is not significantly different from the incubators described above, but has three incubators and two more pinch valves.

  FIG. 9 is a flowchart for explaining the operation of the culture apparatus using the incubator of FIG. Since the operation using the incubator in FIG. 8 is similar to the operation in FIG. 5, differences from FIG. 5 will be described. In FIG. 9, the same components as those in FIG. 5 are denoted by the same reference numerals, and the description thereof is omitted.

  In the operation described in FIG. 5, the medium is discharged at the time of passage (step S61), the incubator is washed with a buffer (step S62), a cell detaching agent is injected (step S63), and a neutralizing agent is injected (step S64). ), Shuffling the incubator, homogenizing and seeding (step S65), discharging the neutralized cell detachment agent (step S66), and injecting a new medium (step S67). That is, at the time of subculture, the cells that had grown in colony were seeded evenly by shuffling the incubator at that location, without being transferred to a new incubator, and cultivated until the cells became target cells again. . On the other hand, in the incubator of FIG. 8, after step S64, the process of “seeding in the lower incubator” in step S90 is executed.

  That is, when it becomes confluent in the incubator 167, the cells are transferred to the incubator 168 below it by feeding. When the incubator 168 becomes confluent, the cells are transferred to the incubator 169 below it by feeding. The amount of the medium to be injected into each incubator is increased every passage, so that it can be cultured on a culture auxiliary plate. That is, in the initial culture, the amount of the medium is an amount in which only the bottom surface of the incubator body 180 is cultured in the incubator 167. The culture after one passage is set to a medium amount that allows the culture auxiliary plate 189 to be immersed in the incubator 168. As a result, the cells can be cultured on both the incubator body 180 and the culture auxiliary plate 189. The culture after the second passage is carried out so that the culture auxiliary plate 190 and the culture auxiliary plate 191 are immersed in the incubator 197. As a result, the cells can be cultured on the three plates of the incubator body 180, the culture auxiliary plate 190, and the culture auxiliary plate 191. By setting the amount to which the culture auxiliary plate is immersed, the incubator 168 is about twice as large as the incubator 167, and the incubator 169 is about three times as large as the incubator 167.

Next, the operation of the pinch valve will be described.
(1) First culture: Pinch valves 186, 187 and 188 are opened when the medium, buffer solution, cell detachment agent and neutralizing agent are discharged.
(2) Culture after the first passage: The pinch valve 186 is opened when the medium, buffer solution, cell detachment agent, and neutralizing agent are injected. In addition, the pinch valves 187 and 188 are opened and closed when the culture medium, the buffer solution, the cell release agent, and the neutralizing agent are discharged.
(3) Culture after 2 passages: Pinch valves 186 and 187 are opened when medium, buffer solution, cell detachment agent and neutralizing agent are injected. Further, the pinch valve 188 is opened and closed when the culture medium, the buffer solution, the cell peeling agent and the neutralizing agent are discharged.

  In the above-described embodiment, the number of incubators and the shape and size of each incubator are not limited, and may be an ellipse or a rectangle, and each size may be changed. Further, the number of culture auxiliary plates is not limited to one or two. In the embodiment shown in FIG. 8, the incubator can be downsized as a whole, and the apparatus can be downsized. In FIG. 8, a plurality of mirrors 278, 279, 282, 283, 284, 285 are disposed between the incubators 167-169. These mirrors are for receiving light emitted from the light source 281 by the CCD camera 280. A filter 286 is provided immediately before the light source 281. The CCD camera 280, the light source 281 and the filter 286 are integrated like a unit 288, and can be moved in the arrow V direction by a drive mechanism (for example, a motor and a feed screw) not shown. It is like that. By moving this unit 288 in the direction of arrow V, even if the incubator has a multi-layer structure, the incubators 167 to 167 can be viewed from the side using the CCD camera 280 and mirrors 278, 279, 282, 283, 284, 285. 169 can be observed. These mirrors 278, 279, 282, 283, 284, and 285 may be moved in the horizontal direction (X-axis direction) in the drawing so that the inside of the incubator can be scanned.

  FIG. 10 is a cross-sectional view showing a third modification of the incubator 38 of the culture apparatus according to the above-described embodiment. The incubator in FIG. 10 is different from the incubator in FIG. 8 in that the incubator can be handled in the same manner as the incubator 38 shown in FIG. Structurally, it has a substantially circular petri dish structure, and a supply tube 197 having the same function as the supply tube 21 of FIG. 2 is connected to the upper surface. The incubator main body 195 is covered with a lid 199, and two auxiliary culture plates 201 and 202 are provided therein. The outline of the operation is the same as that in FIG. 8, but the amount of the medium is increased at each passage so that the culture can be performed on the culture auxiliary plate. That is, the amount of medium in the first culture is only the bottom surface of the incubator body 195. The culture after one passage is set to a medium amount so that the culture auxiliary plate 201 is immersed. As a result, the cells can be cultured on both the incubator body 195 and the culture auxiliary plate 201. The culture after the second passage is carried out so that the culture auxiliary plate 202 is immersed. As a result, the cells can be cultured on the three plates of the incubator body 195, the culture auxiliary plate 201, and the culture auxiliary plate 202. Thereby, as compared with the incubator 38 of FIG. 2, it is possible to realize downsizing similarly to FIG. In the above description, the number of incubators and the shape and size of each incubator are not limited, but may be an ellipse or a rectangle, and the size of each may be variously changed. Further, the number of culture auxiliary plates is not limited to two. According to the embodiment shown in FIG. 10, the incubator can be further miniaturized as a whole, and the apparatus can be miniaturized.

  FIG. 11 is a diagram illustrating an example of a method for uniformly seeding cells. This technique produces a suitable effect when applied to the incubator of the above-described embodiment. In FIG. 11A, the incubator body 300 is provided with a lid 301 of the incubator body, a supply tube connecting member 302 is provided on the upper surface side, and magnets 307, 308, 309, 310 are provided on the lower surface side. Is provided. The inclined portion 381 is intended to reduce the impact of the drop of the liquid supplied from the supply tube connecting member 302 and prevent damage to the cells. These magnets 307, 308, 309, 310 are fixed to the frame 30 in FIG. The spherical members 303, 304, 305, and 306 are placed in the incubator main body 300 (same as the incubator 38 in FIG. 2), and are polymer polymers that are non-toxic to cells on the surface of the spherical magnetic material. , Ceramic, titanium, etc. are coated. When the incubator 300 rotates, the spherical members 303, 304, 305, and 306 also roll in the incubator main body 300 and agitate the medium inside, thereby enabling uniform seeding of cells. In FIG. 11B, the incubator body 312 is provided with a lid 313 of the incubator body, a supply tube connecting member 314 is provided on the upper surface side, and a rod-like member 315 is provided on the lower surface side. The rod-shaped member 315 is placed in the incubator main body 300 (same as the incubator 38 in FIG. 2), and has a non-toxic polymer plastic, ceramic, titanium, or the like on the surface of the rod-shaped magnetic material. Is coated. Even when the rod-shaped member 315 of FIG. 11B is used, the same effect as that of FIG.

  FIG. 12 is a diagram showing an example of a method for connecting an incubator and a tube in the above-described embodiment. In FIG. 2, the incubator, the tube, the reservoir tank, and the like have been described as being connected in advance, but here, the tube cut in the middle is connected to the supply tube connecting member for culturing. A supply tube 320 and a waste liquid tube 326 are connected to the incubator 325 (same as the incubator 38 in FIG. 2). Plugs 321 and 327 made of a flexible material such as rubber are inserted into the cut portions of the respective tubes 320 and 326. Before actually entering the culture, the supply tube 323 having the needles 322 and 328 and the waste liquid tube 329 are connected. Note that the stoppers 321 and 327 may be sterilized with alcohol or the like before the needles 322 and 328 are inserted. By doing so, when the incubator to which a long tube is connected is set in the apparatus, the handling of the tube becomes easy and the operability is improved. In addition, when inject | pouring a cell before culture | cultivation, if a syringe 324 is used, a cell can be directly put into the incubator 325. FIG. Further, the rubber plugs 321 and 327 may be directly attached to the tube connecting member without connecting the supply tube 323 and the waste liquid tube 329 cut in the middle to the incubator 325 in advance.

  FIG. 13 is a diagram showing a sterilization method for a part of the culture apparatus in the above-described embodiment. In this sterilization method, the incubator 38 and each of the tanks 71, 70, 69, 68, 67, 102 are connected by tubes, and the whole is sealed in the sterilization bag as indicated by an arrow S in the state as it is. It is designed to be sterilized. The sterilization bag 340 is made of a material that prevents contact with outside air, and may be a commonly used one. By sterilizing everything that comes into contact with cells in this way, there is no risk of contamination because the tube does not need to be removed during culture. In FIG. 2, the liquid level detecting means 33b detects the liquid level of the incubator 38 using light or ultrasonic waves. When the pump or pinch valve malfunctions, the liquid level deviates from the set value. In such a case, an alarm is issued to the outside.

  FIG. 14 is a diagram showing a detailed view of a mechanism unit according to another embodiment of the culture apparatus to which the present invention is applied. This culture apparatus basically has the same configuration as that of FIG. Therefore, in FIG. 14, since the same code | symbol is attached | subjected to the thing of the same structure as FIG. 2, the description is simplified.

  As in the incubator 38 of FIG. 2, the incubator 140 has cells attached to the bottom surface and culture is performed there. The incubator 140 includes an incubator body and a lid member. The incubator 140 is preferably made of a transparent material because it needs to be able to observe cells in culture with a microscope or the like, and needs to be non-toxic. For these reasons, polystyrene (PS) or polyethylene terephthalate (PET) material is preferable as the material. The lid member is provided with three first ports 141, a second port 142, and a third port 143 for injecting and discharging chemicals.

  The first port 141 is a port for discharging chemicals such as a medium and cells after culturing. A waste liquid tube 141b is connected to the first port 142, and ironing pumps 144 and 145 for discharging the culture medium are arranged so that the culture medium can be discharged. The second port 142 is a port for supplying chemicals such as a medium and cells before culture. A supply tube 142b is connected to the second port 142, and ironing pumps 146 and 147 are arranged in the same manner as the first port 141. The third port 143 is an air suction port into the incubator 140, and an air filter 143b is connected to the outside of the third port 143 via a tube 143a. The third port 143 is an air intake port to the inside of the incubator 140, and need not be provided with a port as long as this purpose is realized. In the culturing apparatus of FIG. 14, the entire incubator 140 is tilted by hooking a hook 149 on the bottom side of the holding ring 148 that holds the incubator 140 and lifting it with a lever 150 and a tilt motor 151.

  The incubator 140 is held by a holding ring 148 fixed to a rotor 153 in a heat insulation box (incubator) 160, and this rotor 153 is an output shaft of an incubator drive motor 29a provided on the top of the heat insulation box 160. And is configured to be rotationally driven in the direction of arrow E. FIG. 14 shows a state in which the rotor 153 is rotated clockwise (leftward) and the holding ring 148 and the incubator 140 are moved to the left side of the heat insulation box 160. Therefore, from this state, the rotor 153 turns counterclockwise (rightward), so that the holding ring 148 and the incubator 140 move to the right side of the heat insulation box 160 through the front in the drawing. The heat insulation box 160 does not have a double box structure as in the prior art, and is configured by a simple method that requires little consideration of airtightness. A detailed configuration of the heat insulation box 160 will be described later.

  One end of the supply tube 142b is connected to a second port 142 provided near the outer periphery of the incubator 140. The supply tube 142b is provided above the rotor 153 in the heat insulation box 160, and freely moves in the interior according to the rotation of the rotor 153. The other end of the supply tube 142b is connected to the heating bag 170. The warming bag 170 warms the temperature of the medium passing through the supply tube 142b from 4 degrees to approximately 20 degrees, and includes a warming heater 171 on the back surface. The shape of the heating bag 170 is not limited as long as the temperature of the liquid can be increased. A tube wound in a spiral shape may be used. When the medium passes through the heating bag 170, the pumps 146 and 147 and the pinch valve 147a are controlled to temporarily retain the medium.

  The culture medium tank 67 stores an unused culture medium, the buffer tank 68 stores a buffer solution, and the cell release agent tanks 69, 70, and 71 store cell release agents. In FIG. 14, only the cell release agent tank 69 is shown, and the cell release agent tanks 70 and 71 are not shown. Each tank 67, 68, 69, 70, 71 is provided in the heat insulation box 80. A heat radiating heat sink 110 is provided on the side surface of the heat insulating box 80 via a Peltier element 109, and a heat absorbing heat sink 111 is provided therein, so that heat is exchanged and maintained at a constant temperature. The pinch valves 72, 105, 73, 74, and 75 control the liquid feeding from the tanks 67, 68, 69, 70, and 71 to the supply tube 142c. The tubes taken out from the tanks 67, 68, 69, 70, 71 are connected to the supply tube 142c and can be fed by the ironing pumps 146, 147. The ironing pumps 146 and 147 are pumps that feed the liquid in the tube by sandwiching the tube with a roller and rotating the roller. Immediately after the squeezing pumps 146 and 147, there are provided pinch valves 146a and 147a for adjusting liquid feeding. Note that the means for measuring the amount of liquid sent by the ironing pumps 146 and 147 and the ironing pumps 144 and 145 is not provided, and the liquid amount is determined by the operating time of these pumps.

  The pinch valves 66a and 66b control the injection of the pre-culture cells into the supply tube 142b, and two pinch valves 66a and 66b are provided in the auxiliary supply tube 142d. The reason why the two pinch valves 66a and 66b are provided side by side is to prevent outside air or the like from flowing in via the auxiliary supply tube 142d after cell injection.

  One end of the supply tube 142c is connected to the heating bag 170, the other end is inserted into the heat insulation box 160 via the pinch valve 172, and an air filter 173 is provided at the end thereof. The air filter 173, the supply tube 142c, the heating bag 170, the supply tube 142b, the third port 143, the tube 143a, and the air filter 143b form an air circulation path. That is, the air in the incubator 140 is circulated by rotating the ironing pumps 146 and 147 and sending out air in the supply tubes 142b and 142c.

  One end of the waste liquid tube 141 b is connected to a first port 141 provided near the outer periphery of the incubator 140. The waste liquid tube 141b is provided above the rotor 153 in the heat insulation box 160, and can move freely in the interior in accordance with the rotation of the rotor 153. The waste liquid tube 141b is bifurcated in the middle, and is sent to the waste liquid tank 102 or the container 84 for storing the cultured cells through the respective paths. That is, one branch of the waste liquid tube 141 b is connected to the waste liquid tank 102 via the pinch valve 176, the pH measuring unit 177, the ironing pump 145, and the pinch valve 178, and the other is waste liquid via the pinch valve 174 and the ironing pump 144. The tank 102 or the pinch valve 174, the ironing pump 144, and the pinch valve 175 are connected to the container 84.

  The old culture medium produced by elution of cell waste products and a decrease in nutrients in the culture medium is stored in the waste liquid tank 102 in the waste liquid recovery box through the waste liquid tube 141b by the peristaltic pump 144. On the other hand, in order to measure the pH of the waste liquid, the old medium passes through the waste liquid tube 141b by the squeezing pump 145, passes through the pH measuring unit 177, and is sent to the waste liquid tank 102 as well.

  15 is a diagram showing a detailed configuration of the heat insulation box of FIG. 14, FIG. 15 (A) shows the internal structure of the heat insulation box in an easy-to-understand manner, and FIG. 15 (B) is an external perspective view of the heat insulation box. FIG. FIG. 16 is a view showing a cross section of the SS plane showing details of the heat insulating structure of FIG. The heat insulation box 160 is a constant temperature bath for culturing and includes an incubator 140 therein. The incubator 140 is attached to a rotor 153 connected to the output shaft of the incubator drive motor 29a, and revolves in the heat insulating box 160 as indicated by arrows A1-A2 according to the revolving operation of the rotor 153. The heat insulation box 160 includes a casing 160a serving as an outer box and an inner box 160b disposed with a predetermined space inside thereof. The material of the housing 160a and the inner box 160b is preferably a plastic such as stainless steel or ABS.

  A first heat insulating material 160c and a second heat insulating material 160d are provided between the outer box 160a and the inner box 160b. As the first heat insulating material 160c, a material having relatively excellent heat insulating performance such as foamed urethane is preferable. However, as will be described later, in order to increase the amount of heat transfer to the inside than the amount of heat transfer to the outside by the second heat insulating material 160d, a foamed urethane is more preferable, and the second heat insulation is more preferable. It is preferable to form it thinner than the material 160d. The heat diffusion plates 160e and 160f are generally U-shaped so as to cover the left and right side portions of the inner box 160b. That is, the light source 34a and the CCD camera 31 are provided above and below the heat insulation box 160, and it is necessary to observe the incubator 140. Therefore, the heat diffusion plates 160e and 160f are not provided for the portions necessary for the observation. is there.

  The material of the heat diffusing plates 160e and 160f is made of aluminum or brass plate having high thermal conductivity. The heat diffusion plates 160e and 160f are bonded with a double-sided tape or the like so as to cover the left and right side surfaces of the first heat insulating material 160c. Further, panel heaters 160g to 160j are similarly bonded to the bottom surface and the side surfaces of the heat diffusion plates 160e and 160f with double-sided tape or the like. The amount of heat transmitted to the outside through the second heat insulating material 160d becomes a leakage loss outside the heat insulation box. Therefore, it is important to improve the heat insulation performance as much as possible for the second heat insulating material 160d. Specifically, a hard foaming urethane or a vacuum heat insulating material such as foaming urethane is put in an aluminum pack, and the inside of the pack is vacuumed. It is preferable to use a plate or the like. As shown in FIG. 15B, a door 160k having a similar heat insulation structure is supported on the heat insulation box 160 by a hinge 160m so as to be opened and closed as indicated by an arrow 160n.

  FIG. 17 is a diagram showing a control block of the heat insulating box 16 of the culture apparatus of FIG. 14, in which parts necessary for the explanation are extracted from FIG. 4 and others are omitted. In FIG. 17, the same components as those in FIG. 4 are denoted by the same reference numerals, and the description thereof is omitted. In this control block, the console 22 includes an operation switch, a temperature setting switch, and the like. The controller 11 is connected to heaters 160g to 160j, an incubator drive motor 29a, and a temperature sensor 106. The temperature sensor 106 may be a temperature sensor using a known technique such as a thermocouple.

  The operation of this control block will be described. When the operator operates the operation switch or the temperature setting switch of the console 22, the control unit 11 takes in the temperature data of the temperature sensor 106, compares it with the set temperature, and supplies the heaters 160g to 160j with power corresponding to the difference. . The acquisition of temperature data and the comparison with the set temperature are performed in a timely manner, and when the temperature inside the heat insulation box 160 is equal to or higher than the set temperature, the power supplied to the heaters 160g to 160j is lowered. On the other hand, the heat quantity of the heaters 160g to 160j whose temperature has risen is transmitted through the heat diffusion plates 160e and 160f and warms the inside of the heat insulating box 160, but the second heat insulating material 160d is transmitted more than the first heat insulating material 160c. Since the amount of heat is small, much of the amount of heat of the heaters 160g to 160j contributes to heating the inside of the heat insulating box 160. The incubator drive motor 29a is for rotating to inoculate the cells inside the incubator 140 evenly.

  According to this embodiment, the heat insulation box can be configured by a simple method and configuration that does not require a conventional double box structure and that hardly requires airtightness. In addition, the amount of heat transmitted through the first heat insulating material 160c is smaller than the amount of heat transmitted through the second heat insulating material 160d, so that energy for heating can be suppressed. Further, the heat diffusion plates 160e and 160f can suppress direct radiant heat to the incubator by forming a notch in the surface perpendicular to the culture surface of the incubator 140, and only the heat retaining box. In addition, the temperature inside the incubator can be made more constant.

  Usually, a culture apparatus using a pinch valve as shown in FIG. 1 and FIG. 14 takes in the pressure for driving the pinch valve as compressed air from an air compressor (air compressor) or the like installed outside the culture apparatus. Or, it was operated using an air compressor built in the culture apparatus. When taking in compressed air from the outside, piping from the air compressor to the culture device is required. Normally, the piping is fixed to the wall, ceiling, etc., so the location of the culture device is restricted or impossible to install. It was not preferable. In addition, the method using an air compressor built in the culture apparatus is not limited, but there are problems such as dust generation, vibration, and noise, but it is not preferable.

  Therefore, in the embodiment shown in FIG. 14, the gas in the carbon dioxide cylinder 210 is used as compressed air for driving a pinch valve provided at various locations on the tube. The gas in the carbon dioxide cylinder 210 is sent to pinch valves provided at various points of the tube via the regulator 211 and the multiple solenoid valve 213. In addition, although the pinch valve cannot be driven when the gas pressure of carbon dioxide becomes low, the use of carbon dioxide is indispensable at the time of culture, so that possibility is low. Further, since the amount of carbon dioxide gas used by the pinch valve is about 0.5 cc per time, it is considerably smaller than the amount of carbon dioxide gas used in the heat insulation box 160. In the case of the embodiment of FIGS. 1 and 2, the pinch valve is driven in the same manner.

  In the culturing apparatus of FIGS. 1 and 14, by opening and closing the pinch valve for an arbitrary time at an arbitrary timing, a necessary amount of a necessary reagent can be injected into the incubator 140 or the medium can be drained. it can. The carbon dioxide phone 210 supplies carbon dioxide gas to each pinch valve through an air tube (pipe shown by a dotted line in FIG. 14). On the other hand, a predetermined amount of carbon dioxide is supplied from the carbon dioxide cylinder 210 into the heat insulation box 160 through the regulator 211 and the electromagnetic valve 212.

  The carbon dioxide gas supplied to each pinch valve is sent as compressed air to an air cylinder for driving the pinch valve, where it is used to drive the pinch valve, and the carbon dioxide gas supplied to the heat insulation box 160 is contained in the heat insulation box 160. It is used to adjust the carbon dioxide concentration. Any number of air cylinders for driving the pinch valve can be added as long as the pressure applied to the air cylinders meets the specifications. In FIG. 14, the carbon dioxide gas from the carbon dioxide cylinder 210 is connected to the regulator 211 by an air tube. It has been. The air cylinder for driving the pinch valve usually has two tubes, and the air cylinder operates by supplying carbon dioxide gas to one side, the air cylinder operates by closing the pinch valve and supplying carbon dioxide gas to the other side. Is configured to operate in the reverse direction and open the pinch valve. The multiple solenoid valve 213 switches the supply of such carbon dioxide gas. On the other hand, the injection and shut-off of the carbon dioxide gas supplied to the heat insulation box 160 is controlled by the electromagnetic valve 212. Normally, the carbon dioxide concentration in the heat insulation box 160 is measured using the carbon dioxide sensor 205. When the carbon dioxide concentration is lower than the target value, the electromagnetic valve 212 is opened. The carbon dioxide concentration is kept constant. In addition, although the carbon dioxide cylinder was demonstrated here as an example, it cannot be overemphasized that it can be used similarly if it is a nonflammable gas.

It is a block diagram which shows the basic composition of the culture apparatus to which this invention is applied. It is a detailed view of the mechanism part of the culture apparatus to which the present invention is applied, and shows an actual configuration in which the system controller 11 in FIG. 1 is omitted. It is a figure which shows the detailed structure of the incubator 38 of FIG. It is a block diagram which shows the detail of the control block diagram of the culture apparatus of FIG. 2, and shows the case where several culture apparatuses are connected and it is planted. It is a flowchart for demonstrating operation | movement of a culture apparatus. It is a figure which shows an example of operation | movement of "shuffling an incubator and homogenizing and seeding | inoculating" of step S55 of FIG. It is a figure which shows the 1st modification of the incubator 38 of the culture apparatus which concerns on the above-mentioned embodiment, FIG. 7 (A) shows a figure seeing from an upper surface, FIG.7 (B) shows the side view. . It is a figure which shows the 2nd modification of the incubator 38 of the culture apparatus which concerns on the above-mentioned embodiment. It is a flowchart for demonstrating operation | movement of the culture apparatus using the incubator of FIG. It is sectional drawing which shows the 3rd modification of the incubator 38 of the culture apparatus which concerns on the above-mentioned embodiment. It is a figure which shows an example of the method of seed | inoculating a cell uniformly. It is a figure which shows an example of the connection method of the incubator and tube in the above-mentioned embodiment. It is a figure which shows the one part sterilization method of the culture apparatus in the above-mentioned embodiment. It is a figure which shows the detailed view of the mechanism part which concerns on another embodiment of the culture apparatus to which this invention is applied. FIG. 15A is a view showing a detailed configuration of the heat insulation box, FIG. 15A shows the internal structure of the heat insulation box in an easy-to-understand manner, and FIG. 15B is an external perspective view of the heat insulation box. It is a figure which shows the cross section of the SS plane which shows the detail of the heat insulation structure of FIG. 15 (A). It is a figure which shows the control block of the heat retention box 16 of the culture apparatus of FIG. 14, extracted the part required for description from FIG. 4, and has abbreviate | omitted and shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Incubator 2 ... Flexible tube member 3 ... Pump 4 ... Reserve tank 5 ... Flexible tube member 6 ... Pump 7 ... Waste liquid tank 8 ... Drive means 9 ... Camera 10 ... Light source 11 ... System controller 15 ... Main body 16 ... gas permeable membrane 17 ... medium 18 ... tube connecting member 19 ... tube connecting member 20 ... weir 21 ... supply tube 22 ... rotor 23 ... waste liquid tube 24 ... pinch valve 25 ... cable drum 26 ... take-up drum 27 ... cam follower 28 ... pinion 29 ... Incubator drive motor 30 ... Insulation box (frame)
31 ... CCD camera 32 ... observation window 33 ... filter 34 ... light source 35 ... guide member 36 ... tube fixing member 37 ... ironing pump 38 ... incubator 381 ... inclined part 39 ... needle 40 ... air filter 41 ... needle 42 ... air filter 50 ... Shutter motor 51 ... Shutter 52 ... Container 53 for storing pre-culture cells 53 ... Needle 56 ... Cell injection tube 55 ... Pipetter arm 54 ... Spindle 57 ... Pipetta rotation motor 58 ... Rotating member 59 ... Pipetta up / down movement motor 60 ... Pulley 62 ... Holder 61 ... Belt 63 ... Motor 65 ... Fans 66a and 66b ... Pinch valve 67 ... Medium tank 68 ... Buffer tank 69, 70, 71 ... Cell releaser tank 72, 105, 73, 74, 75 ... Pinch valve 78 79 ... Air inlet 80 ... Heat insulation box 81 ... Shutter motor 82 ... Shutter 83 ... 84 ... Container 85 for storing cells after culture ... Pipetter arm 87 ... Shaft 88 ... Pipetter rotary motor 89 ... Rotating member 90 ... Pipetter vertical motor 91 ... Pulley 92 ... Belt 93 ... Holder part 94 ... Motor 95 ... Feed screw 96, 97 ... Stand 98 ... Waste liquid collection box 99 ... Guide member 100 ... Tube fixing member 101 ... Iron pump 102 ... Waste liquid tank 103 ... Pinch valve 104 ... Pinch valve 106 ... Temperature sensor 107 ... Joint 108 ... Heater 120 ... I / O
121 ... Bus 122 ... CPU
123 ... console 124 ... memory 125 ... computer network driver 126 ... operation devices 127, 128, 129 ... culture device 130 ... control and monitoring devices 167, 168, 169 ... incubators 170a-170d ... incubator 171 ... tubes 174a-174d ... Tube connection members 175a to 175d ... Tube connection members 182 ... Supply tubes 183, 184 ... Connection tubes 185 ... Waste liquid tubes 186, 187, 188 ... Pinch valves 191, 192 ... Culture auxiliary plate 195 ... Incubator body 197 ... Supply tube 199 ... Lid 201, 202 ... Culture auxiliary plate 250 ... Image capture board 278, 279, 282, 283, 284, 285 ... Mirror 280 ... CCD camera 281 ... Light source 286 ... Filter 288 ... Unit 300 ... Incubator body 301 ... Lid 302 ... Supply For Chu Connecting member 307, 308, 309, 310 ... Magnet 303, 304, 305, 306 ... Spherical member 314 ... Supply tube connecting member 315 ... Bar-shaped member 320 ... Supply tube 321, 327 ... Plug 322, 328 ... Needle 323 ... Supply Tube 324 ... Syringe 325 ... Incubator 326 ... Waste liquid tube 329 ... Waste liquid tube 381 ... Inclined portion 140 ... Incubator 141 ... First port 142 ... Second port 143 ... Third port 141b ... Waste liquid tubes 144, 145 ... Squeezing pump 142b ... Supply tube 142c ... Supply tube 142d ... Auxiliary supply tubes 146 and 147 ... Ironing pumps 146a and 147a ... Pinch valve 143a ... Tube 143b ... Air filter 148 ... Holding ring 149 ... Hook 150 ... Lever 151 ... Tilt motor 153 ... Rotor 16 ... thermal insulation box (incubator)
170 ... Heating bag 172 ... Pinch valve 173 ... Air filter 177 ... pH measuring unit 29a ... Incubator drive motor 109 ... Peltier device 110 ... Heat sink 111 ... Heat sink 201-204 ... Heaters 205, 206 ... Heat sink 205 ... Dioxide Carbon sensor

Claims (2)

Incubator means for culturing inside;
Outer box means for setting the incubator means in a state suitable for culture;
Chemical supply means for supplying unused chemicals from the outside of the outer box means to the incubator means in the outer box means;
Waste liquid discharging means for discharging unnecessary liquid waste from the incubator means in the outer box means to the outside of the outer box means;
A culture apparatus provided with gas concentration adjusting means for supplying gas from the gas cylinder into the outer box means and adjusting the gas concentration in the outer box means,
An culturing apparatus comprising an opening / closing means for opening and closing at least one of the discharge of the chemical supply means and the waste liquid discharge means or the supply path by the gas of the gas cylinder.   The culture apparatus according to claim 1, wherein the path is a tube, and the opening / closing means is a pinch valve.

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