WO2005059091A1 - 細胞培養装置 - Google Patents
細胞培養装置 Download PDFInfo
- Publication number
- WO2005059091A1 WO2005059091A1 PCT/JP2004/018730 JP2004018730W WO2005059091A1 WO 2005059091 A1 WO2005059091 A1 WO 2005059091A1 JP 2004018730 W JP2004018730 W JP 2004018730W WO 2005059091 A1 WO2005059091 A1 WO 2005059091A1
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- WIPO (PCT)
- Prior art keywords
- incubator
- cells
- cell culture
- tube
- container
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/14—Incubators; Climatic chambers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/10—Rotating vessel
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/809—Incubators or racks or holders for culture plates or containers
Definitions
- the present invention relates to a cell culture device for culturing cells, and more particularly, to a cell culture device that can automatically perform operations associated with culturing for several days to several months.
- Cell culture is performed by exchanging the culture medium in an incubator or by re-seeding to optimize the cell density.
- the cleaning is carefully performed in a relatively clean atmosphere in which the concentration of suspended particulates in the air is suppressed by the clean environment generation technology cultivated in the semiconductor manufacturing field.
- the concentration of suspended particulates in the air is suppressed by the clean environment generation technology cultivated in the semiconductor manufacturing field.
- the cleaning is carefully performed in a relatively clean atmosphere in which the concentration of suspended particulates in the air is suppressed by the clean environment generation technology cultivated in the semiconductor manufacturing field.
- the concentration of suspended particulates in the air is suppressed by the clean environment generation technology cultivated in the semiconductor manufacturing field.
- Patent Document 1 USP5, 985, 653
- Patent Document 1 discloses a culture device equipped with a rocking means for uniform seeding, but the risk of contamination is not completely closed because the device is not completely closed. It was left.
- the cell culture operation has a major problem in terms of achieving both mass production and safety.
- An object of the present invention is to provide a cell culture apparatus that can automatically perform operations involving culturing for several days to several months while minimizing the risk of contamination. Means to solve
- a first feature of the cell culture device of the present invention is that the incubator means for culturing cells, a warm box means for arranging the incubator means in a state suitable for culture, and the heat incubator means Drive means for rotating in the box means, chemical supply means for supplying unused chemicals to the incubator means in the heat insulation box means from outside the heat insulation box means, and culturing in the heat insulation box means
- a waste liquid discharging means for discharging unnecessary waste liquid and the like from the incubator means to the outside of the insulated box means; And observation means.
- a new drug can be supplied to the incubator means using a drug supply means that does not remove the incubator means disposed in the insulated box means from the insulated box, or use a waste liquid discharging means. And the wastewater can be drained out of the incubator means, and the culture state can be observed with the incubator means housed in the insulated box means. There is no direct intrusion of outside air, there is no risk of contamination, and the culturing operation can be performed automatically for a long period of time.
- the present invention has the following aspects.
- Incubator means for culturing cells insulated box means for arranging the incubator means in a state suitable for culture and maintaining a predetermined temperature, and rotating the incubator means in the insulated box means Drive means for moving, chemical supply means for supplying unused chemicals to the incubator means in the insulated box means from outside the insulated box means, and incubator means in the insulated box means unnecessary.
- Waste liquid discharging means for discharging the waste liquid to the outside of the heat retaining box means, and culture state observation means for observing the state of cell culture of the incubator means in the heat retaining box means from outside the heat retaining box means.
- a closed cell culture device is also used to observe the state of cell culture of the incubator means in the heat retaining box means.
- a cell culture device wherein a pump, a valve, and a flexible tube member are provided between the incubator means and the drug supply means to supply, culture, and collect cells. .
- the incubator means is a container having a transparent non-toxic material with a smooth central part (may have some unevenness).
- the cell culture according to (5) further comprising: a camera moving unit configured to scan the camera over the entire surface of the incubator unit and to set a focus in the cell incubator unit in an optical axis direction.
- a thin tube whose outside is sealed by a closing member, wherein the thin tube is a cell supply port or a recovery port, and includes a container for accommodating cells, and an upper part of the container is impregnated with a bactericide.
- a member is provided, and the thin tube is inserted into the container after penetrating the sterilizing agent-impregnated member (1). ), (2), or (5).
- the cell culture apparatus further including a drug amount determining unit that determines an amount of the drug supplied from the drug supply unit to the incubator unit during the operation time of the pump.
- the waste liquid discharge means flexible tube member, made of a pump and a waste liquid tank, the cell culture apparatus having a P H measurement unit to the one (1).
- the cell culturing procedure includes control means for storing and executing the timing and contents of cell supply, rotation of the incubator means, supply of a chemical solution, waste liquid and cell supply and recovery, (2) ) Cell culture equipment.
- control means has an interface for exchanging culture information with another control means when a plurality of the cell culture devices are operated.
- FIG. 1 is a block diagram showing a basic configuration of a cell culture device to which the present invention is applied.
- the incubator 1 is a container for culturing cells, and is connected via a pump 3 and a flexible tube member 2 to a reserve tank 4 into which unused chemicals have been injected.
- the waste liquid tank 7 stores used chemicals, and is connected to the incubator 1 via a pump 6 and a flexible tube member 5.
- the driving means 8 is for rotating the incubator 1.
- the camera 9 observes the cultured cells therein by the light emitted from the light source 10 transmitted through the incubator 1.
- the system controller 11 is connected to the pump 3, the pump 6, the driving means 8, the camera 9, and the light source 10, and controls the pump 3, the pump 6, the driving means 8, the camera 9, and the light source 10.
- the incubator 38 is preferably formed 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 should be modified to have hydrophilic properties so that cells can easily adhere to it.
- a tube connecting member 19 for injecting a chemical is provided substantially at the center of the incubator 38, and serves to flow a chemical such as the culture medium 17 into the incubator 38. At this time, the inclined portion 381 reduces the impact of the falling of various liquids and prevents damage to the cultured cells.
- the cells adhere to the bottom surface of the incubator 38, and culture is performed there.
- the tube connecting member 18 is an outlet from which waste products of cells are eluted and the old medium in which nutrients in the medium are reduced is discharged.
- the incubator 38 is fixed on the rotor 22, and the rotor 22 is freely supported at, for example, three circumferential positions so that the rotor 22 can be rotated in the direction of arrow E by the cam follower 27 below. Further, an internal gear (not shown) is formed below the rotor 22, and this gear is connected to a pinion 28 fitted to an output shaft of an incubator drive motor 29 fixed to a heat insulation box (frame) 30. See each other.
- the cable drum 25 winds the wiring of a 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 when the wiring is loosened, automatically winds up the wiring so as not to be entangled with other protrusions. ing.
- it can be realized by applying constant tension to the cable using a spring.
- the supply tube 21 is connected to a tube connection member 19 provided substantially at the 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.
- the tube between the tube fixing member 36 and the tube connecting member 19 is connected to the guide member 35. It is free to move inside.
- the medium tank 67 stores an unused medium
- the buffer solution tank 68 stores a buffer solution
- the cell detachment agent tanks 69, 70, and 71 collect and recycle the Itoda sulphide 1PJ.
- Each of the tanks 67, 68, 69, 70, 71 is provided in a heat insulating box 80 and runs there.
- the pinch valves 72, 105, 73, 74, and 75 control the tanks 67, 68, 69, 70, and 71, respectively.
- the pinch valve 66 is described later. Controls the injection of pre-cultured cells.
- the air inlets 78 and 79 are for introducing air in the air to prevent liquid pooling in the tube, and a filter for removing impurities in the air (having a size of 0.2 ⁇ ⁇ The following is desirable).
- the tubes taken out of each of the tanks 67, 68, 69, 70, 71 are connected to the above-mentioned supply tube 21 so that they can be sent by the ironing pump 37.
- the ironing pump 37 is a pump that sandwiches the tube between rollers, and sends out the liquid in the tube by rotating the rollers.
- the waste liquid tube 23 is connected to the tube connecting member 18 provided on the bottom surface of the incubator 38, and is guided out of the frame 30 by the guide member 99.
- a tube fixing member 100 is provided below the guide member 99, and the waste liquid tube 23 is fixed by the tube fixing member 100.
- the waste liquid tube 23 is provided between the bracket fixing member 100 and the tube connecting member 18. Is free to move.
- the old medium produced by the elution of cell wastes and the decrease of nutrients in the medium is stored in the waste tank 102 in the waste collection box 98 through the waste tube 23 by the ironing pump 101.
- the pinch valve 103 controls liquid supply to the waste liquid tank 102, and the pinch valve 104 controls the liquid supply state when the waste liquid is supplied 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 rotation shaft thereof.
- the shutter 51 can be moved in the direction of arrow A (vertical direction on the drawing).
- the container 52 for storing cells before culturing is supported by the holder 62.
- the holder 62 can be moved in the direction of arrow B (the left-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 from outside air (not shown).
- the needle 53 is connected to the cell injection tube 56 and fixed to the pitcher arm 55.
- the pitcher arm 55 is supported by a shaft 54, and can be rotated in the direction of arrow D1 by a pitcher rotation motor 57.
- the rotating member 58 is a member that rotates together with the shaft 54, and includes a pitcher vertical movement motor 59 and a pulley 60.
- the pulley fixed to the output shaft of the pitcher 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 drive of the pitcher vertical movement motor 59 causes the shaft 54 to move up and down.
- the pinch valve 66 controls the state of liquid supply when the cells before culture are supplied by the ironing pump 37.
- a needle 39 is fixed to the pitter arm 55, and an air filter 40 is provided at one end thereof.
- the function of the needle 39 is to prevent the inside of the container 52 from becoming a printing pressure and making it difficult for cells to be sucked when the container 52 is formed of a hard plastic material.
- the cells before culturing may be supplied by forcing air from the force needle 39 described above to be sucked by the ironing pump 37 to the container 52.
- 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 rotation axis thereof.
- the shutter 82 can be moved in the direction of arrow F (vertical direction on the drawing).
- the container 84 for storing the cells after the culture is supported by the holder 93.
- the holder 93 can be moved in the direction of arrow G (the left-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 outside (not shown).
- the needle 83 is connected to the cell injection tube 84 and is fixed to the pitter arm 85.
- the pitcher arm 85 is supported by a shaft 87 and can be rotated in the direction of arrow D2 by a pitcher rotating motor 88.
- the rotation member 89 is a member that rotates together with the shaft 87, and includes a pitcher vertical movement motor 90 and a pulley 91.
- a pulley fixed to the output shaft of a pitcher vertical movement motor 90 and a pulley 91 are connected by a velvet 92, and a part of the belt 92 is fixed to a shaft 87.
- the shaft 87 moves up and down by driving the pitcher up / down motor 90.
- the pinch valve 103 controls the state of liquid supply when the cells are cultured and the ironing pump 101 supplies the liquid.
- a needle 41 is fixed to the pitter arm 55, and an air filter 42 is provided at one end. The function of the needle 41 is to prevent cells from being difficult to discharge due to a positive pressure inside when the container 84 is formed of a hard plastic material.
- the light source 34 supplies light into the frame 30 from below the frame 30, and includes a filter 33 on the light emission side.
- the CCD camera 31 has a lens, It is used for observing cells cultured in the incubator 38 from the observation window 32 provided on the upper side of the room 30 and for judging the timing of subculture.
- the light source 34 is preferably of a type in which a plurality of LEDs are arranged flat to prevent luminance unevenness in an image. If the light intensity is sufficient, a single LED or a lamp may be used.
- the finale letter 33 includes an ND filter for reducing the amount of light incident on the CCD camera 31, and an appropriate band-pass 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 in front of the CCD camera 31, and the band-pass filter is preferably in front of the light source 34 if it cuts short-wavelength light that harms cells.
- the heater 108 keeps the inside of the frame 30 at a constant temperature based on the temperature detected by the temperature sensor 106.
- the fan 65 stirs the air in the frame 30.
- the stands 96 and 97 are for erecting the entire cell culture device on the floor.
- the joint 107 is provided with a filter for removing impurities when supplying a gas mixture in which the ratio of carbon dioxide, nitrogen and oxygen is controlled.
- the gas permeable membrane 16 attached to the upper surface of the incubator 38 is shown to cover the entire surface, it may be provided partially. Needless to say, it is better to raise 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 the tray with water inside. If the front of the incubator 38 is not covered with the gas permeable membrane 16, the gas mixture supplied from the joint 107 may be supplied directly to the inside of the incubator 38 or may be dissolved in a culture medium or the like. May be.
- the frame 30 may be configured to cover only the peripheral portion of the power incubator 38 so as to cover substantially the entirety. That is, the two sets of pitchers are configured separately from the force frame 30 described in the case where they are provided on the left and right as a part of the frame 30, and the two sets of pitchers are arranged outside the frame 30. You may do it.
- FIG. 3 is a diagram showing a detailed configuration of the incubator 38 of FIG. FIG. 3 (a) is a plan view of the incubator 38 of FIG. 2 as viewed from above, and FIG. 3 (b) is a side sectional view thereof.
- the tube connecting member 19 is provided near the center of rotation at a position away from the center of the circle of the incubator 38 by a distance L1.
- the position and shape of the tube connecting member for discharging the old culture medium 18 ⁇ weir 20 may be the same as in the modified examples 112, 113 and 114. In the modification 112, the weir 20 is omitted.
- the tube connection member 18 is provided on the side surface of the incubator 38, and in the modification 114, the tube connection member 18 is arranged so that the opening of the tube connection member 18 contacts the bottom surface of the incubator 38. It is provided.
- Modified Examples 112 and 114 are different from Modified Example 113 in that in this point, the cells may aggregate in a hollow portion other than the opening of the tube connecting member 18 due to the centrifugal force caused by the rotation of the incubator 38. This is more preferable because the cells can be discharged out of the incubator 38 by centrifugal force.
- the position of the tube connecting member 18 is not particularly limited, and the tube connecting member 18 may be disposed anywhere. Further, the distance L1 is not particularly limited. However, it is preferable that the center of rotation and the center of rotation of the incubator 38 be shifted from the viewpoint of uniform seeding of cells. Aggregation of cells can also be avoided by arranging the position of the tube connecting member 18 near the center of the circle of the incubator 38, for example.
- the term "rotation" includes at least one of rotation, eccentric rotation, parallel movement, and reciprocal parallel movement and a combination thereof, and is particularly useful for agitation and homogenization of cells and liquids.
- the incubator may be tilted to discharge the culture medium and the neutralized cell detachment agent from the incubator.
- the incubator may be vibrated. In manual cell culture, the seeds can be uniformly seeded by operating the incubator to draw a figure-eight locus. In this way, the rotation operation is the most simple and easy to configure, but it is not necessary to limit the rotation to various types of translation operations, or a combination of rotation and translation operations may be used.
- FIG. 4 shows details of a control block diagram of the cell culture device of FIG. 2, and is a block diagram showing a case where a plurality of cell culture devices are connected and planted.
- the cell culture device of FIG. 2 is shown in FIG.
- the CCD camera 31 is connected to the bus 121 via the image capturing board 250 and the I / O 120.
- CPU 121, console 123, controller 126, memory 124, computer network Work driver 125 is connected.
- a computer network is provided outside, and a cell culture device 127 and a plurality of other cell culture devices 128 and 129 are connected to the computer network.
- the plurality of cell culture devices 127, 128, and 129 are monitored and controlled from distant locations by the connected control and monitoring device 130.
- the control and monitoring device 130 can be supported by a general-purpose personal computer.
- 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 cell culture device from a distance, a one-way data communication means may be used.
- the number of cell culture devices connected to this computer network is not particularly limited. When using multiple cell culture devices, large-scale culture can be achieved by connecting the devices using data communication means, since the state of each cell culture device can always be monitored remotely during the culture period, which usually takes several weeks. It is suitable for equipment.
- the function of the control and monitoring device 130 is to provide a function of sequentially monitoring the operation of the cell culture device described in FIG. 5 and issuing an external signal in the event of an abnormality.
- the monitoring function may be performed by each culture apparatus, or the control and monitoring apparatus 130 may contract.
- the easiest method is for the control and monitoring device 130 to check the operation of each cell culture device on a time-division basis, and to notify the cell culture device to the outside when an abnormality occurs in each cell culture device.
- FIG. 5 is a flowchart for explaining the operation of the cell culture device.
- the operation of the cell culture device will be described with reference to FIGS. Since the CPU 122, the memory 124, and the bus 121 shown in FIG. 4 are technologies used in general-purpose computers, detailed operations of the CPU 122, the memory 124, and the bus 121 will be omitted below, and only the operation of each actuator will be described. I will tell.
- Step S51 "Start"
- the control and monitoring device may be configured to press the start switch.
- the chemicals have already been set in the culture device 127 in the incubator 38 and the tanks 67, 68, 69, 70, 71.
- Step S52 “Inject medium”
- the pinch valve 72 is opened, the ironing pump 37 operates, and the medium in the medium tank 67 is sent through the tube 21.
- the medium to be fed flows into the incubator 38 through the paths indicated by arrows J1 and J, and becomes the culture medium 17 in the incubator 38.
- the operation of the ironing pump 37 is stopped, and the pinch valve 72 is closed.
- the set value of the medium volume is stored in the memory 124 in advance.
- Step S53 "Put container 52"
- the shutter motor 50 When the operator operates the corresponding switch on the operating device, the shutter motor 50 operates, and the shutter 51 slides in the direction of arrow A (ascending direction). After the shutter 51 rises by a predetermined amount, the container moving motor 63 operates, and the holder moves in the direction of arrow B (rightward). After this movement, the operator places the container 52 containing the cells before culture in the holder 62. Thereafter, the container moving motor 63 rotates in the direction opposite to the above, and the holder 62 moves in the direction of arrow B (left direction). The shutter motor 50 rotates and the shutter 51 moves in the direction of arrow A (downward direction) and closes.
- Step S54 "Drive the pitta to transfer 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.
- an actuator that vibrates the container 52 may be provided inside the holder 62.
- the pitcher rotating motor 57 operates, and the pitcher arm 55 rotates.
- the pitcher vertical movement motor 59 operates, the pitcher arm 55 descends, and the needle 53 enters the container 52.
- the pinch valve 66 opens, and the ironing pump 37 operates.
- the cells before culture in the container 52 are sucked out, and the cells are fed through the tube 56 in the direction of arrow J2 ⁇ J, and then injected into the incubator 38 through the tube 21.
- the pinch valve 66 closes and the ironing pump 37 stops.
- Step S55 "Shuffle the incubator and homogenize 'seeding"
- the motor 28 rotates, and the cells injected into the incubator 38 are homogenized and suspended for seeding.
- Cell homogenization 'seeding is necessary for efficient cell culture, since excessive cell density may lead to cell denaturation.
- the culture cells are not adhesion-dependent cells (cells that recognize and adhere to solids and are cultured by adhering to the solids). It is better to rotate the motor 28 while the cells before culture are being injected into the incubator 38.
- step S55 there are cases where the process proceeds to step S56 and cases where the process jumps to step S58.
- the case of jumping to step S58 will be described.
- the cells before culturing are forced to enter culturing.
- the temperature inside the frame 30 is controlled to a temperature suitable for culturing (around 37 ° C) by the temperature sensor 106 and the heater 108.
- the atmosphere inside the frame 30 is also agitated by 65 so that there is no temperature unevenness.
- Step S56 “Discharge medium”
- This step can be appropriately executed before executing step S58.
- the pinch valve 24 and the pinch valve 104 are opened, the ironing pump 101 operates, and the medium 17 in the incubator 38 is transferred to the tube 23. , The medium is sent (discharged) to the waste liquid tank 102. After the liquid transfer (discharge) is completed, the ironing pump 101 stops, and the pinch valve 24 and the pinch valve 104 close.
- Step S57 "Inject new medium”
- This step is also a step that can be appropriately executed before executing Step 6, and the pinch valve 72 is opened, the ironing pump 37 is operated, and a new medium is injected into the incubator 38. After the injection of the medium, the pinch valve 72 closes and the ironing pump 37 stops.
- Step S59 "Timing of passage?"
- the time may be determined in advance, or a switch may be provided on the console to allow the operator to instruct the operation. Contributes to stabilization.
- the light source 34 emits light as appropriate, and the CCD camera 31 acquires an image of the cells cultured in the incubator 38. Cells in the early stages of culture often form very dense and partially dense (colonies) in many places. That colony The operation of the incubator drive motor 28 causes the CCD camera 31 to capture and measure. If the cells in the colony have not reached confluence, the cells are subsequently cultured. The determination of whether or not the cell is a confluent is the same as the cell number sensitivity in step S60 described later.
- step S58 the process of steps S56 and S57 is performed, and then the process proceeds to step S58. If the confluence has been reached, the timing of the passage will be the same as that of the passage, and the process proceeds to the next 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 the value set by the operator in advance, the process proceeds to step S68. If the number of cells has not reached the target number of cells, otherwise the process proceeds to step S61.
- Step S61 "Discharge medium"
- Step S61 The process of step S67 is a process executed when the number of cells has not reached the value set by the operator in advance.
- the pinch valve 24 and the pinch valve 104 are opened, the ironing pump 101 operates, and the medium 17 in the incubator 38 passes through the tube 23, and the medium is sent to the waste liquid tank 102. (Discharged). After the liquid transfer (discharge) is completed, the ironing pump 101 stops, and the pinch valve 24 and the pinch valve 104 close.
- Step S62 “Washing incubator with buffer”
- the pinch valve 105 is opened, the ironing pump 37 operates, and the buffer is injected into the incubator 38 from the buffer tank 68. After the injection, the pinch valve 105 closes and the ironing pump 37 stops.
- the incubator drive motor 28 is rotated, and the incubator 38 is rotated to allow the buffer solution to spread to the bottom of the incubator.
- the pinch valve 24 is opened, and the ironing pump 101 is operated to send the buffer solution in the incubator 38 to the waste liquid tank 102.
- Step S63 "Inject cell peeling agent"
- the pinch valve 73 is opened, the ironing pump 37 operates, and the cell releasing agent is injected into the incubator 38 from the cell releasing agent tank 69. After the injection, the pinch valve 73 closes and the ironing pump 37 stops.
- the incubator drive motor 28 rotates to spread the cell detachment agent to the bottom of the incubator.
- Step S64 “Inject neutralizer”
- the medium is a neutralizing agent.
- Various types of cell exfoliants are used as described above. However, it is assumed here that the medium contains serum, and a cell detaching agent that is neutralized by the serum is assumed. Therefore, the operation is the same as that in step S52 described above, and the neutralizing agent is injected from the tank 70 by opening the pinch valve 74.
- Step S65 “Shuffle the incubator and homogenize 'seeding'
- step S55 The same processing as in 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 have adhered, the process proceeds to the next step S66.
- Step S66 "Discharge the neutralized cell detaching agent"
- the pinch valve 24 and the pinch valve 104 are opened, and the ironing pump 101 feeds (discharges) the medium to the operation 102. After the liquid transfer (discharge) is completed, the ironing pump 101 stops, and the pinch valve 24 and the pinch valve 104 close.
- Step S67 "New medium injection"
- step S52 The same processing as in step S52 is performed. That is, the pinch valve 72 is opened, the ironing pump 37 operates, and the medium in the medium tank 67 is sent through the tube 21.
- the medium to be sent flows into the incubator 38 through the paths indicated by arrows Jl and J, and becomes the culture medium 17 in the incubator 38.
- the operation of the ironing pump 37 is stopped, and the pinch valve 72 is closed.
- step S68-step S71 is processing executed when the number of cells has reached the value set by the operator in advance, and is the same as the processing of step S61-step S64 described above.
- Step S68 "Discharge medium"
- step S61 The same processing as in step S61 is performed. That is, the pinch valve 24 and the pinch valve 104 are opened, the ironing pump 101 operates, and the culture medium 17 in the incubator 38 is sent (discharged) to the waste liquid tank 102 through the tube 23. After the liquid transfer (discharge) is completed, the ironing pump 101 stops, and the pinch valve 24 and the pinch valve 104 close.
- Step S69 “Wash the incubator with buffer”
- step S62 The same processing as in step S62 is performed. That is, the pinch valve 105 is opened, the ironing pump 37 operates, and the buffer solution is injected into the incubator 38 from the buffer solution tank 68. After injection , The pinch valve 105 closes and the ironing pump 37 stops. The incubator drive motor 28 rotates, and the incubator 38 is rotated to allow the buffer solution to spread to the bottom of the incubator. Thereafter, the pinch valve 24 is opened, and the ironing pump 101 is operated to supply the buffer solution in the incubator 38 to the waste liquid tank 102.
- Step S70 "Inject cell peeling agent"
- step S63 The same processing as in step S63 is performed. That is, the pinch valve 73 is opened, the ironing pump 37 operates, and the cell releasing agent is injected into the incubator 38 from the cell releasing agent tank 69. After the injection, the pinch valve 73 closes and the ironing pump 37 stops. The incubator drive motor 28 rotates to spread the cell detachment agent to the bottom of the incubator.
- Step S71 "Inject neutralizer"
- step S64 The same processing as in 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 have adhered, the process proceeds to the next step S72.
- Step S72 “Discharge neutralized cell detachment agent”
- step 66 The same processing as in step 66 is performed. That is, the pinch valve 24 and the pinch valve 104 are opened, and the ironing pump 101 feeds (discharges) the medium to the operation 102. After the liquid transfer (discharge) is completed, the ironing pump 101 stops, and the pinch valve 24 and the pinch valve 104 close.
- Step S73 “Private is driven to transfer cells to container”
- the rotation motor 88 operates to rotate the arm 85.
- the pitcher up / down motor 90 operates, the pitcher arm 85 descends, and the needle 83 is inserted into the container 84.
- the pinch valves 24 and 103 are opened, and the ironing pump 101 operates.
- the cells after culture in the incubator 38 are sucked out, and the cells are fed (transferred) through the tube 23 in the direction of arrow P1. Then, it is injected into the cell storage container 84 through the tube 86.
- Step S74 "Transport cell storage container out of device"
- the shutter motor 81 operates, and the shutter 82 rises. After the predetermined amount has risen, 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 has a container containing pure cultured cells without contamination compared to before culture.
- step S53 If the pre-cultured cells placed in the container 52 in step S53 are cells contained in the bone marrow fluid, the following steps are necessary to remove unnecessary cells (blood-related cells) other than the target cells. Between step S56 and step S57.
- Step S62 Step S63 ⁇ Step S64 ⁇ Step S66
- 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. 6 (a). For example, stop slowly once in the forward direction, once in the reverse direction, and at the last stop in the forward direction. That is, by shortening the initial acceleration time and deceleration time (tl, t2, t3, t4, t5), the culture medium 17 of the incubator 38 vibrates violently and becomes suspended. Furthermore, by increasing the deceleration time (t6) of the last operation, the culture medium slows down while continuing to flow in the circumferential direction due to its inertia, and eventually stops. This allows the cells to be spread evenly.
- the last deceleration time (t6) may be an S-shaped curve.
- FIG. 6 (b) is a schematic diagram of a simulation result showing a seeding state of cells.
- the area near the dark center (inner circumference S2) shows that the cells are relatively aggregated due to the low tangential velocity of the flow in the last operation (deceleration time t6), and the outer circumference S1 is This shows that the cells are seeded thinly.
- the number of repetitions of forward / reverse rotation, rotation speed, and angular acceleration (tl, t2, t3, t4, t5, t6) are not particularly limited, and cells may be seeded evenly over the front of the incubator 38 depending on conditions. it can.
- FIG. 7 is a diagram showing a first modification of the incubator 38 of the cell culture device according to the above-described embodiment.
- FIG. 7 (A) is a diagram viewed from the top, and FIG. (B) shows a side view thereof.
- the incubators 170a to 170d in this figure are formed in substantially the same columnar shape, and the tube connecting members 174a to 174d of the incubators 170a to 170d are connected by a tube 171.
- the incubators 170a and 170c are omitted.
- This tube 171 performs the same function as the tube 21 in FIG.
- Tube connecting members 175a-175d discharge the old medium.
- the incubator 170a 174d had four forces.
- the number of incubators is not limited to four, and two, three, and six may be freely selected. Moreover, you may arrange
- the culture area can be freely changed. This makes it possible to adjust the number of cells in cell culture where the number of cells that can be cultured is often in a proportional relationship with the area when the cells are adherent cells (for example, mesenchymal stem cells).
- the operation is the same as the processing flow of FIG. 5.
- the culture medium in the incubator 170a-174d finally flows in the direction of the arrow Q. The cells in 174d will be uniformly seeded.
- FIG. 8 is a diagram showing a second modification of the incubator 38 of the cell culture device according to the above-described embodiment.
- Incubators 167, 168, and 169 shown in FIG. 8 (A) are examples of an incubator suitable for application to cell culture when such characteristics are remarkable.
- the incubator 167 has a substantially circular petri dish structure, and a supply tube 182 having the same function as the supply tube 21 in FIG. 2 is connected to the upper surface thereof.
- the incubator 168 has substantially the same envelope structure as the incubator 167, but has a single culture auxiliary plate 189 provided therein.
- the incubator 169 has substantially the same envelope structure as the incubator 167, but has two culture auxiliary plates 191 and 192 provided therein.
- a waste liquid tube 185 is connected to the bottom of the incubator 169.
- the respective incubators 167, 168, 169 are connected by connecting tubes 183, 184, respectively, and the liquid feeding is controlled by pinch valves 186, 187, 188 provided on the way.
- These culture vessels 167, 168, 169, pinch valves 186, 187, 188 and force S are fixed to the rotor 22 shown in FIG.
- FIG. 8 (B) is a diagram showing the positional relationship between each of the incubators 167, 168, and 169 and the rotation center axis. As shown in FIG. In the direction shown By shuffling, the cells in the incubators 167, 168, 169 are uniformly seeded.
- FIG. 9 is a flowchart for explaining the operation of the cell culture device using the incubator of FIG.
- the operation using the incubator shown in FIG. 8 is similar to that shown in FIG. 5, and therefore differences from FIG. 5 will be described. 9, the same components as those in FIG. 5 are denoted by the same reference numerals, and the description thereof will be omitted.
- the culture medium is drained at the time of passage (step S61), the incubator is washed with a buffer solution (step S62), a cell detaching agent is injected (step S63), and a neutralizing agent is injected. (Step S64), the incubator was shuffled, the seeds were inoculated uniformly (Step S65), the neutralized cell exfoliating agent was discharged (Step S66), and a new medium was injected (Step S67). In other words, at the time of subculture, the cells that had grown in a colony that could not be transferred to a new incubator were evenly seeded by shuffling the incubator at that location, and cultured again until the target cells were obtained. . On the other hand, in the incubator of FIG. 8, after step S64, the process of “seeding the lower incubator” in step S90 is executed.
- the cells become confluent in the incubator 167, the cells are transferred to the incubator 168 thereunder by liquid transfer. Then, when the cells become confluent in the incubator 168, the cells are transferred to the incubator 169 thereunder by liquid transfer.
- the amount of medium to be injected into each incubator increase the amount of medium for each passage so that cultivation on a culture auxiliary plate can be performed. That is, in the first culture, the amount of the culture medium is the amount of culture in the incubator 167 only on the bottom surface of the incubator main body 180. In the culture after one passage, the culture auxiliary plate 189 is immersed in the incubator 168 so that the medium volume is sufficient.
- the cells can be cultured in both the incubator body 180 and the culture auxiliary plate 189.
- the culture medium in the incubator 197 is such that the culture auxiliary plate 190 and the culture auxiliary plate 191 are immersed.
- 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.
- 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.
- the number of incubators, the shape and size of each incubator, and the like are not limited, and each size may be changed to an ellipse or rectangle.
- the number of culture support plates is not limited to:!
- the incubator can be downsized as a whole, and thus the apparatus can be downsized.
- a plurality of mirrors 278, 279, 282, 283, 284, 285 are arranged between the respective incubators 167-169. These mirrors are for receiving the light emitted from the light source 281 at the CCD force camera 280.
- a filter 286 is provided.
- the CCD camera 280, the light beam 281 and the finoletter 286 are integrated like a unit 288, and are moved in the direction of the arrow V by a drive mechanism (for example, composed of a motor and a feed screw) not shown. I can do it.
- a drive mechanism for example, composed of a motor and a feed screw
- the CCD camera 280 and mirrors 278, 279, 282, 283, 284, 285 are used for IJ, and lateral force culture is performed.
- Container 167- 169 Observation power S These mirrors 278, 279, 282, 283, 284, 285f may be moved in the horizontal direction (X-axis direction) 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 cell culture device according to the above-described embodiment.
- the incubator of FIG. 10 differs from the incubator of FIG. 8 in that the incubator of FIG. 8 can be handled in the same manner as the incubator 38 of FIG. Structurally, it has a substantially circular petri dish structure, and a supply tube 197 having the same function as the supply tube 21 in FIG. 2 is connected to the upper surface.
- Incubator body 195 is covered with lid 199
- the inside thereof is provided with two culture auxiliary plates 201 and 202.
- the outline of the operation is the same as in Fig. 8.
- the amount of medium is increased at each power passage so that culturing can be performed using a culture auxiliary plate.
- the amount of medium is only the bottom surface of the incubator body 195.
- the culture after one passage use a medium amount that allows the culture auxiliary plate 201 to be immersed.
- the cells can be cultured in both the incubator main body 195 and the culture auxiliary plate 201.
- the culture medium volume should be such that the culture auxiliary plate 202 is immersed.
- the cells can be cultured on the three plates of the incubator main body 195, the culture auxiliary plate 201, and the culture auxiliary plate 202.
- downsizing can be realized as in FIG.
- the number of incubators, the shape and size of each incubator, and the like are not limited.
- the number of culture auxiliary plates is not limited to two. According to the embodiment shown in FIG. 10, the size of the incubator can be further reduced as a whole, and the size of the apparatus can be reduced.
- FIG. 11 is a diagram showing an example of a technique for uniformly seeding cells. This technique produces a favorable effect when applied to the incubator of the above-described embodiment.
- 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 reduces the impact of the drop of the liquid supplied from the supply tube connecting member 302 and prevents damage to the cells.
- These magnets 307, 308, 309, 310 are fixed to the frame 30 in FIG.
- the spherical spheres 303, 304, 305, and 306 are placed in the incubator body 300 (same as the incubator 38 in FIG. 2), and have a high non-toxicity to cells on the surface of the spherical magnetic material. Molecular plastics, ceramics, titanium, etc. These are S-coated. When the incubator 300 rotates, the spherical members 303, 304, 305, and 306 also roll in the incubator main body 300 to agitate the internal medium, thereby enabling uniform seeding of cells. In FIG.
- 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-shaped member 315 is provided on the lower surface side.
- the rod-shaped member 315 is placed in the incubator body 300 (same as the incubator 38 in FIG. 2).
- the rod-shaped magnetic material has a non-toxic polymer plastic, ceramic, titanium, etc. on the surface of the rod-shaped magnetic material. Is coated.
- Fig. 11 (B In the case where the rod-shaped member 315) is used, the same effect as that of FIG.
- FIG. 12 is a diagram illustrating an example of a method of connecting the incubator and the tube in the above embodiment.
- the incubator, the tube, the reservoir tank, and the like are 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 cut portions of the tubes 320 and 326, for example.
- the supply tube 323 having the needles 322 and 328 and the waste tube 329 are connected.
- 321 and 327 be sterilized with an anore cone before piercing the gold jewels 322 and 328.
- the handling of the tube becomes easier when setting the incubator to which the long tube is connected to the apparatus, and the operability is improved.
- the cells can be directly introduced into the incubator 325 by using the syringe 324.
- the rubber stoppers 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 method of sterilizing a part of the culture device in the above embodiment.
- the incubator 38 and each of the tanks 71, 70, 69, 68, 67, 102 are connected to each of the tubes by force S, and the whole is enclosed in a sterile bag as shown by the arrow S, as it is. It is designed to be sterilized from gamma rays.
- the sterilization bag 340 is made of a material that prevents contact with the outside air, and may be a commonly used one. By sterilizing anything that touches the cells in this way, there is no need to remove the tube during culture, and there is no risk of contamination.
- reference numeral 400 denotes a liquid level detecting means for detecting the liquid level in the incubator 38 using light or ultrasonic waves. If the pump or pinch valve malfunctions, the liquid level deviates from the set position. In such a case, an alarm is issued to the outside.
- FIG. 14 is a diagram showing a detailed view of a mechanism according to another embodiment of the cell culture apparatus to which the present invention is applied.
- This cell culture device has basically the same configuration as that of FIG. Therefore, in FIG. 14, the same components as those in FIG. 2 are denoted by the same reference numerals, and the description thereof will be simplified.
- the incubator 140 includes an incubator body and a lid member.
- the incubator 140 needs to be capable of observing the cells in the culture with a microscope or the like, so that a transparent material is preferable and non-toxic. For these reasons, the material is preferably polystyrene (PS) or polyethylene terephthalate (PET).
- This lid member is provided with three first ports 141, second ports 142, and third ports 143 for injecting and discharging chemicals.
- the first port 141 is a port for discharging a drug such as a medium or cells after culturing.
- a waste liquid tube 141b is connected to the first port 142, and ironing pumps 144 and 145 for discharging the medium are arranged so that the medium can be discharged.
- the second port 142 is a port for supplying a drug such as a culture medium and cells before culturing.
- a supply tube 142b is connected to the second port 142, and ironing pumps 146 and 147 are provided in the same manner as the first port 141.
- the third port 143 is a port for sucking air 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 suction port to the inside of the incubator 140, and may not be particularly provided with a port as long as it achieves this purpose.
- the pulling force of the hook 149 is applied to the bottom of the holding ring 148 that holds the incubator 140, and the lever 150 and the tilt motor 151 lift the inclining device to tilt the entire incubator 140. I am trying to do it.
- the incubator 140 is held by a holding ring 148 fixed to a rotor 153 in an insulated box (incubator) 160.
- the rotor 153 is provided by an incubator drive motor provided above the insulated box 160. It is connected to the output shaft 29a and is configured to be driven to rotate in the direction of arrow E.
- FIG. 14 shows a state in which the rotor 153 has turned clockwise (leftward) and the holding ring 148 and the incubator 140 have moved to the left side of the heat insulation box 160. Accordingly, the rotor 153 rotates counterclockwise (rightward) from this state, so that the retaining ring 148 and the incubator 140 move to the right side of the heat insulation box 160 after passing in front of the drawing.
- the heat insulation box 160 is configured by a simple method that does not have a double box structure as in the related art and does not need to consider airtightness. The 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. It is connected to the.
- the supply tube 142b is provided above the rotor 153 inside the heat insulation box 160, and freely moves inside according to the rotation of the rotor 153.
- the other end of the supply tube 142b is connected to the heating bag 170.
- the heating bag 170 heats the medium passing through the supply tube 142b from 4 degrees to about 20 degrees, and includes a heating heater 171 on the back surface.
- the shape of the heating bag 170 is not limited as long as the temperature of the medium can be increased.
- the tube may be wound in a spiral shape.
- the medium tank 67 stores an unused medium
- the buffer solution tank 68 stores a buffer solution
- the Itoda cell stripping agent tanks 69, 70, and 71 store a cell stripping agent.
- FIG. 15 only the cell detachment agent tank 69 is shown, and illustration of the cell detachment agent tanks 70 and 71 is omitted.
- Each of the tanks 67, 68, 69, 70, 71 is provided in a heat insulating box 80 and is open.
- a heat radiation heat sink 110 is provided outside and a heat absorbing heat sink 111 is provided inside the heat insulation box 80 via a Veltier element 109 on the surface of the heat insulation box 80, and heat is exchanged and maintained at a constant temperature.
- the pinch valves 72, 105, 73, 74, and 75 control the liquid supply to the supply tubes 142c from the tanks 67, 68, 69, 70, and 71.
- Tubes taken out of the tanks 67, 68, 69, 70, 71 are connected to a supply tube 142c, and can be fed by ironing pumps 146, 147.
- the ironing pumps 146, 147 are pumps that sandwich a tube between rollers and feed the liquid in the tube by rotating the rollers. Immediately after the ironing pumps 146, 147, pinch valves 146a, 147a for adjusting the liquid feed are provided.
- No means is provided for measuring the amount of liquid sent by the pumps 144 and 145, and the amount of liquid is determined by the operating time of these pumps.
- the pinch valves 66a and 66b are for controlling the injection of the cells before culture into the supply tube 142b, and are provided two in the auxiliary supply tube 142d.
- the two pinch valves 66a and 66b are provided side by side to prevent outside air from flowing through the auxiliary supply tube 142d after cell injection.
- One end of the supply tube 142c is connected to the heating bag 170, and the other end is connected to the pinch valve 172.
- the air filter 173 is inserted into the heat insulation box 160 via an air filter 173 at its end.
- An air circulation path is formed by 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. That is, the ironing pumps 146, 147 rotate to send out the air in the supply tubes 142b, 142c, thereby circulating the air in the incubator 140.
- One end of the waste liquid tube 141b 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 inside the heat insulation box 160, and freely moves inside according to the rotation of the rotor 153.
- the waste liquid tube 141b is bifurcated on the way, and is sent to the waste liquid tank 102 or the container 84 for storing the cultured cells via each path. That is, one of the branches of the waste liquid tube 141b 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 connected via the pinch valve 174 and the ironing pump 144. Connected to the container 84 via the waste liquid tank 102 or the pinch valve 174, the ironing pump 144, and the pinch valve 175.
- the waste medium from the cells is eluted, and the old culture medium produced by the decrease in nutrients in the culture medium is stored in the waste liquid tank 102 in the waste liquid collection box through the waste liquid tube 141b by the ironing pump 144. .
- the old culture medium is sent to the waste liquid tank 102 by the ironing pump 145 through the waste liquid tube 141b and the pH measuring unit 177.
- the pH measuring section 177 allows the calibration liquid stored in the calibration liquid tanks 161 and 162 provided in the heat insulation box 160 to pass through the pinch valves 163 and 164 before measuring the pH of the waste liquid. In this way, the pH is calibrated to the reference value, and then the waste liquid is passed to measure the pH. The details of the pH measuring section 177 will be described later.
- the container 230 for storing cells before culture is supported by a holder 232 provided eccentrically with respect to the rotation axis of the motor 231. By rotating the motor 231, the pre-cultured cells in the container 230 are sufficiently suspended.
- a cap 233 made of a rubber material is provided on the upper surface of the container 230 to cover the outside air force. Alcohol disinfection inside cap 233
- a nonwoven fabric 234 impregnated with the liquid is provided, and a cover 235 is provided so as to cover the entire cap.
- the needle 236 is connected to the pipe member 238 in the container 230 via the cap 233 and the nonwoven fabric 234, is fixed to an arm (not shown), and can move linearly in the direction of arrow D1.
- the needle 237 supplies the atmosphere into the container 230, and has an air filter 239 at an end thereof.
- the function of the needle 237 is to prevent the cells from being sucked due to negative pressure inside when the container 230 is formed of a hard plastic material.
- the pre-cultured cells are sucked by the ironing pump 147, but the liquid may be sent by forcing air from the needle 237 into the container 230.
- the detailed configuration of the container 230 for storing cells before culture and the needles 236 and 237 will be described later.
- the container 240 for storing cells after culturing is supported by a holder (not shown).
- a cap 241 made of a rubber material is provided on the upper surface of the container 240, and is covered from outside air.
- a nonwoven fabric 244 impregnated with an alcohol disinfectant is provided inside the cap 241, and a cover 245 is provided so as to cover the entire cap.
- the needle 246 penetrates into the container 240 via the cap 241 and the non-woven fabric 244, and is capable of sending cells after culturing.
- the needle 246 is fixed to an arm (not shown) and can move linearly in the direction of arrow G1.
- the needle 247 is for discharging air from the container 240 and has an air filter 249 at the end. The function of the needle 247 is to prevent a negative pressure inside the container 240 from sucking cells when the container 240 is formed of a hard plastic material.
- the cells After the culture, the cells are sucked by an ironing pump 144, but the cells may be sent by discharging the air in the container 240 from the needle 247, or the air may be pumped into the heat insulation box 160. Thus, the liquid may be sent.
- the detailed configuration of the container 240 for storing cells after culturing and the needles 246 and 247 will be described later.
- the light source 34a irradiates light into the heat insulation box 160 above the heat insulation box 160, and includes a filter and the like on the light emission side.
- the CCD camera 31a is equipped with a lens, and is used to observe cells cultured in the incubator 140 from an observation window 32a provided below the heat insulation box 160, and to determine the timing at the time of passage. It is used for The light source 34a In order to prevent uneven brightness of the image, it is preferable to use a type in which a plurality of LEDs are arranged flat, but if there is sufficient light quantity, a single LED or lamp may be used.
- an ND filter for reducing the amount of light incident on the CCD camera 31a and an appropriate bandpass filter for obtaining a contrast suitable for cell observation are configured.
- This filter may be provided on the front of the CCD camera 31a.
- the ND filter is preferably in front of the CCD camera 31a, and the band-pass filter is preferably in front of the light source 34a in the case of cutting short-wavelength light harming cells.
- the light source 34a and the CCD camera 31a are provided so as to be movable in a direction perpendicular to the paper surface of FIG.
- the light source 34a and the CCD camera 31a are provided movably via the rollers 34c, 34d, 31c, 31d with respect to the renoles 34b, 31b extending in the direction perpendicular to the figure.
- a desired position of the incubator 140 can be observed by the rotating incubator 140, the light source 34a moving in the vertical direction, and the CCD camera 31a.
- the heaters 201-204 are for keeping the inside of the heat insulation box 160 at a constant temperature based on the temperature detected by the temperature sensor 106 provided in the heat insulation box 160.
- heat spreaders 205 and 206 for heat diffusion are provided on the heaters 201 and 204 along the side surfaces of the heat insulation box 160.
- the fan 65 is for stirring the air in the heat insulation box 160.
- the joint 107 is provided with a filter for removing impurities when supplying a gas mixture in which the ratio of carbon dioxide, nitrogen and oxygen is controlled.
- the carbon dioxide sensor 205 detects the carbon dioxide in the heat insulation box 160 and keeps it constant.
- the carbon dioxide sensor 205 keeps a predetermined amount of carbon dioxide from the carbon dioxide cylinder 210 through the regulator 211 and the solenoid valve 212. It can be supplied in the box 160.
- the multiple solenoid valve 213 is controlled by using the carbon dioxide gas 210 force and the carbon dioxide gas sent out through the reregulator, and the pinch valves provided at various parts of the tube are controlled. I control it.
- FIG. 15 is a diagram showing details of the incubator used in FIG.
- the incubator 140 serves as an incubator body 140a and a lid member 140b.
- the lid member 140b is provided with three first ports 141, a second port 142, and a third port 143 for injecting and discharging a chemical, and each port has a waste liquid tube 141b and a supply tube 142b. And tube 143a It has been continued.
- FIG. 16 is a diagram showing details of the first port 141, the second port 142, and the third port 143 in the incubator.
- the first port 141 is a port for discharging a drug such as a medium or cells after culturing.
- the first port 141 is provided with a tube member 141a so as to project into the incubator 140.
- the tube member 141a is cut diagonally so that the medium 140c can be sucked even when the tip of the tube member 141a touches the bottom surface of the incubator body 140a.
- a waste liquid tube 141b is connected to the outside of the first port 142, and ironing pumps 144 and 145 for discharging the culture medium are arranged so that the culture medium 140c can be discharged.
- the second port 142 is a port for supplying a drug such as the medium 140c and cells before culturing.
- a supply tube 142b is connected to the outside of 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 a port for inhaling air into the incubator 140, and an air filter 143b is connected to the outside thereof via a tube 143a.
- the air filter 143b has a role of preventing fine particles such as bacteria from entering the inside of the incubator 140, and is configured by enclosing a filter having a pore diameter of about 0.5 ⁇ . . If this pore size completely shuts out the invasion of bacteria, it should be 0.22 / im.
- a filter similar to the air filter 143b may be connected to the tip of the tube 143a, and air may be sent into the incubator 140 by an ironing pump. In this case, air can be positively sent into the incubator 140.
- the third port 143 is an air suction port to the inside of the incubator 140, and may not be provided with any port as long as it achieves this purpose. For example, a part of the lid member may be cut out and a gas permeable membrane may be attached. Further, a plate member may be provided inside the incubator 140 to increase the bottom area. In this way, in the case of adhesion (scaffold) -dependent cells that adhere to the bottom surface and grow in a single layer, the number of cells to be grown can be increased.
- FIG. 17 is a diagram showing a partial cross section of FIG. 15, and is a diagram showing a case where the culture medium in the incubator is discharged.
- the pipe member 141 Raise the side without la relatively to the angle ⁇ with respect to the horizontal plane.
- the culture medium 140c and cells 140d can be discharged without opening the lid member 140b of the incubator 140 by aspirating the liquid such as the culture medium 140c inside the incubator 140 from the tube member 14 la.
- the mechanism for raising the part without the tube member 20 relative to the one with the tube member 141a is not particularly limited, but in the cell culture device of FIG.
- the bottom side of the holding ring 148 holding the incubator 140 is The incubator 140 is inclined by pulling the hook 149 to the holding ring 148 where the tube member 141a does not exist, and lifting the hook 149 with the lever 150 and the tilt motor 151. That is, a fulcrum shaft may be provided on the side where the tube member 141a is located, and the tilting mechanism may be realized by lifting up the side without the tube member 141a, or may be performed manually.
- reference numeral 400 denotes a liquid level detecting means for detecting the liquid level in the incubator 140 using light or ultrasonic waves. If the pump or pinch valve malfunctions, the liquid level will deviate from the set position. In such a case, an alarm is issued to the outside.
- FIG. 18 is a diagram showing a detailed configuration of the heat insulation box in FIG. 14.
- FIG. 18 (A) shows the internal structure of the heat insulation box in an easy-to-understand manner
- FIG. 18 (B) shows the heat insulation box. It is an external appearance perspective view of.
- FIG. 19 is a diagram showing a cross section of the S-S plane showing details of the heat insulation structure in FIG. 18 (A).
- the heat insulation box 160 is a constant temperature bath for cell culture, 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.
- the incubator 140 turns inside the heat insulation box 160 as indicated by arrows A1-A2 according to the turning operation of the rotor 153.
- the heat insulation box 160 includes a housing 160a serving as an outer box, and an inner box 160b disposed inside the housing 160a with a predetermined space.
- the material of the housing 160a and the inner box 160b is preferably plastic such as stainless
- 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.
- the first heat insulating material 160c a material having relatively excellent heat insulating performance such as foamed urethane is preferable.
- soft urethane foams are more preferable even for foamed urethane. It is good to be formed thinner than the heat insulating material 160d.
- Thermal diffusion plate 160e, 160 f has a substantially U shape 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, heat diffusion plates 160e and 160f are not provided for the portions necessary for observation. I am trying to do it.
- the material of the heat diffusion plates 160e and 160f is made of an aluminum or brass plate having a high thermal conductivity.
- the heat diffusion plates 160e and 160f are adhered with double-sided tape or the like so as to cover the left and right side surfaces of the first heat insulating material 160c.
- panel-type heaters 160g and 160j are similarly adhered to the bottom and side surfaces of the heat diffusion plates 160e and 160f with a double-sided tape or the like.
- the amount of heat leaking to the outside by transmitting the second heat insulating material 160d becomes a leakage loss outside the heat insulation box.
- the second heat insulating material 160d it is important for the second heat insulating material 160d to enhance the heat insulation performance as much as possible.
- hard foam urethane or vacuum heat insulating material such as foam urethane
- foam urethane is put in an aluminum pack, and this pack is used. It is preferable to use a material having a vacuum inside and a plate shape.
- a door 160k having the same heat insulation structure is supported by the heat insulation box 160 so as to be freely opened and closed as indicated by an arrow 160 ⁇ by a hinge 16 Om.
- FIG. 20 is a diagram showing a control block of the heat insulation box 16 of the cell culture device of FIG. 14, and shows a portion necessary for explanation from FIG. 4 and omits the others. 20, the same components as those in FIG. 4 are denoted by the same reference numerals, and the description thereof will be omitted.
- the console 22 includes an operation switch, a temperature setting switch, and the like.
- the control unit 11 is connected with heaters 160g-160j, an incubator drive motor 29a, and a temperature sensor 106.
- the temperature sensor 106 is a temperature sensor using a known technology such as a thermocouple.
- the control unit 11 takes in the temperature data of the temperature sensor 106, compares it with the set temperature, and supplies electric power according to the difference to the heater 160g—160j. Give to.
- the acquisition of the 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 larger than the set temperature, the power supplied to the heaters 160g to 160j is reduced.
- the calorific value of the heated heater 160g 160j is transmitted through the heat diffusion plates 160e and 160f, and heats the inside of the heat insulation box 160. Since the heat transfer amount is smaller, most of the heat amount of the heaters 160g-160j contributes to heating the inside of the heat insulation box 160.
- the incubator drive motor 29a is for rotating to inoculate cells inside the incubator 140 evenly.
- the heat insulation box can be configured by a simple method and configuration that does not require the conventional double box structure and hardly considers airtightness. Further, by setting the amount of heat transmitted through the first heat insulating material 160c to be smaller than the amount of heat transmitted through the second heat insulating material 160d, the energy for heating can be suppressed.
- the heat diffusion plates 160e and 160f are provided with cutouts on a surface that vertically overlaps the culture surface of the incubator 140, so that radiant heat directly to the incubator can be suppressed. Not only that, the temperature inside the incubator can be kept more constant.
- FIG. 21 and FIG. 22 are diagrams showing the detailed configuration of the pH measuring unit.
- FIG. 21 is an enlarged view of a part of FIG. 14, and
- FIG. FIG. 3 is a diagram showing a detailed configuration of a unit.
- the pH (pH) measurement is performed by visually judging a change in the color of a cell culture solution containing a pH indicator (phenol red) or by automatically performing a visual judgment (described in JP-A-62-115297).
- the presence of a pH indicator unrelated to cell culture in the cell culture solution was not preferable because it affected the cell culture and was inferior in accuracy.
- a pH measuring unit 177 using a film-shaped pH sensor membrane is provided in the middle of the flow path to the incubator waste liquid tank, and the pH is measured using the waste liquid from the incubator 140. I did it.
- the pH measuring unit 177 includes a light emitting element (LED) 177a that emits light having a wavelength of about 570 [nm] and a light emitting element that emits light having a wavelength of about 770 [nm]. 177b, and a photodetector 177c that receives light from each of the light-emitting elements 177a and 177b, which is transmitted and reflected by the pH sensor film 177d.
- LED light emitting element
- a reflective indicator dye film FR-PR type (phenol red) is used as the pH sensor film 177d.
- This pH sensor membrane 177d changes color according to pH.
- This is a so-called film-type optical chemical sensor that can determine pH by spectroscopically measuring the transmitted light and reflected light.
- the pH sensor membrane 177d is provided on a sensor holder 177e formed of a transmission member between a waste liquid tube 141c branched from the waste liquid tube 141b and a waste liquid tube 141d connected to the waste liquid tank 102.
- the waste liquid from the waste liquid tube 141c passes through the inside of the sensor holder 177e holding the pH sensor membrane 177d and is sent to the waste liquid tube 141d.
- the pH sensor membrane 177d is immersed in the waste liquid.
- the transmitted light and the reflected light from the pH sensor membrane 177d immersed in the waste liquid are received by the photodetector 177c, and the change in the absorption spectrum is measured to measure the pH.
- the calibration liquid is stored in the calibration liquid tanks 161 and 162 provided in the heat retaining box 160, the calibration liquid in the calibration liquid tanks 161 and 162 is sent to the waste liquid tank 102 by the ironing pump 145. As a result, the calibration liquid passes through the pinch valves 163 and 164, and passes through the wafer measuring section 177, so that the pH sensor membrane 177d is calibrated to the pH reference value by the calibration liquid. After calibration, the waste liquid is passed through and the pH is measured.
- the calculation of pH is performed according to a pH value calculation algorithm described below.
- the PD ⁇ current and the offset of the amplifier and ADC are corrected based on the following equation (1).
- Id is the baseline signal at the time of light interruption
- Iraw is raw measurement data
- I and Iraw are both functions of wavelength.
- the relative transmittance T and the raw relative absorbance Araw are calculated based on the following arithmetic expressions (2) and (3).
- 12 is the I value of pH calibration solution 2 (pH2). Therefore, it is a function of the relative transmittance T and the relative absorbance Araw ⁇ .
- S indicates sensitivity
- A1 is the A value of pH standard solution 1 ( ⁇ 1). If pH1 and pH2, A1 will be a negative value. Therefore, the sensitivity S is expressed by the following arithmetic expression (9).
- ⁇ ⁇ ( ⁇ 1- ⁇ 2) / ⁇ 1 + ⁇ 2
- ⁇ is the relative absorbance of the unknown sample.
- A is a negative value for pH1 and pH2.
- I2 (Xl) I2raw ( ⁇ l) -Id
- I2 (2) I2raw ( ⁇ 2) -Id
- Il (l) Ilraw ( ⁇ l) -Id
- AlrawU 1) log (I2 (1) / ⁇ 1 ( ⁇ 1))
- ⁇ Net absorbance ⁇ in the case of the calibration solution 1 is as follows according to the above-mentioned equation (4).
- Al Alraw ( ⁇ 1) -Alraw ( ⁇ 2)
- a calibration curve is derived in accordance with the above arithmetic expressions (6) and (9).
- Iraw (A1) and Iraw (2) are measured for the unknown sample, and the net absorbance A is obtained by the same procedure as described above.
- the pH measurement unit uses waste liquid, the cells in the culture apparatus do not come into contact with the PH sensor. Also, since the calibration liquid flows through the waste liquid flow path, it can be injected into the incubator. None, Therefore, it is possible to avoid direct contact between the cells, the sensor and the calibration solution, and there is no possibility of causing sterilization problems. In addition, since the pH measurement unit is not provided in the culture device but provided in the middle of the waste liquid flow path, the mechanical structure of the device can be reduced in size and simplified.
- the configuration is not limited to providing the pH measurement unit 177 shown in FIG. That is, a change in the color of the cell culture solution may be observed.
- the aforementioned CCD camera 31 You can use a color camera and calculate. Needless to say, the tube 141c, the pinch valves 176, 163, 164, the pump 145, the tube 141d, and the calibration solution tanks 161 and 162 become unnecessary.
- the cell suspension or the cell suspension between the sterilized containers may be removed.
- improvements are being made to the cleanliness of the operating environment, such as inside a clean bench, and to reduce contamination from workers.
- the sterilized container is placed outside a clean environment, such as in a clean bench, that is, in a general environment in which contaminants such as microorganisms are suspended, alcohol may be removed in a clean bench.
- a series of operations such as sterilization of a container placed in such a general environment, extraction of a liquid from a sterilized container, and injection of a liquid into a sterilized container are performed by, for example, regenerative medicine.
- industrial cell production such as cell culture for cell growth, etc. Cell culture. For this reason, the need for skilled technicians for cell culture in such industrial cell production is not desirable because it is one factor that increases the cost of cell culture.
- the pre-cultured cell is used as a configuration that can simplify the operation of extracting a liquid from a container or injecting a liquid into the container while suppressing the generation of contamination.
- a needle 236, 237, and a container 240 and needles 246, 247 for storing cells after culturing will be described with reference to FIGS.
- FIGS. 23 and 24 are diagrams showing a schematic configuration of a stopper for containers 230 and 240, FIG. 23 (A) is a perspective view thereof, and FIG. 23 (B) is a sectional view thereof.
- FIG. 24 (A) is a top view showing a state in which an external closing member is removed
- FIG. 24 (B) is a bottom view showing a schematic configuration of a container stopper.
- FIG. 25 is a perspective view showing an example of a container provided with the container stopper.
- the container stopper (cap) 233 is made of a resin such as rubber or synthetic resin, and has a force at one end surface and a circular passage 233a penetrating the other end surface. — It is formed in a substantially cylindrical shape provided with 233d. Each passage 233a-233d has a different size.
- the hollow needles 236, 237, 246, and 247 for at least one of injecting a liquid into the containers 230 and 240 to which the ⁇ 233 is attached and extracting a liquid from the containers 230 and 240 are provided with the passages 233a to 233d of the 233 233. It becomes a passage for passing through a tubular body or a thin tube.
- the passage 233a is an entrance formed on one end face side of the plug 233.
- the passage 233b is connected to the entrance and is a storage chamber having a diameter larger than that of the entrance.
- the storage chamber 233b accommodates the sterilizing agent-impregnated members (nonwoven fabric) 234 and 244.
- the passage 233c is a through hole formed at the center of the partition plate 233e, which is the bottom of the storage chamber 233b.
- the passage 233d is provided on the other end face side of the stopper 233, and is accommodated through the through hole 233c of the partition plate 233e. It is an outlet communicating with room 233b.
- the outlet of the passage 233d of the stopper 233 also serves to attach the stopper 233 to the containers 230 and 240 in an airtight manner, and
- the inside diameter of 233d is formed to be the same as the outside diameter of cylindrical containers 230 and 240.
- the inner diameter of the outlet 233d can be set appropriately, and the outlet 233d is provided. There can be no configuration.
- the through hole 233c formed in the center of the partition plate 233e of the stopper 233 is formed with a diameter capable of passing through a tubular body such as a hollow metal pipe 236, 237, 246, 247 or a Itoda pipe. .
- the end face of the plug 233 on the side where the inlet part 233a is provided is made of an air-tight film or film so as to cover the end face.
- Occlusion members 235, 245 are attached.
- the external closing members 235 and 245 are provided to prevent evaporation of the germicide, for example, a laminated film obtained by laminating an aluminum foil and a synthetic resin film, a resin film made of rubber and silicon having elasticity and elasticity, and an elastomer resin. It can be formed of various materials that can be suppressed.
- the external closing members 235, 245 when a resin film or the like is used as the external closing members 235, 245, a tubular body or a thin tube such as a hollow needle 236, 237, 246, 247 is punctured into the external closing members 235, 245. Used. Even if a tube or a thin tube, such as a punctured hollow needle 236, 237, 246, 247, is pulled out of the external blocking member 235, 245, if the external blocking member 235, 245 is made of a resin having flexibility and elasticity, it is hollow. The holes formed in the external closing members 235, 245 by the tubes or thin tubes such as Kinju 236, 237, 246, 247 are almost closed.
- the outer closing members 235 and 245 are made of resin, they can be formed integrally with other portions of the plug 233 such as the passages 233a and 233d. In this case, the outer closing members 235 and 245 are formed only at portions corresponding to the entrances of the passages 233a to 233d.
- the disk-shaped germicide-impregnated members 234, 244 housed in the passage 233b of the stopper 233, that is, the storage chamber and formed into a disk shape, are held in the passage 233b by the partition plate 233e.
- the sterilization unit IJ-impregnated members 234 and 244 can penetrate and penetrate a tubular body or a thin tube such as a hollow needle 236, 237, 246, or 247. I have.
- bactericide-impregnated members 234 and 244 are formed from alcohol wool, the ability to impregnate alcohol is used.
- the disinfectant-impregnated members 234 and 244 can be formed by impregnating a nonwoven fabric or the like.
- the germicide impregnated members 234 and 244 can be formed by gelling the germicide, for example, an alcohol gel. If the germicide-impregnated members 234, 244 are formed by alcohol gel or the like, the germicide of the germicide-impregnated members 234, 244 flows out of the germicide-impregnated members 234, 244, and enters the container provided with the stopper 233. The influence on the contents of the container can be suppressed.
- the stopper 233 has a multi-lid-like structure, and is provided at an opening for taking in and out of a liquid in an appropriate container such as the containers 230 and 240 used in a state where the inside is sterilized. It is attached.
- the stopper 233 is not limited to the shape of the containers 230 and 240 illustrated in FIG. 25, but can be applied to containers of various shapes and can be applied to containers for various uses.
- a drug container containing a sterilized drug an incubator containing a culture solution, a centrifuge tube for collecting cells from a cell suspension by centrifugation, a tubular container, a rectangular container,
- the stopper 233 such as a flexible container such as an infusion bag can be applied to containers of various shapes and uses to be used after sterilization.
- a centrifuge tube for recovering cells from a cell suspension by centrifugation is used as a container 230, 240 with a stopper 233
- the interior of the container 230, 240 is sterilized.
- the cell suspension has been stored, but it is taken out of the clean bench for centrifugation and centrifuged in a general environment to be placed in the general environment. , A condition where the outer surface may be contaminated.
- the operator is required to remove the liquid without performing a sterilization operation on the outer surfaces of the containers 230, 240 with alcohol spray or the like.
- a tube or a thin tube such as a hollow needle attached to a syringe or the like for extracting or injecting a liquid is inserted into the passage 233a 233d of FIG. 233 as shown by a wavy arrow in FIG.
- the pipe or the thin tube inserted from the inlet of the passage 233a-233d of the stopper 233 contains a disinfectant. It is pierced by the immersion member 244 and penetrates the germicide impregnated member 244, and enters the inside of the containers 230 and 240 through the through hole 233c of the partition plate 233e.
- the outer surface of the pipe or thin tube may have been contaminated due to the effects of containers 230 and 240 that may have been contaminated on the outside due to being discharged from a clean environment such as a clean bench to the general environment.
- a clean environment such as a clean bench to the general environment.
- the sterilizing agent-impregnated member 244 is impregnated by penetrating the sterilizing agent-impregnated member 244 when a tube or a thin tube such as a hollow needle is passed through the stopper 233, the sterilizing agent-impregnated member 244 is impregnated.
- the outer surface of the tube or tubule can be sterilized with a germicide.
- the worker plugs the tube or thin tube without performing operations such as sterilizing the outer surface of the container by spraying alcohol on the containers 230 and 240 placed in the general environment, and wiping the alcohol around the stopper and opening the stopper.
- the extraction of the liquid from the containers 230 and 240 and the injection of the liquid into the containers 230 and 240 can be performed while suppressing the generation of contamination by merely allowing the liquid to pass through.
- the generation of contamination while simplifying the operation of extracting the liquid from the container and injecting the liquid into the container is simplified. Can be suppressed. Furthermore, by simplifying the operation of extracting liquid from the container and injecting the liquid into the container, the generation of contamination can be suppressed while a skilled technician can extract liquid from the container and It is not necessary to inject the liquid into the liquid. In addition, since operation by a skilled technician is not required, the cost of cell culture in industrial cell production, for example, cell culture for growing cells used for regenerative medicine, can be reduced. In addition, since there is no need for equipment and pipes to supply steam for sterilizing the plug as in the past, restrictions on the use of plugs and containers can be reduced, and the structure of plugs and other components can be simplified. .
- the container is placed in a general environment. Since it is not necessary to sterilize the placed container by spraying alcohol or remove the stopper from the container, it is necessary to automate the extraction of liquid from the container and the injection of liquid into the container using a liquid handling device. Becomes possible. And, by enabling automation, it is possible to prevent contamination by workers themselves and occurrence of contamination due to careless mistakes of workers.
- the end face of the passage 233a of the stopper 233 on the inlet side is capable of piercing a pipe or a thin tube, or is detachable, and the passage 233a is removable.
- the duration of the sterilizing effect of the sterilizing agent-impregnated member can be extended, and there is no need to open the container. For this reason, the operator can perform a series of operations such as injecting the liquid into the container and sampling the liquid from the container, that is, extracting the liquid, with sufficient time. Therefore, there is no need to require the worker to have a high level of skill in performing such a series of aseptic operations as soon as possible.
- FIG. 26 is a cross-sectional view showing a modification of the container stopper of FIGS. 23-25.
- the same components as those in FIGS. 23 to 25 are denoted by the same reference numerals, and the description thereof will be omitted, and different configurations, features, and the like will be described.
- the plug 2331 for the container in FIG. 26 is different from the plug 233 in FIGS. 23 to 25 in that the sterilizing agent impregnated member accommodated in the passage of the plug and the partition plate for holding the sterilizing agent impregnated member in the passage are provided. Is that an internal closing member that closes the passage is provided.
- the container stopper 2331 according to this embodiment has a shape of a passage including the inlet portion 233a, the accommodation room 233b, the through hole 233c formed in the center of the partition plate 233e, and the outlet portion 233d.
- Fig. 23 Same as stopper 233 in Fig. 25.
- a film-like internal closing member is formed so as to cover the surface of the partition plate 233e serving as the bottom surface of the accommodation room 233b and the surface serving as the side surface of the accommodation room 233b.
- 233f is provided.
- the disinfectant impregnated members 234, 244 It is accommodated in the accommodation room 233b while being covered with the internal closing member 233f.
- the inner blocking member 233f is installed between the germicide-impregnated members 234 and 244 and the surface of the partition plate 233e serving as the bottom surface of the storage room 233b and the surface serving as the side surface of the storage room 233b. It is a container-shaped member.
- the internal closing member 233f in Fig. 26 is made of a resin film having flexibility and elasticity, for example, a resin film such as an elastomer resin or a silicone or a natural rubber used for stoppers of containers for injections and the like.
- a resin film such as an elastomer resin or a silicone or a natural rubber used for stoppers of containers for injections and the like.
- the opening of the through hole 233c on the storage chamber 233b side is sealed, and the hole formed in the punctured hollow needle or other tubular body or thin tube remains almost closed even after the tube is pulled out.
- the internal closing member 233f is formed in a container shape so that the liquid can be held inside. It is also possible to dispel sterilizing agent attached to the outer surface of the tubular body or thin tube such as a hollow needle that has penetrated the bactericide-impregnated member 234 P 244.
- the central portion of the internal closing member 233f protrudes in the shape of a truncated cone that tapers toward the tip toward the inside of the storage chamber 233b.
- the portion protruding in the shape of a truncated cone forms a penetration portion 233g through which a tube or a thin tube such as a hollow needle is pierced.
- a hollow state continuous with the through hole 233c is formed so that a tube or a thin tube such as a hollow needle can be easily punctured and penetrated.
- the disinfectant-impregnated members 234 and 244 are set in the storage chamber 233b while being stored in the container-shaped internal closing member 233f. In addition, the germicide-impregnated members 234 and 244 are in a state where a portion corresponding to the penetrating portion 233g of the internal closing member 233f is dented.
- Such a stopper 2331 includes a container-shaped internal closing member 233f having a penetrating portion 233g protruding toward the storage chamber 233b, that is, the sterilizing agent-impregnated members 234, 244, and further includes an internal closing member 233f. Disinfectant impregnated members 234 and 244 are provided therein. Then, a step is formed so that the penetrating part 233g for piercing a tube or a thin tube such as a hollow needle is higher than the other part of the internal closing member 233f, and the top part of the penetrating part 233g having a smaller surface area than the other part. A tube or tubule such as a hollow needle is pierced into the part.
- the bactericide that has flowed out is unlikely to flow into the container through the through-hole 233c, so that a tube or a thin tube such as a hollow needle is punctured.
- the sterilizing agent is prevented from flowing out of the sterilizing agent impregnated member 5, and the discharged sterilizing agent is passed through the through-hole 233c. It is not necessary to perform operations while being careful not to flow into the container, and such operations can be facilitated.
- the entire inner blocking member 233f is formed of a resin having flexibility and elasticity, but at least only a portion through which a tubular body or a thin tube (not shown) such as a hollow needle passes is flexible.
- a configuration made of a resin having properties and elasticity may be employed.
- FIG. 27 is a sectional view showing another modified example of the container stopper of FIGS. 23-25.
- the same components as those in FIGS. 23 to 25 are denoted by the same reference numerals, and the description thereof will be omitted, and different configurations and features will be described.
- the plug 2332 for the container in FIG. 27 is different from the plug 233 in FIG. 23-FIG. 25 in that the sterilizing agent impregnated member accommodated in the passage of the plug and the partition plate for holding the sterilizing agent impregnated member in the passage are provided.
- An internal closing member for closing the passage is provided between the inside and the inside, and the shape of the internal closing member and the disinfectant-impregnated member are different. That is, the stopper 2332 according to this embodiment includes a circular film-like internal blocking member 233h covering the partition plate 233e on the storage chamber 233b side, and a plurality of granular sterilizing agents stored in the storage chamber 233b. And impregnating members 234a and 244a.
- the internal closing member 233h is made of a resin having flexibility and elasticity, for example, a resin such as an elastomer resin, silicone, or natural rubber used for stoppers of containers for injections and the like. It is a circular membrane that seals the opening of the through-hole 233c on the side of the storage chamber 233b, and the hole formed when the tube or thin tube such as a punctured hollow needle is pulled out is almost closed. . Also, in this internal closing member 233h, the through hole 233c is fitted at the center of the surface on the side of the partition plate 233e, that is, at a position corresponding to the through hole 233c of the partition plate 233e. A protrusion 233j is formed.
- the disinfectant-impregnated members 234a and 244a are formed of a granular member having water absorbency, for example, urethane beads, and are impregnated with the disinfectant.
- the internal closing member 233h suppresses the flow of the germicide into the container provided with the stopper 2332, and when the diameter of the germicide-impregnated members 234a and 244a is smaller than the diameter of the through-hole 233c, sterilization is performed.
- the agent impregnating members 234a and 244a are prevented from entering the container through the through holes 233c. This suppresses the effect of the germicide on the contents of the container provided with the stopper 2332.
- a plurality of the sterilizing agent-impregnated members 234a and 244a are accommodated in the accommodation room 233b in a granular form.
- tubes or tubes such as hollow needles that do not require puncturing of a tube or tube such as a hollow needle into a disk-shaped sterilizing agent-impregnated member sterilize the space between the plurality of sterilizing agent-impregnated members 234a and 244a.
- the outer surface of the material impregnated members 234a and 244a is passed through the plug 2332 while the outer surface is in contact therewith.
- FIG. 28 is a cross-sectional view showing still another modified example of the container stopper of FIGS. 23-25.
- the same components as those in FIGS. 23 to 25 are denoted by the same reference numerals, and the description thereof will be omitted, and different configurations and features will be described.
- the difference between the container stopper 2333 of FIG. 28 and that of FIG. 25-FIG. 27 is that the stopper 2333 on the outer surface of a tube or a thin tube such as a hollow needle is located inside the container more than the sterilant-impregnated member in the passage of the stopper. That is, a wiping member for wiping off the adhered liquid is provided. That is, the container stopper 2333 according to this embodiment is provided with a disk-shaped wiping member 233k provided in the outlet of the passages 233a to 233d.
- the wiping member 233k is provided in contact with the surface of the partition plate 233e opposite to the surface on which the germicide-impregnated members 234, 244 are installed, that is, in contact with the inner surface of the container.
- the exit side of the through hole 233c formed in the hole is closed.
- the wiping member 233k can puncture a tubular body such as a hollow needle or a thin tube. It is formed of a material capable of wiping liquid adhering to the outer surface of a tubular body or a thin tube such as an empty needle, for example, styrene foam, cotton, urethane, another filter, or a filter uniform member.
- the container stopper 2333 of this embodiment is configured such that a tube or a thin tube such as a hollow needle is punctured by the germicide-impregnated members 234 and 244 and penetrated by the wiping member 233k.
- a germicide such as alcohol adhered to the outer surface of a tubular body such as a hollow needle or a thin tube when penetrating the germicide-impregnated members 234 and 244. Therefore, the disinfectant can be prevented from being introduced into the container by attaching the disinfectant to the outer surface of a tube or a thin tube such as a hollow needle that penetrates the disinfectant impregnated members 234 and 244. The effect on the contents can be reduced.
- FIG. 29 and FIG. 30 are diagrams illustrating an outline of an operation when extracting a liquid from a container or injecting a liquid into a container.
- the container 230 provided with the container stopper 233 in FIG. 23 is used, and at least one of the functions of injecting liquid into the container and extracting liquid from the container, that is, at least one of the sampling device and the dispensing device is used.
- a liquid handling device that performs a function will be described. This liquid handling device is for transferring cells before culturing from the container 230 for storing cells before culturing to the incubator 140 and for transferring cells after culturing from the incubator 140 to the container 230 in FIG.
- the liquid handling device can be used to sample a cell suspension from the incubator 140, inhale and extract a drug solution, extract a liquid like ivy, and suspend the cell in the incubator 140 containing the culture solution. It fulfills at least one of the functions of a sampling device and a dispensing device, such as liquid injection such as seeding of cells by injection of a liquid or injection of a drug solution.
- a sampling device and a dispensing device such as liquid injection such as seeding of cells by injection of a liquid or injection of a drug solution.
- This embodiment exemplifies a case where a container 230 having a stopper 233 is used as shown in FIG. It is also possible to use a container provided with the holes 2331, 2332 and 2333 shown in FIG. 26 to FIG.
- the container 230 provided with the stopper 233 is held by the holder 292 on the pedestal 291.
- a tube or a thin tube 293 such as a hollow needle for injecting the liquid into the container 230 and / or extracting the liquid from the container 230 is supported by the support member 294.
- the support member 294 is connected via a drive mechanism 295 to a guide rod 295 that guides the vertical movement of the tube or the thin tube 293 by the drive mechanism 295.
- the guide rod 295 extends upward from the base 291 It is fixed to the base 291 in an extended state.
- the tube or the thin tube 293 is connected via a tube 296 to a pump 297 for performing at least one of suction and discharge of the liquid.
- the tube or capillary 293 corresponds to the needles 236 and 246 in FIG. 14
- the pump 297 corresponds to the ironing pumps 144 and 147 in FIG. 14
- the tube 296 corresponds to the tubes 142d and 141b in FIG. Become. 29 and 30, the motor 231 in FIG. 14 is omitted.
- the tube 196 is connected to one end of the tubular body or the thin tube 293 by a connecting portion 294a provided on the lower surface of the support member 294. Further, an opening of a bag-shaped covering member 298 that covers the tubular body or the thin tube 293 is hermetically attached to the connecting portion 294a.
- the covering member 298 is a bag-like member formed of a flexible, elastic and airtight film-like material capable of piercing the tubular body or the thin tube 293, for example, rubber, silicon, or an elastomer resin.
- the tube or the thin tube 293 such as a hollow needle is not inserted through the stopper 233, as shown in FIG. Since 293 is included in the covering member 298, it is in a state of being airtightly isolated from the environment outside the covering member 298. Therefore, even if the liquid handling apparatus is placed in a clean environment, that is, in a general environment that is exposed to the outside air, such as outside a clean bench, the pipe or the thin tube 293 in the covering member 298 is not contaminated by the outside air.
- the driving mechanism 295 moves upward along the guide rod 294.
- the tube or the thin tube 293 returns from the state of FIG. 30 to the state of being included in the covering member 298 as shown in FIG.
- the covering member 298 has flexibility and elasticity, the hole formed when the tubular body or the thin tube 293 is punctured is in a closed state, and the sealing property of the covering member 298 is maintained. Therefore, even after the tube or the thin tube 293 is removed from the container 230, contamination due to the tube or the thin tube 293 coming into contact with the outside air can be prevented.
- a container provided with a stopper 233 as shown in Fig. 23 is used, and further, a tube or a thin tube 293 such as a hollow needle is used. Is covered with a bag-like covering member 298 made of a flexible, elastic and air-tight film-like material capable of piercing the tubing. Since it can be suppressed, the generation of contamination can be suppressed more reliably. Further, this liquid handling apparatus is not limited to those having the configurations shown in FIGS. 29 and 30, and can be implemented in various modes as long as a pipe or a thin tube is covered with a covering member. Is included.
- the container plug 233 and the container 230 are not limited to the container plug of the above-described embodiment, and a passage through which a tube or a thin tube is inserted is formed.
- various types of stoppers can be applied as long as the stopper can penetrate the thin tube and is held by the sterilizing agent impregnated member impregnated with the sterilizing agent.
- a cell culture device using a pinch valve as shown in Figs. 1 and 14 applies a pressure for driving the pinch valve to an air compression device (air-conditioner) installed outside the cell culture device. It was taken in from the plant as compressed air or operated using an air compressor built into the cell culture device. When compressed air is taken in from the outside, piping from the air compressor to the cell culture device is required.In general, the piping is fixed to the wall, ceiling, etc. It was not possible because it became impossible. In addition, in the method using an air compressor built in the cell culture device, the installation place is not limited, but there are problems such as dust generation, vibration, and noise, which are not preferable.
- the gas in carbon dioxide cylinder 210 is A pinch valve provided at the place is used as compressed air for driving.
- the gas in the carbon dioxide cylinder 210 is sent to pinch valves provided at various places in the tube via the regulator 211 and the multiple solenoid valve 213.
- the pinch valve cannot be driven when the gas pressure of carbon dioxide is lowered, but the possibility is small because the use of carbon dioxide is essential during cell culture.
- the amount of carbon dioxide gas used in the pinch valve is about 0.5 cc per operation, which is considerably smaller than the amount of carbon dioxide gas used in the heat insulation box 160. In the embodiment shown in FIG. 1, the pinch valve is similarly driven.
- the pinch valve is controlled to open and close at an arbitrary timing for an arbitrary time, thereby injecting a necessary amount of a necessary reagent into the incubator 140 or draining the culture medium. Or you can.
- the carbon dioxide tube 210 supplies carbon dioxide gas to each pinch valve through an air tube (pipe indicated by a dotted line in FIG. 14).
- a predetermined amount of carbon dioxide is supplied from the carbon dioxide cylinder 210 into the heat insulation box 160 via the regulator 211 and the solenoid 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 the carbon dioxide gas used for driving the pinch valve and supplied to the heat insulation box 160 is It is used to adjust the concentration of carbon dioxide in the heat insulation box 160.
- the number of air cylinders for driving the pinch valve can be increased as long as the pressure applied to the air cylinders meets the specifications.
- the carbon dioxide gas from the carbon dioxide cylinder 210 is connected to the regulator 211 by an air tube, but, although not shown, a sterilizing filter for removing bacteria, a mist filter for removing moisture, and the like are provided. Is provided.
- An air cylinder for driving a pinch valve usually has two tubes, and the air cylinder operates by supplying carbon dioxide gas to one of them, closing the pinch valve and supplying air to the other by supplying carbon dioxide gas to the other.
- the cylinder is configured to operate in the opposite direction and open the pinch valve.
- the multiple solenoid valve 213 switches the supply of such carbon dioxide gas.
- the injection and cutoff of the carbon dioxide gas supplied to the heat insulation box 160 is controlled by the electromagnetic valve 212. Normally, the concentration of carbon dioxide in the insulated box 160 is measured using the carbon dioxide sensor 205, and if it is lower than the target value, the solenoid valve 212 is opened, If it is high, the solenoid valve 212 is closed to keep the carbon dioxide concentration in the heat insulation box 160 constant.
- any non-flammable gas described in the example of the carbon dioxide cylinder can be used in the same manner.
- FIG. 31 is a block diagram showing a schematic configuration of the camera imaging system.
- the camera photographing system 310 performs various processes on image data obtained from the CCD camera 31 for photographing the incubator 140 in the heat insulation box 160 and displays the image data so that cells and the like can be confirmed. 311; an image processing unit 312; a motor controller 313;
- the positional relationship between the incubator 140 and the CCD camera 31 is as shown in FIG. 14, and the light from the light source 34a provided on the upper side of the heat insulation box 160 is transmitted to the observation window 32a provided on the lower side of the heat insulation box 160.
- Through the CCD camera 31a Through the CCD camera 31a.
- For the cell culture device in Fig. 1 Are assumed to have the same positional relationship.
- the converter 311 converts image data captured by the CCD camera 31 into an electric signal to be transferred to the image processing unit 14.
- the image processing unit 14 performs various processes on the electric signal input from the converter 311 and converts the electric signal into an image in which cells can be easily viewed.
- the motor controller 313 controls the relative positional relationship between the CCD camera 31 and the incubator 140 to a desired relationship based on the processed image, and the camera 'incubator driving device 314 controls the CCD camera 31 And the incubator 12 are moved to achieve the desired relationship.
- FIG. 32 is a diagram schematically showing a portion related to the camera photographing system in FIG. 14 extracted. That is, this figure is a diagram showing the most typical control mechanism for controlling the relative positional relationship between the CCD camera 31 and the incubator 140.
- the CCD camera 31 When the incubator 140 is fixed in the insulated box 160, the CCD camera 31 is moved with respect to the incubator 140 to control the relative positional relationship between the two.
- the CCD camera 31 may be fixedly provided as shown in FIG. 1 and the incubator 140 (or the internal mirror) may be moved, or both may be moved simultaneously as shown in FIG. You may.
- FIG. 32 illustrates a case where the CCD camera 31, the incubator 140, and the guide 315 for moving the CCD camera 31 are closed in the heat insulation box 160.
- a light source 34 serving as an illuminating device for illuminating cells in the incubator 140
- a motor 29a driven by the camera's incubator driving device 314 a converter 311 for converting an image signal into an electric signal, etc. May be included in the heat insulation box 160.
- the incubator 140 may be a flask or a culture dish usually used for culturing, or disclosed in Japanese Patent Application No. 2002-180120, Japanese Patent Application No. 2003-027710, or Japanese Patent Application No. 2003-420510. It may be of such a configuration.
- the CCD camera 31 may be an optical imaging device, such as a CCD or a CMOS, or any other device that can acquire electrical, electronic, or optical signals. Les ,.
- the lens 316 can be attached to the CCD camera 31, and the lens 316 can be interchangeable or fixed.
- a lens exchange mount for a CCD camera for example, a screw mount, commonly called a C mount, or a bayonet mount can be applied.
- magnification refers to an imaging magnification determined by the imaging element size of the camera, the focal length of the lens, and the imaging distance.
- the field of view is also determined by the size of the image sensor of the camera, the aperture or shutter aperture of the camera, and the angle of view of the lens.
- the lighting device is not shown, but there are a method of making the incubator transparent and photographing the transmitted light irradiated from the back side, and a method of irradiating the light from the camera side to photograph the reflected light. .
- the lighting device is arranged below the incubator.
- the lower part of the heat insulation box 160 does not necessarily need to be placed in the heat insulation box 160 and has a light transmissive structure, it may be outside the heat insulation box 160. If it is placed outside the heat insulation box 160, it can be repaired and replaced without risk of contamination in the event of a failure or a broken ball.
- the light source preferably does not have a wavelength component harmful to the cells to be cultured.
- UV light is said to damage cellular DNA and trigger UV-induced apoptosis, resulting in cell carcinogenesis. Therefore, when culturing ordinary cells, it is necessary to avoid including such components as a light source.
- infrared rays can generate stress on cells because they generate heat.
- light of a specific wavelength may activate cells, it is possible to actively control the wavelength of the light source to be advantageous for culture. It is desirable that the wavelength of the light source and the component ratio can be changed according to the cells to be cultured and the purpose thereof. Specifically, by interposing a filter between the light source and the incubator to block ultraviolet components that are not desirable for cell culture as described above, or preparing multiple light sources such as strong monochromatic light and LEDs It is also possible to create a light source containing an arbitrary wavelength component by selectively turning it on and off. Alternatively, the wavelength can be selected by using a 3-wavelength fluorescent tube and a filter together.
- the light source may be inside or outside the heat insulation box 160. It may be. In the heat insulation box 160, the distance to the cells is short and the power supply to the light source can be reduced.On the other hand, the light intensity is too strong to damage the cells, and the light is not sufficiently diffused, causing uneven brightness in the image. May also occur. Outside the thermal insulation box 160, the chance of contamination can be reduced for the same reason as described for the transmitted light type, and the maintenance of the light source is easy, but the light from the illumination is blocked by the camera. In some cases, uneven brightness may occur in the image. In such a case, a ring light that surrounds the lens can be used in the heat insulation box. Alternatively, it is also possible to illuminate the cells by illuminating a room outside the heat insulation box without providing a lighting device. Also in this case, the wavelength of the light source is the same as that described for the transmitted light method.
- the illuminating device is capable of adjusting not only the wavelength but also the amount of light and the irradiation angle, the captured image can be made higher in image quality.
- the camera 'incubator driving device 314 is disposed in the heat insulation box 160 as shown in Fig. 32.
- a CCD camera 31, a stand 317 supporting the CCD camera 31, and a carriage 318 serving as a base for supporting the stand 317 move linearly on a rail 315.
- a motor 320a is attached to the carriage 318 via a power transmission unit 319. By driving and rotating the motor 320a, the carriage 318 can be moved linearly via the power transmission unit 319.
- the incubator driving device 314 is composed of a motor 320a, a bogie 3189, and a force composed of the rail 315.
- the incubator 140 is swiveled and the CCD camera 31 is moved linearly to adjust the relative positional relationship between the two.
- FIG. 33 is a diagram showing a state of scanning by the CCD camera.
- FIG. 33 (a) shows a case where the shape of the incubator 140 is a horizontally long rectangle and a lens 316 having a magnification capable of keeping the Y direction within the visual field range 331 is used. In this case, since scanning in the Y direction is unnecessary, it is sufficient to scan only in the X direction.
- the camera side should be moved to XY as shown in Fig. 32.
- Either the configuration for moving the incubator 140 or the configuration for moving the incubator 140 side as shown in FIG. 34 may be adopted.
- Fig. 33 (b) shows a case where the incubator 140 is close to a square and has a large area, so that it is not possible to fit the Y direction into the visual field range 331 only by running in the X direction. In this case, it is sufficient to run in both the X direction and the ⁇ direction.
- the system can be driven with the configuration shown in FIG.
- the camera 31 needs to be configured to be able to run at least in the Y direction by the camera's incubator driving device 314. For example, in the case of FIG.
- the incubator 140 when the incubator 140 has a radius close to a circle as shown in FIG. 1 or FIG. 14 within the visual field range 331, the center of the incubator 140 is rotated. It is only necessary to scan in the ⁇ ⁇ ⁇ direction as the axis. In this case, scanning can be performed by using the configuration shown in FIG. In FIG. 35, the incubator 140 has a circular shape, a motor 351 is attached to the tip of a stand 350, and the camera 351 rotates in a circular shape by rotating the motor 351.
- the incubator 140 may be movable in a circular shape instead of the camera 31.
- FIG. 33 (d) is a diagram showing the relationship between the incubator 140 and the camera 31 in FIG.
- the incubator 140 rotates around the rotor 153 in the heat insulation box 160 as indicated by an arrow 140s.
- the camera 31 moves linearly along the rail 31b as indicated by the arrow 331a, it is possible to run the incubator 140 in the visual field range 331 by adjusting the positional relationship between the two as appropriate. It is.
- FIG. 36 is a diagram showing details of the image processing unit 312 in FIG.
- the image processing unit 312 includes a CPU 362 that performs arithmetic processing via a data bus 361, a main memory 363 that is temporarily used as a storage area by the CPU 362, an external storage device 364 that stores image data and position information, It comprises a communication port 365 for communicating with the motor controller 313, a monitor 366 for displaying an image after cell extraction, and a keyboard 367 for receiving user input.
- the image processing unit 312 captures an image from the CCD camera 31 via the converter 311 and performs various image processing.
- FIG. 37 is a flowchart showing a procedure of colony discrimination performed by the image processing unit 14.
- the size of the incubator 140 may be set in the external storage device 364 in advance, or may be detected by image processing using the camera 31. In this embodiment, the size is set in advance. explain.
- an image capturing position list which is scanning position information for capturing an image of the incubator 140.
- the image position list is, for example, a set of a plurality of XY coordinate points on a plane provided for culturing in the incubator 140 as an XY coordinate system.
- This image shooting position list is stored in the main memory 363, and its contents can be referred to at any time by the motor controller 313 or the like.
- Image shooting location list is stored in the main memory 363, and its contents can be referred to at any time by the motor controller 313 or the like.
- the field of view (angle of view) of the lens As described with reference to FIG. 33, it is determined by the field of view (angle of view) of the lens and the size of the incubator 140.
- step S372 the CPU 362 issues a movement command in accordance with the image shooting position list created in the previous step S371.
- the movement command issued by the CPU 362 reaches the motor controller 367 via the data bus 361 and the communication port 365.
- Motor controller 3 The 13 operates the camera's incubator driving device 314 to stop the camera 31 or the incubator 140 at the photographing position recorded in the image photographing position list.
- step S373 each time the camera reaches the target position corresponding to the image shooting position list, image shooting and multi-value processing are performed. That is, the CPU 362 issues an image capture command to the camera 31.
- the camera 31 converts the image data into an electric signal by the converter 311, and then transfers this signal to the main memory 363 via the data bus 361.
- the CPU 362 executes multi-value processing for displaying this signal on the monitor 366.
- step S374 the CPU 362 executes a histogram calculation process in order to obtain shading information based on the image data stored in the main memory 363.
- step S375 the grayscale information, that is, the pixel value at which the histogram is maximized, is stored in the external storage device 364 together with the imaging position information.
- step S376 it is determined whether or not shooting has been completed for all shooting positions described in the image shooting position list, that is, whether or not shooting has been completed for all measurement points is determined. If it is determined to be (yes), the process proceeds to the next step S377, and if not, the process returns to step S372.
- step S377 the position information and the pixel value stored in the external storage device 364 are read, the pixel value corresponding to the position information is stored in the main memory 363, and the image is displayed on the monitor 366 as an image that can be displayed. prepare.
- step S3708 the grayscale image created in this way is binarized by comparing it with a threshold value previously empirically obtained and stored in the external storage device 364.
- step S379 the presence or absence of the binarized image force colony obtained in the previous step S377, that is, the size, the area, and the perimeter are calculated, and the process ends.
- the size, area, and perimeter of the colony can be obtained as described above.
- the photographing data is stored in the external storage device 364 or the like.
- the information may be recorded together with the scanning position information and the photographing position information in the list, and the photographed image may be called using the corresponding photographing position as a hand.
- a lens with a high magnification can be used, it is possible to evaluate a photographed image cultivation state without having to retake another lens. As a result, Decisions such as imming can be made more reliably in a short time and without the risk of contamination.
- step S371 the region including the incubator 140 is scanned as widely as possible within the range permitted by the design.
- the size of the incubator 140 is obtained from the image thus obtained.
- the CPU 362 creates an image photographing position list from the information of the camera magnification and the visual field range. At this time, information on the magnification of the lens and the field of view of the camera 31 can be recognized from the shooting distance of the camera 31.
- the CPU 362 determines the route of the visual field range 331 that can run the incubator 140 comprehensively as shown in FIGS. 33 (a) to 33 (d). Further, the timing of shooting on the same route is also calculated, and that point is set as the shooting position. It is desirable that this route be traceable in a single word from the viewpoint of minimizing stress on the culture.
- the set of photographing positions thus created is stored in the external storage device 364 or the like as a set of XY coordinates or r-one coordinates. This is the method of creating the image shooting position list. Note that in executing step S371, shooting may be performed using the illumination described in step 373.
- the photographing position is specified by the image processing using the camera 31 in this manner, it is possible to scan the incubators of different sizes without inputting the sizes. For example, even if you forget to measure the surface area of the incubator before culturing in a completely sealed insulated box, the pre-scanning in step 371 can create a list of image capturing positions. For this reason, the burden on workers can be further reduced.
- the image is blurred due to the movement of the camera.
- the blur in this image is due to the shutter speed and running speed
- the degree determines the tolerance. For example, if the size of the cultured cells is about 100 microphones per millimeter, the scanning speed is 1 second per 1 mm, and the shutter speed is 1/1000 second, the moving distance per image is 1 micromillimeter. Results in negligible blurring for the size of the cultured cells.
- the optimal parameters for continuous imaging are determined by changing the running speed and the shutter speed according to the size of the cells to be cultured.
- the density of the image is used for colony discrimination, it is not necessary to capture the edge of the colony clearly. Moreover, since the photographing is completed in a short time, the camera 31 or the incubator 140 is completely stationary at the time of photographing. You don't have to.
- the continuous photographing eliminates the need to repeat the movement stop of the camera 31 and the incubator 140, thereby reducing vibrations applied to the incubator 140 and the like. With such a configuration, the imaging time can be reduced, and in particular, when imaging is performed by moving the incubator 140, there is an advantage that stress on cells can be reduced.
- the optical system of the camera uses the optical system of the phase difference method described in the method of detecting scattered light or transmitted light on the cell surface using simple light. You may.
- a threshold value is calculated based on the distribution of pixel values, and pixels that are equal to or larger than the threshold value or smaller than the threshold value are extracted as cells.
- filter processing such as noise elimination, smoothing filter, and edge enhancement is essential.
- the filter processing before the cell extraction processing has a great effect on the cell extraction accuracy.
- this filter processing it is possible to remove to a certain extent the difference in brightness between the central part and the peripheral part of the image, noise contained in the image, etc.
- the filter described in Patent Document 1 also removes noise contained in the image to some extent. be able to.
- the effect is small or the cell outline becomes unclear. There were cases.
- the basic configuration of this camera photographing system is the same as that of the block diagram shown in FIG. 36. In this embodiment, however, the CCD camera 31 moves up and down along a force moving guide to set an arbitrary focal point. This is different from the above-described one in that the configuration is such that an image of the incubator 140 is taken.
- FIG. 38 is a diagram schematically showing an arrangement of each constituent means in the cell culture device.
- the light source 381 is attached to the upper surface of the culture device.
- An incubator 140 is arranged below the light source 381, and a CCD camera 31 having an objective lens 382 is arranged below the center of the incubator 140 below.
- the CCD camera 31 is configured to be controlled to move up and down along a movement guide 383 by a camera's incubator driving device 314, so that an image of the incubator 140 at an arbitrary focal point can be taken. I have.
- FIG. 39 is a flowchart illustrating an example of a cell extraction process performed by the image processing unit when capturing an image of the incubator 140 at an arbitrary focus.
- an instruction is issued to the motor controller 313 to execute an image acquisition process of capturing an image while moving the CCD camera 31 up and down.
- the image data acquired by this image acquisition processing is stored in the external storage device 364 via the converter 311.
- an image selection process is executed.
- this image selection process at least two or more images with clear cell edges are selected.
- the change in pixel value is larger than in an image in which the edges are not clear. Therefore, the absolute value of the difference between adjacent pixel values is calculated, and the sum of them is calculated and stored in the main memory 363.
- FIG. 40 is a diagram showing the relationship between the movement distance of the camera and the sum of differences between neighboring pixel values.
- FIG. 41 and FIG. 42 are diagrams illustrating an example of an image corresponding to the two peaks described above.
- FIG. 41 is a diagram illustrating an example of an image when the focal position of the objective lens 382 is located on the bottom surface of the incubator 140.
- FIG. 43 is a diagram illustrating an example of an image in the case of being on the front side, and
- FIG. 43 is a diagram illustrating an example of an image in the case where the focal position of the objective lens 382 is behind the bottom surface of the incubator 140.
- an image in which the edges of the cells are clearer can be obtained when the focal position of the objective lens 382 is shifted to one of the front and rear.
- step S392 two or more images corresponding to the above-mentioned two peaks are selected, and it is determined whether or not the positions have been calculated. If yes, the process proceeds to the next step S394, and no In the case of, the process returns to step S392.
- one of the two images is shifted by one pixel in the X and Y directions to obtain a difference, and a difference / position adjustment process for adjusting the position is performed.
- Difference of the previous step S394. As a result of the alignment processing, it is determined whether the number of cells is the minimum and the total length of the cells is the maximum, and if yes, two cells are determined at that position. After the difference 'positioning process of the image is completed, the process proceeds to step S396. If no, the process returns to step S394, and the difference' positioning process is performed until the determination in step S395 becomes yes.
- FIG. 44 shows a binarization process for a difference image in which the number of cells is the smallest and the total length of the cells is the largest as a result of the difference 'positioning process for the images in FIGS. 42 and 43. It is a figure showing an image at the time of giving. Based on the image of FIG. 44, it is possible to easily analyze the length, number, shape, etc. of the cells.
- a diagram showing the incubator is displayed on the operation terminal, and a mark indicating the current position of the camera, a mark indicating the position to be moved, and a mark in which images have been stored in the past are displayed on the drawing. Then, by issuing a movement start instruction on the drawing of the incubator on the drawing of the incubator by the control means that receives the input of the movement start instruction, the camera can be moved to the target photographing position. Configuration was adopted. After the movement is completed, the mark indicating the position to be moved changes to the mark indicating the current camera position.
- FIG. 45 is a diagram schematically showing a camera photographing system provided with a camera position adjusting function capable of moving a camera to a desired position.
- the camera imaging system 450 includes an insulated box 160 that provides the optimal temperature and carbon dioxide concentration for cell culture, an incubator 140 for culturing cells, and an objective lens 382 for imaging cells.
- a CCD camera 31 for digitizing the data of the objective lens 382, a converter 311 for transferring image data obtained from the CCD camera 31 to an image processing unit 312, and a camera drive for moving the CCD camera 31 It comprises an apparatus 314a, an incubator drive 314b for moving the incubator 140, and a motor controller 313 for controlling the camera drive 314a and the incubator drive 314b.
- the detailed configuration of the image processing unit 312 in FIG. 45 is almost the same as that shown in FIG.
- the image processing unit 312 includes a CPU 362 that performs arithmetic processing via a data bus 361, a main memory 363 that is temporarily used as a storage area by the CPU 362, an external storage device 364 that stores image data and position information, Communication to communicate with motor controller 313 It comprises a port 365, a monitor 366 that displays an image after cell extraction, and a keyboard 367 that accepts user input.
- the image processing unit 312 captures images from the CCD camera 31 via a converter 311 and performs various image processing.
- a description will be given assuming that a mouse is connected in addition to the keyboard 367 as a device for receiving user input.
- FIG. 46 is a diagram schematically showing the relationship between the incubator 140 in the heat insulation box 160 in FIG. 14 and the camera 31.
- the incubator 140 moves around the rotor 153 so as to swirl inside the insulated box 160, but in FIG. 46, the incubator 140 is moved by the motor 320a as in the case of FIG. The description will be made assuming that the linear drive is performed.
- FIG. 47 is a diagram showing an example of an operation screen of “Shooting position setting” display.
- the imaging position setting 'display screen 470 shows a circular incubator 471 imitating the incubator 140.
- a camera position marker 472 indicating the current position of the camera
- a movement position marker 473 indicating the movement position of the camera
- stored image position markers 474-476 indicating the positions of the images already stored are shown.
- the image capturing position setting 'display screen 470 includes a background selection controller 477 for selecting the background of the incubator 471, a camera movement controller 478 for moving the camera to the movement position marker 473, and an image for displaying an image.
- a display controller 479, a position storage controller 47A for storing the position of the camera, a position call controller 47B for calling the stored position, and an end controller 47C for ending the processing are displayed.
- the stored image position marker 474 has a selected / non-selected state, which is used for determining a display image on an image display screen described later.
- FIG. 48 is a diagram showing a display example of an image displayed on the monitor, and is displayed by the image processing unit 312.
- This display image is stored in an image display area 480 for displaying the image at the current position of the camera and the stored image, a display image storage controller 481 for storing the image displayed in the image display area 480, and The image reading controller 482 for reading the image that was read, the image feed controller 483 for changing the image displayed in the image display area 480, the image return controller 484, and the image displayed in the image display area 480 at the same position.
- An image comparison controller 485 for issuing an instruction to compare images captured at different times is displayed.
- FIG. 49 is a flowchart showing an example of processing of the “shooting position setting” display processing software.
- the operation of the “photographing position setting” display process will be described with reference to this flowchart.
- the image processing unit 312 monitors the operator for any input in an input waiting loop.
- step S491 the input from the end controller 47C shown in FIG. 47 is determined, and if there is an input (yes), the process ends, the process proceeds to step S49M, and an input waiting loop process is executed. On the other hand, when there is no input (no), the process proceeds to the next step S492.
- step S492 the current position of the camera is calculated, and the camera current position marker 472 is displayed on the image.
- step S493 the background selection controller 477 determines whether or not a background has been specified. If the background has been specified (yes), the process proceeds to step S494. If not (no), the process proceeds to step S494. Proceed to S495.
- step S494 since the background is specified by the background selection controller 477,
- step S495 when an image that has already been stored exists, a stored image position marker 474—476 indicating the position of the image is displayed on the incubator 471 as shown in FIG.
- step S496 whether the operator has selected one of the stored image position markers 474-476 or has read the stored position of the image stored in advance using the position recall controller 47B. A determination is made as to whether or not the data is read. If the data is being read (yes), the process proceeds to step S497 (go forward). If the data is not read (no), the process proceeds to step S498.
- step S497 since the storage position is read, the movement position marker
- a movement position display process for displaying 473 on the incubator 471 is executed. If the movement position marker 473 is already displayed on the incubator 471 as shown in FIG. 47, one of the stored image position markers 474 476 is changed to the movement position marker 473. In addition, when the operator sets the movement position marker 473 using the mouse or the keyboard. The same processing is executed for
- step S4908 it is determined whether or not a camera movement command has been issued by the camera movement controller 478. If the command has been issued (yes), the process proceeds to step S499; if not (no), Proceed to step S49C.
- step S499 since a camera movement instruction has been issued by the camera movement controller 478, camera movement processing is executed according to the movement instruction.
- the CPU 362 in FIG. 36 converts the coordinates, issues a command to the motor controller 313 via the communication port 365, and outputs the camera driving device 314a and the incubator driving device 314b in FIG. Drive.
- the movement position marker 473 changes to the camera current position marker 472.
- step S49A it is determined whether an instruction to save the current position has been issued from the position save controller 47A. If the instruction has been issued (yes), the process proceeds to step S49B. Proceeds to step S49C.
- step S49B since the command to save the current position has been issued from the position saving controller 47A, the process of saving the current position is executed according to the save instruction.
- the storage position is displayed as a new storage image position marker on the incubator 471 in FIG. 47 by this storage processing.
- step S49C it is determined whether an image display instruction has been issued from the image display controller 479. If the instruction has been issued (yes), the process proceeds to step S49D; if not (no), the process proceeds to step S49D. Proceed to.
- step S49D it is determined whether or not any of the stored image position markers 474-476 is in the selected state. If it is in the selected state (yes), the flow advances to step S49E; ), Go to step S49F.
- step S49D a selected position image display process for displaying an image stored at any position of the selected stored image position marker 474 476 is executed. At this time, if a plurality of images exist for one stored image position marker, all images at that position can be displayed using the image forward controller 483 and the image return controller 483. Te, ru. [0250] In step S49E, the image at the current camera position is displayed because it corresponds to the case where an image display command is issued from the image display controller 479 without selecting any of the stored image position markers 474-476. A current position image display process is performed, and the process proceeds to step S49G.
- step S49G it is determined whether or not an image reading instruction has been issued from the image reading controller 482 in Fig. 48. If it has been issued (yes), the process proceeds to step S49H; if not (no), Goes to step S49K.
- step S49H the image specified by the operator in the previous step S49G is read from the external storage device 364 in Fig. 36 into the main memory 363, and an image reading process to be displayed in the image display area 480 is executed. Proceed to step S49J.
- step S49J the stored image position markers 474 476 at the corresponding positions are set to the selected state, and an image position display update process is performed to clearly indicate to the operator at which position the image was captured. Proceed to S49K.
- step S49K it is determined whether an image comparison instruction has been issued from the image comparison controller 485 in Fig. 48. If the instruction has been issued (yes), the flow advances to step S49L; otherwise (no). In this case, the flow advances to step S49M to execute an input waiting loop process.
- step S49L a monotone image of a different color is created, weighted according to the transmittance set for each image, an addition process is performed, and a monotone / addition process for displaying a screen showing the result is performed. Is executed, and the flow advances to step S49M to execute an input waiting loop process.
- step S49M similar to step S490, the image processing unit 312 performs input waiting loop processing, and monitors the operator for any input.
- FIG. 50 is a diagram showing details of the monotone Z addition processing.
- the image data in this monotone / addition processing has three components of RGB per pixel.
- a color image determination is performed to determine whether the input is a color image or a grayscale image. If yes, the process proceeds to step S501; if no, the process proceeds to the next step S502.
- step S501 since the input is a color image, grayscale conversion processing for converting the color image into a black and white image is performed, and the process proceeds to step S502.
- step S502 it is determined whether or not the processing target is the image displayed in the image display area 480, that is, whether or not the image is currently being displayed. If yes, the process proceeds to step S503. If no, go to step S505.
- step S503 of the RGB components of the image data, the pixel value after black and white conversion is substituted (copied) into the R component, and 0 is substituted into the other G and B components. Execute component substitution processing. At this point, the image is represented as a shade of R component.
- step S504 a transparency calculation process for weighting the composite ratio of the currently displayed image and the comparison target image, that is, the currently displayed image, is performed, and the flow advances to step S505.
- step S505 it is determined whether or not the processing target is a comparison target image. If yes, the process proceeds to step S506 (if no, the process proceeds to step S508).
- step S506 the pixel value of the image after black and white conversion is substituted for the G component in the RBG component, and 0 is set for the other R and B components in the comparison target image, as in step S503.
- the G component substitution processing is executed. At this point, the image to be compared is represented as a shade of G component.
- step S507 similarly to step S504, a transparency calculation process of weighting the composite ratio of the currently displayed image and the comparison target image, that is, the comparison target image, is performed.
- the comparison target image is emphasized if the transparency in the processing of step S504 is high, and the current display is that the transparency in the processing of step S507 is high.
- the image inside is emphasized and displayed.
- step S508 the result image of the transparency calculation processing in steps S504 and S507 is added.
- the image after the transparency calculation processing in step S504 has only the R component
- the image after the transparency calculation processing in step S507 has only the G component.
- the overlapping pixel portion of the image data becomes an image having an RG component, and a pixel that does not overlap between the two has a pixel value of only the R component or the G component. In this manner, the difference between the two images can be expressed by the color components by the monotone / addition processing.
- FIG. 51 is a diagram showing a schematic configuration of a cell culture device that can be operated automatically and with high reliability over a long period of time.
- the automatic culture device 511 includes a device control means 512, a user interface means 513, a culture schedule management means 514, and a UPS (uninterruptible power supply) 515.
- the UPS 515 is connected to the culture schedule management means 514.
- communication is performed at predetermined intervals between the culture schedule management means 514 and the apparatus control means 512 and between the culture schedule management means 514 and the user interface means 513, and the communication is performed.
- the predetermined interval may be a fixed time interval or a variable time, which means that communication is performed periodically.
- FIG. 52 is a flowchart showing an example of a predetermined monitoring communication process executed by the automatic culture device of FIG. 51.
- monitoring communication between the culture schedule management means 514 and the apparatus control means 512 will be described with reference to the flowchart in FIG.
- a response request signal is transmitted from the culture schedule management means 514 to the device control means 512 in step S520.
- the culture schedule management unit 514 determines whether or not a response confirmation signal has been received from the device control unit 512 at a predetermined interval. If the response confirmation signal has been received (yes), the device control unit 512 Upon receiving the response confirmation signal from the schedule management means 514, the culture schedule A response confirmation signal is sent to the rule management means 514. Upon receiving the response confirmation signal from the device control unit 512, the culture schedule management unit 514 terminates the monitoring communication from the culture schedule management unit 514 to the device control unit 512, and returns. On the other hand, if the response confirmation signal has not been received (no), the process proceeds to the next step S522.
- step S522 the culture schedule management unit 514 determines whether or not the number of times the response request signal has been transmitted to the device control unit 512 is equal to or less than a predetermined value a. If it is less than a (yes), the process returns to step S520 and resends the response request signal. On the other hand, if the number of response request signal transmissions is larger than the predetermined value a, the process proceeds to the next step S523.
- Step S523 is processing performed when a response confirmation signal has not been received. This processing is performed when a failure occurs in the device control unit 512 and the culture schedule unit 514 receives a response confirmation signal from the device control unit 512 at a predetermined interval. Although the response request signal is transmitted several times (variable setting is possible), this process is executed when the response confirmation signal from the device control means 512 cannot be received even after that. A restart command is transmitted to the control means 512, and the device control means 512 is restarted by software.
- step S524 it is determined whether or not the time required for restarting the device control means 512 (variable setting possible) has passed, and the culture schedule management means 514 has received a response request signal from the device control means 512. Is determined, and if yes, it is recognized that the device control means 512 has been restarted normally, and the process jumps to step S529.
- step S529 the culture schedule data is transmitted to the device control means 512, the monitoring communication process ends, and the routine returns.
- step S525 the culture schedule management means 514 It is determined whether or not the number of times the restart command signal is transmitted is equal to or less than a predetermined value b. If the number of times the restart command signal is transmitted is equal to or less than the predetermined value b (yes), the process returns to step S523, and the restart instruction is performed. Retransmit the signal. On the other hand, when the number of times of restart command signal transmission is larger than the predetermined value b, the process proceeds to the next step S526.
- step S526 the culture schedule management means 514 has transmitted the restart instruction b times to the device control means 512, but no response request signal has been sent from the device control means 512 to the culture schedule management means 514.
- the culture schedule management unit 514 performs a process of forcibly turning off (OFF) and turning on (ON) the power of the device control unit 512, that is, a forced restart process. .
- step S527 the culture schedule management means 514 passes the time required for restarting the device control means 512 (variable setting possible), and sends a response request signal from the device control means 512 to the culture schedule management means 514. It is determined whether or not the device control unit 512 has been received. If yes, it is recognized that the device control unit 512 has been normally restarted, and the process jumps to step S529.
- step S529 the culture schedule data is transmitted to the device control means 512, the monitoring communication process ends, and the process returns.
- the power is turned on from the culture schedule management means 514 to the apparatus control means 512.
- step S528 the culture schedule management unit 514 determines whether or not the number of times the device control unit 512 has performed the forced restart by turning the power on and off is equal to or less than a predetermined value c. If the number of starts is equal to or smaller than the predetermined value c (yes), the process returns to step S526, and the forced restart is executed again. On the other hand, if the number of forced restarts is larger than the predetermined value c, the process proceeds to the next step S52A.
- step S52A the culture schedule management means 514 executed c-forced restarting of the device control means 512 by turning on / off the power. However, the culture schedule management means 514 from the device control means 512 still performed. In this case, it is reported to the user that a system error has occurred. . This report is made by displaying that fact on a buzzer or monitor.
- Monitoring communication from the device control means 512 to the culture schedule management means 514 is performed in a manner reverse to that described above. In addition, monitoring communication between the user interface unit 513 and the culture schedule management unit 514 is performed in the same manner. If the power is cut off, the culture schedule management means 514 transmits culture schedule data to the device control means 512 and the user interface means 513 when the power is restored because the UPS (uninterruptible power supply) 515 is connected.
- UPS uninterruptible power supply
- the device control means 512, the user interface means 513, and the culture schedule management means 514) perform communication between a plurality of units during the culture period, and perform an abnormality detection process for detecting an abnormality of the opposing unit. In addition, even if one of the plurality of units becomes abnormal due to a failure, the abnormality of the automatic culture apparatus can be notified to the outside. Furthermore, if any one of the multiple units detects an abnormal state, loading the culture schedule data from the other units in the normal state can automatically return the abnormal unit to the normal state. In addition, any one of the multiple units is connected to the uninterruptible power supply, so even if the power is cut off, any of the multiple units is operating. The operation of the entire automatic culturing device can be restored, centering on the unit, and the reliability of the automatic culturing device is further improved.
- an incubator means for culturing cells typically, according to the present invention, an incubator means for culturing cells, and an insulated box for arranging the incubator means in a state suitable for culturing and maintaining at a predetermined temperature Means, a driving means for rotating the incubator means in the insulated box means, and a drug supply for supplying unused chemicals to the incubator means in the insulated box means from outside the insulated box means.
- Means waste liquid discharging means for discharging unnecessary waste liquid from the incubator means in the insulated box means to the outside of the insulated box means, and the cell culture state of the incubator means in the insulated box means.
- a closed system cell culture device is provided, which comprises a culture state observation means for observing from outside the heat insulation box means.
- the cell culture device includes a pump, a valve, and a pump between the incubator means and the drug supply means.
- a flexible tube member is provided to supply, culture and recover the cells.
- the incubator means is preferably a non-toxic container made of a non-toxic material with a smooth central portion, but the central portion may have some irregularities.
- the transparent non-toxic material is preferably polystyrene or polyethylene terephthalate.
- the culture state observation means includes a camera.
- the cell culture device preferably includes a camera moving unit that allows the camera to run over the entire surface of the incubator unit and that can set the focus in the cell incubator unit in the optical axis direction.
- the cell culture device includes a storage means for storing a photographing position of the camera on the incubator means, and the camera moving means reproduce the same photographing position as stored in the storage means. .
- the cell culture device includes a thin tube whose outside is sealed with a closing member, the thin tube is a supply port or a collection port for cells, and includes a container for accommodating cells.
- a germicide impregnating member is provided, and the thin tube is inserted into the container after penetrating the germicide impregnating member.
- the cell culture device is provided with a gas cylinder for supplying an atmosphere into the heat insulation box means, and the valve be opened and closed using the gas pressure of the gas cylinder as a drive source.
- the cell culture device has a drug amount determining means for determining the amount of the drug supplied from the drug supply means to the incubator means during the operation time of the pump.
- the waste liquid discharging means includes a flexible pipe member, a pump, and a waste liquid tank, and any one of them includes a pH measuring unit.
- the pH measurement unit has an object whose color changes due to a change in pH, and a light receiving element that reads the color of the object.
- the cell culture apparatus may include control means for storing and executing the timing and contents of cell supply, rotation of the incubator means, supply of a drug solution, waste liquid, and cell supply / recovery as a cell culture procedure. preferable.
- control means may communicate with other control means. It is preferable to have an interface to exchange information.
- FIG. 1 is a block diagram showing a basic configuration of a cell culture device to which the present invention is applied.
- FIG. 2 is a detailed view of a mechanism section of the cell culture apparatus to which the present invention is applied, showing an actual configuration in which the system controller 11 in FIG. 1 is omitted.
- FIG. 3 is a diagram showing a detailed configuration of an incubator 38 of FIG. 2.
- FIG. 4 is a block diagram showing details of a control block diagram of the cell culture device of FIG. 2, showing a case where a plurality of cell culture devices are connected to form a plant.
- FIG. 5 is a flowchart for explaining the operation of the cell culture device.
- FIG. 6 is a diagram showing an example of an operation of “shuffling the incubator and homogenizing 'seeding” in step S55 of FIG. 5.
- FIG. 7 is a diagram showing a first modification of the incubator 38 of the cell culture device according to the above-described embodiment, where FIG. 7 (A) is a diagram viewed from above, and FIG. The side view is shown.
- FIG. 8 is a view showing a second modification of the incubator 38 of the cell culture device according to the above-described embodiment.
- FIG. 9 is a flowchart for explaining the operation of the cell culture device using the incubator of FIG. 8.
- FIG. 10 is a cross-sectional view showing a third modification of the incubator 38 of the cell culture device according to the above-described embodiment.
- FIG. 11 is a diagram showing an example of a method for uniformly seeding cells.
- FIG. 12 is a diagram showing an example of a method of connecting a culture vessel and a tube in the above embodiment.
- FIG. 13 is a view showing a method of sterilizing a part of the culture device in the above embodiment.
- FIG. 14 is a view showing a detailed view of a mechanism according to another embodiment of the cell culture apparatus to which the present invention is applied.
- FIG. 15 is a diagram showing details of an incubator used in FIG.
- FIG. 16 is a view showing details of a first port 141, a second port 142, and a third port 143 in the incubator.
- FIG. 17 is a view showing a partial cross section of FIG. 15, and showing a case where the culture medium in the incubator is discharged.
- FIG. 18 A diagram showing a detailed configuration of the heat insulation box of FIG. 14, in which FIG. 18 (A) shows the internal structure of the heat insulation box for easy understanding, and FIG. 18 (B) is a perspective view of the appearance of the heat insulation box.
- FIG. 18 (A) shows the internal structure of the heat insulation box for easy understanding
- FIG. 18 (B) is a perspective view of the appearance of the heat insulation box.
- FIG. 19 is a view showing a cross section of an S—S plane showing details of the heat insulating structure in FIG. 18 (A).
- FIG. 20 is a diagram showing a control block of a heat insulation box 16 of the cell culture device of FIG. 14, in which parts required for explanation are extracted from FIG. 4, and others are omitted.
- FIG. 21 is a diagram showing a detailed configuration of a pH measuring section, and is an enlarged view of a part of FIG.
- FIG. 22 is a diagram illustrating a detailed configuration of a pH measuring unit, and is a diagram illustrating a detailed configuration of a sensor unit of the pH measuring unit.
- FIG. 23 is a diagram showing a schematic configuration of a stopper for containers 230 and 240, FIG. 23 (A) is a perspective view thereof, and FIG. 23 (B) is a sectional view thereof.
- FIG. 24 is a diagram showing a schematic configuration of a stopper for containers 230 and 240
- FIG. 24 (A) is a top view showing a state where an external blocking member is removed
- FIG. 24 (B) is a container
- FIG. 2 is a bottom view showing a schematic configuration of a plug for use.
- FIG. 25 is a perspective view showing an example of a container provided with the container stopper of FIG. 24.
- FIG. 26 is a sectional view showing a modified example of the container stopper of FIGS. 23-25.
- FIG. 27 is a cross-sectional view showing another modification of the container stopper of FIGS. 23-25.
- FIG. 28 is a cross-sectional view showing still another modification of the container stopper of FIGS. 23-25.
- FIG. 29 is a diagram illustrating an outline of an operation when extracting a liquid from a container or injecting a liquid into the container.
- FIG. 30 is a diagram schematically illustrating an operation for extracting a liquid from a container or injecting a liquid into the container.
- FIG. 31 is a block diagram illustrating a schematic configuration of a camera imaging system.
- FIG. 32 is a diagram schematically showing a portion related to the camera photographing system in FIG. 14 extracted.
- FIG. 33 is a diagram showing a state of scanning by a CCD camera.
- FIG. 34 is a diagram showing a state of scanning by a CCD camera.
- FIG. 35 is a diagram showing a state of scanning by a CCD camera.
- FIG. 36 is a diagram showing details of an image processing unit 312 in FIG. 31.
- FIG. 37 is a flowchart showing a processing procedure of colony discrimination performed by the image processing unit 14.
- FIG. 38 is a diagram schematically showing an arrangement of each constituent means in the cell culture device.
- FIG. 39 is a flowchart showing an example of a cell extraction process executed by the image processing unit when capturing an image of the incubator 140 at an arbitrary focus.
- FIG. 40 is a diagram showing the relationship between the moving distance of the camera and the sum of differences between adjacent pixel values.
- FIG. 41 is a diagram showing an example of an image when the focal position of the objective lens 382 is located on the bottom surface of the incubator 140.
- FIG. 42 is a view showing an example of an image when the focal position of the objective lens 382 is located on the front side of the bottom surface of the incubator 140,
- FIG. 43 is a diagram showing an example of an image when the focal position of the objective lens 382 is located behind the bottom surface of the incubator 140.
- FIG. 45 is a diagram schematically showing a camera photographing system having a camera position adjusting function capable of moving a camera to a desired position.
- FIG. 46 A diagram schematically showing the relationship between the incubator 140 in the heat insulation box 160 in FIG. 14 and the camera 31.
- FIG. 47 is a diagram showing an example of an operation screen for “shooting position setting” display.
- FIG. 48 is a diagram showing a display example of an image displayed on the monitor, which is displayed by the image processing unit 312.
- FIG. 49 is a flowchart showing an example of processing of a shooting position setting ′ display processing software.
- FIG. 50 is a diagram showing details of a monotone Z addition process.
- FIG. 51 is a diagram showing a schematic configuration of a cell culture device that can be automatically and reliably operated for a long period of time.
- FIG. 52 is a flowchart showing an example of a predetermined monitoring communication process executed by the automatic culture device of FIG. 51.
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
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CN2004800377036A CN1894397B (zh) | 2003-12-18 | 2004-12-15 | 细胞培养装置 |
EP04807089.0A EP1696024B1 (en) | 2003-12-18 | 2004-12-15 | Device for cell culture |
KR1020067011117A KR101139090B1 (ko) | 2003-12-18 | 2004-12-15 | 세포 배양 장치 |
US10/583,511 US7754478B2 (en) | 2003-12-18 | 2004-12-15 | Device for cell culture |
JP2005516322A JPWO2005059091A1 (ja) | 2003-12-18 | 2004-12-15 | 細胞培養装置 |
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JP2003-420510 | 2003-12-18 | ||
JP2003420510 | 2003-12-18 |
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US (1) | US7754478B2 (ja) |
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JP (2) | JPWO2005059091A1 (ja) |
KR (1) | KR101139090B1 (ja) |
CN (1) | CN1894397B (ja) |
WO (1) | WO2005059091A1 (ja) |
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Also Published As
Publication number | Publication date |
---|---|
CN1894397B (zh) | 2011-07-13 |
US7754478B2 (en) | 2010-07-13 |
EP1696024B1 (en) | 2015-03-04 |
JP5285666B2 (ja) | 2013-09-11 |
KR20070006680A (ko) | 2007-01-11 |
JP2010268813A (ja) | 2010-12-02 |
US20070148764A1 (en) | 2007-06-28 |
EP1696024A4 (en) | 2008-05-07 |
KR101139090B1 (ko) | 2012-04-30 |
JPWO2005059091A1 (ja) | 2007-12-13 |
EP1696024A1 (en) | 2006-08-30 |
CN1894397A (zh) | 2007-01-10 |
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