WO2014141477A1 - 細胞培養装置 - Google Patents
細胞培養装置 Download PDFInfo
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- WO2014141477A1 WO2014141477A1 PCT/JP2013/057510 JP2013057510W WO2014141477A1 WO 2014141477 A1 WO2014141477 A1 WO 2014141477A1 JP 2013057510 W JP2013057510 W JP 2013057510W WO 2014141477 A1 WO2014141477 A1 WO 2014141477A1
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- culture
- culture vessel
- cell
- flow path
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- 0 CC(C)C(C1)C(C2C)C(C3)C2[C@@]32[C@@](C)C3*(CC*)CC1C23 Chemical compound CC(C)C(C1)C(C2C)C(C3)C2[C@@]32[C@@](C)C3*(CC*)CC1C23 0.000 description 1
- XAZKFISIRYLAEE-KNVOCYPGSA-N C[C@H]1C[C@@H](C)CC1 Chemical compound C[C@H]1C[C@@H](C)CC1 XAZKFISIRYLAEE-KNVOCYPGSA-N 0.000 description 1
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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
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
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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
- C12M23/50—Means for positioning or orientating the apparatus
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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
- C12M23/58—Reaction vessels connected in series or in parallel
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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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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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
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Definitions
- the present invention relates to a culture apparatus for culturing cells or tissues by automatic operation.
- Regenerative medicine that restores the function of organs and the like using biological samples such as regenerative tissues produced using cells as raw materials is expected as a radical treatment method for diseases for which there has been no conventional treatment method.
- Regenerated tissue is manufactured in accordance with SOP (Standard Operating Procedure) by CPC (Cell Processing Center), which provides a clean manufacturing environment, by manufacturing workers with specialized cell culture technology.
- SOP Standard Operating Procedure
- CPC Cell Processing Center
- automatic culture equipment is an alternative to manual labor, but it is necessary to comply with GMP (Good Manufacturing Practice: Practice) for manual labor.
- GMP Good Manufacturing Practice: Practice
- Development guidelines for clinical culture automatic culture equipment regenerative medicine field (design guidelines for human cell culture processing equipment [revised]), 2009) have been presented by the Ministry of Economy, Trade and Industry, and it is necessary to comply with them.
- the automatic culture device can produce high-quality regenerative tissue with high reproducibility in a clean environment based on scientific evidence, taking into account GMP for manual labor and development guidelines for automatic culture devices. Is required.
- Patent Document 1 For a plurality of culture vessels arranged in a matrix, the flow tube lengths from the solenoid valves are aligned in units of columns, and a plurality of culture vessels in the same matrix are used.
- An apparatus for making the quality of regenerated tissue uniform by culturing is disclosed.
- Patent Document 2 discloses an apparatus in which components necessary for culture such as a culture vessel and a medium bottle are always connected by a flow tube or the like to reduce the risk of biological contamination, thereby forming a closed culture space. ing.
- Patent Document 1 since the method for adjusting the flow path length in Patent Document 1 merely aligns the flow path lengths to the culture vessels arranged in the same row, cell loss is reduced when the cell suspension used for culture is fed. The degree differs depending on the culture container in the row unit, and there is a problem that the quality cannot be made uniform among a plurality of culture containers cultured at the same time.
- the present invention has been made in view of such problems, and provides a closed-system cell culture apparatus that enables high-quality and reproducible cell culture. Specifically, the present invention provides a cell culturing apparatus that makes it possible to uniformize the quality of cells after production and to suppress user work mistakes in channel installation.
- a cell culture apparatus for culturing cells in order to solve the above-described problem, a cell culture apparatus for culturing cells, a solution holding unit for holding a solution used for culture, a plurality of units each connecting a solution holding unit and a plurality of culture containers
- the plurality of flow paths provide a cell culture device having a structure having the same length from the solution holding unit to each of the plurality of previous culture containers.
- the cell culture device using the closed culture vessel according to the present invention enables high-quality cell culture. Specifically, high quality and homogenization of cultured cells are realized in each of a plurality of culture containers. In addition, a mechanism that normally performs the flow path setting operation on the user's device suppresses cell quality degradation due to the user's erroneous operation.
- regeneration tissue which shows a 1st Example.
- Example 1 the basic configuration and operation flow of the cell culture device according to each example including Example 1 will be described in detail with reference to the drawings.
- the basic configuration and the operation flow are not limited to this, and a configuration may be added or the operation flow may be changed as appropriate depending on the application.
- an automatic culture apparatus comprising 12 components will be described in detail below.
- the twelve components are a culture vessel section 1, a flow path section 2, a rotary valve mechanism 3, a culture cell and feeder cell bottle section 4, a medium bottle section 5 including a refrigerator, and a preheating bottle section 6. , Drainage bag unit 7, observation unit 8, incubator unit 9, gas supply unit 10, humidifying bottle unit 11, and control unit 12.
- the control unit 12 includes a control terminal 13.
- the automatic culture apparatus uses the cell bottle installed by the user, the cell suspension in the medium bottle, and the medium for the closed system flow path that is a closed culture space, and the flow path unit of the present apparatus.
- the control unit 12 controls a solenoid valve (not shown), a tube pump, and the like provided in 2 and the like, so that the cells are seeded into the culture vessel of the culture vessel unit 1 and cultured.
- control unit 12 controls the observation unit 8 including a microscope provided in the apparatus to capture a cell image in the culture vessel. Except at the time of automatic photographing, cell seeding, medium exchange, and gas exchange, it is possible to control the position of the microscope based on the input from the operation screen of the control terminal and to photograph and store the cell image by manual observation of the microscope.
- FIG. 2 shows a closed circuit flow path circuit of the above-described automatic culture apparatus.
- the closed system flow path includes the culture container section 1, the flow path section 2, the rotary valve mechanism 3, the cell bottle section 4, the culture bottle section 5, the preheating bottle section 6, and the drainage bag section among the components shown in FIG. 7 includes a gas supply unit 10 and a humidification bottle unit 11.
- Example 1 will be described by way of example for the purpose of producing a regenerative tissue of epithelial cells such as corneal epithelial cells, oral mucosal epithelial cells, and epidermal cells.
- cell types that can be cultured by this cell culture device Is not limited to this.
- the flow path circuit uses two types of cells. However, when only one type of cells such as cardiomyocytes and fibroblasts are to be cultured, one cell is used. It is good also as a flow path circuit of the cell bottle and the flow path with respect to it.
- a channel circuit that uses two types of cells only a channel circuit that targets one type of cell may be used.
- the flow path circuit of FIG. 2 uses 10 culture vessels, it is also possible to use a flow path circuit composed of different numbers of culture vessels by installing or removing the culture vessels in parallel.
- the closed system flow path shown in FIG. 2 mainly has the following components.
- ten culture vessels 201 are provided to produce ten regenerated tissues.
- the whole culture vessel 201 is installed on a culture vessel base 202 which is a flat plate, and an actuator 203 for changing the inclination is attached to the culture vessel base 202.
- two cell bottles 204 and 205 are used.
- One type of cell is placed in each cell bottle 204,205.
- the entire culture vessel has a two-layer structure, and one type of cell is cultured in each layer.
- the flow path circuits from the cell bottles 204 and 205 to each layer of the culture container use different flow path circuits (1) and (2) in order to prevent cells from being mixed during the liquid feeding.
- the cell suspension in the cell bottle 204 passes through the solid line channel circuit (1) 206 and is fed to a layer on one side of each culture vessel, for example, the upper layer of all the culture vessels.
- the cell suspension in the cell bottle 205 passes through the dotted channel circuit (2) 207, and is sent to one layer of each culture vessel, for example, the lower layer of all the culture vessels.
- reference numeral 222 denotes a multi-branch unit which will be described later.
- the cell bottle is different for each cell type, since the medium is common, one medium bottle 208 is used.
- the medium bottle is stored at 4 ° C. using a refrigerator as described in FIG.
- the amount necessary for one medium exchange is transferred to the preheating bottle 209, and after heating to 36 ° C., for example, it is used for medium exchange.
- the flow path circuit (1) 206 and the flow path circuit (2) 207 are appropriately branched via the bifurcation 210. Thereafter, the medium sent to the channel circuit (1) 206 is sequentially sent to one layer of each culture vessel. The same applies to the medium fed to the flow path circuit (2) 207.
- the tube pump 211 gives a driving force for liquid feeding and air feeding in the flow path.
- the liquid feeding direction is controlled by the electromagnetic valve 212 and the rotary valve mechanism 213 corresponding to the rotary valve mechanism 3 of FIG.
- oxygen and carbon dioxide are supplied to each culture vessel 201 as gas exchange. This is because cells consume oxygen and discharge carbon dioxide.
- a gas cylinder 216 filled with air containing 5% CO 2 is adjusted to a predetermined air supply speed with a gas flow meter 217, and then passed through a humidifying bottle 218 containing sterilized water. Saturate and air. Gas is sent to each culture vessel through an air supply circuit 219 located in parallel with the tube pump 211.
- the flow path circuit of the present embodiment there is a sterile desorption part 220 and a sterile connection part 221.
- the aseptic desorption part 220 is installed on the flow channel tube in the vicinity of each culture vessel 201.
- one culture container can be removed aseptically to inspect as a sample the day before transplantation.
- the removed culture container and the remaining culture containers and flow paths after removal can each maintain sterility.
- the culture vessel 201 is removed using the aseptic desorption part 220.
- the aseptic desorption part 220 is a flow-path tube which can be heat-welded as an example, and cuts between two places sandwiching the cut portion after heat-welding.
- the aseptic connection part 221 is installed on the flow path tubes in the vicinity of the cell bottles 204 and 205, the medium bottle 208, and the humidification bottle 218.
- the cell bottles 204 and 205, the medium bottle 208, and the humidification bottle 218 are brought into a CPC (Cell Processing Center) in an empty state, and the user enters a predetermined cell suspension, medium and sterilized water into a closed system flow. Install on the road. At that time, aseptic connection is made using the aseptic connection part 221.
- CPC Cell Processing Center
- FIG. 3 shows an example of the culture container base 202 of the apparatus of the present embodiment and a state in which ten culture containers 201 are installed on the culture container base 202.
- the culture vessel base 202 of this example has a horseshoe shape. That is, the culture vessel base 202 has a structure in which a gap is formed by hollowing out a central portion with a rectangular flat plate, and a notch is provided at an end of the flat plate on the side to be inserted into the apparatus, that is, a part of the outer periphery. And has a so-called U-shaped shape.
- the inner side of the central part of the culture vessel base 202 is circular, and the culture vessel 201 is arranged in a circle around it.
- the culture vessel base 202 is formed with holding means such as a recess for holding each culture vessel 201.
- a microscope of the observation unit 8 to be described later is disposed in the hollow space portion that is hollowed out.
- An observation hole 301 for microscopic observation is provided in a portion that holds the culture vessel 201.
- the installation direction in the apparatus is uniquely determined. That is, when the culture vessel base 202 is installed, the culture vessel base 202 is brought close to the microscope from the scraping side formed on one side of the culture vessel base 202, the microscope is guided into the gap, and installed in the actuator or the like. With the shape having the cut-out gap, the culture vessel base 202 can be arranged at the center of the plurality of culture vessels 201. In addition, since the user can easily install and remove the plurality of culture vessels 201 on the culture vessel base 202 without contacting the microscope, the quality deterioration of the cell culture can be suppressed by damaging the flow path or the like due to human error. It becomes possible.
- another culture vessel installation base (not shown) having the same shape as the culture vessel base 202 or divided into a plurality of pieces is provided in the apparatus, and the culture vessel base 202 is placed on the other culture vessel installation base.
- the installation work may be simplified.
- FIG. 3 shows a state in which ten culture vessels 201 are installed on the culture vessel base 202.
- four channel tubes 302 are connected to each of the culture vessels 201, and the four channel tubes 302 are arranged outwardly with respect to the culture vessel base 202.
- the channel tube 302 By disposing the channel tube 302 outside the culture vessel base 202, that is, on the outer periphery, it is possible to suppress deterioration in cell quality due to contact between the microscope, the culture vessel base 202, the channel tube, and the like when the microscope is driven.
- the sterilization / removal part 303 for enabling the removal of each culture vessel.
- the culture surface of the culture vessel is subjected to a temperature response when the temperature of the culture vessel 201 falls below the phase transition temperature of the temperature-responsive culture surface, for example, 32 ° C.
- the nature of the surface of the sex culture changes from hydrophobic to hydrophilic, and the cells that have adhered, spread and proliferated spontaneously at 37 ° C. are detached.
- the culture conditions change greatly, and the quality of the cells at the time of transplantation also changes.
- a small door for taking out the work before the day before the completion of the culture for the purpose of quality confirmation in the culture process Can be prepared in the upper part of the door of the incubator unit 9, and the time and range of exposing the inside of the incubator unit 9 to the outside air can be reduced to reduce the temperature drop during operation.
- a small door for taking out the day before with a transparent material such as glass it is possible to check the progress of the culture, for example, pH due to the color of the medium, biological contamination due to turbidity, etc. from the small door. Is also possible.
- a tube pump 401, a solenoid valve 402, a flow tube 403, cell bottles 404 and 405, a preheating bottle 406, and the like are installed in the flow channel unit 2.
- a tube pump 401, a solenoid valve 402, a flow tube 403, cell bottles 404 and 405, a preheating bottle 406, and the like are installed in the flow channel unit 2.
- it may be set as a solution holding part as a general term for these bottles.
- the tube pump 401 squeezes the flow tube 403 from the outside of the flow tube and supplies liquid or air.
- the electromagnetic valve 402 opens and closes the flow path tube 403 by energization to control the liquid feeding or air feeding direction.
- a filter having a pore diameter of 0.22 ⁇ m, for example is used so that the pressure inside and outside the flow path is adjusted so that bacteria and the like do not enter from the outside of the flow path.
- the positions of the tube pump, the solenoid valve, the filter, and the branching portion of the flow path unit 2 are determined according to the following priority conditions.
- the first condition is that the flow path length from the cell bottle of the cell bottle portion 4 containing the cell suspension of epithelial cells to each culture vessel 201 is the shortest and equal length.
- the highest priority is to minimize the influence of the epithelial cells to be cultured on the culture process during the seeding process.
- the second condition is that the flow path length from the cell bottle in which the cell suspension of the feeder cells of the cell bottle part 4 is placed to each culture vessel 201 is the shortest and equal length. It is also desirable that feeder cells for calculating growth factors given to epithelial cells have a minimum effect on the culture during the seeding process.
- the third condition is that the flow path length from the medium bottle in which the medium of the medium bottle part 5 is put to each culture vessel 201 is the shortest and equal length. This is because the medium is fed through the shortest flow path to avoid a decrease in liquid feed accuracy due to adhesion of protein as a medium component in the flow path tube or residual liquid droplets.
- the solution holding unit to the multi-branch unit 222 are common to each culture vessel, By making the length from the multi-branch portion 222 to each culture vessel 201 equal, it is possible to achieve the purpose of making each channel length equal. Details of the method for equalizing the length from the multi-branch portion 222 to each culture vessel 201 will be described later.
- FIG. 5 shows an example of a rotary valve mechanism as the solution supply mechanism shown in FIG. 1 in the present embodiment.
- the solution supply mechanism in the present embodiment is not limited to this rotary valve mechanism, and is a supply mechanism that can control the supply of the solution that has passed through the multi-branch portion 222 to each of the culture vessels 201. Needless to say, this is applicable.
- the rotary valve mechanism 3 shown in FIG. 5 collectively controls medium and gas supply to a plurality of culture vessels 201 to be cultured at the same time.
- this is a valve mechanism that opens and closes a plurality of flow channel tubes, and a clip portion 502 that closes the flow channel tube 501 and a multiple cam portion 503 that opens the clip portion 502 by contact.
- the clip portion 502 includes a spring 505 that closes the clip and a clip contact portion 506 that contacts the multiple cam portion 503. Since the clip contact portion 506 can change the clip to be closed by the rotation of the multiple cam portion 503, the liquid feeding and air feeding directions can be controlled.
- two cell bottles 404 and 405, which will be described later, and a preheating bottle 406 are located in a state where they are connected to each other by a flow path portion and a flow path tube. It is installed on the top.
- the amount of liquid to be fed is confirmed from the change in weight after feeding the cells to each culture vessel 201 when the cell suspension is fed.
- the amount of liquid delivered is controlled by the operating time of the tube pump, the amount of liquid delivered can be grasped more reliably by monitoring the change in weight.
- the preheating bottle 406 grasps the amount of liquid to be fed by two items of tube pump operating time and weight change.
- FIG. 6 shows an example of a cell bottle, a preheating bottle, and a humidification bottle of the solution holding unit included in the flow path circuit used in the apparatus of this example.
- the cell bottle 601 of this embodiment includes a cell bottle main body 602 and a cell bottle lid 603.
- the cell bottle lid 603 is provided with a liquid-feeding channel tube 604 and an internal pressure adjusting channel tube 605, and one end of each is in the cell bottle main body 602.
- the end on the cell bottle side of the flow channel tube 604 is in contact with the bottom surface of the cell bottle main body 602.
- the cell suspension can be used efficiently, and as a result, the amount of cells collected from the patient is reduced and the burden on the patient is reduced.
- a sterile connection portion 606 is attached to the end of the flow channel tube 604 for liquid feeding opposite to the cell bottle.
- the end on the cell bottle side of the internal pressure adjusting channel tube 605 is in a portion that becomes a gas phase when the cell suspension is put into the cell bottle main body 602.
- a filter 607 is attached to the end opposite to the cell bottle side of the internal pressure adjusting flow channel tube 605, and for example, a filter that does not pass particles of 0.22 ⁇ m or more is used.
- the medium bottle is built in the refrigerator, because the medium is common to the two types of cells.
- the medium bottle has the same configuration as the above-described cell bottle 601, and a medium is used instead of the cell suspension.
- the medium is stored in a refrigerator at a low temperature of 4 ° C., for example, in order to prevent deterioration of growth factors and the like in the medium.
- the preheating bottle portion of this embodiment includes a preheating bottle 608 and a receiving portion 609.
- the medium is stored at 4 ° C. until use, but since the culture is performed at 37 ° C., the medium is preheated in the preheating bottle section 6 before the medium replacement.
- this method does not require temperature control of the heater, and it is possible to avoid an increase in the number of parts and an increase in channel tube length.
- the preheating bottle 608 includes a preheating bottle main body 610 and a preheating bottle lid 611.
- the periphery of the preheating bottle 608 is surrounded by a receiving portion 609 made of a material such as aluminum having high thermal conductivity.
- a supply channel tube 612 and a liquid supply channel tube 613 are attached to the preheating bottle lid 611, and one end of each is in the preheating bottle main body 610.
- the end of the pre-heating bottle side of the liquid-feeding channel tube 613 is in contact with the bottom surface of the pre-heating bottle main body 610.
- the end of the supply channel tube 612 on the preheating bottle side is in a portion that becomes a gas phase when the culture medium is put into the preheating bottle main body 610.
- a preheating bottle is installed in the back of a cell bottle, for example. Since both the cell bottle and the preheating bottle measure the weight at the time of liquid feeding, the installation space of the weigh scale may be reduced by placing all containers on the same weigh scale as necessary.
- the humidifying bottle portion of this embodiment is composed of a humidifying bottle 614.
- the humidifying bottle 614 includes a humidifying bottle main body 615 and a humidifying bottle lid 616.
- An air supply channel tube 617 and a gas supply channel tube 618 are attached to the humidifying bottle lid 616, and one end of each is in the humidifying bottle main body 615.
- the end of the air supply channel tube 617 on the cell bottle side is in a portion that becomes a gas phase when sterilized water is put into the humidifying bottle main body 615.
- a filter 619 is attached to the end of the air flow channel tube 617 opposite to the humidifying bottle side.
- the filter 619 is of a quality that does not pass particles of, for example, 0.22 ⁇ m or more.
- a sterile connection 620 is attached further outside the filter 619.
- the filter may be attached at any location as long as it is between the sterile connection portion 620 and the gas supply portion 10.
- the end of the gas supply flow channel tube 618 on the humidifying bottle side is in contact with the bottom surface of the humidifying bottle main body 615.
- the purpose of the humidification bottle is to saturate the water with respect to the gas delivered. Therefore, the efficiency of saturating the gas is improved when the time of contact with the sterilized water is increased.
- a filter may be installed at the end of the gas supply channel tube 618 on the humidifying bottle side to make the bubbles fine. Since the surface area increases, the saturation efficiency also increases.
- a connecting portion 621 is installed at the end of the gas supply channel tube 618 opposite to the humidifying bottle side and used for connection to the gas supply portion 10.
- the drainage bag portion 7 of this embodiment shown in FIG. 1 collects drainage which is an old medium used for culture.
- the flow path of the present embodiment shown in FIG. 2 separates the upper and lower drainage of the culture vessel 201 and collects the next drainage in a batch. In this way, the medium of each layer is analyzed by collecting the upper and lower layers separately at each medium exchange, and determining whether the cells are cultured normally during the culture process Is possible.
- the drainage bag 7 is provided in the container. However, when the component analysis of the drainage is performed, the drainage bag 7 is also stored in the refrigerator to maintain the quality of the drainage. Also good.
- the waste liquid when the component analysis of the waste liquid is not performed, the waste liquid may be collected in a lump without dividing the upper layer and the lower layer.
- a sterile desorption part is introduced immediately before the drainage bag and removed aseptically.
- a solenoid valve is introduced immediately before each drainage bag.
- the rotary valve mechanism described above is introduced, or a clip that is handled manually and does not require energization because it is used only once. You may do it.
- This apparatus does not move the stage on which the culture vessel is placed and observes the cells in the observation. Instead, the phase contrast microscope 700 is applied to the culture vessel base 202 on which the culture vessel 201 is installed. Rotate and observe the direction of rotation and the horizontal direction of the installation surface.
- the culture container itself is moved back and forth in the horizontal direction with respect to the installation surface of the culture container base 202, a mechanism for independently moving each culture container is required, which complicates the apparatus.
- the relative position of the flow tube with respect to the culture vessel base 202 also changes when each culture vessel 201 is moved independently.
- the length of the flow path tube becomes longer, and there is a case where the risk of cell loss or occurrence of damage to the cell membrane increases at the time of sowing. Furthermore, when the culture vessel itself is driven, the connected flow channel tube is twisted, and there is a risk of cell loss or the like as described above.
- the phase contrast microscope 700 moves with respect to the culture vessel base 202 as described above.
- the culture vessel base can be fixed and handled during cultivation, so that twisting of the flow tube caused by rotation of the culture vessel base for observation can be avoided, and the risk of flow channel damage can be avoided. It is.
- the phase-contrast microscope 700 has an operating part 702 that is a driving means for operating the culture vessel base 202. Therefore, rotational movement in the rotation direction of the phase-contrast microscope 700 and back-and-forth movement in the horizontal direction with respect to the installation surface of the culture vessel base 202 are possible. Note that another configuration example related to microscopic observation shown in FIG. 7B will be described later in Example 6.
- FIG. 8 shows a state where the door 802 of the incubator 801 in the incubator section of the automatic culture apparatus is closed.
- a monitor monitor 803 is installed in the incubator 801.
- the monitor monitor 803 displays the temperature in the incubator and the operation status indicating whether the operation of the solenoid valve, tube pump, microscope, or the like is normal.
- Incubator 801 maintains the interior at 37 ° C.
- the gas supply unit 804 corresponding to the gas supply unit 10 of FIG. 1 includes a gas cylinder and a gas flow controller.
- the gas cylinder is filled with air containing 5% CO2.
- the type of gas to be filled can be changed according to the medium used for culture and the cell type to be cultured. During the gas exchange, the gas flow rate is adjusted by the gas flow controller.
- the incubator 801 is on a desk 805.
- a refrigerator 806 for storing medium bottles at, for example, 4 ° C., and a drainage bag for the drainage bag unit 7 are provided under the desk. It has a storage 807 for storage.
- the incubator 801, the refrigerator 806, and the storage 807 are connected by a flow channel tube 808.
- This means that the incubator 801 maintained at 37 ° C. and the refrigerator 806 maintained at about 4 ° C. are not spatially close to each other with a CPC air-conditioned space generally being about 25 ° C. between them. Yes.
- This configuration eliminates the need for advanced heat insulating material between the incubator 801 and the refrigerator 806, and enables cost reduction and temperature maintenance performance to be improved by simplifying the device configuration.
- the fully automatic culture apparatus can be controlled by a single control apparatus.
- a management monitor that enables management from outside the CPC will be installed if necessary.
- the components inside the apparatus such as the flow path section 2 and the culture vessel base 202, which are installed in the incubator 801, are connected to the rails installed in the incubator 801. Can be pulled out from the door 802 in a lump.
- the user connects and installs the flow channel to the flow channel unit etc. at the start of culture, etc., it becomes possible to install with the mounting table pulled out, reducing the complexity when installing the flow channel, It is possible to suppress human error due to.
- it is desirable that the flow path unit 2 and the microscope 700 of the observation unit 8 are not completely taken out from the incubator 801 but only a part thereof.
- the microscope 700 may be fixed in the apparatus.
- the part to be removed from the incubator 801 may be the flow path unit 2 and the culture vessel base 202, and the system without removing the microscope may be used. In this case, since the number of components to be pulled out is reduced as compared with the above-described method, the drawing operation becomes easier.
- a bundle of channel tubes 902 is arranged on the outer peripheral side of the culture vessel base 202 from the culture vessel base 202 toward the rotary valve mechanism 903 corresponding to the rotary valve mechanism 3 of FIG. This is because the driving of the microscope 700 is not hindered as described above.
- a bundle 902 of flow tube is a bundle of 20 flow tubes for supplying various solutions to the culture vessel and 20 flow tubes for discharging.
- a sterile desorption part is also arranged on the inner peripheral side with respect to each culture container.
- the rotary valve mechanism 903 is positioned on the rotary valve mechanism base 904, and 40 channel tubes are installed on the rotary valve mechanism base 904.
- the bundle 902 of the flow tube is positioned near the center of the 40 flow tubes arranged in the rotary valve mechanism 903. This is to make the length of each flow tube uniform while making it easy for the user to handle the bundled flow tube.
- the ten culture vessels on the culture vessel base 202 are arranged symmetrically with respect to the bundle 902 of flow channel tubes. This makes it possible to minimize the difference in the distance between the culture vessel 201 located farthest from the rotary valve mechanism 903 and the culture vessel located closest.
- the lengths of the flow tube to each culture container equal. Specifically, the length from the multi-branch portion branching from one channel tube to 10 to each culture vessel is made uniform.
- the flow path tube in order to make the manufactured cells uniform in the plurality of culture vessels 201, it is important to equalize the length of the flow path tube to each culture vessel 201. In order to improve the quality of the regenerated tissue, it is desirable to configure the flow path tube having a uniform length between the culture vessels so as to be the shortest.
- the rotary valve mechanism 903 is installed on the door side and below the culture vessel base 202 as shown in (A) on the left side of FIG.
- the reason for installing on the door side is to make it easier for the user to attach a flow path or the like to the rotary valve mechanism 903.
- the reason for installing the culture vessel base 202 below the culture vessel base 202 is to prevent the culture vessel base 202 from becoming an obstacle when inserted into the apparatus.
- the portion farthest from the rotary valve mechanism 903 is a notch portion where the culture vessel 201 cannot be disposed in the culture vessel base 202.
- the culture vessel 201 is not arranged on the extended line in the direction from the rotary valve mechanism 3 side to the microscope 700 side.
- the culture vessel 201 can be configured not to be disposed at a position farthest from the rotary valve mechanism 903. Therefore, the culture vessel 201 that is the farthest from the rotary valve mechanism 903, which serves as a reference for equalizing the length of the flow path, is disposed closest to the cut portion, such as 201-1, 201-10. Culture container.
- the rotary valve mechanism 903 is arranged at a position on the flow path side or the like, the position farthest from the rotary valve mechanism 903, that is, the extension in the direction from the rotary valve mechanism 903 side to the microscope side 700. Since there is no notch portion on the line, the culture vessel is arranged on the side facing the rotary valve mechanism 903 via the microscope 700. Therefore, since this culture container serves as a standard for making the length of the flow path uniform, the flow path becomes longer than the configuration of the present embodiment.
- the cells produced in the culture vessel are more uniform and more uniform than other arrangement relationships. Higher quality can be achieved.
- the flow path tube bundle 902 shown in (A) on the left side of FIG. By installing the jig 905 to be arranged between the rotary valve mechanism 903 and the culture vessel base 202, troublesomeness when the culture vessel base 202 is attached to and detached from the apparatus is eliminated, and it is also easier to check whether there is a wiring mistake during manufacturing. It becomes.
- the 20 channel tubes supplied to the culture vessel 201 are branched from one for each cell type. That is, one flow tube 403 for the purpose of supplying various solutions from the cell bottle 404 of epithelial cells to the upper layer of the culture vessel 201 via the electromagnetic valve 402 and the tube pump 401 is divided into ten in the multi-branch portion 222. Divided. The same applies to the feeder cell cell bottle 405. Although not shown in the figure, the medium is sent from the medium bottle stored at 4 ° C. in the refrigerator to the preheating bottle 406, and is sent to each culture vessel 201 after preheating.
- the two cell bottles 404 and 405 are each branched into ten at the multi-branch portion 222, but the length from the multi-branch portion 222 to each culture vessel 201 is equal as described above. Thereby, it becomes possible to make the liquid feeding conditions at the time of cell seeding and medium exchange the same. If the flow path length to each culture vessel is uniform, the distance from the multi-branch portion branching from 1 to 10 to each culture vessel differs for each culture vessel, so the flow path from the multi-branch portion to each culture vessel Among the lengths, the flow path tubes corresponding to different lengths may be installed as adjustment paths in an adjustment path installation area provided in the apparatus.
- FIG. 10 is a diagram showing a three-dimensional arrangement of only the portion related to the flow path of the apparatus of the present embodiment and the state of the apparatus when the flow path is installed. If the flow tube is arranged in a straight line according to the distance from the multi-branch portion to each culture vessel, the difference between the length of the flow tube and the distance is generated as the deflection of the flow tube. A group of these portions is an adjustment path 1001 shown in FIG. In order to prevent the adjustment path 1001 from being affected by potential energy during liquid feeding, the adjustment path 1001 is arranged so that the flow path tubes are on the same plane. Thereby, the influence on the liquid feeding speed etc. by the difference in potential energy can be suppressed.
- the position of the adjustment path 1001 is installed between the multi-branch portion and each culture vessel, the purpose of equalizing the distance to each culture vessel is achieved.
- the flow path tubes constituting the adjustment path 1001 are located on the same plane, but the plane to be installed is most preferably on the plane where the rotary valve mechanism 903 is installed. This is because the rotary valve mechanism 903 is located below the culture vessel base 202 on which the user works and is also outside the driving range of the microscope 700, so that it is a position that is unlikely to become an obstacle during cell culture.
- an area where the adjustment path 1001 is arranged may be referred to as an adjustment area.
- the rotary valve mechanism 903 is positioned on the rotary valve mechanism base 904.
- the rotary valve mechanism installation plate 1002 When the rotary valve mechanism installation plate 1002 is installed, the rotary valve mechanism 903 is rotated in the horizontal direction with respect to the rotary valve mechanism base 904 as shown in FIG.
- the rotary valve mechanism seems to close vigorously when the rotary valve mechanism installation plate is installed. In this case, it is possible to suppress the risk of injury to the operator.
- FIG. 11 shows the movement of the culture vessel base immediately after cell seeding and at the time of medium exchange and the movement of the microscope at the time of cell observation in the apparatus of this example.
- the culture vessel base 202 changes the installation angle with respect to the horizontal plane by three actuators 901.
- the cell distribution in the culture vessel 201 can be made uniform by swinging the culture vessel base 202.
- the culture vessel 201 and feeding the solution there is an effect that the generation of bubbles in the vessel can be suppressed.
- the time of arrangement replacement as shown in the sectional view of FIG.
- FIG. 12 shows different apparatus configuration examples for equalizing the channel lengths from the multi-branch portion to the respective culture vessels in the apparatus of the present embodiment.
- the flow channel length may be equal to each flow tube while minimizing the flow channel length from the multi-branch portion for feeding epithelial cells and feeder cells to each culture vessel.
- 12A to 12D a microscope 700, a rotary valve mechanism 902, a flow path unit 2, and a culture vessel base 202 which are main components of the apparatus are shown, and the others are omitted.
- FIG. 13 is a functional block diagram illustrating the functional configuration of the above-described automatic culture apparatus.
- Each component controlled by the control device 1301 corresponding to the control unit in FIG. 1 is arranged inside the incubator unit / refrigerator / housing 1303 and connected to the culture vessel 201. Needless to say, what is placed in the incubator / refrigerator / storage 1303 is a culture vessel installed in the automatic culture apparatus.
- the control device 1301 includes a temperature adjusting unit 1304 that controls the temperature of the incubator, refrigerator, and storage 1303, a temperature sensor 1305, and a gas supply unit 1306 that supplies gas into the culture vessel corresponding to the previous gas supply unit 10.
- a temperature adjusting unit 1304 that controls the temperature of the incubator, refrigerator, and storage 1303, a temperature sensor 1305, and a gas supply unit 1306 that supplies gas into the culture vessel corresponding to the previous gas supply unit 10.
- fluid movement control unit 1308 for automatically feeding the liquid and gas in the channel corresponding to the previous channel unit 2, and the previous microscope 700
- a cell observation microscope 1309 is connected.
- FIG. 14 is a flowchart for explaining the operation of the automatic culture apparatus.
- the operation of the automatic culture apparatus of this example will be described with reference to FIG.
- the culture procedure may be carried out by sequentially performing the following operations on each culture vessel.
- Step S1 Start> Activate the automatic culture device.
- the operation is started when the operator presses the start switch of the operation unit in the control device.
- a value related to the internal environment of the automatic culture apparatus is displayed on the operation screen of the display of the control unit 12.
- Step S2 Schedule determination> An automatic culture schedule to be executed by an automatic culture apparatus is input according to the type and amount of cells to be cultured. Conditions such as date, frequency, fluid volume, etc. for operations such as cell seeding, medium exchange, microscopic observation, drainage collection, examination tissue collection, transplantation tissue collection, etc. are controlled from the control terminal 13 connected to the control unit 12 or the like. input.
- Step S3 Cell seeding> After opening and closing the appropriate solenoid valve, operate the tube pump to draw the cell suspension from the cell bottle.
- the cell suspension is composed of oral mucosal epithelial cells suspended in KCM medium (keratinocyte culture medium) and 3T3-J2 cells or NIH suspended in KCM medium. -3T3 cells and the like. Each is in a different cell bottle.
- each cell suspension is sent to the culture vessel 201 from two cell bottles.
- the state is set so that the liquid can be fed by opening the culture vessel and the electromagnetic valve connected to the flow path.
- the solenoid valve connected to the culture vessel and the flow path that are not the liquid supply target is closed so that the liquid cannot be supplied.
- Cell seeding is sequentially performed on the upper and lower layers of 10 culture vessels.
- Step S4 Cell Culture> Incubation is performed for a predetermined time in a state where the culture vessel 201 is left horizontally.
- the stationary period is about 3 days after seeding.
- the inside is maintained at 37 ° C. by an incubator.
- the air in the apparatus is constantly stirred by a fan so that the temperature distribution is always uniform.
- gas exchange is performed by supplying a predetermined component gas into the culture vessel. Gas exchange is also performed several times a day during the culture period.
- air containing 5% CO2 is supplied into the culture container.
- the gas is supplied from a gas supply unit, and water molecules are saturated by passing the humidifying bottle unit 11 before supplying the gas to each culture vessel. Thereby, it is avoided that water is evaporated from the culture medium in each culture vessel, and as a result, the culture medium components are changed. Further, the gas is directly supplied to each culture vessel through a gas supply circuit in parallel with the tube pump without using a tube pump.
- the air supply speed can be increased as compared with the case through the tube pump, and the gas exchange efficiency is improved. Also, the load on the tube pump is eliminated. Unnecessary gas after being supplied to the culture vessel is discharged out of the flow path through the filter. Further, the air pressure in the flow path is adjusted through a filter as necessary. For example, a filter having a quality that does not pass particles of 0.22 ⁇ m or more is used.
- the culture vessel 201 used in the apparatus of the present embodiment does not distinguish between a channel tube used for liquid feeding and a channel tube used for air feeding. That is, the air supply function is used in combination in the flow channel tube used for liquid supply.
- the number of channel tubes connected to the culture vessel is reduced as compared with the case where the channel tube used for liquid feeding and the channel tube used for air feeding are made independent. As a result, the flow path can be simplified.
- ⁇ Step S5 Observation with a microscope>
- Cell images are acquired using a microscope placed in an automatic culture device.
- the light source installed in the automatic culture apparatus is appropriately illuminated, and the cells are focused and imaged with a microscope. If necessary, set a fixed point on the culture surface and take a picture.
- the acquired cell image is stored in a database and viewed on a control terminal installed outside the apparatus. Judging from the information regarding the growth state of the cells obtained by microscopic observation, the frequency and timing of medium replacement are adjusted. For example, when the cell adhesion is insufficient, the culture of S4 cells is continued without performing the medium exchange of S6.
- ⁇ Step S6 Medium replacement> Medium exchange is performed once every few days during the culture period.
- the medium stored at 4 ° C. in the refrigerator is fed to the preheating bottle and preheated. It heats by the heat conduction by the contact with the receiving part arrange
- the old medium is discharged from the culture vessel 201.
- the culture vessel is tilted to the outlet side by the actuator so that the entire amount of the old medium is discharged.
- a new pre-heated new medium is supplied into the culture vessel. This avoids cell drying and temperature drop on the culture surface.
- the old medium is finally discharged to the drainage bag unit 7. Assuming that it is used for medium component analysis, in this embodiment, the upper and lower layers of the culture container are collected in a separated state.
- the cell suspension and medium flow in one direction in the channel circuit shown in FIG. That is, the old medium used for culture in the culture container and the new medium not used for culture are not mixed.
- an old medium and a new medium have different amounts of glucose consumed by cells and lactic acid produced, which means that the culture environment changes when they are mixed at the time of medium exchange.
- the effect of improving the reproducibility of cell culture can be obtained by preventing both from being mixed by the apparatus configuration of the present embodiment.
- the effect of improving the accuracy of medium component analysis can be obtained because the new medium and the old medium are not mixed.
- Step S7 Collection of examination tissue>
- one culture container is aseptically removed from the apparatus for testing for determining whether or not transplantation is possible. If necessary, the number of culture vessels taken out for testing may be plural.
- a sterile desorption part installed in a flow channel tube in the vicinity of each culture vessel is used.
- the aseptic desorption part is, for example, a heat-sealable flow channel tube, and cuts between two parts sandwiching the cut part after heat welding. This makes it possible to maintain the sterility of the removed culture container and the culture container and the flow channel that have not been removed even after the removal. Thereafter, the removed culture container is promptly inspected to determine whether transplantation is possible.
- Step S8 Culture and medium exchange just before transplantation> As in steps S4 and S6, cell culture and medium exchange are performed.
- Step S9 Collection of transplanted tissue>
- the fact that the culture is completed is displayed on the display of the control unit.
- the tissue is aseptically removed from the apparatus, transported to the operating room, and used for regenerative medical treatment.
- the length of the flow path from the culture vessel to the multi-branch portion or the cell bottle is shortened as much as possible, and the length of each flow path is made equal to each culture.
- Treatment with a regenerated tissue with uniform quality of cells cultured in the container can be performed.
- Example 1 In the automatic culture apparatus of Example 1, the configuration in which the flow tube connected to each culture vessel is bundled by using a jig or the like is shown. However, in the automatic culture apparatus of Example 2, the flow tube is not bundled. An embodiment in which the valves of the corresponding rotary valve mechanisms are installed independently will be described.
- FIG. 23 shows a perspective view of a configuration example of an automatic culture apparatus capable of suppressing the crossing and twisting of the flow paths of this example.
- the flow channel on the right side of the culture vessel base 202 is shown for explanation, but the flow channel is similarly arranged on the left side of the vessel base 202.
- each culture vessel 201 is sequentially connected from the culture vessel 201-1 farthest from the rotary valve mechanism 903 to the valve on the center side from the end side valve of the rotary valve mechanism 903. That is, the culture vessel 201-5 located near the center as viewed from the door side is placed on the end side of the grip of the valve 2001 provided near the center of the rotary valve mechanism 903. 201-1 are respectively connected to the grips of the valve 2002 provided on the end side.
- the flow path length is made uniform with reference to the length of the flow path 2301 disposed in the grip of the valve 2002 on the endmost side of the rotary valve mechanism 903.
- the adjustment path connected to the same plane as the rotary valve mechanism 903 is longer in the center position of the rotary valve mechanism, that is, the culture container arranged at a position closer to the rotary valve mechanism. Therefore, the arrangement area of the adjustment path arranged in the adjustment region is large, and the installation area can be reduced toward the end.
- a notch 1501 for stably arranging the flow paths in the culture vessel base 202 is provided. You may provide in both right and left. One notch 1501 may be provided on each of the left and right sides, or may be provided for each flow path connected to each culture vessel 201.
- a partition 1502 may be provided on the culture vessel base 202 so as to partition the flow paths, and the installation space for arranging the flow paths may be divided.
- the notch 1501 and the partition 1502 in each flow path, it becomes possible to reduce the risk of tangling with a flow path tube connected to another culture vessel 201.
- a holding means for holding the flow path such as forming the notch 1501 or forming a partition, may be provided.
- Example 3 describes an example of observing the microscope by driving the outer peripheral side of the culture vessel base.
- the flow tube is arranged on the inner peripheral side of the culture vessel base 202 described in Example 1, there is no flow tube that can interfere with driving the microscope on the outer peripheral side. It becomes possible to make it.
- FIG. 16 shows an example in which a flow tube is arranged toward the center of the culture vessel base 202 and the microscope 700 moves and observes the outer peripheral side of the culture vessel base 202 as a configuration of the present embodiment. Since the bundle of flow channel tubes has a volume smaller than that of the microscope 700, the inner space of the culture vessel base 202 can be made smaller than that of the first embodiment, and the entire apparatus can be downsized.
- Examples 1, 2, and 3 the configuration in which the culture vessel base is arranged at the center or the outer periphery has been described, but the shape of the culture vessel base and the arrangement position of the microscope are not limited thereto.
- a fourth embodiment an arrangement of a microscope and a culture vessel base different from the above embodiments will be described.
- FIG. 17A shows an example of the configuration.
- This is a system in which a CCD camera microscope 1701 having a lens and illumination is installed on an incubator upper surface 1702, and each culture vessel is observed from above.
- the ceiling has a rail for operating the microscope 1701.
- the flow channel tubes are arranged so as to converge toward the inner peripheral side, the flow channels can be bundled closer to each culture vessel.
- the curvature of the flow tube can be made uniform as compared with the case where it is arranged on the outer peripheral side of the culture vessel base, so that the shear stress caused by the flow tube can be made uniform.
- Cell quality can be made uniform.
- FIG. 17B shows an example in which the flow channel tube bundle 1704 is bundled and the notch 1705 provided on the outer periphery of the culture vessel base 202 on the door side is passed. Also in this configuration example, since the microscope 1701 as the observation unit is suspended from above the apparatus, there is no need to provide a gap or the like in the culture container base 202. As a result, the culture container base becomes small and the installation area of the apparatus is small. It becomes possible to do. In the case of this example, since the shape of the culture vessel base and the arrangement position of the culture vessel are not dependent on the arrangement position of the observation unit, the arrangement of the culture vessel 201 may be other than the circumferential shape.
- the culture vessels 201 may be arranged in a line and arranged in parallel in a plurality of lines.
- the aseptic detachment part 1706 of each culture vessel 201 is arranged on the same side as the flow tube bundle 1703 installed on one side.
- the distance of each culture vessel to the flow channel tube can be shortened. It is desirable to arrange in the direction.
- Example 5 an example of another shape configuration of the culture vessel base shape when the microscope is arranged on the floor or side surface of the incubator will be described with reference to FIG. Needless to say, the culture vessel base shape described in the present embodiment may be applied to a configuration in which the microscope is suspended on the upper surface of the incubator described in the second embodiment.
- the L-shaped culture vessel base 1801 shows a case where the culture vessel base is refracted to form an L-shaped crank shape (hereinafter referred to as “L-shaped”).
- L-shaped culture vessel base 1801 can narrow the driving range of the microscope while securing the observation unit arrangement region 1802, that is, the microscope arrangement space. Since the culture vessel 201 is disposed along the L-shaped culture vessel base 1801, the rotation angle during observation with a microscope is 90 °. In the U-shaped arrangement shown in the first embodiment, the rotation angle of the microscope is approximately 360 °, but the rotation mechanism of the microscope can be reduced as the angle becomes smaller.
- Example 6 As shown in FIG. 19, an example of another configuration when using a U-shaped culture vessel base as in Example 1 will be described.
- the culture vessel 201 in the culture vessel base 202 shown in Example 1 is installed twice or more on the same plane. Thereby, a plurality of culture vessels can be integrated.
- (B) of FIG. 17 is a case where the culture container base 202 shown in Example 1 is installed in multiple stages. Similarly, integration is realized.
- Example 1 Although the example which slides a U-shaped culture container base and arrange
- Example 7 describes an example in which another photographing means is installed around the microscope in the microscope of Example 1.
- FIG. 7B shows a phase contrast microscope disposed on the front surface of the microscope 705 and a pH evaluation device based on color temperature measurement disposed on the back surface.
- the pH evaluation apparatus includes a web camera 703 and a white plate 704.
- the pH is determined by the color temperature of the medium.
- phenol red is used as a pH indicator of a medium, it is close to yellow when acidic, and close to red when alkaline.
- the white plate 704 as a background, a clear image can be taken by the web camera 703.
- the pH is evaluated based on the quantified RBG component value.
- evaluation is performed by mounting an absorptiometer.
- the microscope 705 since the microscope 705 has an operation unit 702 as a driving means as a rotation function like the microscope 700 of the previous embodiment, the microscope 705 can be implemented without using another individual microscope, and the installation space is small. can do.
- Example 8 as shown in FIG. 20, an example will be described in which the length of the channel tube connected to each culture vessel and the rotary valve mechanism is made more uniform and shorter.
- each flow tube is bundled on the rotary valve mechanism 903 side. Since each culture vessel 201 is circumferentially arranged on the culture vessel base 202, the length from the bundled point (referred to as a convergence point) 2001 to each culture vessel 201 is different.
- the valves (clips) for opening and closing the respective flow path tubes in the rotary valve mechanism 903 are arranged in a row facing the culture vessel base 903. The length of the flow tube up to the convergence point 2003 is also different.
- a flow path tube indicated by a broken line connected to the culture vessel 201-5 having a short distance from the convergence point 2003 is disposed on the valve 2002 far from the convergence point 2003, that is, on the end side of the rotary valve mechanism 903,
- the flow path tube shown by the dotted line connected to the culture vessel 201-1 far from the convergence point is arranged on the valve 2001 having a short distance from the convergence point 2003, that is, on the center side of the rotary valve mechanism 903.
- Example 9 an example in which the inflow conditions of cells when passing through each flow tube are made uniform will be described with reference to FIG.
- the multi-branch part for dispersing the solution from the cell bottle to each culture vessel branches to the power of 2 through the two branch parts in order, Thus, a configuration with ten branched flow paths is adopted.
- the number of bifurcated portions 2101 relayed for each branched flow path is different.
- the connecting portion between the bifurcated portion 2101 and the channel tube has a step difference because the channel diameter is different.
- the friction applied to the solution at the bifurcated portion 2101 is different from the friction with the channel tube. That is, shear stress is generated in the cell every time it passes through the bifurcated portion 2101. Therefore, if the number of bifurcated portions 2101 relayed by each branched flow path is different, such as 3 or 4, the conditions of the cells to be cultured also differ. There is a possibility that the quality of the cells after culturing becomes non-uniform.
- the two branch portions 2101 that relay each branched flow path are connected so as to have the same number of four, so that the conditions for inflow of the solution into the culture vessel 201 are uniform.
- the closed part 2102 is formed in the path for the branch that does not connect the flow path tube by heat welding or the like.
- a relay member using the same material and flow path diameter as that of the bifurcated portion may be used without using the bifurcated portion with the one-side path closed as described above.
- the present embodiment is an embodiment having a configuration in which the normality of the flow channel installation to the measure can be determined more accurately by providing a pressure sensor in the flow channel in the flow channel installation to the apparatus in the first embodiment. .
- the normality and flow of the flow path are set for each solenoid valve by performing pressurization and depressurization while sequentially opening and closing the solenoid valves provided in the rotary valve mechanism. Evaluate whether the road is damaged.
- the electromagnetic valve in the route is opened and gas is supplied from the gas cylinder. Whether the pressure has increased thereby is evaluated by the pressure sensor 2201.
- everything except the pressure sensor 2201 is the same as the configuration of FIG. 2, and thus the figure numbers described in FIG. 2 are not shown.
- the same check is performed on the solenoid valve outside the shortest route. That is, the gas is supplied from the gas cylinder to the second route including the shortest route and the flow path to the solenoid valve located outside the shortest route, and it is confirmed whether the pressure increases. Subsequently, the solenoid valve is opened and it is confirmed whether the pressure decreases.
- This operation is sequentially performed on all the solenoid valves by a signal from the control unit.
- the present invention is useful as a culture apparatus for automatically culturing cells or tissues using a culture vessel, particularly as an automatic culture apparatus capable of producing a regenerated tissue that can be used for regenerative medicine.
- this invention is not limited to the above-mentioned Example, Various modifications are included.
- the above-described embodiments have been described in detail for better understanding of the present invention, and are not necessarily limited to those having all the configurations described.
- a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment.
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Abstract
Description
細胞ボトル601の場合は、先に説明したように、上皮系細胞の培養では上皮系細胞とフィーダー細胞に対応した2個を使用する。図6左側の(A)が示すように、本実施例の細胞ボトル601は、細胞ボトル本体部602と細胞ボトル蓋部603から成る。細胞ボトル蓋部603には送液用流路チューブ604と内圧調整用流路チューブ605が取り付けられ、それぞれの片方の端は細胞ボトル本体部602内にある。送液用流路チューブ604の細胞ボトル側の端は細胞ボトル本体部602の底面に接している。これにより送液時、送液過程が進むにつれ細胞懸濁液の量が少なくなっても送液を可能とする。細胞懸濁液を効率良い使用が可能となり、結果として患者からの細胞採取量が少なくなり患者への負担は減少する。
予熱ボトル608は、予熱ボトル本体部610と予熱ボトル蓋部611から成る。予熱ボトル608の周囲は熱伝導性の高いアルミ等の素材から成る受け部609により囲われている。予熱ボトル蓋部611には、供給用流路チューブ612と送液用流路チューブ613が取り付けられ、それぞれの片方の端は予熱ボトル本体部610内にある。送液用流路チューブ613の予熱ボトル側の端は予熱ボトル本体部610の底面に接している。これにより送液時、予熱ボトル内の培地量が少なくなっても送液可能ある。供給用流路チューブ612の予熱ボトル側の端は、予熱ボトル本体部610に培地を入れた際、気相となる部分にある。予熱ボトルは、例えば細胞ボトルの奥に設置する。細胞ボトル及び予熱ボトルは共に送液に際し重量を計測するため、必要に応じ全容器を同一の重量計に乗せることで重量計の設置スペースを小さくしても良い。
本実施例では、図8右側の(B)の矢印で示すように、培養容器ベース202を、培養容器ベース202に設けられた切りかき側から、図8左側の(A)に示す扉802を介して装置の筐体内に挿入する。顕微鏡700を、培養容器ベース202が接触しないように空隙部分に導くためである。
まず、図13は前述した自動培養装置の機能構成を説明する機能ブロック図である。図1の制御部に対応する制御装置1301により制御される各構成要素が、インキュベータ部・冷蔵庫・収容庫1303の内部に配置され培養容器201に接続されている。尚、インキュベータ・冷蔵庫・収容庫1303中に配置されるものは自動培養装置内に設置された培養容器であることは言うまでもない。
自動培養装置を起動させる。操作者が制御装置にある操作部のスタートスイッチを押すことにより起動する。制御部12のディスプレイの操作画面には自動培養装置の内部環境に関する値が表示されている。
培養する細胞の種類と量に合わせ、自動培養装置により実施する自動培養スケジュールを入力する。細胞播種、培地交換、顕微鏡観察、排液回収、検査用組織回収、移植用組織回収等の操作を行う日時、頻度、液量等の条件を制御部12に接続された制御用端末13などより入力する。
適切な電磁弁の開閉を行った後、チューブポンプを作動させ細胞ボトルより細胞懸濁液を吸引する。細胞懸濁液は、食道再生の例では口腔粘膜上皮細胞を培養するため、KCM培地(keratinocyte culture medium)に懸濁した口腔粘膜上皮細胞と、同じくKCM培地に懸濁した3T3-J2細胞またはNIH-3T3細胞等である。それぞれ異なる細胞ボトル内に入っている。
培養容器201を水平に静置した状態で所定時間、培養する。例として口腔粘膜上皮細胞の場合、静置期間は播種後3日間程度とする。培養中はインキュベータにより内部を37℃に維持する。装置内の空気はファンにより常に攪拌し、温度分布が常に一様となるようにする。尚、本例では示していないが装置内にパーティクルカウンタや生菌数計測装置を取り付け、清浄度をモニタリングし、製造の安全性を増すことが可能である。
自動培養装置内に設置した顕微鏡を用い細胞画像を取得する。自動培養装置内に設置した光源を適宜発光させ、顕微鏡により細胞に焦点を合わせ撮像する。必要に応じ培養表面に定点を任意に定め撮影する。取得した細胞画像はデータベースに保存し、装置外に設置した制御用端末上で閲覧する。顕微鏡観察により得た細胞の生育状態に関する情報から判断し、培地交換の頻度、時期の調整を行う。例えば細胞の接着が不十分な場合、S6の培地交換は実施せずS4の細胞の培養を継続する。
培地交換は培養期間中、数日に一度の頻度で実施する。最初に冷蔵庫内で4℃にて保管されている培地を予熱ボトルまで送液し予熱を行う。予熱ボトルの周囲に配置した受け部との接触による熱伝導と、インキュベータによる37℃の気相により加温する。例えば数時間~1日程度予熱を行うことで温度を36℃以上まで上昇させ、その培地を培地交換に用いる。
顕微鏡700によって観察した細胞の培養状況を考慮し、移植可否を判定する検査用として1個の培養容器を無菌的に装置から取り出す。尚、必要に応じ検査用に取り出す培養容器数は複数個であっても良い。取り出しに際しては各培養容器の近傍の流路チューブ内に設置された無菌脱着部を使用する。無菌脱着部は例として熱溶着可能な流路チューブであり、切断箇所を挟む2ヶ所を熱溶着後にその間を切断する。これにより取り外した培養容器内及び、取り外さなかった培養容器と流路内は、取り外し後も無菌性を維持可能となる。取り出した培養容器に対しその後、速やかに検査を実施し移植可否を判定する。
ステップS4及びS6と同じく、細胞の培養と培地交換を実施する。
ステップS7による検査の結果により移植可能と判断された場合、制御部のディスプレイに培養が完了した旨を表示する。その後ステップS7と同じく組織を装置から無菌的に取り出し手術室まで運び、再生医療治療に使用する。
このとき、回転式弁機構903と同一平面に接続される調整路は、回転式弁機構の中央位置、つまりは回転式弁機構からの距離が近い位置に配置された培養容器ほど調整路は長くなるため、調整領域に配置される調整路の配置面積が大きく、端に向かうほど設置面積が小さくすることができる。これは、対応する回転式弁機構の弁のグリップから培養容器ベースまでの距離が遠いほど退避させる流路長が短いため調整路の設置面積が小さく、近いほど長く調整路の設置面積は大きくなるためである。回転式弁機構903の端側、つまり培養容器ベース202の端側の調整路の設置面積を小さくすることで、ユーザの手等が接触し易い培養容器ベース202の端付近において、調整路の流路チューブとユーザとが接触することによる品質低下及び流路破損危険性を低減可能となる。
2、1203 流路部
3、213、903、1202 回転式弁機構
4 細胞ボトル部
5 培地ボトル部
6 予熱ボトル部
7 排液バッグ部
8 観察部
9 インキュベータ部
10、1306 気体供給部
11 加湿ボトル部
12 制御部
13 制御用端末
201 培養容器
202、1201、1204 培養容器ベース
203、504、901、1202 アクチュエータ
204、205、601 細胞ボトル
206 流路回路(1)
207 流路回路(2)
208 培地ボトル
209、608 予熱ボトル
210 分岐部
211、401 チューブポンプ
212、402 電磁弁
214、215 排液バッグ
216、804 ガスボンベ
217 気体フローメータ
218、614 加湿ボトル
219 送気用回路
220、303、1706 無菌脱着部
221、606、620 無菌接続部
222 多分岐部
301、701 観察孔
302、403、501、808、2201 流路チューブ
404、405、601 細胞ボトル
406 予熱ボトル
502 クリップ部
503 多連式カム部
505 バネ
506 クリップ接触部
602 細胞ボトル本体部
603 細胞ボトル蓋部
604、613 送液用流路チューブ
605 内圧調整用流路チューブ
607、619 フィルタ
609 受け部
610 予熱ボトル本体部
611 予熱ボトル蓋部
612 供給用流路チューブ
615 加湿ボトル本体部
616 加湿ボトル蓋部
617 送気用流路チューブ
618 気体供給用流路チューブ
621 接続部
700、705、1201、1309 顕微鏡
702 稼働部
703 ウェブカメラ
704 白色板
801 インキュベータ
802 扉
803 監視モニタ
805 机
806 冷蔵庫
807 収容庫
902 流路チューブ
904 回転式弁機構台
905 治具
1001 調整路
1002 回転式弁機構用設置プレート
1301 制御装置
1302 表示画面
1303 インキュベータ他
1304 温度調節部
1305 温度センサ
1306 気体供給部
1307 溶液保持部他
1308 流体移動制御機構部
1501 切り込み
1502 仕切り
1701、1705 切りかき
1702 敷居
1703、1704 流路チューブ束
1801 L字型培養容器ベース
1802 観察部配置領域
1803 S字型培養容器ベース
2001、2002 弁
2003 収束点
2101 二分岐部
2102 閉鎖部
2201 圧力センサ
2301 流路
Claims (15)
- 細胞を培養する細胞培養装置であって、
培養に用いる溶液を保持する溶液保持部と、
前記溶液保持部と複数の培養容器とを夫々接続させる複数の流路と、
を備え、
前記複数の流路は、前記溶液保持部から前記複数の培養容器夫々まで長さが等しい
ことを特徴とする細胞培養装置。 - 前記複数の培養容器は、前記複数の流路の夫々が接続される多分岐部を介して前記溶液保持部に接続され、
前記複数の流路は、前記多分岐部から前記複数の培養容器夫々までの長さが等しい
ことを特徴とする請求項1記載の細胞培養装置。 - 筐体内に着脱可能な、前記複数の培養容器を保持する培養容器ベースと、
前記複数の培養容器内を観察する観察部と、
を有し、
前記培養容器ベースには空隙が形成され、
当該培養容器ベースを前記筐体内に設置した場合、前記空隙内に前記観察部が設置され、
前記複数の流路は、前記培養容器ベースの外側に配置される
ことを特徴とする請求項1または2記載の細胞培養装置。 - 前記培養容器ベースの外周の一部に、前記観察部を前記空隙側へ通過させるための切りかきを有する
ことを特徴とする請求項3記載の細胞培養装置。 - 前記溶液保持部から前記複数の培養容器の夫々へ、前記複数の流路を介して前記溶液を供給させる供給機構を有する
ことを特徴とする請求項3または4記載の細胞培養装置。 - 前記培養容器ベースを前記筐体内に挿入させる扉部をさらに有し、
前記培養容器ベースは、前記切りかき側から、前記細胞培養容器内に挿入可能であり、
前記供給機構は、前記培養容器ベースに対して、扉部側かつ下方に配置される
ことを特徴とする請求項5記載の細胞培養装置。 - 前記供給機構は、前記複数の流路の夫々を設置させる弁機構を有し、
前記弁機構によって、前記複数の培養容器夫々に供給される前記溶液を制御させ、
前記弁機構から前記複数の培養容器の夫々との間に位置する流路の一部を調整路として設置させる調整領域を有する
ことを特徴とする請求項5または6記載の細胞培養装置。 - 前記調整領域は、前記培養容器ベースの下方かつ前記供給機構の上方に形成される
ことを特徴とする請求項7記載の細胞培養装置。 - 前記複数の流路を束状に収束させる収束手段を有する
ことを特徴とする請求項3記載の細胞培養装置。 - 前記細胞培養装置内に着脱可能な、前記複数の培養容器を保持する培養容器ベースと、
前記複数の培養容器内を観察する観察部と、
を有し、
前前記培養容器ベースには空隙が形成され、
前記複数の流路は、前記空隙を介して配置される
ことを特徴とする請求項1または2記載の細胞培養装置。 - 前記培養容器ベースに、前記複数の流路夫々を保持する保持手段が形成されている
ことを特徴とする請求項3記載の細胞培養装置。 - 前記複数の培養容器は、前記空隙の周囲に円周上に配置され、
前記観察部を自転方向と水平方向に駆動させる駆動手段を有する
ことを特徴とする請求項3から9までの何れかに記載の細胞培養装置。 - 前記複数の培養容器ベースに接続され、前記培養容器ベースの設置角度を変更させるアクチュエータを有する
ことを特徴とする請求項3から12までの何れかに記載の細胞培養装置。 - 前記扉部は、前記培養容器ベースより高い位置に形成される小扉を有する
ことを特徴とする請求項6記載の細胞培養装置。 - 細胞を培養する細胞培養装置であって、
培養に用いる溶液を保持する溶液保持部と、
前記溶液保持部から供給された前記溶液を保持する複数の培養容器と、
前記溶液保持部と接続され、前記溶液を通過させる多分岐部と、
前記多分岐部と前記複数の培養容器とを夫々接続させ、前記多分岐部を通過した溶液を前記複数の培養容器夫々に供給させる複数の流路と、
を備え、
前記複数の流路は、前記多分岐部から前記複数の培養容器夫々までの長さが等しい
ことを特徴とする細胞培養装置。
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WO2020009017A1 (ja) * | 2018-07-05 | 2020-01-09 | 富士フイルム株式会社 | 細胞培養装置及び撹拌方法 |
CN118240658A (zh) * | 2024-03-26 | 2024-06-25 | 江苏育瑞康生物科技有限公司 | Pbmc细胞激活用nk培养基自动添加装置 |
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JP6097817B2 (ja) | 2017-03-15 |
US10138450B2 (en) | 2018-11-27 |
JPWO2014141477A1 (ja) | 2017-02-16 |
EP2975110A4 (en) | 2016-11-16 |
EP2975110A1 (en) | 2016-01-20 |
US20160002584A1 (en) | 2016-01-07 |
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