WO2016006680A1 - 細胞培養システム - Google Patents
細胞培養システム Download PDFInfo
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- WO2016006680A1 WO2016006680A1 PCT/JP2015/069869 JP2015069869W WO2016006680A1 WO 2016006680 A1 WO2016006680 A1 WO 2016006680A1 JP 2015069869 W JP2015069869 W JP 2015069869W WO 2016006680 A1 WO2016006680 A1 WO 2016006680A1
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- solution
- cell culture
- medium
- container
- culture container
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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
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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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
- C12M41/44—Means for regulation, monitoring, measurement or control, e.g. flow regulation of volume or liquid level
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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
- C12M23/34—Internal compartments or partitions
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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
-
- 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
- C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
Definitions
- the present invention relates to a cell culture system for exchanging a medium in a cell culture container.
- the present invention has been made in view of the above-described circumstances, and is capable of easily replacing the medium in the cell culture container in the cell culture space and reducing the risk of contamination of the cell culture system.
- the purpose is to provide.
- the present invention provides the following means.
- the first aspect of the present invention is: Solution holding means for holding a medium for culturing cells; Medium supply means connected to the solution holding means and supplying the medium supplied from the solution holding means to a cell culture container;
- a cell culture system comprising: The cell culture container is connected to the medium supply means at a position below the medium supply means in the direction of gravity; The medium supply means has a space for dropping the medium supplied from the solution holding means;
- the cell culture container has a discharge port for discharging the medium at a desired height position on the side surface of the container, and the discharge port has a height of the upper surface of the medium when the medium is supplied from the medium supply means.
- a cell culture system for discharging a culture medium to the outside when the discharge port is reached.
- the second aspect of the present invention is: Solution holding means for holding a medium for culturing cells; Medium supply means connected to the solution holding means and supplying the medium supplied from the solution holding means to a cell culture container;
- a cell culture system comprising: The cell culture container is provided with a supply port through which the medium is supplied from the medium supply means on the upper surface of the container, and a discharge port for discharging the medium is provided at a desired height on the side of the container, The medium supplied from the supply port is dropped onto the upper surface of the medium from the supply port through the space in the cell culture container, and the height of the upper surface of the medium is increased by supplying the medium from the medium supply means to the discharge port.
- a cell culture system for discharging a culture medium to the outside when the discharge port is reached.
- a solution such as a medium can be supplied to and discharged from the cell culture container.
- the medium is dropped in the cell culture container, it is possible to reduce the risk of contamination of the medium in the solution holding means and the medium supply means located upstream of the medium.
- a dropping speed adjusting means for controlling the dropping speed of the solution may be provided.
- the old medium in the cell culture container can be replaced with an arbitrary replacement efficiency, and the deterioration rate of the medium can be slowed down.
- a controller for remotely controlling the dropping speed adjusting means may be provided.
- a pressure applying unit for applying pressure to the medium of the medium storage unit may be provided.
- the adjustment range of the dropping speed is widened, and the dropping speed can be controlled more accurately.
- save means and a temporary culture medium holding means will go up.
- a negative pressure supply means for applying a negative pressure to the discharge port of the cell culture container may be provided.
- the degree of freedom of the installation position of the discharge port of the cell culture container is increased, and the number of usable cell culture containers can be increased.
- the apparatus further comprises waste liquid holding means connected to the discharge port of the cell culture container and holding the medium supplied from the cell culture container, and the medium discharged from the cell culture container is the waste liquid
- the space in the holding means may be dropped.
- the third aspect of the present invention is: Solution holding means for holding a solution such as a medium for culturing cells; A solution supply means connected to the solution holding means and supplying a solution supplied from the solution holding means to a cell culture container; A cell culture system comprising a solution discharge means for discharging the solution from the cell culture container, The cell culture vessel is connected to the solution supply means at a position below the solution supply means in the direction of gravity; Provided is a cell culture system in which the solution supply means has a space for dropping the solution supplied from the solution holding means.
- the solution is dripped in the solution supply means, it is possible to reduce the risk of contamination of the solution such as the medium of the solution holding means upstream thereof.
- the fourth aspect of the present invention is: Solution holding means for holding a solution such as a medium for culturing cells; A solution supply means connected to the solution holding means and supplying a solution supplied from the solution holding means to a cell culture container; A cell culture system comprising a solution discharge means for discharging the solution from the cell culture container, The cell culture container is connected to the solution supply means at a position below the solution supply means in the direction of gravity, and includes a supply port through which the solution is supplied from the solution supply means on the upper surface of the cell culture container, Provided is a cell culture system in which a solution supplied from the supply port is dropped from the supply port into a space in the cell culture container.
- the solution is dripped in the cell culture container, it is possible to reduce the risk of contamination of the solution such as the medium of the solution holding means upstream thereof.
- the solution supply unit includes a dropping rate adjusting unit that controls the dropping rate of the solution
- the solution discharging unit includes a waste liquid rate adjusting unit that controls the discharging rate of the solution.
- a control unit for remotely controlling the solution dropping speed control by the dropping speed adjusting means and the solution discharging speed control by the waste liquid speed adjusting means may be provided. This allows the operator to remotely change the culture medium, reducing the number of times that the operator enters the work space, reducing labor and costs associated with the work, and contaminating the culture system. The chance of being played can also be reduced.
- a pressure applying means for applying pressure to the solution of the solution holding means may be provided.
- the adjustment range of the dropping speed is widened, and the dropping speed can be controlled more accurately. Further, the degree of freedom of the installation position of the solution storage means is increased.
- the cell culture container includes a discharge port connected to the solution discharge unit, and a negative pressure supply unit for applying a negative pressure to the discharge port of the cell culture container. May be provided.
- the adjustment range of the discharge rate of the solution from the cell culture container is expanded, and the discharge rate can be controlled with higher accuracy.
- the degree of freedom of the installation position of the outlet of the cell culture container is increased, and the number of usable cell culture containers can be increased.
- the solution discharging means includes waste liquid holding means that is connected to the cell culture container and holds the solution discharged from the cell culture container, and is discharged from the cell culture container.
- the solution thus prepared may be dropped in the space in the waste liquid holding means.
- the waste liquid is dripped in the waste liquid holding means, it is possible to prevent backflow and to reduce the risk of contamination of the cell culture container.
- Solution holding means for holding a solution such as a medium for culturing cells
- a solution supply means connected to the solution holding means and supplying a solution supplied from the solution holding means to a cell culture container
- a cell culture system comprising: Provided is a cell culture system in which the solution supply means supplies a solution to the cell culture container through a step of dropping the solution.
- Solution holding means for holding a solution such as a medium for culturing cells
- a solution supply means connected to the solution holding means and supplying a solution supplied from the solution holding means to a cell culture container
- Solution discharging means for discharging the solution from the cell culture vessel
- a cell culture system comprising: Provided is a cell culture system in which the solution discharge means discharges the solution from the cell culture container through a step of dropping the solution.
- a solution such as a medium can be supplied to and discharged from the cell culture container.
- Medium storage means for maintaining a medium for culturing cells at a temperature suitable for storage of the medium;
- a temporary medium holding means connected to the medium storage means and holding the medium supplied from the medium storage means at a temperature suitable for cell culture;
- Medium supply means connected to the temporary medium holding means and supplying the medium supplied from the temporary medium holding means to the cell culture container;
- a cell culture vessel connected to the medium supply means at a position below the medium supply means in the direction of gravity and culturing cells in the medium supplied from the medium supply means;
- a cell culture system comprising:
- the medium supply means includes a dropping container having a space for dropping the medium supplied from the temporary medium holding means, and supplies the medium dropped in the dropping container to the cell culture container;
- the cell culture container includes a discharge port for discharging the medium at a desired height position on the side surface of the container, and the discharge port has a height of the upper surface of the medium when the medium is supplied from the medium supply unit.
- a cell culture system for discharging a culture medium to the
- a solution such as a medium can be automatically supplied to and discharged from the cell culture container.
- the culture medium is dropped in the dropping container, the risk of contamination of the culture medium in the culture medium storage means and the temporary culture medium holding means upstream thereof can be reduced.
- Another aspect of the present invention is: Medium storage means for maintaining a medium for culturing cells at a temperature suitable for storage of the medium; A temporary medium holding means connected to the medium storage means and holding the medium supplied from the medium storage means at a temperature suitable for cell culture; Medium supply means connected to the temporary medium holding means and supplying the medium supplied from the temporary medium holding means to the cell culture container; A cell culture vessel connected to the medium supply means at a position below the medium supply means in the direction of gravity and culturing cells in the medium supplied from the medium supply means; A cell culture system comprising: The cell culture container is provided with a supply port supplied from the medium supply means on the upper surface of the container, and a discharge port for discharging the medium at a desired height position on the side of the container, and the medium supplied from the supply port However, when the space in the cell culture container is dropped from the supply port onto the upper surface of the medium, and the height of the upper surface of the medium reaches the discharge port when the medium is supplied from the medium supply means.
- a solution such as a medium can be automatically supplied to and discharged from the cell culture container.
- the medium is dripped in the cell culture container, the risk of contamination of the medium in the medium storage means, the temporary medium holding means, and the medium supply means located upstream thereof can be reduced.
- the present invention it is possible to remotely change the medium of the cell culture system even when the operator is not present, and the number of times the operator enters the work space can be reduced. This can reduce labor and costs such as changing to disposable work clothes, and can reduce the risk of contamination of the cell culture system by bacteria and the like.
- a cell culture system 100 according to the present embodiment is a system having the configuration shown in FIGS. 1A and 1B and is a system for exchanging a medium in a cell culture container (culture container) installed in an incubator.
- FIG. 1A shows a side view
- FIG. 1B shows a front view.
- the medium storage means 3 is installed outside the incubator 1 and stores the medium (cell culture solution) inside.
- a temperature control means 2 is provided in order to maintain the temperature of the culture medium at a temperature suitable for storage (for example, 4 ° C.).
- the medium storage means 3 is connected to a temporary holding means 4 installed inside the incubator 1 via a tubular member (tube or the like). Since the medium storage means 3 is installed at a higher position in the direction of gravity than the temporary holding means 4, the medium in the medium storage means 3 is supplied to the temporary holding means 4 via a tubular member (tube or the like) by gravity. .
- the medium supplied to the temporary holding means 4 is warmed to a temperature in an incubator (for example, 37 ° C.) that is a temperature suitable for cell culture.
- the medium heated to a temperature suitable for cell culture (for example, 37 ° C.) by the temporary holding means 4 is supplied to the cell culture container 11 through the medium supply means 6 in the incubator 1.
- the medium supply means 6 includes a dropping speed adjusting means 7 and a dropping container 8.
- the dropping speed adjusting means 7 is installed in a tubular member that connects the temporary holding means 4 and the dropping container 8, and can control the flow rate of the medium flowing in the tubular member.
- the dripping container 8 is a container having a supply port 9 connected to the tubular member connected from the temporary holding means 4 and a discharge port 10 connected to the tubular member connected to the cell culture container 11.
- the supply port 9 is disposed in the upper part in the gravity direction, and the discharge port 10 is disposed in the lower part in the gravity direction from the supply port 9.
- the medium supplied to the supply port 9 is dropped into the space in the dropping container 8, discharged from the discharge port 10, and supplied to the cell culture container 11 through the tubular member. As described above, the medium is dripped into the space in the dropping container 8 to prevent the back flow of the medium, and the medium in the temporary holding means 4 and the medium storage means 3 is contaminated (contamination occurs). This can be prevented.
- the cell culture container 11 has a supply port 12 connected to a tubular member connected from the dropping container 8 and a discharge port 13 for discharging the medium outside the cell culture container.
- the discharge port 13 is disposed on the side surface of the cell culture container 11 so that when the amount of medium (height of the medium) in the cell culture container exceeds a certain value, the medium is discharged to the waste liquid holding means 14 through the discharge port 13. It has become.
- the amount of the medium retained in the cell culture container is determined by the installation height of the discharge port 13.
- the installation position of the supply port 12 is arbitrary, the conversion efficiency of the culture medium can be increased by separating the discharge port 13 as far as possible.
- the waste liquid holding means 14 has a waste liquid supply port 15 connected to a tubular member connected from the cell culture container 11 and a waste liquid discharge port 16 for discharging the medium outside the waste liquid holding means 14 and functions as a medium discharge means. .
- the waste liquid supply port 15 is installed on the upper surface of the waste liquid holding means 14, and the medium supplied to the waste liquid supply port 15 is dropped into the space in the waste liquid holding means and discharged through the waste liquid discharge port 16. In this way, the medium is dropped into the space in the waste liquid holding means 14, so that the back flow of the medium can be prevented and the inside of the cell culture container 11 can be prevented from being contaminated (contamination occurs). it can.
- FIG. 1B shows an example of a cell culture system including four sets of the medium supply means 6 and the cell culture container 11.
- the dropping speed adjusting means 7 is disposed on a tubular member (tube or the like) that connects the discharge port 5 of the temporary holding means 4 and the supply port 9 of the dropping container 8, and is deformed by applying an external force to the tubular member.
- a tubular member tube or the like
- the flow rate of the solution is limited to suppress the flow rate.
- the dripping speed adjusting means 7 adjusts the flow rate of the solution flowing in the tubular member by the strength of the external force with respect to the tubular member.
- FIGS. 2A to 2D An example of how to apply an external force to the tubular member by the dropping speed adjusting means 7 is shown in FIGS. 2A to 2D.
- 2A shows an example in which the tubular member 20 is sandwiched by two plate-like members 21
- FIG. 2B shows an example in which the tubular member 20 passed through the through hole 23 is sandwiched by a plurality of spherical (or cylindrical) members 22
- FIG. 2D shows an example in which the inner diameter of the through hole 26 through the tubular member 20 is reduced and the tubular member is deformed.
- a liquid feed pump such as a peristaltic pump may be used as the dropping speed adjusting means 7.
- the user of this system first sets the system to the state where the dropping speed is 0, that is, the state where the dropping is stopped, by the dropping speed adjusting means 7.
- the culture medium storage means 3 and the temporary holding means 4 are supplemented with a culture medium, and the temperature of the medium in the temporary holding means 4 is set to the temperature in the incubator.
- a cell culture container 11 containing a culture medium and cells is prepared, and in the incubator, the supply port 12 of the cell culture container 11 is connected to the discharge port 10 of the dropping container 8 via a tubular member, and the discharge port 13 of the culture container 11 is tubular.
- Each is connected to a waste liquid supply port 15 of the waste liquid holding means 14 via a member.
- the user first adjusts the dropping speed adjusting means 7 to set the dropping speed to an appropriate speed.
- the dropping speed is released from the state of 0 by the dropping speed adjusting means 7, the medium starts to drop by gravity in the dropping container 8.
- the adjustment of the dropping speed may be set to a predetermined speed, or may be adjusted to an appropriate dropping speed while being visually observed by the user.
- the medium When dripping is started, the medium is supplied to the cell culture container 11 through the dripping container 8, and the medium exceeding the specified amount in the cell culture container 11 is discharged from the discharge port 13 of the cell culture container 11 to hold the waste liquid. It is discharged out of the incubator through the means 14. As a result, the old medium in the cell culture vessel 11 is replaced with a new medium, and the rate at which the medium deteriorates can be reduced.
- the cell culture system 200 As shown in FIGS. 3A and 3B, the cell culture system 200 according to the present embodiment is not provided with the dropping container 8, and is the first implementation in that the supply port 12 of the cell culture container 11 is arranged on the upper surface of the cell culture container 11. It is different from the form. The rest is the same as in the first embodiment. In this case, since the supply port 12 of the cell culture container 11 is located on the upper surface of the cell culture container 11, the supplied medium is dropped toward the medium surface through the space in the cell culture container 11. It has the same function. Therefore, also in the present embodiment, the back flow of the medium can be prevented, and the medium in the temporary holding means 4 and the medium storage means 3 can be prevented from being contaminated (contamination occurs).
- the dropping container 8 since the dropping container 8 is not installed, the system can be made compact. Since the dropping container 8 is not provided, the discharge port 5 of the temporary holding means 4 and the supply port 12 of the cell culture container 11 are directly connected via a tubular member, and the dropping speed adjusting means 7 is installed on the tubular member. ing.
- a cell culture system 300 shown in FIGS. 4A and 4B can be exemplified.
- This modification employs the dropping container 8 in the second embodiment as in the first embodiment.
- the medium is dropped in the dropping container 8 and the cell culture container 11, and the medium in the temporary holding means 4 and the medium storing means 3 is contaminated (contamination occurs) due to the back flow of the medium. Can be further reduced.
- the cell culture system 400 according to the present embodiment is different from the above embodiments in that it includes a pressure applying unit 60 for applying pressure to the medium in the medium storage unit 3. ing.
- a pressure applying unit 60 for applying pressure to the medium in the medium storage unit 3.
- 5A and 5B are examples corresponding to the first embodiment, but other embodiments are also the same.
- Examples of the pressure applying means 60 include a means for sending gas into the medium storage means 3 by a pump. Thereby, the pressure of the air layer in the culture medium storage means 3 is increased, and an external pressure can be applied to the culture medium. It is preferable to use a sterilized gas to be fed into the culture medium storage means 3.
- gas (or liquid) is fed into the bag-like member 61, and pressure is applied to the culture medium with the bag having an increased volume.
- the isolation is movable while ensuring airtightness.
- the member 62 may be used to block the air layer 63 and the medium, and a gas (or liquid) may be sent to the air layer 63 to apply pressure to the medium using the isolation member 62.
- the weight 65 having a predetermined weight is put on the upper portion of the separating member 64 for isolation.
- a constant pressure may be applied to the member 64.
- the medium holding means and the partition member are structured like a syringe cylinder and a pusher (plunger), respectively, and the partition member, which is a pusher (plunger), is mechanically moved to apply pressure to the medium inside the medium holding means. It is okay to spend.
- the movement of the partition member may be controlled by the control unit in a wired or wireless manner.
- the medium holding means has a cylindrical structure
- the partition wall member has a disk-like structure that fits on the inner wall of the cylindrical structure
- the screw structure that meshes with the inner wall of the cylindrical structure and the outer periphery of the disk-like structure.
- the pressure applying means 60 is preferably controlled so that the pressure in the medium storage means 3 does not rise above a certain level.
- the culture medium storage unit 3 does not necessarily have to be installed at a higher position than the temporary holding unit 4.
- the temporary holding means 4 is not necessarily installed at a position higher than the medium supply means 6.
- the cell culture system 500 includes the above-described embodiments in that it includes negative pressure supply means 71 for applying a negative pressure to the discharge port 13 of the cell culture container 11. It is different from the form. Other than that is the same as each said embodiment. 7A and 7B are examples corresponding to the first embodiment, but other embodiments are also the same.
- the negative pressure supply means 71 As an example of the negative pressure supply means 71, a means provided with a pump 81 and a waste liquid container 82 as shown in FIG.
- the suction port 83 of the negative pressure supply means 71 is connected to the waste liquid discharge port 16 of the waste liquid holding means 14 so that the inside of the waste liquid holding means 14 has a negative pressure, and the discharge port 13 of the culture container 11 has a negative pressure. It is possible to aspirate the culture medium.
- a liquid feed pump such as a peristaltic pump may be used as a pump for the negative pressure supply means. In this case, a liquid feed pump may be installed on the tubular member of the suction port 83.
- the cell culture system 600 As shown in FIGS. 9A and 9B, the cell culture system 600 according to the present embodiment is configured such that, in each of the above embodiments, the dropping rate adjusting means 7 is remotely or wirelessly connected by the control unit 19 installed outside the incubator. Can be operated manually. Other than that is the same as each said embodiment. 9A and 9B are examples corresponding to the first embodiment, but other embodiments are also the same.
- the drip rate adjusting means 7 is capable of exchanging information with the control unit 19 installed outside the incubator wirelessly or by wire, and can be used to remotely transfer a solution such as a medium flowing in a tubular member (tube or the like). The flow rate can be controlled.
- the user adjusts the dropping speed adjusting means 7 remotely by the control unit 19 to set the dropping speed to an appropriate speed.
- the adjustment of the dropping speed may be set to a predetermined speed, or may be adjusted to an appropriate dropping speed while monitoring the dropping speed with a monitoring system (not shown).
- the user can remotely start medium exchange at any timing during cell culture or can remotely change the dropping rate.
- a system not shown
- it can start medium exchange remotely at any time during cell culture according to the state of cells, or remotely The dripping speed can be changed.
- the user can work without entering the work space, so that the labor and cost of changing to disposable work clothes can be reduced, and contamination of the cell culture system by bacteria etc. can be reduced. Risk can be reduced.
- the control unit 19 can exchange information with the temperature control unit 2, the pressure application unit 60, and the negative pressure supply unit 71 in each of the above embodiments wirelessly or by wire, and can control them remotely. Also good. As a result, the efficiency of remote work by the user can be increased.
- the cell culture container 91 shown in FIGS. 10A and 10B can also be used.
- the cell culture container 91 has a supply port 93 and a discharge port 94 in the lid portion 92 of the container.
- the supply port 93 is connected to the culture medium supply means 6 through a tubular member, and a supply pipe portion 95 extends from the supply port 93 toward the inside of the cell culture container.
- the culture medium supplied from the culture medium supply means 6 passes through the supply port 93 and enters the supply pipe part 95.
- the tip of the supply pipe part 95 is disposed above the medium surface, and the medium that has exited the supply pipe part 95 is dropped into the cell culture container 91.
- the tip of the supply pipe part 95 does not necessarily have to be disposed above the medium surface.
- the discharge port 94 is connected to the waste liquid holding means 14 through a tubular member, and a discharge pipe portion 96 extends from the discharge port 94 toward the inside of the cell culture container.
- the tip of the discharge tube portion 96 is arranged at a predetermined height from the bottom surface inside the culture vessel. When the medium in the cell culture vessel reaches the height of the tip of the discharge tube portion 96, cell culture is performed. It is discharged out of the container. The amount of the medium retained in the cell culture container can be adjusted by the height at which the tip of the discharge pipe portion 96 is arranged. At this time, the replacement efficiency of the culture medium is improved when the tip of the supply pipe portion 95 and the tip of the discharge pipe portion 96 are separated as much as possible.
- the lid portion 92 is formed by cutting a screw engaged between the lid main body 112 to be attached to the neck portion 97 of the cell culture container main body, the disk member 111 having the supply port 93 and the discharge port 94, and the inside of the lid main body 112 to the outer periphery.
- the ring-shaped member 113 is formed.
- the inner periphery of the lid main body 112 and the outer periphery of the neck portion 97 of the cell culture container main body are provided with screws that mesh with each other, and the lid main body 112 can be fixed to the neck portion 97 by rotating the lid main body 112.
- the disk member 111 is a disk having a diameter that can be independently rotated in the circumferential direction inside the lid main body 112, and the disk member 111 is rotated so that the supply pipe part 95 and the discharge pipe part 96 are positioned at appropriate positions in the cell culture vessel. Can be adjusted to place. In order to arrange the disk member 111 so as to be able to rotate independently with respect to the lid main body 112, for example, as shown in FIGS.
- the disk member 111 is first fitted inside the lid main body 112, and then the ring-shaped member 113 is inserted.
- the disk member 111 is rotatably attached to the lid body 112 by screwing the inside of the lid body 112.
- the disk member 111 is tightened in such a manner as to be sandwiched between the neck portion 97 and the ring-shaped member 113, so that the lid main body 112 is positioned while positioning the supply pipe portion 95 and the discharge pipe portion 96. Is screwed into the neck 97 to tighten the disk-shaped member.
- the inside of the cell culture container is opened by loosening the screwing of the lid body 112 and the neck 97 or loosening the screwing of the ring-shaped member 113 and the lid body 112.
- a convex portion is provided inside the lid portion 92, and the neck portion 97 of one container body is provided with a concave portion into which the convex portion is fitted.
- the convex portion and the concave portion are positioned so that the supply pipe portion 95 and the discharge pipe portion 96 can be arranged at appropriate positions in the cell culture container by engaging the convex portion and the concave portion. If you do.
- FIG. 1 A more specific example of the structure near the lid is shown in FIG.
- the neck 97 is provided with two ring-shaped members 102 at intervals in the circumferential direction, and a recess 103 is provided between the two ring-shaped members 102.
- the lid portion 92 is fitted into the neck portion 97, the convex portion 101 inside the lid portion 92 fits between the two ring-shaped members 102.
- the lid portion 92 is rotatable in the circumferential direction of the neck portion 97. It becomes a state.
- the supply pipe portion 95 and the discharge pipe portion 96 can be arranged at appropriate positions in the cell culture container.
- the part 96 may be arranged at an appropriate position in the cell culture container.
- a flask-like culture container is shown in the drawings as a culture container to be used.
- a petri dish-like culture container may be used.
- the same reference numerals are given to the components corresponding to the cell culture container of FIG.
- a cell culture container having a threshold for forming a flow path may be used. If the supply port 141 and the discharge port 142 are installed at positions separated along the flow path using the container, the exchange efficiency of the medium is improved.
- the waste liquid discharged from the discharge port of the culture vessel has been shown as being discharged through the waste liquid holding means.
- the waste liquid holding means is not provided. Alternatively, it may be discharged directly from the outlet of the cell culture container.
- 15A and 15B are examples corresponding to the first embodiment, but the other embodiments are also the same.
- the suction port of the negative pressure supply means may be connected to each discharge port of the culture container.
- the medium storage unit may not be employed.
- the medium may be directly replenished to the temporary holding means, and the temporary holding means is preferably provided with a supply port therefor.
- the temporary holding means may be used up without a supply port.
- 16A and 16B are examples corresponding to the first embodiment, but the other embodiments are also the same.
- the pressure applying unit 60 may be configured to apply pressure to the medium in the temporary holding unit instead of the medium storage unit.
- the medium storage means 3 is connected to the temporary holding means 4 via a tubular member (tube or the like), and the medium storage means 3 is installed at a higher position in the direction of gravity than the temporary holding means 4.
- the medium in the medium storage means 3 is supplied to the temporary holding means 4 through a tubular member (tube or the like) by gravity.
- a liquid feeding pump 121 such as a peristaltic pump is installed on a tubular member (tube or the like) connecting the medium storage means and the temporary holding means, and the medium is transferred from the medium storage means to the temporary holding means. You may supply.
- the culture medium holding means 3 does not necessarily need to be installed at a position higher in the direction of gravity than the temporary holding means 4, and the degree of freedom of the installation place of the culture medium holding means 3 is increased.
- the liquid feed pump 121 such as a peristaltic pump may be remotely operated by the control means. The remote operation may be wired or wireless. 17A and 17B are examples corresponding to the first embodiment, but other embodiments are also the same.
- the temporary holding unit when the medium rises to a temperature suitable for cell culture before the medium is supplied to the cell culture container, the temporary holding unit is necessarily employed. There is no need. For example, if the dropping speed is sufficiently slow or the capacity of the dropping container is sufficiently large, the temperature of the medium will be suitable for cell culture until the medium is supplied to the cell culture container. It is also possible.
- the medium storage means and the medium supply means may be directly connected by a tubular member (tube or the like).
- the medium storage means may include a plurality of outlets, and the plurality of outlets may be connected to different medium supply means.
- FIG. 21A the medium storage means may include a plurality of outlets, and the plurality of outlets may be connected to different medium supply means.
- the outlet of the medium storage means The tubular member connected to may be branched into a plurality of tubular members on the way, and the plurality of tubular members may be connected to different medium supply means.
- the set temperature of the medium solution in the medium storage means may be a temperature suitable for storage (for example, 4 ° C.) or a temperature suitable for cell culture (for example, 37 ° C.), and is set to an optimum temperature depending on the culture conditions. can do.
- a liquid feed pump such as a peristaltic pump is installed in a tubular member (tube or the like) connecting the medium storage means and the medium supply means, and the medium is transferred from the medium storage means to the medium supply means. May be supplied.
- the culture medium holding means does not necessarily need to be installed at a higher position in the direction of gravity than the culture medium supply means, and the degree of freedom of the installation location of the culture medium storage means is increased.
- the liquid feed pump functions as the dropping speed adjusting means of the medium supply means
- the dropping speed adjusting means may not be provided.
- a liquid feed pump such as a peristaltic pump may be installed either inside or outside the incubator, and may be remotely operated by a control means by wire or wirelessly.
- the dropping container may not be used, and the supply port of the cell culture container may be installed on the upper surface of the container, and a solution such as a medium may be dropped from there.
- FIG. 21A and FIG. 21B are examples corresponding to 1st Embodiment, other embodiment is also the same.
- FIG. 22A and FIG. 22B are examples corresponding to 2nd Embodiment, other embodiment is also the same.
- FIGS. 18A and 18B a mode in which one medium holding unit and one temporary holding unit are provided has been described.
- a plurality of medium holding units and a plurality of temporary holding units may be provided. good.
- a plurality of types of media can be supplied to the cell culture container.
- the medium supply means connected to the temporary holding means may be provided in each of a plurality of temporary holding means as shown in FIGS. 18A and 18B, for example, or a plurality of temporary holdings as shown in FIGS. 19A and 19B, for example.
- the aspect connected with one culture medium supply means from the means may be sufficient.
- 18A and 18B and FIGS. 19A and 19B are examples corresponding to the first embodiment, but the other embodiments are also the same.
- the dropping container may include a plurality of outlets, and the plurality of outlets may be connected to supply ports of different cell culture containers, respectively.
- the tubular member connected to the outlet may be branched into a plurality of tubular members on the way, and the plurality of tubular members may be connected to supply ports of different cell culture containers.
- a liquid feed pump such as a peristaltic pump may be used as the dropping speed adjusting means.
- the liquid feeding pump may be remotely controlled by a control unit by wire or wirelessly.
- the medium temperature monitoring means for monitoring the medium temperature may be installed in the temporary holding means. At this time, information on the medium temperature monitored by the medium temperature monitoring means may be sent to the control unit remotely. As a result, it is possible to prevent a medium having a temperature not suitable for cell culture from being erroneously supplied.
- the cell culture system of the present invention can be applied not only to the culture of adherent cells but also to the culture of non-adherent cells.
- the medium exchange speed can be reduced, and the risk of non-adherent cells flowing out of the outlet can be reduced because the outlet of the cell culture container is arranged upward in the height direction. Because.
- exchange efficiency of a culture medium can be improved by making the quantity of the culture medium hold
- control unit can exchange information with the temperature control unit, the pressure applying unit, the negative pressure supply unit, and the liquid feed pump in each of the above embodiments wirelessly or by wire, and can control them remotely. May be. As a result, the efficiency of remote work by the user can be increased.
- control unit may remotely operate the medium replacement based on a schedule (program) set in advance by the user.
- the temporary holding means may be installed outside the incubator.
- a temperature control means for maintaining the solution in the temporary holding means at a temperature suitable for cell culture (for example, 37 ° C.).
- the waste liquid holding means may be installed outside the incubator.
- the cell culture container shown in FIG. 23A can be used.
- the cell culture container has a shelf-like structure composed of plate-like members 130 inside the container.
- the plate-like member is fixed inside the container to form a shelf-like structure, and a solution stopper 131 is provided on the upper surface portion so that a predetermined amount of solution can be held.
- the plate-like member 130 is fixed inside the container so that the upper and lower spaces partitioned thereby communicate with each other through the opening 132.
- a supply port 133 is provided on the upper surface of the container, and the solution supplied from the supply port 133 is supplied to a shelf formed by the plate member 130.
- a predetermined amount of solution is held on the plate-like member 130 by the solution stopper 131, and the solution exceeding the predetermined amount passes through the solution stopper 131 and is dropped from the opening 132 into the lower space.
- a discharge port 134 is provided on the side surface of the container at a predetermined height from the bottom surface. When the solution held in the lower space reaches the height of the discharge port 134, the discharge port 134 is discharged.
- the supply port 133 may be at a position where the solution can be supplied to the shelf formed by the plate-like member 130, and may be a side surface of the container. Alternatively, as shown in FIG. 23B, a supply port 133 ′ may be further provided at a position where the solution can be directly supplied to the lower space.
- one shelf portion is formed by one plate-like member
- two or more shelf portions may be formed by two or more plate-like members. According to such a cell culture container, it is possible to earn a culture area for culturing cells and efficiently increase the number of cells.
- the waste liquid speed adjusting means may be provided on the tubular member that connects the discharge port of the cell culture container and the waste liquid supply port of the waste liquid holding means.
- the waste liquid speed adjusting means can control the flow rate of the waste liquid flowing in the tubular member, and the structure thereof is the same as the dropping speed adjusting means shown in FIGS. 2A to 2D.
- a liquid feed pump such as a peristaltic pump may be employed as the waste liquid speed adjusting means.
- the waste liquid speed adjusting means may be operable remotely or wirelessly by a control unit installed outside the incubator.
- control unit of the present invention is a PC, and the PC can perform control executed by the control unit in each of the above embodiments.
- the culture medium storage means and the temporary culture medium holding means are means capable of holding a culture medium and other solutions (for example, a washing solution), and each function as a solution holding means. That is, in the aspect provided with only the culture medium storage means or the temporary culture medium holding means, each functions as a solution holding means, and in the aspect provided with the culture medium storage means and the temporary culture medium holding means, both means function as a solution holding means.
- a medium storage means for maintaining a medium for culturing cells at a temperature suitable for storage of the medium, and a medium connected to the medium storage means and supplied from the medium storage means are suitable for cell culture.
- Temporary medium holding means for holding at a temperature, medium connected to the temporary medium holding means, medium supply means for supplying the medium supplied from the temporary medium holding means to a cell culture container, connected to the medium supply means,
- a cell culture system comprising a cell culture container for culturing cells in a medium supplied from a medium supply means, wherein the medium supply means supplies the medium to the cell culture container through a step of dropping the medium.
- a medium storage means for maintaining a medium for culturing cells at a temperature suitable for storage of the medium, and a medium connected to the medium storage means and supplied from the medium storage means are suitable for cell culture.
- Temporary medium holding means for holding at a temperature, medium connected to the temporary medium holding means, medium supply means for supplying the medium supplied from the temporary medium holding means to a cell culture container, connected to the medium supply means,
- a cell culture system comprising a cell culture container for culturing cells in a medium supplied from a medium supply means, and a medium discharge means connected to the cell culture container and discharging the medium supplied from the cell culture container.
- the medium discharging means can provide a cell culture system characterized in that the medium is discharged through a step of dropping the medium.
- a cell culture system comprising a solution holding means for holding a medium, and a medium supply means connected to the solution holding means and for supplying the medium supplied from the solution holding means to a cell culture container.
- the medium supply means can provide a cell culture system that supplies the medium to the cell culture container through a step of dropping the medium.
- a solution holding means for holding a medium for holding a medium
- a medium supply means connected to the solution holding means
- a medium supply means for supplying the medium supplied from the solution holding means to the cell culture container, and the cell culture container
- a cell culture system comprising a medium discharge means for discharging a medium supplied from the cell culture container, wherein the medium discharge means discharges the medium through a step of dropping the medium.
- a cell culture system 1800 according to the present embodiment is a system having the configuration shown in FIG. 24 and is a system for exchanging a medium in a cell culture container (culture container) installed in an incubator.
- the solution holding means 3 (the first solution holding means 3a and the second solution holding means 3b) are installed inside the incubator 1, and store a solution such as a culture medium (cell culture solution) or a washing solution therein. It is.
- FIG. 24 shows an example in which a medium (cell culture solution) is held in the first solution holding means 3a and a cleaning solution is held in the second solution holding means 3b.
- the solution held in the solution holding means 3 is warmed to a temperature (for example, 37 ° C.) in the incubator that is a temperature suitable for cell culture.
- the solution heated to a temperature suitable for cell culture (for example, 37 ° C.) by the solution holding unit 3 is transferred to the cells via the solution supply unit 6 (first solution supply unit 6a, second solution supply unit 6b) in the incubator 1. Supplied to the culture vessel 11.
- the solution supply means 6 includes a dropping speed adjusting means 7 (first dropping speed adjusting means 7a, second dropping speed adjusting means 7b) and a dropping container 8 (first dropping container 8a, second dropping container 8b).
- the dropping speed adjusting means 7 is installed on a tubular member (tube or the like) that connects the solution holding means 3 and the dropping container 8, and can control the flow rate of the solution flowing in the tubular member.
- the first dropping speed adjusting means 7a is installed on a tubular member that connects the first solution holding means 3a and the first dropping container 8a
- the second dropping speed adjusting means 7b is a second solution holding means 3b, a second dropping container 8b, It is installed in the tubular member which connects.
- the dropping speed adjusting means 7 can be remotely operated wirelessly or by wire via a control unit 19 installed outside the incubator, and can control the flow of a solution such as a medium that remotely flows in a tubular member (tube or the like). It is possible to stop (state where the flow rate is 0) and control the flow rate.
- the user adjusts the dropping speed adjusting means 7 remotely by the control unit 19 to set the dropping speed to an appropriate speed.
- the adjustment of the dropping speed may be set to a predetermined speed, or may be adjusted to an appropriate dropping speed while monitoring the dropping speed with a monitoring system (not shown).
- the dripping container 8 is a container having a supply port 9 connected to a tubular member connected from the solution holding means 3 and a discharge port 10 connected to a tubular member (tube or the like) connected to the cell culture container 11.
- the supply port 9 is disposed in the upper part in the direction of gravity
- the discharge port 10 is disposed in the lower part in the direction of gravity than the supply port 9, and the solution supplied to the supply port 9 is dropped into the space in the dropping container 8. And is supplied to the cell culture container 11 through a tubular member.
- the cell culture container 11 has a supply port 12 (first supply port 12a, second supply port 12b) connected to a tubular member connected from the dropping container 8, and a discharge port 13 for discharging the solution out of the cell culture container.
- the first supply port 12a is connected to the discharge port 10 of the first dropping container 8a via a tubular member
- the second supply port 12b is connected to the discharge port 10 of the second dropping container 8b via a tubular member.
- the solution that has passed through the supply port 12 is dropped in the space in the culture vessel and supplied into the culture vessel.
- a tubular member (tube or the like) may be installed from the supply port 12 toward the inside of the culture vessel and supplied without dropping the solution.
- the supply port 12 is preferably installed on the lid of the cell culture container as shown in FIG. 24, but may be installed on the side of the cell culture container, for example.
- the discharge port 13 is installed on the lid of the cell culture container as shown in FIG. 24, for example, and has a structure in which a tubular member (tube or the like) extends toward the inside of the cell culture container.
- the tubular member has a length that reaches the substantially bottom surface of the cell culture container, and can discharge the solution on the bottom surface of the cell culture container. This makes it possible to discharge almost the entire amount of the solution in the culture vessel.
- the tubular member extending from the discharge port 13 to the inside of the cell culture container may have a hole 28 as shown in FIG. 25 on the side surface, and the solution may be discharged through the hole 28.
- the solution can be discharged even if the distal end of the tubular member is arranged so as to lie over the bottom surface of the culture vessel.
- the installation position of the discharge port 13 is arbitrary as long as the solution on the bottom surface of the cell culture container can be discharged. For example, it may be installed on the side surface or the bottom surface of the cell culture container. In that case, there may be no tubular member extending into the cell culture container.
- the discharge port 13 of the cell culture vessel 11 is connected to the solution discharge means via a tubular member (tube or the like).
- the solution discharge means includes a waste liquid holding means 14 and a negative pressure supply means 71.
- the waste liquid holding means 14 is connected to the discharge port 13 of the cell culture container 11 through a tubular member, and the waste liquid supply port 15 connected to the tubular member and the waste liquid drain for discharging the waste liquid to the outside of the waste liquid holding means 14. And an outlet 16.
- the installation position of the waste liquid holding means 14 may be inside or outside the incubator.
- the waste liquid supply port 15 is installed on the upper surface of the waste liquid holding means 14, and the waste liquid supplied to the waste liquid supply port 15 is dropped into the space in the waste liquid holding means and discharged through the waste liquid discharge port 16.
- the tubular member that connects the discharge port 13 of the cell culture container 11 and the waste liquid supply port 15 includes a waste liquid speed adjusting means 17.
- the waste liquid speed adjusting means 17 can control the flow rate of the waste liquid flowing in the tubular member.
- the waste liquid speed adjusting means 17 can be remotely operated wirelessly or wired by a control unit 19 installed outside the incubator, and can remotely stop the flow of waste liquid in a tubular member (tube or the like) (flow speed). 0 state), the flow rate can be controlled.
- the control unit 19 When the medium needs to be replaced, the user remotely adjusts the waste liquid speed adjusting means 17 by the control unit 19 to set the waste liquid speed to an appropriate speed.
- the waste liquid speed may be adjusted to a predetermined speed, or may be adjusted to an appropriate speed while monitoring the waste liquid speed with a monitoring system (not shown).
- the structure of the waste liquid speed adjusting means 17 is the same as the dropping speed adjusting means 7 shown in FIGS. 2A to 2D.
- the waste liquid discharge port 16 of the waste liquid holding means 14 is connected to the negative pressure supply means 71 so that the waste liquid holding means 14 can be set to a negative pressure.
- the negative pressure supply means 71 include a means provided with a pump 31 and a waste liquid container 32 as shown in FIG.
- the suction port 33 of the negative pressure supply means 71 is connected to the waste liquid discharge port 16 of the waste liquid holding means 14 so that the inside of the waste liquid holding means 14 has a negative pressure, and the discharge port 13 of the culture container 11 is set to a negative pressure. It is possible to aspirate the medium from 11.
- a liquid feed pump such as a peristaltic pump may be used as the negative pressure supply means 71. In this case, a liquid feed pump may be installed on the tubular member of the suction port 33.
- the dropping speed adjusting means 7 is disposed on a tubular member (tube or the like) that connects the discharge port 5 of the solution holding means 3 and the supply port 9 of the dropping container 8, and is deformed by applying an external force to the tubular member.
- a tubular member tube or the like
- the flow rate of the solution is limited to suppress the flow rate.
- the tubular member returns to its original state by the elastic force of the tubular member, and the flow velocity can be increased. In this way, the dropping speed adjusting means 7 adjusts the flow rate of the solution flowing in the tubular member by the strength of the external force on the tubular member.
- the method of applying an external force to the tubular member by the dropping speed adjusting means 7 is the same as in the first embodiment shown as an example in FIGS. 2A to 2D.
- the structure of the waste liquid speed adjusting means is the same as that of the dropping speed adjusting means 7.
- a liquid feed pump such as a peristaltic pump may be employed as the dropping speed adjusting means 7.
- a liquid feed pump such as a peristaltic pump may be employed as the waste liquid speed adjusting means.
- the liquid feed pump may be remotely operated by the control unit 19.
- the user of this system first sets the system by the dropping speed adjusting means 7 so that the dropping speed is 0, that is, the dropping is stopped.
- the solution holding means 3 is supplemented with a solution such as a medium, and the temperature of the medium and the solution in the solution holding means 3 is set to the temperature in the incubator.
- a cell culture container 11 containing a culture medium and cells is prepared.
- the supply port 12 of the cell culture container 11 is connected to the discharge port 10 of the dropping container 8 via a tubular member, and the discharge port 13 of the cell culture container 11 is tubular.
- Each is connected to a waste liquid supply port 15 of the waste liquid holding means 14 via a member.
- the user When the medium needs to be replaced, the user first operates the waste liquid speed adjusting means 17 by the control unit 19 to set the waste liquid speed to an appropriate speed.
- the waste liquid speed is canceled by the waste liquid speed adjusting means 17, the negative pressure of the waste liquid holding means 14 is transmitted to the discharge port 13 of the cell culture container, and the medium in the cell culture container is sucked into the waste liquid holding means 14 as waste liquid.
- the waste liquid speed is returned to zero by the waste liquid speed adjusting means 17.
- the user operates the second dropping speed adjusting means 7b by the control unit 19 to set the dropping speed to an appropriate speed.
- the dropping speed is released from the state of 0 by the dropping speed adjusting means 7b
- the cleaning liquid in the second solution holding means 4b starts to drop by gravity in the dropping container 8b and is supplied into the cell culture container.
- the user operates the second dropping rate adjusting means 7b by the control unit 19 to return the dropping rate to 0.
- the controller 19 operates the waste liquid speed adjusting means 17 to set the waste liquid speed to an appropriate speed and discharge the washing liquid in the culture vessel.
- the user operates the first dropping speed adjusting means 7a by the control unit 19 to set the dropping speed to an appropriate speed.
- the dropping speed is released from the state of 0 by the dropping speed adjusting means 7a
- the medium in the first solution holding means 4a starts to drop by gravity in the dropping container 8a and is supplied into the cell culture container.
- the user operates the first dropping rate adjusting means 7a by the control unit 19 to return the dropping rate to 0.
- each of the first dropping container 41a and the second dropping container 41b has a plurality of discharge ports.
- the plurality of discharge ports provided in the first dropping container 41a and the second dropping container 41b are connected to the supply ports 12 (first supply port 42a and second supply port 42b) of different cell culture containers via tubular members.
- the FIG. 27A shows an embodiment using two cell culture containers, but it is possible to install as many cell culture containers as the number of outlets provided in each dropping container.
- each dripping container has one outlet as shown in FIG. 27B, and a tubular member connected to the outlet branches into a plurality of parts in the middle to supply different cell culture containers. It may be connected to the port 12 (first supply port 42a, second supply port 42b). Others are the same as in the first embodiment.
- the cell culture system 2100 is an embodiment in which the first dropping container 8a and the second dropping container 8b are used as one dropping container 8c.
- the dropping container 8c has two supply ports (first supply port 43a and second supply port 43b) for supplying the solution to the inside, and one discharge port 44 for discharging the solution from the inside.
- the first supply port 43a and the second supply port 43b are connected to the first solution holding unit 3a and the second solution holding unit 3b, respectively, through tubular members.
- the discharge port 44 is connected to the supply port 45 of the cell culture container via a tubular member. Others are the same as in the first embodiment. In this case, as shown in FIG.
- the first dropping container 8c may have a plurality of discharge ports.
- the plurality of discharge ports provided in the dropping container 8c are connected to supply ports 45 of different cell culture containers through tubular members.
- FIG. 29A is an embodiment using two cell culture containers, but it is possible to install three or more cell culture containers. Instead of providing a plurality of outlets for the dropping container, each dropping container has one outlet as shown in FIG. 29B, and a tubular member connected to the outlet is branched into a plurality of parts in the middle to supply different cell culture containers. 45 may be connected.
- the dropping container may be provided with one supply port, and the tubular members from the first solution holding unit 3a and the second solution holding unit 3b may be joined together and connected to the one supply port.
- the cell culture system 2400 does not include the dripping container 8 as shown in FIG. 30, and the arrangement position of the supply port 12 (first supply port 12a, second supply port 12b) of the cell culture container 11 is determined as a cell.
- This is different from the sixth embodiment in that it is limited to the upper surface of the culture vessel 11. The rest is the same as in the sixth embodiment.
- the supply port 12 of the cell culture container 11 is located on the upper surface of the cell culture container 11, the supplied solution is dropped in the space in the cell culture container 11, and the function similar to that of the dropping container 8. Will have. Therefore, also in this embodiment, the back flow of the culture medium can be prevented, and the culture medium in the solution storage means 3 can be prevented from being contaminated (contamination occurs). Further, since the dropping container 8 is not installed, the system can be made compact.
- the dropping container 8 Since the dropping container 8 is not provided, the discharge port 5 of the solution holding means 3 and the supply port 12 of the cell culture container 11 are directly connected via a tubular member, and the dropping speed adjusting means 7 is installed on the tubular member. ing. That is, the first solution holding means 3a and the second solution holding means 3b are connected to the first supply port 12a and the second supply port 12b of the cell culture container via the tubular members, respectively. A dropping speed adjusting means 7a and a second dropping speed adjusting means 7b are installed.
- the cell culture system 2500 includes solution stock means 51 (first solution stock means 51a, second solution stock means 51b) for supplying a solution to the solution holding means 3.
- solution stock means 51 first solution stock means 51a, second solution stock means 51b
- the solution stock means 51 includes a temperature control means 52 in order to maintain a solution such as a medium held therein at a temperature suitable for storage of the solution (for example, 4 ° C.).
- the installation place of the solution stock means 51 is arbitrary as long as it is higher in the direction of gravity than the solution holding means 3, and may be installed either inside or outside the incubator.
- the solution stock means 51 (first solution stock means 51a, second solution stock means 51b) is connected to the solution holding means 3 (first solution holding means 3a, second solution holding means 3b) via a tubular member (tube or the like). It is connected. Since the solution stock means 51 is installed at a position higher than the solution holding means 3 in the direction of gravity, the solution in the solution stock means 51 is supplied to the solution holding means 3 through a tubular member (tube or the like) by gravity. . The medium supplied to the solution holding means 3 is warmed to a temperature in the incubator (for example, 37 ° C.) that is a temperature suitable for cell culture.
- a temperature in the incubator for example, 37 ° C.
- FIG. 31 shows a mode in which the solution is moved from the solution stock means 51 to the solution holding means 3 by using gravity.
- a tubular member (tube) connecting the solution stock means 51 and the solution holding means 3 is used.
- Etc. may be provided with a liquid feeding pump 53 (first liquid feeding pump 53a, second liquid feeding pump 53b) such as a peristaltic pump, and the solution may be moved from the solution stock means 51 to the solution holding means 3.
- the installation location of the solution stock means 51 may not be higher than the solution holding means 3 in the direction of gravity.
- the cell culture system 2700 according to this embodiment is different from the above embodiments in that it includes a pressure applying means 60 for applying pressure to the solution in the solution holding means 3.
- a pressure applying means 60 for applying pressure to the solution in the solution holding means 3.
- FIG. 33 is an example corresponding to the sixth embodiment, but the same applies to other embodiments.
- a means for sending a gas into the solution holding means 3 by a pump can be cited as an example. Thereby, the pressure of the air layer in the solution storage means 3 increases, and it becomes possible to apply an external pressure to the solution.
- the gas sent into the solution holding means 3 is preferably sterilized.
- the pressure applying means the same one as illustrated in FIGS. 6A to 6C can be used.
- the pressure applying means 60 is preferably controlled so that the pressure in the solution holding means 3 does not rise above a certain level.
- the solution holding means 3 does not necessarily have to be installed at a position higher than the solution supply means 6.
- the solution stock means 51 (first solution stock means 51a, What is necessary is just to make it the aspect which applies a pressure to the solution in the 2nd solution stock means 51b).
- the solution stock means 51 does not necessarily have to be installed at a position higher than the solution holding means 3.
- the solution holding means 3 does not necessarily have to be installed at a higher position than the solution supply means 6.
- the cell culture container 91 shown in FIGS. 38A and 38B can also be used.
- the cell culture container 91 has a supply port 93 and a discharge port 94 in the lid portion 92 of the container.
- the supply port 93 is connected to the solution holding means 6 via a tubular member, and a supply pipe portion 95 extends from the supply port 93 toward the inside of the cell culture container.
- the solution supplied from the solution supply means 6 passes through the supply port 93 and enters the supply pipe portion 95.
- the tip of the supply tube portion 95 is disposed at a predetermined height above the bottom surface inside the cell culture container, and the solution exiting the supply tube portion 95 is dropped into the cell culture container 91. .
- the tip of the supply pipe part 95 does not necessarily have to be disposed at a predetermined height above the bottom surface inside the cell culture container.
- the discharge port 94 is connected to the waste liquid holding means 14 through a tubular member, and a discharge pipe portion 96 extends from the discharge port 94 toward the inside of the cell culture container.
- the tip of the discharge pipe portion 96 is arranged at a position reaching the bottom surface inside the culture vessel, and almost all of the solution in the cell culture vessel can be discharged.
- the discharge pipe portion 96 may have a hole 28 as shown in FIG. 25 on its side surface, and the solution may be discharged through the hole 28.
- the solution can be discharged even if the tip of the discharge tube portion 96 is arranged so as to be placed on the bottom surface of the culture vessel.
- the installation position of the discharge port 94 is arbitrary as long as it can discharge almost the entire amount of the solution in the cell culture container. For example, it may be installed on the bottom surface or side surface of the cell culture container. In this case, the discharge pipe portion 96 may not be provided.
- the waste liquid discharged from the discharge port of the cell culture container is shown as being discharged through the waste liquid holding means.
- the discharge port of the cell culture container may be connected to the negative pressure supply means 71 through a tubular member.
- FIG. 35 is an example corresponding to the first embodiment, but the other embodiments are also the same.
- solution temperature monitoring means for monitoring the solution temperature may be installed in the solution holding means. At this time, information on the solution temperature monitored by the solution temperature monitoring means may be sent to the control unit remotely. This can prevent a solution having a temperature not suitable for cell culture from being erroneously supplied.
- the mode in which two solution holding means (and solution stock means) are provided is shown, but three or more solution holding means (and solution stock means) may be provided. In this case, a plurality of media having different compositions can be used. Alternatively, the solution holding means (and solution stock means) may be integrated into one, and the solution holding means (and solution stock means) for the cleaning liquid may not be used.
- control unit 19 can exchange information with the temperature control unit 2, the pressure applying unit 60, and the negative pressure supply unit 71 in each of the above embodiments wirelessly or by wire, and can control them remotely. May be. As a result, the efficiency of remote work by the user can be increased.
- each of the above embodiments one (or two) cell culture containers have been described, but in reality, a plurality of cell culture containers can be installed.
- the description of each of the above embodiments and modifications applies to each cell culture container when a plurality of cell culture containers are installed.
- the negative pressure supply means is employed to suck the waste liquid.
- the waste liquid may be discharged by gravity without using the negative pressure supply means.
- the discharge port of the cell culture container may be disposed on the bottom or side surface of the cell culture container, and the solution in the cell culture container may be discharged from the discharge port by gravity.
- the cell culture container is tilted so that the solution flows in the direction of the discharge port.
- a liquid feed pump 53 such as a peristaltic pump may be installed on a tubular member connected to the discharge port of the cell culture container.
- a mode is also possible in which the installation position of the outlet of the cell culture container is used as a lid on the upper surface of the cell culture container, and the tubular member extends from there to the inside of the cell culture container.
- the mode in which the solution holding means is installed in the incubator is shown, but the solution holding means may be installed outside the incubator. In that case, it is preferable to install a temperature control means for maintaining the solution in the solution holding means at a temperature suitable for cell culture (for example, 37 ° C.).
- control unit of the present invention is a PC, and the PC can perform control executed by the control unit 19 in each of the above embodiments.
- the one supply port is provided after the tubular members connected from the two (plurality) solution supply means are merged. It may be connected to the mouth.
- a bag-shaped cell culture bag may be used as the cell culture container used in the present invention.
- the lid 151 has a structure in which at least one hole 154 is formed in the lid main body 152 and the tubular member 153 passes therethrough. 39A and 39B show a case where there are two holes 154 and two tubular members 153, respectively, but there may be three or more or one.
- the lid portion 151 is made of a material that can withstand high heat during sterilization by dry heat treatment or autoclave. Examples of materials that can withstand high heat include metals such as aluminum and glass.
- the container body 155 may not be a material that can withstand high heat.
- the tubular member 153 is preferably configured to be movable in the insertion direction via the hole 154. This makes it possible to adjust the length of the tubular member 153 protruding into the container, and it is possible to appropriately adjust the optimum conditions according to the type of the container used and the culture system. According to the said culture container, a cover part can be reused and it becomes effective at the point of cost. In addition, since the container body can be exchanged, an optimum container can be selected as appropriate according to the culture system, and variations of the culture system are widened. In addition, the use of a commercially available container body is also effective in terms of cost.
- a solution holding unit that holds a solution such as a medium for culturing cells, and a solution supply that is connected to the solution holding unit and supplies the solution supplied from the solution holding unit to the cell culture container And a solution discharge means for discharging the solution from the cell culture container, wherein the solution supply means supplies the solution to the cell culture container through a step of dropping the solution.
- a cell culture system can be provided.
- a solution holding unit that holds a solution such as a medium for culturing cells, and a solution supply that is connected to the solution holding unit and supplies the solution supplied from the solution holding unit to the cell culture container
- Cell culture system comprising: means; and solution discharging means for discharging the solution from the cell culture container, wherein the solution discharging means discharges the solution through a step of dropping the solution.
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Abstract
Description
細胞を培養するには定期的に培地(細胞培養液)の交換が必要となるが、培地交換は培養系の汚染(コンタミネーション)のリスクを伴い、可能な限り人手を介さずに自動で行えることが望ましい。自動で培地を交換するシステムとして培地を培養系に連続還流するシステムが知られている(特許文献1)。
細胞を培養するための培地を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された培地を細胞培養容器に供給する培地供給手段と、
を備えた細胞培養システムであって、
前記細胞培養容器が、前記培地供給手段より重力方向で下の位置で前記培地供給手段と連結され、
前記培地供給手段が、前記溶液保持手段から供給された培地を滴下する空間を有し、
前記細胞培養容器が、培地を排出するための排出口を容器側面の所望の高さの位置に備え、前記排出口が、前記培地供給手段から培地が供給されることによって培地上面の高さが前記排出口に達したときに培地を外部に排出する細胞培養システムを提供する。
細胞を培養するための培地を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された培地を細胞培養容器に供給する培地供給手段と、
を備えた細胞培養システムであって、
前記細胞培養容器が、容器上面に前記培地供給手段から培地を供給される供給口を備えるとともに、培地を排出するための排出口を容器側面の所望の高さの位置に備え、
前記供給口から供給された培地が、細胞培養容器内の空間を前記供給口から培地上面に滴下され、前記排出口が、前記培地供給手段から培地が供給されることによって培地上面の高さが前記排出口に達したときに培地を外部に排出する細胞培養システムを提供する。
細胞を培養するための培地等の溶液を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された溶液を細胞培養容器に供給する溶液供給手段と、
前記細胞培養容器から溶液を排出する溶液排出手段とを備えた細胞培養システムであって、
前記細胞培養容器が、前記溶液供給手段より重力方向で下の位置で前記溶液供給手段と連結され、
前記溶液供給手段が、前記溶液保持手段から供給された溶液を滴下する空間を有する細胞培養システムを提供する。
細胞を培養するための培地等の溶液を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された溶液を細胞培養容器に供給する溶液供給手段と、
前記細胞培養容器から溶液を排出する溶液排出手段とを備えた細胞培養システムであって、
前記細胞培養容器が、前記溶液供給手段より重力方向で下の位置で前記溶液供給手段と連結され、前記細胞培養容器上面に前記溶液供給手段から溶液を供給される供給口を備え、
該供給口から供給された溶液が、前記細胞培養容器内の空間を前記供給口から滴下される細胞培養システムを提供する。
このことにより、作業者が遠隔的に培養系の培地を交換することができるので、作業者が作業空間に立ち入り作業をする回数を減らし、作業にともなう手間とコストを減らすとともに、培養系が汚染される機会も減らすことができる。
細胞を培養するための培地等の溶液を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された溶液を細胞培養容器に供給する溶液供給手段と、
を備えた細胞培養システムであって、
前記溶液供給手段が、溶液を滴下する工程を経て前記細胞培養容器に溶液を供給する細胞培養システムを提供する。
細胞を培養するための培地等の溶液を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された溶液を細胞培養容器に供給する溶液供給手段と、
前記細胞培養容器から溶液を排出する溶液排出手段と、
を備えた細胞培養システムであって、
前記溶液排出手段が、溶液を滴下する工程を経て前記細胞培養容器から溶液を排出する細胞培養システムを提供する。
細胞を培養するための培地を培地の保存に適した温度で保持する培地保存手段と、
該培地保存手段と連結され、前記培地保存手段から供給された培地を細胞培養に適した温度で保持する一時培地保持手段と、
該一時培地保持手段と連結され、前記一時培地保持手段から供給された培地を細胞培養容器に供給する培地供給手段と、
該培地供給手段より重力方向で下の位置で前記培地供給手段と連結され、前記培地供給手段から供給された培地中で細胞を培養する細胞培養容器と、
を備えた細胞培養システムであって、
前記培地供給手段が、前記一時培地保持手段から供給された培地を滴下する空間を有する滴下容器を備え、該滴下容器内で滴下された培地を前記細胞培養容器に供給し、
前記細胞培養容器が、容器側面の所望の高さの位置に培地を排出するための排出口を備え、該排出口が、前記培地供給手段から培地が供給されることによって培地上面の高さが前記排出口に達したときに培地を外部に排出する細胞培養システムを提供する。
細胞を培養するための培地を培地の保存に適した温度で保持する培地保存手段と、
該培地保存手段と連結され、前記培地保存手段から供給された培地を細胞培養に適した温度で保持する一時培地保持手段と、
該一時培地保持手段と連結され、前記一時培地保持手段から供給された培地を細胞培養容器に供給する培地供給手段と、
該培地供給手段より重力方向で下の位置で前記培地供給手段と連結され、前記培地供給手段から供給された培地中で細胞を培養する細胞培養容器と、
を備えた細胞培養システムであって、
前記細胞培養容器が、容器上面に前記培地供給手段から供給される供給口と、容器側面の所望の高さの位置に培地を排出するための排出口を備え、前記供給口から供給された培地が、細胞培養容器内の空間を前記供給口から培地上面に滴下され、前記排出口が、前記培地供給手段から培地が供給されることによって培地上面の高さが前記排出口に達したときに培地を外部に排出する細胞培養システムを提供する。
(第1実施形態)
本実施形態に係る細胞培養システム100は、図1A及び図1Bに示される構成のシステムであり、インキュベータ内に設置された細胞培養容器(培養容器)内の培地を交換するシステムである。図1Aに側面図、図1Bに正面図を示す。
培地供給手段6は、滴下速度調整手段7と滴下容器8とを備えている。滴下速度調整手段7は、一時保持手段4と滴下容器8とをつなぐ管状部材に設置されており、管状部材内を流れる培地の流速を制御することができる。
このように、培地が滴下容器8内の空間に滴下されることで、培地の逆流を防ぐことができ、一時保持手段4や培地保存手段3内の培地が汚染される(コンタミネーションが起こる)ことを防止することができる。
図1Bには、培地供給手段6、細胞培養容器11を4組備えた細胞培養システムの一例を示している。
滴下速度調整手段7は、一時保持手段4の排出口5と滴下容器8の供給口9をつなぐ管状部材(チューブ等)に配置されており、管状部材に外力を加えて変形させ、管状部材内腔の断面積を小さくすることで溶液の流量を制限して流速を抑制する。逆に外力を解除すると、管状部材の弾性力により管状部材が元の状態に戻り、流速を上げることができる。このように、滴下速度調整手段7は、管状部材に対する外力の強弱により管状部材内に流れる溶液の流速を調整する。滴下速度調整手段7による管状部材への外力の加え方について図2Aから図2Dに例を示す。図2Aは2枚の板状部材21により管状部材20を挟む例、図2Bは複数の球状(または円柱状)部材22により貫通穴23に通した管状部材20を挟む例、図2Cはシャッター状部材24で貫通穴25に通した管状部材20を挟む例、図2Dは管状部材20を通した貫通穴26の内径が小さくなり管状部材を変形させる例である。これ以外でも外力を加えて管状部材を変形させることができる機構であれば良い。
また、滴下速度調整手段7としてペリスタポンプ等の送液ポンプを使用しても良い。
本システムのユーザは、まず滴下速度調整手段7により、滴下速度が0の状態、つまり、滴下が止まった状態にシステムを設定する。培地保存手段3および一時保持手段4に培地を補充し、一時保持手段4内の培地温度をインキュベータ内の温度にする。培地及び細胞が入った細胞培養容器11を用意し、インキュベータ内で、細胞培養容器11の供給口12を管状部材を介して滴下容器8の排出口10に、培養容器11の排出口13を管状部材を介して廃液保持手段14の廃液供給口15に、それぞれ接続する。
培地交換が必要になった際、ユーザはまず滴下速度調整手段7を調整して滴下速度を適当な速度にする。滴下速度調整手段7により滴下速度が0の状態を解除すると、滴下容器8内で培地は重力により滴下し始める。滴下速度の調整はあらかじめ決められた速度に設定しても良いし、ユーザが目視しながら適当な滴下速度に調整しても良い。
本実施形態に係る細胞培養システム200は、図3A及び図3Bに示すように滴下容器8を備えず、細胞培養容器11の供給口12を細胞培養容器11の上面に配置させる点で第1実施形態と異なっている。それ以外は第1実施形態と同様である。
この場合、細胞培養容器11の供給口12は細胞培養容器11の上面に位置するため、供給された培地は細胞培養容器11内の空間を培地表面に向け滴下されることになり、滴下容器8と同様の機能を有することになる。したがって、本実施形態においても培地の逆流を防ぐことができ、一時保持手段4や培地保存手段3内の培地が汚染される(コンタミネーションが起こる)ことを防止することができる。
また、滴下容器8を設置しないのでシステムをコンパクトにすることができる。
なお、滴下容器8を備えていないため、一時保持手段4の排出口5と細胞培養容器11の供給口12は管状部材を介して直接連結され、その管状部材に滴下速度調整手段7が設置されている。
本変形例は、第2実施形態において、第1実施形態と同様に滴下容器8を採用するものである。このことにより、培地の滴下を滴下容器8と細胞培養容器11とで行うことになり、培地の逆流により一時保持手段4や培地保存手段3内の培地が汚染される(コンタミネーションが起こる)リスクをさらに減少させることができる。
本実施形態に係る細胞培養システム400は、図5A及び図5Bに示すように、培地保存手段3内の培地に圧力をかけるための圧力付加手段60を備えている点で上記各実施形態と異なっている。それ以外は上記各実施形態と同様である。図5A及び図5Bは第1実施形態に対応する例であるが、他の実施形態も同様である。
また、培地保持手段が円柱状構造をし、隔壁部材がその円柱状構造の内壁に気密性を保ちながらはまる円盤状構造をし、円柱状構造の内壁と円盤状構造の外周に互いにかみ合うネジ構造を有する態様にしても良い。この場合、隔壁部材を培地保持手段の内部に取り付けて円盤周方向に回転させると隔壁部材が円柱構造の高さ方向に移動して培地保持手段内部の培地に圧力をかる(または圧力を除く)ことができる。隔壁部材の回転を機械的行い、制御部によって有線または無線で隔壁部材の回転を制御しても良い。
なお、本実施形態においては、培地保存手段3は一時保持手段4よりも必ずしも高い位置に設置されている必要はない。また、一時保持手段4は培地供給手段6よりも必ずしも高い位置に設置されている必要はない。
本実施形態に係る細胞培養システム500は、図7A及び図7Bに示すように、細胞培養容器11の排出口13に陰圧をかけるための陰圧供給手段71を備えている点で上記各実施形態と異なっている。それ以外は上記各実施形態と同様である。図7A及び図7Bは第1実施形態に対応する例であるが、他の実施形態も同様である。
陰圧供給手段のポンプとしてペリスタポンプ等の送液ポンプを使用しても良い。この場合、送液ポンプを吸引口83の管状部材に設置しても良い。
本実施形態に係る細胞培養システム600は、図9A及び図9Bに示すように、上記各実施形態において、滴下速度調整手段7が、インキュベータ外に設置された制御部19によって、無線または有線により遠隔的に操作可能となっている。それ以外は上記各実施形態と同様である。図9A及び図9Bは第1実施形態に対応する例であるが、他の実施形態も同様である。
培地交換が必要になった際、ユーザは制御部19により遠隔的に滴下速度調整手段7を調整して滴下速度を適当な速度にする。滴下速度の調整はあらかじめ決められた速度に設定しても良いし、図示しないモニタリングシステムで滴下速度を監視しながら適当な滴下速度に調整しても良い。
供給口93は管状部材を介して培地供給手段6に接続されているとともに、供給口93から細胞培養容器内部に向かって供給管部95が伸びている。培地供給手段6から供給された培地は、供給口93を通過し、供給管部95に進入する。図10Aに示すように、供給管部95の先端は培地表面より上方に配置されており、供給管部95を出た培地は細胞培養容器91内に滴下される。なお、滴下容器8を採用する態様においては、図10Bに例を示すように、必ずしも供給管部95の先端が培地表面より上方に配置されている必要はない。
排出口94は管状部材を介して廃液保持手段14に接続されているとともに、排出口94から細胞培養容器内部に向かって排出管部96が伸びている。排出管部96の先端は培養容器内部の底面から所定の高さの位置に配置されるようになっており、細胞培養容器内の培地が排出管部96の先端の高さに達すると細胞培養容器の外に排出される。細胞培養容器内に保持する培地の量は、排出管部96の先端を配置する高さにより調整可能となる。
このとき、供給管部95の先端と排出管部96の先端は可能な限り距離を隔てたほうが培地の交換効率は向上する。
蓋部92は、細胞培養容器本体の首部97に取り付けるための蓋本体112と、供給口93と排出口94があいた円盤部材111と、蓋本体112の内側との間でかみ合うねじを外周に切ってあるリング状部材113から構成される。蓋本体112の内周と細胞培養容器本体の首部97の外周には、互いにかみ合うねじがきってあり、蓋本体112を回転させることで首部97に蓋本体112を固定することができる。円盤部材111は、蓋本体112の内側で周方向に独立して回転できる直径の円盤であり、円盤部材111を回転させ供給管部95や排出管部96が細胞培養容器内で適当な位置に配置するように調整することができる。円盤部材111を蓋本体112に対して独立して回転できるように配置するには、例えば図11A及び図11Bに示すように、まず蓋本体112の内側に円盤部材111をはめ込み、リング状部材113を蓋本体112の内側にねじ込むことで円盤部材111を蓋本体112に対し回転可能に取り付ける。その状態で蓋本体112を首部97にねじ込むと、円盤部材111が首部97とリング状部材113に挟まれるかたちで締め付けられるので、供給管部95と排出管部96の位置決めをしながら蓋本体112を首部97にねじ込み、円盤状部材の締め付けを行う。なお、細胞培養中は蓋本体112と首部97のねじ込みを緩めたり、リング状部材113と蓋本体112のねじ込みを緩めたりすることで、細胞培養容器内を開放系とする。
蓋部92の内側には凸部を備え、一方の容器本体の首部97は凸部がはまる凹部を備えている。蓋部92を首部97にはめた時、凸部と凹部をかみ合わせることで、供給管部95や排出管部96が細胞培養容器内で適当な位置に配置できるように凸部と凹部の位置決めをしておけば良い。
図12においては、蓋部の凸部と首部の凹部をかみ合わせる態様を示したが、蓋部および首部がともに凸部を有し、それら凸部が互いに突き当たる位置で供給管部95や排出管部96が細胞培養容器内で適当な位置に配置できるようになっていても良い。
また、溶液を供給する供給口、溶液を排出する排出口を備えた袋状の細胞培養バッグを用いても良い。
さらに、図14Aから図14Dに示すように、流路を形成するしきいを有した細胞培養容器を用いても良い。当該容器を用い、供給口141と排出口142を流路に沿って離れた位置に設置すれば培地の交換効率が向上する。
この場合、第3実施形態では、培養容器の各排出口に陰圧供給手段の吸引口を接続すれば良い。
また、例えば図22A及び図22Bに示すように、培地保存手段と培地供給手段とをつなぐ管状部材(チューブ等)にペリスタポンプなどの送液ポンプを設置して、培地保存手段から培地供給手段へ培地を供給しても良い。こうすることで、培地保持手段は必ずしも培地供給手段より重力方向で高い位置に設置する必要がなく、培地保存手段の設置場所の自由度が上がる。この場合、送液ポンプが培地供給手段の滴下速度調整手段として機能するため、滴下速度調整手段がなくても良い。ペリスタポンプなどの送液ポンプは、インキュベータの内外どちらに設置されていても良く、制御手段により有線または無線で遠隔的に操作されても良い。また、図22A及び図22Bに示すように、滴下容器を採用せず、細胞培養容器の供給口を容器上面に設置してそこから培地等の溶液を滴下させても良い。
図21A及び図21Bは第1実施形態に対応する例であるが、他の実施形態も同様である。また図22A及び図22Bは第2実施形態に対応する例であるが、他の実施形態も同様である。
また、細胞培養容器内に保持する培地の量を可能な限り少量とすることで、培地の交換効率を向上させることができる。細胞培養容器内に保持する培地の量は、培養する細胞の種類により最適化すれば良い。
また、上記各実施形態において、廃液保持手段がインキュベータ内に設置された態様を示したが、例えば図22A及び図22Bに示すように、廃液保持手段がインキュベータ外に設置されていても良い。
このような細胞培養容器によれば、細胞を培養する培養面積をかせぐことができ、効率的に細胞を増やすことができる。
(第6実施形態)
本実施形態に係る細胞培養システム1800は、図24に示される構成のシステムであり、インキュベータ内に設置された細胞培養容器(培養容器)内の培地を交換するシステムである。
溶液供給手段6は、滴下速度調整手段7(第1滴下速度調整手段7a、第2滴下速度調整手段7b)と滴下容器8(第1滴下容器8a、第2滴下容器8b)とを備えている。滴下速度調整手段7は、溶液保持手段3と滴下容器8とをつなぐ管状部材(チューブ等)に設置されており、管状部材内を流れる溶液の流速を制御することができる。第1滴下速度調整手段7aは第1溶液保持手段3aと第1滴下容器8aとをつなぐ管状部材に設置され、第2滴下速度調整手段7bは第2溶液保持手段3bと第2滴下容器8bとをつなぐ管状部材に設置されている。
培地交換が必要になった際、ユーザは制御部19により遠隔的に滴下速度調整手段7を調整して滴下速度を適当な速度にする。滴下速度の調整はあらかじめ決められた速度に設定しても良いし、図示しないモニタリングシステムで滴下速度を監視しながら適当な滴下速度に調整しても良い。
このように、溶液が滴下容器8内の空間に滴下されることで、溶液の逆流を防ぐことができ、溶液保持手段3内の溶液が汚染される(コンタミネーションが起こる)ことを防止することができる。
供給口12を通過した溶液は、培養容器内の空間を滴下され培養容器内に供給される。このとき、供給口12から培養容器内部に向け管状部材(チューブ等)を設置して溶液を滴下させずに供給しても良い。また、供給口12の設置位置は溶液を滴下させる場合は、図24に示すように細胞培養容器の蓋に設置することが望ましいが、例えば細胞培養容器側面に設置しても良い。
排出口13の設置位置は、細胞培養容器の底面部の溶液を排出することが可能であれば任意であり、例えば細胞培養容器の側面や底面に設置しても良い。その場合、細胞培養容器内部に伸びる管状部材がなくても良い。
培地交換が必要になった際、ユーザは制御部19により遠隔的に廃液速度調整手段17を調整して廃液速度を適当な速度にする。廃液速度の調整はあらかじめ決められた速度に設定しても良いし、図示しないモニタリングシステムで廃液速度を監視しながら適当な速度に調整しても良い。廃液速度調整手段17の構造は、図2Aから図2Dに示す滴下速度調整手段7と同様である。
陰圧供給手段71としては、図26に示すようなポンプ31と廃液容器32とを備えた手段を挙げることができる。陰圧供給手段71の吸引口33を廃液保持手段14の廃液排出口16と接続して廃液保持手段14内を陰圧にし、培養容器11の排出口13を陰圧にすることで細胞培養容器11から培地を吸引することが可能である。
また、陰圧供給手段71としてペリスタポンプなどの送液ポンプを使用しても良い。この場合、送液ポンプを吸引口33の管状部材に設置しても良い。
滴下速度調整手段7は、溶液保持手段3の排出口5と滴下容器8の供給口9とをつなぐ管状部材(チューブ等)に配置されており、管状部材に外力を加えて変形させ、管状部材内腔の断面積を小さくすることで溶液の流量を制限して流速を抑制する。逆に外力を解除すると、管状部材の弾性力により管状部材が元の状態に戻り、流速を上げることができる。このように、滴下速度調整手段7は管状部材に対する外力の強弱により管状部材内に流れる溶液の流速を調整する。滴下速度調整手段7による管状部材への外力の加え方については、図2Aから図2Dに例を示した第1実施形態と同様である。
なお、廃液速度調整手段の構造は、滴下速度調整手段7と同様である。
また、滴下速度調整手段7としてペリスタポンプなどの送液ポンプを採用しても良い。廃液速度調整手段としてペリスタポンプなどの送液ポンプを採用しても良い。送液ポンプは制御部19により遠隔的に操作されても良い。
本システムのユーザは、まず滴下速度調整手段7により、滴下速度が0の状態つまり滴下が止まった状態にシステムを設定する。溶液保持手段3に培地等の溶液を補充し、溶液保持手段3内の培地や溶液の温度をインキュベータ内の温度にする。培地及び細胞が入った細胞培養容器11を用意し、インキュベータ内で細胞培養容器11の供給口12を管状部材を介して滴下容器8の排出口10に、細胞培養容器11の排出口13を管状部材を介して廃液保持手段14の廃液供給口15に、それぞれ接続する。
本変形例に係る細胞培養システム1900は、第1滴下容器41aおよび第2滴下容器41bがそれぞれ複数の排出口を有する態様である。第1滴下容器41aおよび第2滴下容器41bが備えた複数の排出口は、各々異なる細胞培養容器の供給口12(第1供給口42a、第2供給口42b)に管状部材を介して接続される。図27Aは2つの細胞培養容器を用いる態様であるが、各滴下容器が備えた排出口の数だけ細胞培養容器を設置することが可能である。
各滴下容器が複数の排出口を備える代わりに、図27Bに示すように各滴下容器が1つの排出口を備え、そこにつながる管状部材が途中で複数に分岐し、各々異なる細胞培養容器の供給口12(第1供給口42a、第2供給口42b)に接続されても良い。
他は第1実施形態と同様である。
本変形例に係る細胞培養システム2100は、第1滴下容器8aおよび第2滴下容器8bを一つの滴下容器8cとする態様である。滴下容器8cは溶液を内部に供給するための2つの供給口(第1供給口43a、第2供給口43b)と、溶液を内部から排出するための1つの排出口44とを有している。第1供給口43a、第2供給口43bは、それぞれ管状部材を介して第1溶液保持手段3a、第2溶液保持手段3bと接続している。排出口44は管状部材を介して細胞培養容器の供給口45に接続している。他は第1実施形態と同様である。
この場合において、図29Aに示すように第1滴下容器8cが複数の排出口を有する態様でも良い。滴下容器8cが備えた複数の排出口は各々異なる細胞培養容器の供給口45に管状部材を介して接続される。図29Aは2つの細胞培養容器を用いる態様であるが、3以上の細胞培養容器を設置することが可能である。
滴下容器が複数の排出口を供える代わりに、図29Bに示すように各滴下容器が1つの排出口を備え、そこにつながる管状部材が途中で複数に分岐し、各々異なる細胞培養容器の供給口45に接続されても良い。
滴下容器が1つの供給口を備え、第1溶液保持手段3a、第2溶液保持手段3bからの管状部材が途中で合流し、当該1つの供給口に接続していても良い。
本実施形態に係る細胞培養システム2400は、図30に示すように滴下容器8を備えず、細胞培養容器11の供給口12(第1供給口12a、第2供給口12b)の配置位置を細胞培養容器11の上面に限定する点で第6実施形態と異なっている。それ以外は第6実施形態と同様である。
本実施形態に係る細胞培養システム2500は、図31に示すように、溶液保持手段3に溶液を供給するための溶液ストック手段51(第1溶液ストック手段51a、第2溶液ストック手段51b)を備えている点で上記各実施形態と異なっている。それ以外は上記各実施形態と同様である。図31は第1実施形態に対応する例であるが、他の実施形態も同様である。
溶液ストック手段51は、内部に保持した培地等の溶液を溶液の保存に適した温度(例えば4℃)に維持するために温度制御手段52を備えている。溶液ストック手段51の設置場所は、溶液保持手段3よりも重力方向で高い位置であれば任意であり、インキュベータの内外どちらに設置しても良い。
本実施形態に係る細胞培養システム2700は、図33に示すように、溶液保持手段3内の溶液に圧力をかけるための圧力付加手段60を備えている点で上記各実施形態と異なっている。それ以外は上記各実施形態と同様である。図33は第6実施形態に対応する例であるが、他の実施形態も同様である。
本実施形態においては、溶液保持手段3は溶液供給手段6よりも必ずしも高い位置に設置されている必要はない。
供給口93は管状部材を介して溶液保持手段6に接続されているとともに、供給口93から細胞培養容器内部に向かって供給管部95が伸びている。溶液供給手段6から供給された溶液は供給口93を通過し供給管部95に進入する。図38Aに示すように、供給管部95の先端は細胞培養容器内部の底面より所定の高さ上方に配置されており、供給管部95を出た溶液は細胞培養容器91内に滴下される。なお、滴下容器8を採用する態様においては、図38Bに例を示すように、必ずしも供給管部95の先端が細胞培養容器内部の底面より所定の高さ上方に配置されている必要はない。
排出口94は管状部材を介して廃液保持手段14に接続されているとともに、排出口94から細胞培養容器内部に向かって排出管部96が伸びている。排出管部96の先端は培養容器内部の底面に達する位置に配置されるようになっており、細胞培養容器内の溶液をほぼ全量排出することが可能である。
排出管部96は、その側面に図25に示すような穴28を有し、当該穴28を通して溶液を排出しても良い。この場合、排出管部96の先端を培養容器底面に這わせるように配置しても溶液の排出が可能となる。
排出口94の設置位置は、細胞培養容器内の溶液をほぼ全量排出することができる場所であれば任意であり、例えば細胞培養容器の底面や側面に設置しても良い。この場合、排出管部96がなくても良い。
また、図37に示すように、細胞培養容器の排出口につながる管状部材にペリスタポンプ等の送液ポンプ53を設置しても良い。この場合、細胞培養容器の排出口の設置位置を細胞培養容器上面の蓋にし、そこから細胞培養容器内部に向け管状部材が伸びた態様も可能となる。
当該培養容器によれば、蓋部を再利用することができコストの点で有効となる。また、容器本体部が交換可能なので培養系に応じて最適な容器を適宜選択することができ培養系のバリエーションが広がるとともに、市販の容器本体部を使用することでコストの点でも有効である。
2 温度制御手段
3 培地保存手段(溶液保持手段)
4 一時保持手段
6 培地供給手段
7 滴下速度調整手段
8 滴下容器
11 細胞培養容器
14 廃液保持手段
17 廃液速度調整手段
19 制御部
20 管状部材(チューブ)
21 板状部材
22 球状(円柱状)部材
24 シャッター状部材
23、25、26 貫通穴
31 ポンプ
32 廃液容器
33 吸引口
41 滴下容器
51 溶液ストック手段
52 温度制御手段
53 ポンプ
60 圧力付加手段
61 袋状部材
62、64 隔離部材
65 重り
71 陰圧供給手段
81 ポンプ
82 廃液容器
83 吸引口
91 培養容器
92 蓋部
95 供給管部
96 排出管部
97 首部
101 凸部
102 リング状部材
103 凹部
111 円盤状部材
112 蓋本体
113 リング状部材
121 送液ポンプ
130 板状部材
131 溶液ストッパ
132 開口部
133 供給口
134 排出口
151 蓋部
152 蓋本体
153 管状部材
154 穴部
155 容器本体
Claims (16)
- 細胞を培養するための培地等の溶液を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された溶液を細胞培養容器に供給する溶液供給手段と、
を備えた細胞培養システムであって、
前記溶液供給手段が、溶液を滴下する工程を経て前記細胞培養容器に溶液を供給する細胞培養システム。 - 前記細胞培養容器が、前記溶液供給手段より重力方向で下の位置で前記溶液供給手段と連結され、
前記溶液供給手段が、前記溶液保持手段から供給された溶液を滴下する空間を有し、
前記細胞培養容器が、溶液を排出するための排出口を容器側面の所望の高さの位置に備え、
溶液が前記溶液供給手段から前記細胞培養容器に供給されることによって、前記細胞培養容器内での溶液上面の高さが前記排出口に達したときに、溶液が前記排出口から前記細胞培養容器の外部に排出される請求項1に記載の細胞培養システム。 - 前記細胞培養容器が、容器上面に前記溶液供給手段から溶液を供給される供給口を備えるとともに、溶液を排出するための排出口を容器側面の所望の高さの位置に備え、
前記供給口から供給された溶液が、前記細胞培養容器内の空間で溶液上面に向けて滴下され、
溶液が前記溶液供給手段から供給されることによって、前記細胞培養容器内での溶液上面の高さが前記排出口に達したときに、溶液が前記排出口から前記細胞培養容器の外部に排出される請求項1に記載の細胞培養システム。 - 前記溶液供給手段が、
前記溶液保持手段から供給された溶液を滴下する空間を有する滴下容器と、
該滴下容器内における溶液の滴下速度を制御する滴下速度調整手段と、を備える請求項1に記載の細胞培養システム。 - 前記細胞培養容器内における溶液滴下速度を制御する滴下速度調整手段を備える請求項1に記載の細胞培養システム。
- 前記滴下速度調整手段を遠隔的に制御する制御部を備えた請求項4または5に記載の細胞培養システム。
- 前記溶液保持手段の溶液に圧力をかけるための圧力付加手段を備えた請求項1から6のいずれかに記載の細胞培養システム。
- 前記細胞培養容器の前記排出口に陰圧をかけるための陰圧供給手段を備えた請求項1から7のいずれかに記載の細胞培養システム。
- 前記細胞培養容器の排出口と連結され、前記細胞培養容器から排出された培地を保持する廃液保持手段をさらに備え、
前記細胞培養容器から排出された培地が、前記廃液保持手段の中の空間に滴下される請求項1から8のいずれかに記載の細胞培養システム。 - 前記細胞培養容器から溶液を排出する溶液排出手段をさらに備え、
前記細胞培養容器が、前記溶液供給手段より重力方向で下の位置で前記溶液供給手段と連結され、
前記溶液供給手段が、前記溶液保持手段から供給された溶液を滴下する空間を有する請求項1に記載の細胞培養システム。 - 前記細胞培養容器から溶液を排出する溶液排出手段をさらに備え、
前記細胞培養容器が、容器上面に前記溶液供給手段から溶液を供給される供給口を備え、
前記溶液供給手段から該供給口に供給された溶液が、前記供給口から、前記細胞培養容器内の空間に滴下される請求項1に記載の細胞培養システム。 - 前記溶液供給手段が、溶液の滴下速度を制御する滴下速度調整手段を備え、
前記溶液排出手段が、溶液の排出速度を制御する廃液速度調整手段を備え、
前記滴下速度調整手段による溶液の滴下速度の制御と、前記廃液速度調整手段による溶液の排出速度の制御とを遠隔的に操作する制御部を備えた請求項10または11に記載の細胞培養システム。 - 前記溶液保持手段の溶液に圧力をかけるための圧力付加手段を備えた請求項10から12のいずれかに記載の細胞培養システム。
- 前記細胞培養容器が、前記溶液排出手段と連結する排出口を備え、
前記細胞培養容器の前記排出口に陰圧をかけるための陰圧供給手段を備えた請求項10から13のいずれかに記載の細胞培養システム。 - 前記溶液排出手段が、前記細胞培養容器と連結され前記細胞培養容器から排出された溶液を保持する廃液保持手段を備え、
前記細胞培養容器から排出された溶液が、前記廃液保持手段の中の空間に滴下される請求項10から14のいずれかに記載の細胞培養システム。 - 細胞を培養するための培地等の溶液を保持する溶液保持手段と、
該溶液保持手段と連結され、前記溶液保持手段から供給された溶液を細胞培養容器に供給する溶液供給手段と、
前記細胞培養容器から溶液を排出する溶液排出手段と、
を備えた細胞培養システムであって、
前記溶液排出手段が、溶液を滴下する工程を経て前記細胞培養容器から溶液を排出する細胞培養システム。
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| WO2018186426A1 (ja) * | 2017-04-07 | 2018-10-11 | オリンパス株式会社 | 培地交換装置および培養システム |
| WO2019124446A1 (ja) * | 2017-12-19 | 2019-06-27 | オリンパス株式会社 | 計測装置および培養システム |
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| CN109517732B (zh) * | 2017-09-19 | 2022-12-16 | 德诺杰亿(北京)生物科技有限公司 | 一体化dna分析系统 |
| JP7001516B2 (ja) * | 2018-03-23 | 2022-01-19 | 住友ベークライト株式会社 | 培養容器及び細胞培養装置 |
| CN110229752B (zh) * | 2019-06-24 | 2024-03-19 | 上海长海医院 | 一种微负压细胞培养装置 |
| EP4303295A4 (en) * | 2021-03-26 | 2024-11-06 | TERUMO Kabushiki Kaisha | CELL CULTURE SYSTEM |
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| EP3168291A4 (en) | 2018-03-28 |
| CN106488979A (zh) | 2017-03-08 |
| JP6675309B2 (ja) | 2020-04-01 |
| JPWO2016006680A1 (ja) | 2017-07-06 |
| US20170107475A1 (en) | 2017-04-20 |
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