WO2010103748A1 - 細胞培養方法、細胞培養装置、容器内の計数対象物の計数方法、及び計数用装置 - Google Patents
細胞培養方法、細胞培養装置、容器内の計数対象物の計数方法、及び計数用装置 Download PDFInfo
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- WO2010103748A1 WO2010103748A1 PCT/JP2010/001414 JP2010001414W WO2010103748A1 WO 2010103748 A1 WO2010103748 A1 WO 2010103748A1 JP 2010001414 W JP2010001414 W JP 2010001414W WO 2010103748 A1 WO2010103748 A1 WO 2010103748A1
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
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- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
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- 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
- C12M23/02—Form or structure of the vessel
- C12M23/14—Bags
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/18—Rollers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Definitions
- the present invention relates to a cell culture method for culturing cells such as cells, tissues and microorganisms, a cell culture device, a counting method for counting objects in a container, and a counting device.
- shaking culture in which the culture vessel is constantly stirred is also widely performed.
- the cell culture device described in Patent Document 1 it is possible to move the stage on which the culture container is loaded in various patterns such as rotation and shaking, and to stir the culture solution in the culture container.
- the cell culture apparatus described in Patent Document 2 or Patent Document 3 is a mechanism for supplying oxygen by the movement of waves so that the liquid medium in the culture container is shaken so as not to generate bubbles and the cells are not damaged. It has become.
- the entire medium is vigorously stirred, so that the cells are separated individually, and oxygen and nutrients are diffused throughout and can be sufficiently supplied to each cell. It has become.
- Patent Literature 4 discloses a cell culture device that can appropriately maintain the cell density in a culture solution in conjunction with cell proliferation.
- a culture solution containing cultured cells is collected from a sampling port connected to the culture vessel, and a predetermined buffer is added to the cells in the culture solution. After adjusting the density to a density suitable for measurement, it was injected into a counting board, and the cell density was calculated by reading the number with a human or a machine.
- Patent Document 5 discloses a culture apparatus provided with photographing means. According to this culture apparatus, cell images can be periodically acquired and stored.
- a cell image can be obtained, but it has been difficult to accurately measure the number of cells based on the image and obtain a cell density. That is, when photographing the cells in the culture vessel by the photographing means described in Patent Document 5, the number of cells in the cell image is measured and divided by the volume of the culture solution in the field of view of the photographing means, Cell density can be calculated. However, when directly observing cells in the cell container in this way, as shown in FIG. 24, when the cell density is large and the cells overlap, the number of cells cannot be measured accurately. Further, when the cell density is too small, it is difficult to predict the whole density, and there is a problem that the accuracy of the calculated cell density is lowered.
- the thickness of the counter is usually about 0.1 mm compared to the case of measuring using a conventional counter.
- the thickness of the culture vessel is about 1 to 2 cm, and the thickness is 100 to 200 times.
- the number of cells that can be seen from the imaging means is 100 to 200 times that when actually measured using a counter even at the same volume density. Therefore, the cells in the culture container often overlap each other, and it is difficult to directly measure the number of cells by directly observing the cells in the culture container. Therefore, as a result of diligent research, the present inventors have adjusted the thickness of the culture vessel so that the number of cells visible from the imaging means can be measured, and then directly measuring the number of cells in the culture vessel. The cell density close to the measured value was successfully obtained.
- the present invention has been considered in view of the above circumstances, and by controlling the formation of aggregates of cells in a culture vessel and controlling the decomposition of aggregates, the aggregates are adjusted to an appropriate size, and cell proliferation
- An object is to provide a cell culture apparatus and a cell culture method capable of improving efficiency.
- the present invention provides a counting method for counting objects in a container capable of measuring the number of cells in any density range in a culture environment without opening the culture system and irrespective of the density of the grown cells. And it aims at providing the device for counting.
- the cell culturing method of the present invention is a cell culturing method using a culture container, and controls the formation of aggregates of cells in the culture container by applying an external force to the culture container. This is a method for culturing cells by controlling at least one of the degradation control of aggregates.
- the cell culture device of the present invention is a cell culture device for culturing cells using a culture vessel, pressing a culture vessel with a predetermined pushing amount, And a stirring member that can move in the horizontal direction, and by applying an external force to the culture vessel by moving the stirring member, at least one of formation and decomposition of the cell aggregate in the culture vessel is controlled.
- the counting method of the counting object in the container of the present invention is a method for measuring the number of counting objects in the liquid in the sealed container, and the adjusted range is obtained by adjusting the thickness of at least a part of the container. This is a method of measuring the number of counting objects in the measurement target range using at least a part of the measurement target range. Moreover, the counting method of the counting object in the container of the present invention is a method of adjusting the thickness of at least a part of the container after stirring the liquid in the container and equalizing the counting object in the liquid.
- the counting device of the present invention is a counting device for measuring the number of counting objects in a liquid in a sealed container, and includes at least a loading table on which the container is loaded and a measurement target range in the container. An adjustment member that partially adjusts the thickness to a predetermined thickness is provided.
- the counting apparatus of the present invention may further include a stirring member that stirs the liquid in the container before the thickness of the container is adjusted by the adjusting member.
- the counting device of the present invention further includes a photographing means for photographing the counting object in the container, a counting means for measuring the number of counting objects in the photographed image, and the number of counting objects as a result of the measurement by the counting means. And a driving device that drives the adjustment member to adjust at least a part of the container to a predetermined thickness so that the number of objects to be counted in the image falls within the predetermined range. It is also possible to adopt a configuration.
- ADVANTAGE OF THE INVENTION when performing cell culture of a cell, a structure
- the number of cells can be measured without opening the culture system and irrespective of the density of the proliferated cells.
- FIG. 1st embodiment of this invention It is a figure which shows the structure of the cell culture apparatus of 1st embodiment of this invention. It is a figure which shows the structure of the drive device in the cell culture apparatus of 1st embodiment of this invention. It is a schematic side view of the cell culture device of the first embodiment of the present invention. It is a figure which shows formation of the aggregate of a cell in this invention, and decomposition
- FIG. 4 It is a figure which shows the structure of the apparatus for counting of 4th embodiment of this invention. It is a figure which shows the thickness adjustment method (when reducing thickness) of the container by the counting apparatus of 4th embodiment of this invention. It is a figure which shows the thickness adjustment method (when increasing thickness) of the container by the counting apparatus of 4th embodiment of this invention. It is a figure which shows the structure of the apparatus for counting of 5th embodiment of this invention. It is a figure which shows the basic position of the apparatus for counting of 6th embodiment of this invention. It is a figure which shows the stirring state of the apparatus for counting of 6th embodiment of this invention. It is a figure which shows the thickness adjustment of the apparatus for counting of 6th embodiment of this invention, and the waiting state for precipitation (when reducing thickness).
- FIG. 3 is a view showing a culture vessel used in Example 1-5 and Comparative Example 1 of the present invention. It is a figure which shows the image of the cell in Example 1 and Comparative Example 1 of this invention. It is a figure which shows the result of Example 1 and Comparative Example 1 of this invention.
- FIG. 5 is a diagram showing images of cells in Examples 2 to 5 of the present invention.
- FIG. 6 is a diagram showing the results of Examples 2 to 5 of the present invention. It is a figure which shows the counting method of the counting target object in the conventional container.
- FIG. 1 is a diagram showing the configuration of the cell culture device of the present embodiment.
- FIG. 2 is a diagram illustrating a configuration of a driving device in the cell culture device of the present embodiment.
- FIG. 3 is a schematic side view of the cell culture device of the present embodiment.
- FIG. 4 is a diagram showing the formation of cell aggregates and the decomposition of cell aggregates in the present invention.
- the cell culture device 10 of the present embodiment includes a culture vessel 11, a loading table 13, and a stirring member 14, and a container 11-1 in the culture vessel 11 contains a culture solution (medium) and Cells are encapsulated and the tube 12 is connected.
- the culture container 11 is a container formed in a bag shape (back shape) using soft packaging material as a material.
- soft packaging material include, for example, Japanese Patent Application Laid-Open No. 2002-255277 (a food packaging body using a soft packaging material film sheet and a method for taking out food), and Japanese Patent Application Laid-Open No. 2004-323077 (a pressure extraction type bag shape). Those described in (Containers) can be used.
- the culture vessel 11 has gas permeability necessary for cell culture, and part or all of the culture vessel 11 is transparent so that the contents can be confirmed.
- the material for the culture container satisfying such conditions include polyolefin, ethylene-vinyl acetate copolymer, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, silicone rubber and the like.
- the tube 12 is for injecting the culture solution and cells in the culture vessel 11 from the outside or collecting them outside, and each side of the culture vessel 11 is sealed, but at least two or more of them are sealed.
- a tube 12 is connected. One of these is for injection for injecting cultured cells and medium from outside into the culture container 11, and the other is for recovery for recovering cultured cells and medium from the culture container 11. Further, as shown in FIG. 1, when three tubes 12 are attached, the third tube can be used for sampling for taking out cultured cells and culture media from the culture vessel 11 as samples.
- the material of the tube 12 may be appropriately selected according to the usage environment.
- silicone rubber soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, Use SBS (styrene, butadiene, styrene), SIS (styrene, isoprene, styrene), SEBS (styrene, ethylene, butylene, styrene), SEPS (styrene, ethylene, propylene, styrene), polyolefin resins, fluorine resins, etc. Can do.
- the loading table 13 is a flat table on which the culture vessel 11 is placed on the upper surface, and the stirring member 14 is disposed on the upper surface of the culture vessel 11.
- Stop members 13-1 are erected at the four corners of the upper surface of the loading table 13 where the culture vessel 11 is placed.
- holes 11-2 into which the stopper members 13-1 are engaged are formed. By passing each hole 11-2 of the culture vessel 11 through each of the stopper members 13-1, the culture vessel 11 can be placed on the upper surface of the loading table 13. Further, it is possible to prevent the culture vessel 11 from being displaced with the movement of the stirring member 14.
- the stop member is not limited to the above member, and various members can be used as long as they have a mechanism for preventing the culture vessel 11 from shifting.
- the stirring member 14 controls the formation of cell aggregates in the culture container 11 and the decomposition of the cell aggregates by applying an external force to the culture container 11.
- the stirring member 14 is moved in parallel with the loading table 13 at a predetermined speed while pressing the culture vessel 11 with a predetermined pushing amount by the stirring member 14. . This movement is repeatedly executed in a predetermined cycle.
- a roller can be used as the stirring member 14.
- the stirring member 14 is used to apply an external force to the culture vessel, so that the stirring in the culture vessel 11 can be finely adjusted. It is possible to perform agitation suitable for performing and decomposing cell clumps.
- the support table 15 connects the bearing units to a bearing unit that is erected upward on each side of the loading table 13 and supports both ends of the stirring member 14 so as to be rotatable. It is formed by the connecting part.
- the support base 15 can be moved up and down by a rod-type electric cylinder 17 (vertical operation actuator) attached below the connecting portion, and by the stirring member 14 attached to the support base 15. It is possible to finely adjust the push-in amount with respect to the culture vessel 11 by 0.1 mm.
- the rod-type electric cylinder 17 is attached to the moving table 16 on the slider-type electric cylinder 21 (horizontal operation actuator), and moves in the horizontal direction with respect to the loading table 13. Further, the moving speed of the stirring member 14 attached to the support table 15 is adjusted by controlling the moving speed of the moving table 16 in the horizontal direction.
- the drive device in the cell culture device 10 of the present embodiment includes a support base 15, a moving base 16, a rod-type electric cylinder 17, a slider-type electric cylinder 21, and the like.
- the rod-type electric cylinder 17 and the slider-type electric cylinder 21 are used to adjust the pushing amount of the stirring member 14 with respect to the culture container 11 and the moving speed of the stirring member 14. This makes it possible to optimally adjust the size of the cell aggregate by controlling the stirring of the culture solution in the culture vessel 11.
- an actuator using air pressure, hydraulic pressure, electromagnetic force, or a motor or cam is used instead of the electric actuator such as the rod type electric cylinder 17 or the slider type electric cylinder 21. It is also possible to make a configuration that was suitable.
- a cultured cell has a property that a single cell has a low division rate, and it only starts to divide enough when it adheres to each other to form a certain aggregate.
- the rate is relatively slow.
- a well plate is used to forcibly collect cells in one place at a high density to facilitate cell adhesion, or a small volume container is used to proliferate cells. Therefore, the culture was performed while preventing a decrease in cell density by using a larger container.
- the cells floating on the bottom surface in the culture vessel 11 are actively moved by stirring, the probability that the cells adhere to each other is increased, and an appropriately sized aggregate can be formed more quickly. ing.
- the cells when the cells are in an individual state at the start of the cell culture, the cells come into contact with each other to form an appropriately sized aggregate. Can be promoted, and cell proliferation efficiency can be improved.
- control of the formation of cell aggregates is not limited to the initial stage of culture (during seeding).
- the aggregates can be suitably performed even when the cells have collapsed and the cells have become individual, and the cell proliferation efficiency can be improved.
- the stirring member 14 is pressed against the loading container 14 while the culture container 11 is pressed by the stirring member 14 with a predetermined pushing amount. 13 can be moved at a predetermined speed in parallel with 13, and the strength of the external force applied to the culture vessel can be appropriately controlled. For this reason, the agitation in the culture vessel 11 can be finely adjusted to perform agitation suitable for the formation of cell agglomerates, and the agglomerates can be decomposed so that the cells do not fall apart. It is possible to adjust the mass to a size suitable for growth.
- FIG. 1 is a diagram showing the configuration of the cell culture device of the present embodiment.
- the culture part Is different from the first embodiment in that it can be stirred by a stirring member (stirring roller) 14.
- a stirring member stir roller
- both ends of the culture vessel 11 are fixed by the clamp members 23.
- the clamp members 23 about another point, it is the same as that of 1st embodiment.
- the cell culture device 10 of the present embodiment divides the culture vessel 11 using a partition roller (partition member) 22, a culture unit that encloses the culture solution and cells, and the partition roller 22. And an expandable part for expanding the volume of the culture part.
- the partition roller 22 is provided in parallel with the stirring member (stirring roller) 14 and can move in parallel with the loading table 13.
- the volume of the culture part can be continuously changed, and the cell density can be adjusted appropriately by moving the partition roller 22 along with cell proliferation and increasing the volume of the culture part. It is possible to maintain within the range.
- the culture vessel 11 is sandwiched and partitioned from above and below using two partition rollers 22, but the present invention is not limited to this, and the culture vessel 11 is viewed from above by one partition roller 22. It is also possible to have a configuration in which the culture vessel 11 is partitioned into a culture part and an expandable part by pressing against the loading table 13.
- the technique for controlling the culture volume and improving the culture efficiency using such a partition member is described in detail in International Publication No. 2008/136371 and International Publication No. 2008/136339 by the applicant. Yes.
- the size of the culture volume can be controlled using the partition roller 22 so that high cell proliferation efficiency can be obtained, and the culture unit The size of the cell aggregates in can be adjusted so as to obtain high cell growth efficiency. For this reason, it is possible to further improve the cell proliferation efficiency.
- FIG. 1 is a diagram showing a configuration of the cell culture device 10 of the present embodiment.
- This embodiment is the first in that the cells in the culture vessel 11 are photographed to automatically determine whether the size of the aggregate is within a predetermined range, and the aggregate is adjusted to an appropriate size based on the determination result. Different from the embodiment. About another point, it is the same as that of 1st embodiment.
- the cell culture device 10 of this embodiment is provided with the imaging device 30 and the control device 40 in addition to the configuration of the first embodiment, as shown in FIG.
- the imaging device 30 images the cells in the culture vessel 11 and transmits the obtained image to the control device 40.
- the instruction information for photographing by the photographing device 30 can be automatically transmitted from the control device 40 at a predetermined timing.
- a CCD camera can be attached to a lens barrel of a phase contrast microscope.
- the control device 40 is an information processing device that controls the driving device for moving the stirring member 14 in the cell culture device 10 and the imaging device 30. As shown in FIG. 6, the control device 40 includes an imaging device control unit 41, an aggregate size determination unit 42, and a drive device control unit 43.
- the imaging device control unit 41 transmits instruction information for causing the imaging device 30 to perform imaging at a predetermined timing, and receives an image captured from the imaging device 30.
- the aggregate size determination unit 42 determines whether the size of the cell aggregate in the image information is within a predetermined range.
- the predetermined range may be 100 ⁇ m to 600 ⁇ m.
- an average value of the photographed aggregate can be used. Then, as a result of the determination, when the size of the cell aggregate is smaller or larger than the predetermined range, the determination result is output to the drive device control unit 43.
- the drive device control unit 43 determines the pushing amount, moving speed, and cycle of movement of the stirring member 14, and the rod-type electric cylinder 17 according to these driving conditions. And the slider type electric cylinder 21 is controlled. Thereby, when the size of the cell aggregate is smaller than the predetermined range, the cell aggregate in the culture vessel 11 is formed to an appropriate size, and the size of the cell aggregate is larger than the predetermined range. If so, it can be broken down to the appropriate size and adjusted to the optimal size for growth.
- control device 40 or the drive device control unit 43 has various agglomerate sizes, the pushing amount of the stirring member 14 suitable for each case, the moving speed, and the cycle for moving. It is preferable to have a table or the like stored in association.
- the size of the aggregate in the culture vessel 11 can be automatically adjusted, and the cell proliferation efficiency can be stably performed. Can be improved.
- FIG. 7 shows a state in which cells in a culture container are directly observed using a microscope and the number thereof is measured.
- FIG. 7 (A) shows a conventional method of adjusting the thickness of the container. It shows the case of observing without.
- a culture medium whose specific gravity is smaller than the specific gravity of the cells, the cells can be submerged in the bottom of the container and can be made suitable for microscopic observation.
- a culture solution having a large specific gravity may be used to collect cells at the top of the container for observation.
- the present invention when the number of cells is too large to be measured accurately, as shown in FIG. 7B, by reducing the thickness of the container, in the observation range (measurement target range). Reduce the number of cells and adjust to the number of cells suitable for measurement. Thereby, even when culturing a large amount of cells in a culture container, the number of cells can be measured without opening the culture system by directly observing the cells in the culture container. In addition, even when the number of cells in the culture vessel is small and it is difficult to predict the density of the entire culture vessel, increasing the vessel thickness increases the number of cells in the observation range and adjusts the number of cells suitable for measurement. can do.
- FIG. 1 is a diagram showing the configuration of the counting apparatus of the present embodiment.
- the counting device 50 of the present embodiment includes a thickness adjusting member 51, a loading table 13, a photographing unit 52, a driving device 53, a driving device 54, and an illumination 55.
- the culture vessel 11 is placed on top and the number of cultured cells in the culture vessel 11 is counted.
- the thickness adjusting member 51 adjusts the thickness of the culture vessel 11 on the loading table 13.
- the thickness adjusting member 51 is composed of a pressing plate having a flat portion for pressing the culture vessel 11, and the thickness of the thickness adjusting member 51 is reduced by pressing a part of the culture vessel 11 made of soft packaging material from above. It is possible to reduce this.
- the thickness adjusting member 51 is made of a transparent material, the part is illuminated from above by the illumination 55, and observed from below by the photographing means 52 comprising a microscope and a CCD camera. It is like that.
- the thickness adjusting member 51 is not limited to a pressing plate that presses the culture vessel 11 with a flat portion as shown in FIG. 8, and can also be configured using a roller or a tension member.
- the thickness of the culture vessel 11 can be increased by reducing the horizontal area of the culture vessel 11 by moving the culture vessel 11 from both the upper and lower sides or from one side with a roller.
- the thickness of the culture vessel 11 can be decreased by increasing the horizontal area of the culture vessel 11.
- the thickness of the culture container 11 can also be reduced by extending
- the loading table 13 is a flat table on which the culture vessel 11 is placed, and constitutes a loading device for counting together with the thickness adjusting member 51.
- the lower part of the measurement target range in the loading table 13 is configured by a transparent member such as a glass plate 56, and the culture vessel 11 can be observed from below by the photographing means 52.
- the driving device 53 moves the thickness adjusting member 51 up and down by a ball screw screw. As a result, the thickness adjusting member 51 is pressed against the culture vessel 11 on the loading table 13 so that the thickness of the culture vessel 11 can be adjusted.
- a rod-type electric cylinder vertical direction actuator
- the thickness of the culture vessel 11 can be finely adjusted in units of 0.01 mm.
- the driving device 54 moves the photographing means 52 in the horizontal direction with respect to the loading table 13 by a ball screw screw.
- the photographing means 52 is arranged outside the loading table 13 except when the cells in the culture container 11 are photographed, and the photographing means 52 is moved below the measurement target range of the culture container 11 when photographing.
- the driving device 53 and the driving device 54 can be configured using an actuator using air pressure, hydraulic pressure, or electromagnetic force in addition to an electric actuator, or can be configured using a motor or a cam.
- the illumination 55 illuminates the measurement target range in the culture vessel 11 through the thickness adjusting member 51 and provides brightness necessary for photographing the cells by the photographing means 52.
- the amount of transmitted light varies depending on the thickness of the culture solution adjusted at this time, and a difference in brightness occurs in the photographed image, it is preferable to adjust the amount of light according to the thickness of the culture solution.
- the culture vessel 11 can be the same as in the first embodiment.
- FIG. 9 shows a case where the thickness of the container is reduced
- FIG. 10 shows a case where the thickness of the container is increased.
- the thickness adjustment of the vessel is performed by pressing the thickness adjusting member 51 from below the culture vessel 11 as shown in FIG. Can be performed.
- the culture vessel 11 When the culture vessel 11 is made of a material that can be extended horizontally, as shown in FIG. 9B, the culture vessel 11 is pressed using a thickness adjusting member 51 that can cover the entire surface of the culture vessel 11, The thickness of the culture vessel 11 can also be reduced. In addition, as shown in FIG. 9C, the thickness of the culture vessel 11 can be extended by the thickness adjusting member 51 to reduce the thickness thereof. In this case, one end of the edge of the culture vessel 11 is fixed and the edge on the opposite side in the horizontal direction is pulled by the thickness adjusting member 51 made of a tensile member, or two thickness adjusting members 51 are used. The culture vessel 11 can be stretched by pulling both ends in the horizontal direction of the culture vessel 11.
- silicone rubber can be used as a material of such a flexible culture vessel 11.
- a roller or the like is used as the thickness adjusting member 51, and by moving this, the horizontal area of the culture vessel 11 is increased and the thickness of the culture vessel 11 is reduced. You can also
- the thickness adjusting member 51 when increasing the thickness of the culture vessel 11, for example, as shown in FIG. 10E, a part of the upper surface of the culture vessel 11 is pressed by the thickness adjusting member 51 and pressed in the culture vessel 11. The thickness other than the portion can be increased. Further, as shown in FIG. 10 (f), a roller or the like is used as the thickness adjusting member 51, and this is moved to reduce the horizontal area of the culture vessel 11 and increase the thickness of the culture vessel 11. You can also
- the imaging unit 52 is moved below the measurement target range of the culture vessel 11 using the driving device 54.
- the thickness adjusting member 51 is lowered to adjust the thickness of the culture vessel 11 to a predetermined thickness.
- the predetermined thickness can take various values depending on the cell type, the size of the culture vessel, the area of the measurement target range, the culture period, and the like.
- the area to be measured is illuminated by the illumination 55, and the image is taken by the imaging means 52. Then, the cells on the photographed image are counted. At this time, the photographed image can be transmitted from the photographing means 52 to the counting means, and the number of cells can be automatically measured by the counting means.
- a counting means a known cell counting analyzer, cell number measuring device, or the like can be used.
- the cultured cells are precipitated on the bottom of the culture vessel 11, and the photographing means is focused on the precipitated cells. Can be taken.
- the cell density is large and the cells in the measurement target range overlap, the number of cells cannot be counted correctly. Therefore, in the counting method of the counting object in the container of the present embodiment, when the cells in the measurement target range overlap in this way, the thickness adjusting member 51 is moved to reduce the thickness of the culture container 11, and the measurement is performed. By reducing the number of target cells, the cells can be measured.
- the maximum number of cells that can be observed without overlapping in the measurement target range can be considered to be less than the number obtained by dividing the area of the measurement target range by the average horizontal area of the cultured cells. Therefore, when the number of cells is calculated to be greater than or equal to the maximum number of cells as a result of counting the cells, it is preferable to further reduce the thickness of the culture vessel 11 and perform counting again. Whether or not the thickness of the culture vessel 11 is adjusted with other values, such as more accurately estimating the number when cells are closely arranged in the measurement target range and using the estimated value as the maximum number of cells. It can also be used for judgment.
- the accuracy of the obtained cell density is lowered. Therefore, it is preferable to count again after increasing the thickness of the culture vessel 11.
- the measuring device for increasing the thickness of the culture vessel 11 will be described in detail in the sixth embodiment.
- the cell density can be calculated by dividing the number of cells by the volume of the measurement target range. Furthermore, by multiplying the volume of the culture vessel 11 by the obtained cell density, the number of cells in the entire culture vessel 11 can be calculated. Such cell density and the total number of cells in the culture vessel 11 can also be automatically calculated by the counting means.
- the thickness of the culture vessel 11 is reduced even when the number of cells cannot be measured accurately.
- the number of cells can be measured, and the cell density in the culture vessel 11 can be calculated.
- FIG. 11 is a diagram showing the configuration of the counting apparatus according to the present embodiment.
- FIG. 2 is a diagram showing a configuration of a drive device (for a stirring member) in the cell culture device of the first embodiment, and the same device can also be used in this embodiment.
- the counting device according to the present embodiment further includes a stirring member in addition to the configuration of the counting device according to the fourth embodiment.
- the agitation member is used to agitate the culture solution in the culture vessel 11 so that the cells can be dispersed for easy observation. Other points can be the same as in the fourth embodiment.
- the agitating member 14 moves while being pressed against the culture vessel 11 to agitate the culture solution in the culture vessel 11 and disperse the cultured cells in the culture solution.
- a roller can be used as shown in FIG.
- the stirring member 14 is repeatedly moved at a predetermined speed and a predetermined cycle in parallel with the loading table 13, thereby Is configured to be stirred.
- the support table 15 connects the bearing units to a bearing unit that is erected upward on each side of the loading table 13 and supports both ends of the stirring member 14 so as to be rotatable. It is formed by the connecting part. Further, as shown in FIG. 2, the support base 15 can be moved up and down by a rod-type electric cylinder 17 (vertical direction actuator) attached below the connecting portion, and a stirring member attached to the support base 15. It is possible to finely adjust the amount of pressing of 14 against the culture vessel 11 by 0.1 mm.
- the rod-type electric cylinder 17 is attached to the moving table 16 on the slider-type electric cylinder 21 (horizontal operation actuator), and moves in the horizontal direction with respect to the loading table 13. Further, the moving speed of the stirring member 14 attached to the support table 15 is adjusted by controlling the moving speed of the moving table 16 in the horizontal direction.
- an actuator using air pressure, hydraulic pressure or electromagnetic force is used instead of an electric actuator such as the rod type electric cylinder 17 or the slider type electric cylinder 21, or a motor or cam is used. It is also possible to use the configuration used.
- the stirring member 14 is pressed against the culture container 11 with a predetermined pushing amount before the number of cells in the culture container 11 is measured.
- the stirring member 14 can be reciprocated in the horizontal direction for a certain time.
- the culture solution in the culture vessel 11 can be stirred, and the cultured cells in the culture vessel 11 can be dispersed so as to be easily counted.
- FIGS. 14 are schematic diagram showing a case where it is desired to reduce FIG. 14), a microscope observation state (FIG. 15), a thickness adjustment and a waiting state for precipitation (when it is desired to increase the number of cells to be counted).
- the counting device of the present embodiment further includes a thickness adjusting member for increasing the thickness of the culture vessel 11.
- Other points can be the same as in the fifth embodiment.
- the operation procedure of the counting apparatus in the counting method of the counting object in the container according to the present embodiment will be described including the operation for increasing the thickness of the culture container 11 and the operation for decreasing the thickness.
- the culture vessel 11 is disposed on the loading table 13, and a thickness adjusting member 51-1 (pressing plate) is disposed above the observation hole of the loading table 13.
- the thickness adjusting member 51-1 is moved downward to press the culture vessel 11 to reduce its thickness.
- a thickness adjusting member 51-2 (roller) is disposed at one end of the edge of the culture vessel 11, and by moving this, the horizontal area of the culture vessel 11 can be reduced and the thickness can be increased. It has become.
- a stirring member 14 (roller) is disposed above the culture vessel 11, and moved in the horizontal direction in a state where the stirring member 14 is moved downward and pressed against the culture vessel 11. It is possible to stir the culture medium inside.
- the microscope 52-1, the CCD camera 52-2, and the illumination 55 that constitute the imaging means 52 for imaging the cell are arranged outside the loading table 13.
- the photographing means 52 including a microscope 52-1 and a CCD camera 52-2, and illumination 55 is moved in the horizontal direction from the basic position shown in FIG. 12, and the photographing means 52 is disposed directly below the observation hole and the illumination 55 is disposed above the observation hole. Then, the cells precipitated in the culture vessel 11 are photographed by the CCD camera 52-2.
- the image thus obtained is input to a counting device (not shown), the number of cells in the image is measured by this counting device, and the obtained number of cells is measured by a measurement target range (observed by the CCD camera 52-2).
- the cell density in the culture vessel 11 can be calculated by dividing by the volume of the region in the culture vessel 11. As a result, when cells in the culture vessel 11 grow and precipitate with the same thickness, even if they are overlapped and cannot be counted accurately, the thickness can be counted by adjusting the thickness of the culture vessel 11. It is possible to calculate the cell density in the culture vessel 11 without opening the culture system.
- the stirring member 14 is moved upward to return to the basic position in FIG. 12, and the thickness adjusting member 51-2 made of a roller is moved in the direction of the measurement target range to press the culture vessel 11. Then, the thickness of the culture vessel 11 is increased. At this time, in order to make the thickness of the measurement target range in the culture vessel 11 constant, the thickness adjusting member 51-1 is moved to a position where the thickness adjustment member 51-1 is brought into contact with the upper surface of the culture vessel 11 on the measurement target range. And it is made to stand until the cell in the culture container 11 settles.
- the number of cells in the measurement target range can be increased, and when the number of cells in the culture vessel 11 is small, the cell density can be calculated more accurately.
- the specific number of cells when the number of cells in the culture vessel 11 is small is appropriately determined based on the type of cells, the area of the measurement target range, and the like, but can be a number such as 0 or less than 10, for example.
- the cells in the measurement target range are overlapped, or the cells are not seen or almost seen. If not, the thickness of the culture vessel 11 is adjusted again according to the procedures (1) to (5), and the number of cells is counted.
- FIG. 17 is a diagram showing the types of stirring conditions performed using the cell culture device 10 of the present embodiment
- FIG. 18 is a diagram summarizing the results of the cell state under each stirring condition.
- the culture vessel 11 is pressed from above using a stirring member 14, and this stirring member 14 is moved in parallel with respect to the loading table 13. Stir the medium inside.
- the pushing amount when the stirring member 14 is pressed against the culture vessel 11 can take various values. For example, as shown in the figure, when the thickness of the culture vessel 11 is 10.5 mm, Values such as 2 mm, 4 mm, 6 mm, and 8 mm can be taken. And according to each pushing amount, the moving speed of the stirring member 14 is adjusted, respectively, so that the culture solution in the culture vessel 11 can be stirred, which is suitable for the formation of cell aggregates, or cell aggregates Control to one suitable for disassembly.
- the diameter of the stirring member 14 is not particularly limited, but is preferably 0.5 to 3.0 times the thickness of the culture vessel 11 in order to accurately control stirring.
- FIG. 18 shows how much the culture solution in the culture vessel 11 is stirred when stirring is performed under the stirring conditions as described above.
- the culture vessel 11 All of the cells remained submerged.
- stirring is referred to as “weak stirring”. By performing such “weak stirring” stirring, it is possible to promote the formation of cell aggregates.
- the culture solution in the culture vessel 11 is stirred to some extent, Almost lifted up.
- Such agitation under the agitation conditions in the vicinity of the boundary between the state in which the cells are mostly submerged and the state in which the cells are almost lifted is referred to as “medium agitation” in this specification.
- Example 1 As the culture vessel 11, a bag made of LDPE (linear low density polyethylene) and having a film thickness of 0.15 mm was used. As shown in FIG. 19, the culture vessel 11 is partitioned by a partition member (rubber roller), so that the long side of the culture part, which is the culture area, is 250 mm and the short side is 210 mm, and 640 ml of the culture solution is placed in the culture part. I put it in and used it. In addition, the container thickness when the culture container 11 was laid flat at this time was approximately 16 mm, although the upper surface of the container was not completely horizontal. The size of the horizontal plane of the observation range (measurement target range) is a square with a side of 0.5 mm.
- LDPE linear low density polyethylene
- AlyS505N-0 medium of Cell Science Laboratory was used, and as a cultured cell, a human leukemia T lymphoid Jurkat E6.1 strain grown in a necessary amount in a dish for cell culture was used.
- a roller having a diameter of 12 mm was used as the stirring member 14, and stirring was performed under a stirring condition of a pushing amount of 13 mm, a speed of 50 mm / s, and a reciprocation number of 10 times.
- the thickness adjusting member made of an acrylic plate having a width of 50 mm, a thickness of 3 mm, and a length larger than the long side of the culture vessel 11 was lowered to adjust the thickness of the bag to 3.1 mm.
- the observation range was image
- the cell density was calculated based on the number of cells and the volume of the observation range. The photograph taken is shown in FIG. 20, and the number of cells in the observation range, cell density, measured density, and measured value ratio are shown in FIG.
- the measured density is determined by measuring the number of cells in the culture solution collected from the bag using a counting board (One Cell Counter (manufactured by One Cell)) by a conventional method, and dividing this by the volume of the observation range. Obtained.
- Example 2 Similar to Example 1, except that a culture solution having a cell density different from that of Example 1 was used, and that the number of cells was measured, the bag thickness was 11.0 mm, and the resting time was 45 minutes thereafter. The experiment was conducted. The types of the culture solution and the cultured cells are the same as those in Example 1. However, in this example, those having fewer cultured cells than Example 1 were used. The results are shown in FIGS.
- Example 3 In measuring the number of cells, the experiment was conducted in the same manner as in Example 2 except that the thickness of the bag was 7.0 mm and the resting time was 30 minutes. The results are shown in FIGS.
- Example 4 In measuring the number of cells, the experiment was performed in the same manner as in Example 2 except that the thickness of the bag was 4.0 mm and the subsequent standing time was 12 minutes. The results are shown in FIGS.
- Example 5 In measuring the number of cells, the experiment was performed in the same manner as in Example 2 except that the thickness of the bag was 3.1 mm and the resting time was 12 minutes. The results are shown in FIGS.
- the culture vessel 11 has a shape with rounded corners so that no corners are formed, and the stirring member 14 is not cylindrical, and the cross section shown in FIG. It is possible to make appropriate changes such as obtaining an effect.
- cultivation cells are made into measurement object, it is not limited to this,
- other organic bodies, such as plankton, or inorganic substance can also be made into measurement object.
- the “liquid” in the culture vessel includes a liquid such as a culture solution and a semi-fluid.
- the cultured cells can be positioned in the upper portion of the culture vessel 11 and may be counted. . In addition to counting the cells in the above embodiment and examples, it is also possible to observe the growth state of the cells.
- the present invention can be suitably used in the fields of biopharmaceuticals, regenerative medicine, immunotherapy and the like that require culturing a large amount of cells.
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Abstract
Description
このような細胞の大量培養で、特に浮遊系細胞を培養する際は、通常、撹拌翼を備えた培養槽を用いての撹拌培養が一般的である。しかし、特に外力によるダメージに弱い細胞や、凝集塊を形成しながら増殖する細胞の場合、撹拌翼を用いず、細胞を培養容器に封入し、静置して(細胞が底面に沈んだ状態で)培養を行い、細胞の増殖に応じて、容器底面積の大きなものへの移し替えや、容器の数を増やしていく方法が広く用いられている。しかし、この静置培養では、細胞の増殖に伴い細胞の凝集塊が大きくなると、次第に各細胞への酸素や栄養分の供給が不足していき、増殖効率が低下するという問題があった。
また、容器を移し替える際に、一旦培養液は撹拌され、酸素や栄養分の偏りは解消されるが、その都度、移し替え操作時に生じる細胞へのダメージによって増殖効率が低下するといった問題があった。
例えば、特許文献1に記載の細胞培養装置によれば、回転や振とうなど種々のパターンで培養容器を積載した台を動かすことができ、培養容器内の培養液を攪拌することが可能である。
また、特許文献2や特許文献3に記載の細胞培養装置は、培養容器内の液体培地を気泡が発生しないように振とうさせ、細胞が損傷しないように、波の動きにより酸素を供給するしくみになっている。
これらのような細胞培養装置による振とう方法では、培地全体が激しく攪拌されるため、細胞は個々バラバラに分離し、酸素や栄養分は全体に拡散されて、各細胞に十分に供給することが可能となっている。
すなわち、培養液における細胞密度が大きすぎると各細胞に十分な酸素や栄養を供給することができなくなり、細胞増殖効率が低下する。また、培養液における細胞密度が小さすぎても十分な増殖効率が得られない。
そこで、細胞培養においては、培養中に細胞密度を把握するため、適宜培養容器内における培養液中の細胞数を計測する必要があった。
このような細胞培養装置を用いて細胞数の計測を行う場合、従来は培養容器内につながるサンプリング用ポートから培養細胞が含まれる培養液を採集し、所定の緩衝液を加えて培養液における細胞を測定に適する密度に調整した後、計数盤へ注入し、人や機械によってその数を読み取って、細胞密度を算出するということが行われていた。
また、特許文献5には、撮影手段を備えた培養装置が開示されている。この培養装置によれば、細胞画像を定期的に取得し、保存することが可能になっている。
そのような細胞の場合、従来の静置培養では、移し替えタイミングごとに、培養容器を激しく攪拌するため、その度に細胞は個々バラバラとなり、細胞の増殖効率が低下するという問題があった。
また、特許文献1~3に記載の細胞培養装置等などにより振とう培養を行う場合も、同様に培地全体が激しく攪拌され、培地中にバラバラに浮遊した状態となるため、細胞は適正なサイズの凝集塊になりにくく、細胞の増殖効率を最適化することは難しいという問題があった。
すなわち、特許文献5に記載の撮影手段により培養容器内の細胞を撮影する場合、細胞画像の細胞数を計測して、これを当該撮影手段の視野内における培養液の容積で除算することで、細胞密度を算出することはできる。
しかし、このように細胞容器内の細胞を直接観察する場合、図24に示すように、細胞の密度が大きく細胞が重なり合うときは、細胞数を正確に計測することができない。また、細胞の密度が小さすぎるときは、全体の密度予測が難しく、算出される細胞密度の精度が低くなってしまうという問題があった。
このため、撮影手段から見える細胞数は、同じ体積密度でも計数盤を用いて実測する場合の100~200倍となってしまう。したがって、培養容器内の細胞は互いに重なり合う場合が多く、培養容器内の細胞を直接観測して細胞数を計測することは、困難であった。
そこで、本発明者らは鋭意研究した結果、培養容器の厚みを調整して、撮影手段から見える細胞数を計測可能な数にした後に、培養容器内の細胞数を直接観測により計測することで、実測値に近い細胞密度を得ることに成功した。
また、本発明は、培養系を開放することなく、かつ増殖した細胞の密度に拘わらず、培養環境下のあらゆる密度範囲で細胞数を計測することが可能な容器内の計数対象物の計数方法、及び計数用装置を提供することを目的とする。
また、本発明の容器内の計数対象物の計数方法は、容器の液体を攪拌し、液体中の計数対象物を均等化した後に、容器の少なくとも一部の厚みを調整する方法としてある。
また、本発明の計数用装置に、さらに調整部材により容器の厚みを調整する前に、容器内の液体を攪拌する攪拌部材を備えた構成とすることもできる。
また、本発明によれば、培養系を開放することなく、また増殖した細胞の密度に拘わらず、細胞数を計測することが可能となる。
まず、図1~図4を参照して、本発明の第一実施形態について説明する。図1は、本実施形態の細胞培養装置の構成を示す図である。図2は、本実施形態の細胞培養装置における駆動装置の構成を示す図である。図3は、本実施形態の細胞培養装置の概略側面図である。図4は、本発明における細胞の凝集塊の形成と細胞の凝集塊の分解を示す図である。
図1に示すように、本実施形態の細胞培養装置10は、培養容器11、積載台13、攪拌部材14を備えており、培養容器11における収容部11-1には培養液(培地)と細胞が封入され、チューブ12が接続されている。
この積載台13の上面のうち、培養容器11が載置される部分の四隅には止め部材13-1が立設されている。一方、培養容器11の四隅には、止め部材13-1が係入する孔11-2が穿設されている。
止め部材13-1のそれぞれに培養容器11の各孔11-2を通すことで、培養容器11を積載台13の上面に定置させることができる。また、攪拌部材14の移動にともなって培養容器11がずれるのを防ぐことができる。
なお、止め部材は、上記部材に限る必要はなく、培養容器11がずれるのを防ぐ機構を有するものであれば種々のものを用いることができる。
本実施形態の細胞培養方法では、図3に示すように、攪拌部材14により培養容器11を所定の押込量で押し当てつつ、この攪拌部材14を積載台13と平行に所定の速度で移動させる。この移動は、所定のサイクルで繰り返し実行される。この攪拌部材14としては、例えばローラを使用することができる。
本実施形態の細胞培養装置10における駆動装置は、支持台15、移動台16、ロッド型電動シリンダ17、スライダ型電動シリンダ21等によって構成されている。
なお、撹拌部材の動作制御には、ロッド型電動シリンダ17やスライダ型電動シリンダ21のような電動アクチュエータにかえて、空気圧や油圧、電磁力を利用したアクチュエータを使用したり、モータやカムを用いた構成にしたりすることもできる。
ここで、図4に示すように、細胞培養においては、細胞の種類に応じて培養に適切な細胞の凝集塊のサイズがある。
すなわち、培養細胞には、単体での分裂速度は低く、互いに接着して一定の凝集塊を形成してはじめて十分な分裂を開始するという性質がある。
通常の静置培養において、培養初期の細胞密度が低い状態では、拡散により細胞が接着して次第に凝集塊を形成するが、その速度は比較的遅い。
これに対し、本実施形態では、攪拌することにより培養容器11内の底面に漂う細胞を積極的に動かして、細胞同士が接着する確率を高め、適正なサイズの凝集塊をより早く形成可能にしている。
また、このような細胞の凝集塊の形成制御は、培養の初期(播種時)に限られるものではなく、例えば細胞の培養中に培養容器11に対して過剰な外力を加えた結果、凝集塊が崩壊し、細胞が個々バラバラになってしまった場合などにおいても好適に行うことができ、細胞増殖効率を向上させることが可能となっている。
一方、細胞の凝集塊が大きくなりすぎると、凝集塊の内部が酸素不足かつ栄養不足となり、増殖効率は低下する。
このため、凝集塊が巨大化したときは、培養液に強い流れ(乱流)を起こして凝集塊を分解させることが好ましい。
本実施形態の細胞培養装置10を用いた培養方法によれば、凝集塊が巨大化したときは、攪拌部材14の押込量と移動速度を調整することで、培養液に強い流れを起こせるように攪拌を制御し、凝集塊を適切なサイズに分解することが可能となっている。
このため、培養容器11内における攪拌を微調整して細胞の凝集塊の形成に適する攪拌を行うことができるとともに、細胞が個々バラバラにならないように凝集塊を分解することもでき、細胞の凝集塊を増殖に適するサイズに調整することが可能となっている。
次に、本発明の第二実施形態について、図5を参照して説明する。同図は、本実施形態の細胞培養装置の構成を示す図である。
本実施形態は、培養容器11を仕切部材を用いて培養部と拡張可能部とに分割し、培地容積を細胞の増殖に合わせて適切な大きさに調整可能な細胞培養装置10において、培養部を攪拌部材(撹拌ローラ)14により攪拌可能とした点で第一実施形態と異なる。また、本実施形態では、培養容器11の両端をクランプ部材23で固定している。その他の点については、第一実施形態と同様である。
このような仕切ローラ22を用いることで、培養部の容積を連続的に変化させることができ、細胞増殖に伴って仕切ローラ22を移動させ、培養部の容積を増やすことで、細胞密度を適正な範囲内に維持することが可能となっている。
なお、このような仕切部材を用いて培養容積を制御し、培養効率を向上させる技術については、本出願人による国際公開2008/136371号パンフレット及び国際公開2008/136339号パンフレットに詳細に記載されている。
このため、細胞の増殖効率をより一層向上させることが可能となっている。
次に、本発明の第三実施形態について、図6を参照して説明する。同図は、本実施形態の細胞培養装置10の構成を示す図である。
本実施形態は、培養容器11内の細胞を撮影して凝集塊のサイズが所定の範囲内であるかを自動判定し、この判定結果にもとづき凝集塊を適切なサイズに調整する点で第一実施形態と異なる。その他の点については、第一実施形態と同様である。
撮影装置30は、制御装置40から撮影の指示情報を受信すると、培養容器11内の細胞を撮影し、得られた画像を制御装置40へ送信する。撮影装置30による撮影の指示情報は所定のタイミングで制御装置40から自動的に送信することができる。
この撮影装置30としては、例えば位相差顕微鏡の鏡筒にCCDカメラを取り付けて用いることができる。
撮影装置制御部41は、所定のタイミングで、撮影装置30に対して撮影を行わせるための指示情報を送信し、撮影装置30から撮影された画像を受信する。
そして、判定の結果、細胞の凝集塊のサイズが、所定範囲よりも小さい場合又は大きい場合に、その判定結果を駆動装置制御部43へ出力する。
これによって、細胞の凝集塊のサイズが、所定範囲よりも小さい場合は、培養容器11内の細胞の凝集塊を適切なサイズにまで形成し、細胞の凝集塊のサイズが、所定範囲よりも大きい場合は、これを適切なサイズにまで分解して、増殖に最適なサイズに調整することができる。
同図は、顕微鏡を用いて培養容器内の細胞を直接観察し、その数を計測する様子を示したものであり、図7(A)は、従来のように、容器の厚さを調節することなく、観測する場合を示している。なお、培養液としてその比重が細胞の比重よりも小さいものを使用することで、細胞を容器の底に沈め、顕微鏡観察に適したものとすることができる。また反対に比重の大きい培養液を使用して細胞を容器の上部に集めて観察するようにしても良い。
これによって、培養容器により細胞を大量に培養する場合でも、培養容器内の細胞を直接観察することで、培養系を開放することなく、細胞数を計測することが可能となっている。
また、培養容器内の細胞数が少なく、培養容器全体の密度予測が難しい場合にも、容器の厚みを増大させることで、観測範囲内における細胞数を増加させ、計測に適した細胞数に調整することができる。
次に、本発明の第四実施形態の容器内の計数対象物の計数方法、及び計数用装置について、図8を参照して説明する。同図は、本実施形態の計数用装置の構成を示す図である。
本実施形態の計数用装置50は、同図に示すように、厚さ調整部材51、積載台13、撮影手段52、駆動装置53、駆動装置54、及び照明55を備えており、積載台13上に培養容器11を配置して、培養容器11内の培養細胞数を計測する。
なお、厚さ調整部材51は、図8に示すような平面部分により培養容器11を押圧する押圧板に限定されるものではなく、ローラや引張部材を用いて構成することもできる。
この積載台13における測定対象範囲の下方部分は、ガラス板56などの透明な部材により構成され、撮影手段52により下方から培養容器11を観測できるようになっている。
この駆動装置53には、例えばロッド型電動シリンダ(垂直方向動作用アクチュエータ)を用いることができ、これによって、培養容器11の厚さを0.01ミリ単位できめ細かく調整することが可能である。
なお、駆動装置53や駆動装置54を、電動アクチュエータの他、空気圧や油圧、電磁力を利用したアクチュエータを用いて構成したり、あるいはモータやカムを用いて構成したりすることもできる。
培養容器11は、第一実施形態と同様のものを用いることができる。
また、図9の(d)に示すように、厚さ調整部材51としてローラなどを使用し、これを移動させることで、培養容器11の水平面積を増加させ、培養容器11の厚みを減少させることもできる。
また、図10の(f)に示すように、厚さ調整部材51としてローラなどを使用し、これを移動させることで、培養容器11の水平面積を減少させ、培養容器11の厚みを増加させることもできる。
まず、培養容器11内の細胞数を計測する工程に先だって、培養容器11内の培養液を攪拌することが好ましい。なお、培養液の攪拌手段については、第五実施形態において、詳細に説明する。
次に、駆動装置53を用いて、厚さ調整部材51を降下させ、培養容器11の厚さを所定の厚さに調整する。
このとき、所定の厚さは、細胞の種類、培養容器のサイズ、測定対象範囲の面積、及び培養期間などにより種々の値を取り得る。
そこで、本実施形態の容器内の計数対象物の計数方法では、このように測定対象範囲における細胞が重なり合う場合は、厚さ調整部材51を移動させて培養容器11の厚さを減少し、計測対象の細胞数を減らすことで、細胞を計測可能にしている。
このような細胞密度及び培養容器11全体の細胞数も、計数手段により自動的に算出させることができる。
次に、本発明の第五実施形態の容器内の計数対象物の計数方法、及び計数用装置について、図11及び図2を参照して説明する。図11は、本実施形態の計数用装置の構成を示す図である。図2は、第一実施形態の細胞培養装置における駆動装置(攪拌部材用)の構成を示す図であり、本実施形態でも同様のものを用いることができる。
本実施形態の計数用装置は、第四実施形態の計数用装置の構成に加えて、さらに攪拌部材を備えている。本実施形態では、この攪拌部材を用いて培養容器11内の培養液を攪拌することにより、細胞を観測しやすいように分散できるようになっている。その他の点は、第四実施形態と同様のものとすることができる。
同図の例では、攪拌部材14により培養容器11を所定の押込量で押し当てつつ、この攪拌部材14を積載台13と平行に所定の速度及び所定のサイクルで繰り返し移動させることで、培養液を攪拌する構成となっている。
また、この支持台15は、図2に示すように、連結部下に取り付けたロッド型電動シリンダ17(垂直方向動作用アクチュエータ)によって上下に移動させることができ、支持台15に取り付けられた攪拌部材14による培養容器11に対する押込量を0.1ミリ単位できめ細かく調整することが可能となっている。
なお、撹拌部材14の動作制御には、ロッド型電動シリンダ17やスライダ型電動シリンダ21のような電動アクチュエータにかえて、空気圧や油圧、電磁力を利用したアクチュエータを使用したり、モータやカムを用いた構成にしたりすることもできる。
これによって、培養容器11内の培養液を攪拌することができ、培養容器11内における培養細胞を計数し易いように分散させることが可能となる。
次に、本発明の第六実施形態の容器内の計数対象物の計数方法、及び計数用装置について、図12~図16を参照して説明する。これらの図はそれぞれ、本実施形態の容器内の計数対象物の計数方法における計数用装置の基本位置(図12)、攪拌状態(図13)、厚さ調整及び沈殿待ち状態(計数する細胞を減らしたい場合 図14)、顕微鏡観察状態(図15)、厚さ調整及び沈殿待ち状態(計数する細胞を増やしたい場合 図16)を示す模式図である。
以下、本実施形態の容器内の計数対象物の計数方法における計数用装置の動作手順について、培養容器11の厚さを増加させる動作及び減少させる動作を含めて説明する。
まず、図12を参照して、本実施形態の計数用装置の構成要素の基本位置について説明する。
同図において、積載台13には顕微鏡観察用の観測孔が設けられ、この観測孔の上部に積載台13の上面の一部を構成するガラス板56がはめ込まれている。
また、培養容器11の縁の一端には厚さ調整部材51-2(ローラ)が配置され、これを移動させることで、培養容器11の水平面積を減少させ、厚さを増加させることが可能になっている。
また、基本位置では、細胞を撮影するための撮影手段52を構成する顕微鏡52-1とCCDカメラ52-2、及び照明55は、積載台13の外側に配置されている。
次に、図13を参照して、容器内の計数対象物の数の計数を行うに先だち、培養容器11内の培養液を攪拌する手順について説明する。
まず、図12の基本位置から攪拌部材14を下方に移動させて、培養容器11を所定の押込量で押し当てる。次に、この攪拌部材14を積載台13と平行に、所定の速度及び所定のサイクルで繰り返し移動させる。
これにより、培養容器11内の培養液を攪拌し、培養液中の細胞を均等化することが可能となる。
次に、図14を参照して、培養容器11の厚さを減少させ、計数する細胞を減らす手順について説明する。
まず、攪拌部材14を上方に移動させて図12の基本位置に戻し、厚さ調整部材51-1を下方に移動させて、培養容器の厚さを減少させる。このとき、厚さ調整部材51-1により、積載台13の観測孔上の領域を含む培養容器11の一部を押圧し、培養容器11の測定対象範囲の厚さを所定のサイズに調整する。このとき、培養環境下において、培養容器11の厚さが小さいほど、計数する細胞を減らすことができる。
培養容器11の厚さを減少させた後、培養容器11内の細胞が沈殿するまで静置させる。
次に、図15を参照して、細胞計数を行う手順について説明する。
培養容器11の厚さを減少させた状態で、培養容器11内の細胞が沈殿した後、図15に示すように、顕微鏡52-1とCCDカメラ52-2とからなる撮影手段52、及び照明55を図12の基本位置から水平方向に移動させ、撮影手段52を観測孔の真下に、照明55を観測孔の上方に配置させる。そして、CCDカメラ52-2により培養容器11内に沈殿している細胞を撮影する。
これにより、培養容器11内の細胞が増殖して、そのままの厚さで沈殿させると、重なり合いが生じて正確に計数できない場合でも、培養容器11の厚さを調整することで計数可能にすることができ、培養容器11内の細胞密度を、培養系を開放することなく、算出することが可能となる。
一方、培養の初期段階などにおいては、細胞数が少ないため、計数はできるものの、得られる細胞密度の精度が低くなる場合がある。
このため、観測される細胞数が少ない場合は、図16に示すように、培養容器11の厚さを増加させ、測定対象範囲における細胞数を増やしてから、(4)で説明した顕微鏡観察を行う。
培養容器11内の細胞数が少ない場合の具体的な細胞数は、細胞の種類や測定対象範囲の面積等にもとづき適宜決定されるが、例えば0又は10未満などの数にすることができる。
この攪拌部材14を培養容器11に押し当てる際の押込量は、種々の値をとることができるが、例えば同図に示すように、培養容器11の厚さが10.5mmの場合には、2mm,4mm,6mm,8mmなどの値をとることができる。
そして、各押込量に応じて、攪拌部材14の移動速度をそれぞれ調整することで培養容器11内の培養液の攪拌を、細胞の凝集塊の形成に適したもの、又は、細胞の凝集塊の分解に適したものに制御する。
なお、攪拌部材14の直径は、特に限定されないが、攪拌を的確に制御するため、培養容器11の厚さに対して、0.5倍~3.0倍とすることが好ましい。
同図に示すように、押込量が2mmで移動速度が2.5mm/s,12.5mm/sのとき、及び押込量が4mmで移動速度が2.5mm/sのときは、培養容器11内の細胞は全て沈んだままであった。
本明細書において、このような攪拌を「弱撹拌」と呼ぶ。このような「弱撹拌」の攪拌を行うことで、細胞の凝集塊の形成を促すことが可能となる。
このような細胞がほとんど沈んだままである状態と、ほとんど浮き上がる状態との境界付近の攪拌条件における攪拌を本明細書において、「中撹拌」と呼ぶ。このような「中撹拌」の攪拌を行うことで、細胞の凝集塊を分解しすぎることなく、適切なサイズに調整することが可能となる。
従来の細胞培養における攪拌では、通常培養容器が激しく攪拌される結果、「強撹拌」に相当する攪拌が行われており、増殖効率の低下を招くという問題があったが、本発明によりこのような問題を解消することが可能となっている。
培養容器11として、LDPE(直鎖状低密度ポリエチレン)製、フィルム厚0.15mmのバッグを使用した。培養容器11は、図19に示すように、仕切り部材(ゴムローラ)で仕切ることにより、培養を行う領域である培養部の長辺を250mm、短辺を210mmとし、当該培養部に培養液640mlを入れて使用した。なお、このときの培養容器11を平置きした際の容器厚みは、容器上面が完全な水平面にならないが、およそ16mmであった。観察範囲(測定対象範囲)の水平面のサイズは一辺0.5mmの正方形である。
攪拌部材14として直径12mmのローラを使用し、押込量13mm、速度50mm/s、往復回数10回の攪拌条件により攪拌を行った。
なお、実測密度は、従来の方法により計数盤(ワンセルカウンター(ワンセル社製))を用いて、バッグから回収した培養液中の細胞数を計測し、これを観測範囲の容積で除算して得た。
実施例1と同一の培養容器11及び培養液を用いて、同一の攪拌条件で攪拌を行った。
次に、バッグの厚さを調節することなく、60分間静置させた後、観測範囲を撮影した。バッグの上面は押圧を行っていないため波状の凹凸が生じており、バッグの厚みはおよそ16mmであった。
この場合、細胞に重なり合いが見られ、細胞数の計数を行うことはできず、密度の算出も行うことができなかった。その結果を図20,図21に示す。
実施例1とは細胞密度の異なる培養液を用いるとともに、細胞数の計測にあたり、バッグの厚さを11.0mmとし、その後の静置時間を45分間にした点以外は、実施例1と同様にして、実験を行った。培養液と培養細胞の種類は、実施例1と同じであるが、本実施例では実施例1に比較して培養細胞数が少ないものを使用した。その結果を図22及び図23に示す。
細胞数の計測にあたり、バッグの厚さを7.0mmとし、その後の静置時間を30分間にした点以外は、実施例2と同様にして、実験を行った。その結果を図22及び図23に示す。
細胞数の計測にあたり、バッグの厚さを4.0mmとし、その後の静置時間を12分間にした点以外は、実施例2と同様にして、実験を行った。その結果を図22及び図23に示す。
細胞数の計測にあたり、バッグの厚さを3.1mmとし、その後の静置時間を12分間にした点以外は、実施例2と同様にして、実験を行った。その結果を図22及び図23に示す。
これに対して、実施例1では、バッグの厚さを減少させることで、細胞数を計測することができ、培養容器11内における細胞密度を算出することが可能であった。
よって、細胞数が多く、その計数ができない場合、細胞密度が大きい程、バッグの厚さをより大きく減少させることで、細胞を計測可能にできることがわかった。
これらの実験結果から、本発明の計数用装置を用いた容器内の計数対象物の計数方法によれば、培養系を開放することなく、また細胞密度に拘わらず、培養容器内の細胞数を計測でき、細胞密度を算出できることが明らかとなった。
例えば、培養容器11をコーナー部分が生じないように角を丸くした形状としたり、攪拌部材14を円柱状ではなく、図17に示す断面を星形などの種々の形状に構成し、様々な攪拌効果が得られるようにしたりするなど適宜変更することが可能である。
また、上記実施形態及び実施例では、培養細胞を計測対象としているが、これに限定されるものではなく、例えばプランクトンなどその他の有機体、又は無機物を計測対象とすることも可能である。培養容器内の「液体」には、培養液などの液体の他、半流動体も含まれる。また、培養容器11内の培養液として、培養細胞の比重よりも大きいものを使用することで、培養細胞を培養容器11内の上部に位置させることができ、これを計数するようにしても良い。また、上記実施形態及び実施例において細胞を計数するのみならず、細胞の生育状態等を観察することもできる。
Claims (20)
- 培養容器を用いた細胞の培養方法であって、前記培養容器へ外力を付与することにより、前記培養容器内の培養液中にある細胞の凝集塊の形成制御及び凝集塊の分解制御の少なくとも一方の制御を行って細胞の培養を行うことを特徴とする細胞の培養方法。
- 前記培養容器への外力の付与を、平置きした培養容器の上面から攪拌部材を所定の押込量で押し当て、所定の速度で水平方向に移動させることにより行い、かつ、前記培養容器への外力の付与によって、前記培養容器内の培養液を攪拌することで、前記培養容器内の細胞の凝集塊の形成及び分解の少なくとも一方を制御することを特徴とする請求項1記載の細胞の培養方法。
- 前記細胞の凝集塊の形成が、培養の開始時に、前記培養容器内に播種された細胞に対して行われることを特徴とする請求項1又は2記載の細胞の培養方法。
- 前記細胞の凝集塊の分解により、前記細胞の凝集塊のサイズが、所定範囲内の大きさとなるように制御されることを特徴とする請求項1又は2記載の細胞の培養方法。
- 培地及び細胞を含む培養液を封入した培養容器を載置する積載台と、
前記培養容器に所定の押込量で押し当て、所定の速度で水平方向に移動可能な攪拌部材とを備え、
前記攪拌部材を移動させて前記培養容器内の培養液を攪拌することで、前記培養容器内の細胞の凝集塊の形成及び分解の少なくとも一方を制御することを特徴とする細胞培養装置。 - 前記攪拌部材がロール形状であることを特徴とする請求項5記載の細胞培養装置。
- 前記培養容器内の細胞を撮影する撮影装置と、
前記撮影装置により撮影された画像を入力して、前記細胞の凝集塊のサイズが、所定範囲内の大きさであるか否かを判定する判定手段と、
前記判定手段による判定の結果、前記細胞の凝集塊のサイズが所定範囲よりも小さい場合は、前記攪拌部材を、所定の押込量かつ所定の速度で水平方向に移動させて前記細胞の凝集塊を形成させ、また、前記細胞の凝集塊のサイズが所定範囲よりも大きい場合には、前記攪拌部材を、所定の押込量かつ所定の速度で水平方向に移動させて前記細胞の凝集塊を分解させる駆動装置と、を備えたことを特徴とする請求項5又は6記載の細胞培養装置。 - 前記培養容器を培養部と拡張可能部を含む二室以上に仕切り、前記培養部における細胞数の増加に合わせて前記培養部の容積を拡張する仕切部材を備え、
前記培養部内の培地を前記攪拌部材により攪拌することを特徴とする請求項5~7のいずれかに記載の細胞培養装置。 - 密封された容器内の液体中の計数対象物の数を計測する方法であって、前記容器の少なくとも一部の厚みを調整し、調整した範囲の少なくとも一部を測定対象範囲として、当該測定対象範囲における計数対象物の数を計測する
ことを特徴とする容器内の計数対象物の計数方法。 - 前記容器内の液体を攪拌し、前記液体中の前記計数対象物を均等化した後に、前記容器の少なくとも一部の厚みを調整する
ことを特徴とする請求項9記載の容器内の計数対象物の計数方法。 - 計測された前記計数対象物の数を用いて、前記液体中の計数対象物の密度を算出し、及び/又は、前記液体全体における計数対象物の数を算出する
ことを特徴とする請求項9又は10記載の容器内の計数対象物の計数方法。 - 前記計数対象物の数の計測が、前記測定対象範囲を撮影した画像中の前記計数対象物の数を計測することにより行われる
ことを特徴とする請求項9~11のいずれかに記載の容器内の計数対象物の計数方法。 - 前記測定対象範囲における前記計数対象物の数が所定値以上である場合、前記容器の少なくとも一部の厚みを減少させた後、前記計数対象物の数を計測する
ことを特徴とする請求項9~12のいずれかに記載の容器内の計数対象物の計数方法。 - 厚み調整部材を用いて、前記容器を押圧し、又は、前記容器を引き伸ばし、前記容器の少なくとも一部の厚みを減少させる
ことを特徴とする請求項13記載の容器内の計数対象物の計数方法。 - 前記測定対象範囲における前記計数対象物の数が所定値未満である場合、前記容器の少なくとも一部の厚みを増加させた後、前記計数対象物の数を計測する
ことを特徴とする請求項9~12のいずれかに記載の容器内の計数対象物の計数方法。 - 厚み調整部材を用いて、前記容器を押圧し、前記容器の少なくとも一部の厚みを増加させる
ことを特徴とする請求項15記載の容器内の計数対象物の計数方法。 - 前記容器が培養容器であり、前記計数対象物が細胞である請求項9~16いずれかに記載の容器内の計数対象物の計数方法。
- 密封された容器内の液体中の計数対象物の数を計測するための計数用装置であって、
前記容器を積載する積載台と、前記容器における測定対象範囲を含む少なくとも一部を所定の厚みに調整する調整部材とを備えたことを特徴とする計数用装置。 - 前記調整部材により前記容器の厚みを調整する前に、前記容器内の液体を攪拌する攪拌部材を備えたことを特徴とする請求項18記載の計数用装置。
- 前記容器内の前記計数対象物を撮影する撮影手段と、撮影された画像内の前記計数対象物の数を計測する計数手段と、前記計数手段による計測の結果、前記計数対象物の数が所定の範囲内に無い場合、前記画像内の計数対象物の数が、所定の範囲内になるように、前記調整部材を駆動して、前記容器の少なくとも一部を所定の厚みに調整させる駆動装置とを備えたことを特徴とする請求項18又は19記載の計数用装置。
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Also Published As
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US20110318725A1 (en) | 2011-12-29 |
EP2407533A4 (en) | 2013-09-25 |
CN103276046A (zh) | 2013-09-04 |
EP2407533B1 (en) | 2016-06-15 |
US20140011186A1 (en) | 2014-01-09 |
KR101773601B1 (ko) | 2017-08-31 |
US9388376B2 (en) | 2016-07-12 |
CN103276046B (zh) | 2015-07-29 |
KR20140071440A (ko) | 2014-06-11 |
EP2772533A2 (en) | 2014-09-03 |
EP2772533A3 (en) | 2014-10-01 |
CN102348794B (zh) | 2014-09-03 |
EP2772533B1 (en) | 2017-02-01 |
KR20150043561A (ko) | 2015-04-22 |
EP2407533A1 (en) | 2012-01-18 |
CN102348794A (zh) | 2012-02-08 |
KR20130031897A (ko) | 2013-03-29 |
KR101828916B1 (ko) | 2018-03-29 |
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