Liquid-filled culture bag, method for producing liquid-filled culture bag, and method for using liquid-filled culture bag

Abstract

The present invention provides a liquid-filled culture bag that makes it possible to efficiently remove air bubbles that are in fine recesses in a culture bag, without the need for extensive labor or dedicated equipment, and that makes it possible to reduce burdensome work and the risk of contamination at the site of production. Provided is a liquid-filled culture bag in which a culture bag that has fine structures formed in at least part of a culture part thereof is accommodated in an exterior package part. A space that can be reduced in pressure exists between the exterior package part and the outside of the culture part. The liquid-filled culture bag is packaged in a degassed manner. A plurality of first protrusions are preferably provided to the outside of the culture part. A plurality of second protrusions are preferably provided to a region of the exterior package part that is opposite from the outside of the culture part. Furthermore, preferably provided is a spacer part which forms the space that can be reduced in pressure between the exterior package part and the outside of the culture part.

Description

Liquid-filled culture bag, method for manufacturing liquid-filled culture bag, and method for using liquid-filled culture bag

[0001] The present invention relates to cell culture technology, and more particularly to a culture bag having a minute recess or the like in the culture section.

[0002] When culturing spheroids or organoids of adherent cells such as iPS cells or ES cells, culture bags with many micro-wells formed in the culture area are sometimes used. When such culture bags are filled with culture medium, air bubbles are trapped and accumulated in the micro-wells, hindering cell culture. Specifically, if air bubbles remain in the micro-wells of the culture bag, the formation of spheres (cell aggregates) composed of iPS cells or the like is hindered. However, it is extremely difficult to remove all of the air bubbles from the micro-wells.

[0003] International Publication No. 2021 / 241665 Pamphlet Japanese Patent Application Laid-Open No. 2020-80670

[0004] One method for removing bubbles accumulated in the micro-recesses is to use a micropipette or similar device to spray liquid onto the bubbles, thereby removing the bubbles with water pressure. However, this method using a micropipette may not be sufficient to remove the bubbles, especially when the recesses are small. Furthermore, this method is only possible in open containers and is not suitable for removing bubbles accumulated in the culture area of ​​a culture bag.

[0005] Here, Patent Document 1 discloses that a culture bag having micro-recesses in the culture section is filled with a culture medium, and then pressure is applied to the culture bag to remove air from the culture bag. Patent Document 2 discloses that a culture bag having micro-recesses in the culture section is filled with a degassed culture medium filling solution to remove air bubbles that have accumulated in the micro-recesses. This document also discloses that the culture bag having micro-recesses in the culture section is placed in a vacuum exhaust device and exposed to a vacuum to remove air from the culture bag.

[0006] However, these methods have problems such as the time-consuming and labor-intensive process of removing air bubbles and the need for specialized equipment.Furthermore, in the production of regenerative medicine and cell medicines, adjustments are required on-site to remove air bubbles from the culture bags, which can complicate the manufacturing process and raise the risk of contamination.

[0007] The present inventors conducted extensive research to develop a liquid-filled culture bag that can solve these problems, and have completed the present invention. That is, the present invention was made in consideration of the above circumstances, and aims to provide a liquid-filled culture bag, a method for manufacturing a liquid-filled culture bag, and a method for using a liquid-filled culture bag that can efficiently remove air bubbles that have accumulated in the micro-recesses in the culture bag without requiring much effort or dedicated equipment, and that can reduce cumbersome work and the risk of contamination at the manufacturing site.

[0008] In order to achieve the above-mentioned object, the liquid-filled culture bag of the present invention is a liquid-filled culture bag in which a culture bag having a microstructure formed in at least a part of the culture section is contained in an outer packaging section, and a space that can be depressurized exists between the outside of the culture section and the outer packaging section, and the bag is configured to be vacuum-packaged.

[0009] Furthermore, it is preferable that the liquid-filled culture bag of the present invention is configured to have a plurality of first protrusions on the outside of the culture section, and it is also preferable that the liquid-filled culture bag of the present invention is configured to have a plurality of second protrusions in the area of ​​the outer packaging section that faces the outside of the culture section, and it is also preferable that the liquid-filled culture bag of the present invention is configured to have a spacer section that forms a space that can be depressurized between the outside of the culture section and the outer packaging section.

[0010] In addition, it is preferable that the liquid-filled culture bag of the present invention is configured such that the microstructure is a plurality of recesses or a plurality of grooves. Furthermore, in the liquid-filled culture bag of the present invention, the oxygen permeability of the material in the region where the culture section is provided in the culture bag is 3,000 ml / m or less. 2 ・It is preferable to have a configuration that is at least 1 / day and 1 / 2 ATM.

[0011] Furthermore, it is preferable that the liquid-filled culture bag of the present invention is configured such that the oxygen permeability of the member of the region of the outer packaging part facing the outside of the culture part is equal to or lower than the oxygen permeability of the culture part, and the oxygen permeability of the member of the region of the outer packaging part facing the outside of the culture part is equal to or lower than 1,000 ml / m 2 It is more preferable that the liquid-filled culture bag of the present invention has a gas barrier property of 1000 kJ / day atm or less. Furthermore, it is also preferable that the liquid-filled culture bag of the present invention is configured by combining the above-mentioned liquid-filled culture bags in various ways.

[0012] The method for manufacturing a liquid-filled culture bag of the present invention is a method for manufacturing a liquid-filled culture bag in which a culture bag having a microstructure formed in at least a part of the culture section is contained in an outer packaging section, and a space that can be depressurized exists between the outside of the culture section and the outer packaging section, and the culture bag is filled with liquid and vacuum-packaged.

[0013] Furthermore, it is preferable that the method for manufacturing a liquid-filled culture bag of the present invention is a method in which a plurality of first protrusions are provided on the outside of the culture section, and / or a plurality of second protrusions are provided in an area of ​​the outer packaging section facing the outside of the culture section, and / or a spacer section is provided that forms a space that can be depressurized between the outside of the culture section and the outer packaging section.

[0014] The method for using the liquid-filled culture bag of the present invention is a method for storing or transporting the liquid-filled culture bag at room temperature, refrigerated, or frozen. The method for using the liquid-filled culture bag of the present invention is a method for injecting cells, culture medium, and cytokines such as cell adhesion factors and cell growth factors into the liquid-filled culture bag to perform culture.

[0015] According to the present invention, it is possible to provide a liquid-filled culture bag, a method for manufacturing a liquid-filled culture bag, and a method for using a liquid-filled culture bag that can efficiently remove air bubbles that have accumulated in the micro-recesses in the culture bag without requiring much effort or dedicated equipment, thereby reducing cumbersome work and the risk of contamination at the manufacturing site.

[0016] FIG. 1 is a schematic diagram showing the configuration of a liquid-filled culture bag according to an embodiment of the present invention; FIG. 2 is a schematic cross-sectional view showing the configuration of a liquid-filled culture bag according to an embodiment of the present invention; FIG. 3 is a schematic cross-sectional view showing the configuration of a liquid-filled culture bag according to an embodiment of the present invention; FIG. 4 is a schematic cross-sectional view showing the configuration of a liquid-filled culture bag according to an embodiment of the present invention; FIG. 5 is a schematic cross-sectional view showing the configuration of a liquid-filled culture bag according to an embodiment of the present invention; FIG. 6 is a diagram showing a photograph representing the results of test 1; FIG. 7 is a diagram showing a photograph representing the results of test 2; FIG. 8 is a diagram showing a photograph representing the results of test 3; and FIG. 9 is a diagram showing a photograph representing the results of test 4.

[0017] Hereinafter, embodiments of the liquid-filled culture bag, the method for manufacturing the liquid-filled culture bag, and the method for using the liquid-filled culture bag of the present invention will be described in detail, but the present invention is not limited to the specific contents of the following embodiments and examples.

[0018] First, a liquid-filled culture bag according to an embodiment of the present invention will be described in detail with reference to the drawings. The liquid-filled culture bag of this embodiment is a liquid-filled culture bag in which a microstructure is formed in at least a portion of the culture section and is housed in an exterior packaging section, and is characterized in that a space that can be depressurized exists between the outside of the culture section and the exterior packaging section, and the liquid-filled culture bag is vacuum-packaged. That is, this liquid-filled culture bag is a culture bag in which a microstructure is formed in at least a portion of the culture section, filled with liquid, and the culture bag is housed in an exterior packaging section.

[0019] Specifically, as shown in Figures 1 and 2, the liquid-filled culture bag of this embodiment comprises a culture bag 10 and an exterior packaging part 20, and the culture bag 10 is sealed in the exterior packaging part 20. The culture bag 10 is a bag-shaped container made of a soft packaging material for culturing cells or the like, and is provided with a culture part 11 therein having a culture surface, which is an area for culturing cells. The outside of the culture part 11 is the side opposite to the culture surface (the side provided with the first protrusion part 112 in Figure 2) on the bag wall part (the bottom surface part of the bag in Figure 2) on which the culture part 11 is provided. In other words, the outside of the culture part 11 is the area corresponding to the culture surface on the outside of the culture bag 10.

[0020] 2, the culture unit 11 is provided only on the bottom side of the culture bag 10, but the culture unit 11 may be provided only on the top plate side of the culture bag 10, or on both the bottom side and the top plate side. In these cases, the outside of the culture unit 11 on the top plate side is the area corresponding to the culture surface on the top plate side on the outside of the culture bag 10, and the outside of the culture unit 11 on the bottom plate side is the area corresponding to the culture surface on the bottom side on the outside of the culture bag 10. The same applies to the modified examples shown in FIGS. 3 to 5.

[0021] The culture bag 10 can be produced, for example, by sandwiching one or more ports between two sheets of film and heat-sealing the periphery. In Fig. 1, the culture bag 10 includes a bag seal 12 formed by heat sealing, one port 13, a tube 14 connected to the port 13, and a cap 15 that seals the tube 14. Note that the method for producing the culture bag 10 is not limited to this, and the culture bag 10 can also be produced by other methods, such as using a vacuum-pressure formed film or an inflation-formed film.

[0022] The number of ports provided in the culture bag 10 is not limited to one, but may be two or more. Furthermore, the method of sealing the tube 14 is not limited to the method using the cap 15. For example, the tube 14 may be sealed using a connector with a cap function, or a general connector may be connected to the tube 14 and a cap may be provided to seal the connector.

[0023] A microstructure 111 is formed in the culture section 11 of the culture bag 10. This microstructure 111 may be, for example, a plurality of recesses (wells) or a plurality of grooves. Furthermore, the culture section 11 having this microstructure 111 can be subjected to a cell low-adhesion treatment, a cell adhesion treatment, or the like.

[0024] The shape of the plurality of recesses is not particularly limited, but may be, for example, a hemisphere, a cone, a pyramid, a shape consisting of a cylinder and a cone, or a shape consisting of a prism and a pyramid, etc. The shape of the plurality of grooves is also not particularly limited, but may be, for example, a V-shaped cross section perpendicular to the longitudinal direction of the groove, and may be linear or curved.

[0025] The openings of the recesses are preferably circular or rectangular, and the diameter of the circle or inscribed circle can be, for example, 2 mm or less, or 1.5 mm or less, or 1.0 mm or less. The depth of the recesses can be, for example, 1.5 mm or less, or 1.0 mm or less, or 0.5 mm or less, or 0.35 mm or less. Similarly, the width and depth of the grooves can each be, for example, 1.5 mm or less, or 1.0 mm or less, or 0.5 mm or less, or 0.35 mm or less.

[0026] By forming the microstructure 111 in the culture section 11 into a plurality of recesses, it becomes possible to suitably form spheres consisting of a plurality of iPS cells or other adherent cells using the culture bag 10. Furthermore, by forming the microstructure 111 in the culture section 11 into a plurality of grooves, it becomes possible to adhere and suitably culture adherent cells to the culture section 11 while increasing the area of ​​the culture surface of the culture bag 10.

[0027] The culture bag 10 is filled with a liquid L. As the liquid L, it is preferable to use a culture medium, water for injection, phosphate buffer solution, physiological saline, or the like. In the liquid-filled culture bag of this embodiment, once air bubbles are removed from the microstructure 111 in the culture section 11 and the surface of the microstructure 111 becomes wet, air bubbles do not usually reappear in the microstructure 111. For this reason, it is also preferable to fill the liquid L with not only a culture medium but also other liquids such as water for injection.

[0028] In the culture bag 10, it is preferable that at least the material in the region where the culture section 11 is provided is made of a gas-permeable material so that air bubbles accumulated in the microstructure 111 can be removed to the outside of the culture bag 10. For example, the oxygen transmission rate of the material in the region of the culture section 11 at 37°C is 3,000 ml / m 2 ・day・atm or more is preferable, 5,000 ml / m 2 ・day・atm or more is more preferable, and 8,000 ml / m 2 ・It is even more preferable that it is at least 1 / 2 day.

[0029] In the liquid-filled culture bag of this embodiment, a space S that can be depressurized (decompressed space) exists between the outside of the culture unit 11 and the outer packaging unit 20. As described above, the outside of the culture unit 11 is the area that corresponds to the culture surface on the outside of the culture bag 10.

[0030] 2, the liquid-filled culture bag of this embodiment is provided with a plurality of first protrusions 112 on the outside of the culture unit 11, so that a reduced pressure space S can be preferably formed between the outside of the culture unit 11 and the outer packaging unit 20. In addition, as shown in Modification 1 of FIG. 3, it is also preferable that the liquid-filled culture bag of this embodiment has a shape in which the bottom portion of the culture bag 10 is a continuous U-shaped body, and has both a recessed portion (microstructure 111) inside the bag and a protrusion (first protrusion 112) outside the bag.

[0031] Furthermore, as shown in Modification 2 of Fig. 4 , the liquid-filled culture bag of this embodiment can be provided with a plurality of second protrusions 22 in an area of ​​the exterior packaging part 20 facing the outside of the culture part 11, thereby preferably forming a reduced pressure space S between the outside of the culture part 11 and the exterior packaging part 20. The first protrusions 112 and the second protrusions 22 may be any protrusions that can form a reduced pressure space S between the outside of the culture part 11 and the exterior packaging part 20, and there are no particular limitations on their shape, size, or number.

[0032] Furthermore, as shown in Modification 3 of Fig. 5 , the liquid-filled culture bag of this embodiment can also be provided with a spacer part 30 that forms a space that can be depressurized between the outside of the culture part 11 and the exterior packaging part 20, thereby preferably forming a depressurized space S between the outside of the culture part 11 and the exterior packaging part 20. The spacer part 30 may be any part that can form a space that can be depressurized between the outside of the culture part 11 and the exterior packaging part 20, and the specific structure thereof is not particularly limited, but for example, a plate-like member or a porous member having a plurality of through holes can be preferably used.

[0033] The liquid-filled culture bag of this embodiment may be double-packaged by enclosing the culture bag 10 in another exterior packaging part, and enclosing this exterior packaging part in the exterior packaging part 20. Even if the liquid-filled culture bag of this embodiment is configured in this manner, it is possible to suitably remove air bubbles from the culture part 11, for example, if the other exterior packaging part fulfills the function of the spacer part 30 described above, thereby forming a reduced-pressure space S between the outside of the culture part 11 and the exterior packaging part 20.

[0034] Furthermore, in the liquid-filled culture bag of this embodiment, it is also preferable to have a plurality of first protrusions 112 on the outside of the culture section 11 and a plurality of second protrusions 22 in the area of ​​the outer packaging section 20 facing the outside of the culture section 11; it is also preferable to have a plurality of first protrusions 112 on the outside of the culture section 11 and a spacer section 30 that forms a space that can be depressurized between the outside of the culture section 11 and the outer packaging section 20; it is also preferable to have a plurality of second protrusions 22 in the area of ​​the outer packaging section 20 that faces the outside of the culture section 11 and a spacer section 30 that forms a space that can be depressurized between the outside of the culture section 11 and the outer packaging section 20; and it is also preferable to have all of these configurations simultaneously.

[0035] According to the liquid-filled culture bag of this embodiment, by forming such a reduced pressure space S, the outside of the culture section 11 and the outer packaging section 20 do not ultimately come into complete contact with each other, and a vacuum or reduced pressure space is created within the outer packaging section 20, making it possible to efficiently remove air bubbles accumulated in the microstructure 111 into the reduced pressure space S.

[0036] The liquid-filled culture bag of this embodiment is vacuum-packaged with the culture bag 10 sealed in the exterior packaging unit 20. That is, in the liquid-filled culture bag of this embodiment, the interior of the exterior packaging unit 20 is in a vacuum or reduced pressure state, or is in a substantially vacuum state. This vacuum packaging can be performed using a commercially available heat sealing device or the like. For example, a commercial vacuum sealer manufactured by Fuji Impulse Co., Ltd. can be suitably used. In the liquid-filled culture bag of this embodiment, an exterior sealing unit 21 is formed around the periphery of the exterior packaging unit 20.

[0037] In the liquid-filled culture bag of this embodiment, the outer packaging part 20 does not have to have gas barrier properties, but preferably has gas barrier properties. As will be described later in the examples, by using a material with gas barrier properties for the outer packaging part 20, the time required to remove air bubbles accumulated in the microstructure 111 can be shortened compared to when a material without gas barrier properties is used.

[0038] Specifically, in the liquid-filled culture bag of this embodiment, the oxygen permeability of the member of the region of the outer packaging part 20 facing the outside of the culture part 11 is preferably equal to or lower than the oxygen permeability of the culture part 11, and is preferably 8,000 ml / m 2 ・day・atm or less is more preferable, and 1,000 ml / m 2 It is more preferable that the viscosity is 100 ml / m or less. 2 It is particularly preferable that the temperature is below 1 / day atm.

[0039] Here, Test 3 described below shows that even if the oxygen permeability of the film in the culture section and the oxygen permeability of the outer packaging section are the same, it takes time to remove air bubbles from the culture section. Furthermore, the greater the difference between the oxygen permeability of the outer packaging section and the oxygen permeability of the film in the culture section, the greater the amount of gas leaving the reduced pressure space S from the culture section side compared to the amount of gas entering the reduced pressure space S from outside the outer packaging section, making it possible to shorten the time required to remove air bubbles accumulated in the culture section.

[0040] In the liquid-filled culture bag of this embodiment, resin films and the like can be suitably used as the material for the culture bag 10, and polyolefin resins such as polyethylene, polypropylene, and polymethylpentene can be used. Examples include polyethylene, copolymers of ethylene and α-olefins, copolymers of ethylene and vinyl acetate, and ionomers using ethylene, acrylic acid, or methacrylic acid copolymers and metal ions. Styrene-based elastomers and polyester-based thermoplastic elastomers can also be used. Furthermore, soft vinyl chloride resin, polybutadiene resin, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomers, silicone-based thermoplastic elastomers, styrene-based elastomers such as SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), and fluorine-based resins can also be used.

[0041] In the liquid-filled culture bag of this embodiment, the outer packaging part 20 can be made of, for example, polyethylene, polypropylene, a laminated film having a sealant layer and a barrier layer, or a vapor-deposited film (examples of barrier layers: ethylene-vinyl alcohol copolymer (EVOH), nylon, polyamide; examples of vapor deposition: aluminum vapor deposition, alumina vapor deposition, silica vapor deposition, etc.).

[0042] According to the liquid-filled culture bag of this embodiment, air bubbles accumulated in the micro-recesses in the culture bag can be efficiently removed without much effort or the need for dedicated equipment, thereby reducing the cumbersome work and risk of contamination at the manufacturing site.

[0043] The method for manufacturing a liquid-filled culture bag of this embodiment is a method for manufacturing a liquid-filled culture bag in which a culture bag having a microstructure formed in at least a portion of a culture section is contained in an outer packaging section, and is characterized in that a space that can be depressurized exists between the outside of the culture section and the outer packaging section, and the culture bag is filled with liquid and vacuum-packaged. Furthermore, the method for manufacturing a liquid-filled culture bag of this embodiment is preferably a method in which a plurality of first protrusions are provided on the outside of the culture section, and / or a plurality of second protrusions are provided in an area of ​​the outer packaging section facing the outside of the culture section, and / or a spacer portion is provided that forms a space that can be depressurized between the outside of the culture section and the outer packaging section.

[0044] According to the method for manufacturing a liquid-filled culture bag of this embodiment, air bubbles that existed in the microstructure formed in the culture section of the culture bag can be removed into the space between the outside of the culture section and the outer packaging section, making it possible to obtain a liquid-filled culture bag from which air bubbles have been removed from the microstructure. In addition, by providing a space that can be depressurized between the outside of the culture section and the outer packaging section and vacuum-packaging the culture bag while it is filled with liquid, it is also possible to check for liquid leakage from the culture bag.

[0045] The method of using the liquid-filled culture bag of this embodiment is the same as the method of using the liquid-filled culture bag of this embodiment described above, characterized in that it is stored or transported at room temperature, refrigerated, or frozen. It is also preferable that the method of using the liquid-filled culture bag of this embodiment be a method of injecting target cells, culture medium, cell adhesion factors, cytokines such as cell growth factors, etc. into the liquid-filled culture bag of this embodiment described above and culturing them. It is also preferable that the method be a method of once removing the filled liquid from the culture bag and then injecting target cells and culture medium, etc.

[0046] According to the method of using the liquid-filled culture bag of this embodiment, the culture bag can be stored or transported while being filled with liquid, and air bubbles can be removed during storage and transport. Furthermore, since air bubbles have been removed from the liquid-filled culture bag of this embodiment in this usage method, cell culture can be performed suitably without the need to remove air bubbles from the wells prior to cell culture.

[0047] Below, we will explain the liquid-filled culture bag according to an embodiment of the present invention, the method for manufacturing the liquid-filled culture bag, and tests conducted to confirm the effect of removing bubbles using the liquid-filled culture bag.

[0048] (Test 1) A test was conducted to confirm that this embodiment can provide a liquid-filled culture bag from which air bubbles have been removed. Specifically, the culture bag shown in Figures 1 and 3 was first produced. Polyethylene film (manufactured by Toyo Seikan Group Holdings, Ltd.) was used as the material for the culture bag, and two sheets of film measuring 100 mm x 70 mm x 0.10 mm were produced.

[0049] In addition, multiple hemispherical recesses were formed in the culture area of ​​the culture bag. The opening diameter of the recesses was 0.5 mm and the depth was 0.25 mm, and approximately 18,000 of these recesses were formed. On the outside of the culture area of ​​the culture bag, hemispherical protrusions were formed, each 250 μm high and spaced approximately 500 μm apart. The oxygen permeability of the film of the culture bag was 8,500 ml / m. 2・day・atm (37℃-50%RH).

[0050] The film with the culture surface formed on it was then attached to the other film by heat sealing, forming one port, to which a needleless connector (manufactured by Qosina) was connected via a tube.

[0051] Next, the prepared culture bag was filled with 15 mL of culture medium (StemFit AK02N (Ajinomoto Co., Inc., product number RCAK02N)), and only large air bubbles were removed by sucking them out with a syringe. The culture bag filled with the culture medium was then sealed in an outer packaging part (Co-pack® ST1525, Asahi Kasei Pax Co., Ltd.), and vacuum packaging was performed using a commercially available vacuum sealer (V301, Fuji Impulse Co., Ltd.) to produce the liquid-filled culture bag of this embodiment. The film configuration of the outer packaging part was ONy#15 / LDPE20 / L-LDPE#40 (total 75 μm), and its oxygen permeability was 23 ml / m 2 ・day・atm (23℃-50%RH).

[0052] The resulting liquid-filled culture bag was then stored in a refrigerator at 4°C, and micrographs of the recesses on the culture surface of the liquid-filled culture bag were taken at the start of storage, 2.5 hours later, and 5.5 hours later. The results are shown in Figure 6.

[0053] The photograph of Figure 6 taken at the start of storage (0 hr) shows air bubbles accumulating within the black circle in the recess. The white circle in the center is due to light reflection, and air bubbles are present in this area. Next, the photograph of Figure 6 taken 2.5 hours later (2.5 hr) shows roughly crescent-shaped particles in the recess, which are smaller air bubbles and indicate that some of the air bubbles have been removed. Note that external water droplets caused by condensation are also visible in this photograph.

[0054] Furthermore, in the photograph of Figure 6 taken 5.5 hours later (5.5 hr), no black circles are visible in the recesses, indicating that air bubbles have been completely removed. This result demonstrates that this embodiment can suitably produce liquid-filled culture bags from which air bubbles have been removed.

[0055] (Test 2) A test was conducted to confirm that cell culture could be performed using the liquid-filled culture bag obtained according to this embodiment. Specifically, cell culture was performed using the liquid-filled culture bag prepared in Test 1 that had been stored for three months.

[0056] The culture medium was completely removed from the liquid-filled culture bag that had been stored for 3 months, and the culture bag was filled with 20 ml of StemFit AK02N (Ajinomoto Co., Inc., product number RCAK02N) containing 10 mM Y-27632 (Wako Pure Chemical Industries, Ltd.). Human induced pluripotent stem (iPS) cells (1231A3 strain) were seeded so that each well contained approximately 150 cells.

[0057] Then, 24 hours after the start of culture, a micrograph was taken of the recessed portion of the culture surface of the liquid-filled culture bag. The results are shown in Figure 7. The photograph on the left side of Figure 7 shows the state of the culture surface before the medium was removed from the liquid-filled culture bag stored for three months. As shown in this photograph, no air bubbles were present in the recessed portion of the culture surface.

[0058] The top and bottom photographs in the center of Figure 7 show the state of the culture surface at the start of culture (0 hr), and the small particles in the wells in the enlarged view below are cells. The top and bottom photographs on the right side of Figure 7 show the state of the culture surface 24 hours (24 hr) after the start of culture, and spheroids can be observed forming in the center of each well.

[0059] These results demonstrate that with the liquid-filled culture bag of this embodiment, air bubbles are removed in advance, allowing cell culture to be carried out effectively without the need to remove air bubbles from the recesses prior to cell culture.

[0060] (Test 3) A test was conducted to confirm whether air bubbles could be removed when a gas-permeable material was used as the outer packaging material for the liquid-filled culture bag of this embodiment. Specifically, the same culture bag as in Test 1 was first produced. Polyethylene film (manufactured by Toyo Seikan Group Holdings, Ltd.) was used as the culture bag material, and two films measuring 100 mm x 70 mm x 0.10 mm were produced.

[0061] In addition, multiple hemispherical recesses were formed on the culture surface of the culture bag. The opening diameter of the recesses was 0.5 mm and the depth was 0.25 mm, and approximately 18,000 of these recesses were formed. On the outside of the culture area of ​​the culture bag, hemispherical protrusions were formed, each 250 μm high and spaced approximately 500 μm apart. The oxygen permeability of the film of the culture bag was 8,500 ml / m. 2 ・day・atm (37℃-50%RH).

[0062] The film with the culture surface formed on it was then attached to the other film by heat sealing, forming one port, to which a needleless connector (manufactured by Qosina) was connected via a tube.

[0063] Next, the prepared culture bag was filled with 15 mL of culture medium (StemFit AK02N (Ajinomoto Co., Inc., product number RCAK02N)), and only large air bubbles were removed by sucking them out with a syringe. The culture bag filled with culture medium was then sealed in a gas-permeable outer packaging section, and vacuum packaging was performed using a commercially available vacuum sealer (V301, Fuji Impulse Co., Ltd.), to produce a liquid-filled culture bag of this embodiment. The outer packaging section was made of the same polyethylene film (manufactured by Toyo Seikan Group Holdings Co., Ltd.) used for the culture bag, which was heat-sealed to the same size as the Corpac® ST1525 used in Test 1. The oxygen permeability of this outer packaging film was 8,500 ml / m 2 ・day・atm (37℃-50%RH).

[0064] The resulting liquid-filled culture bag was then stored in a refrigerator at 4°C, and micrographs of the recesses on the culture surface of the liquid-filled culture bag were taken at the start of storage, 2.5 hours, 6 hours, and 70 hours after storage. The results are shown in Figure 8.

[0065] The black circle in the recess in the photograph taken at the start of storage (0 hr) in Figure 8 shows air bubbles accumulating. The black circle in the recess in the photographs taken 2.5 hours (2.5 hr) and 6 hours (6 hr) in Figure 8 also shows air bubbles still accumulating. On the other hand, the black circle indicating air bubbles is not visible in the recess in the photograph taken 70 hours (70 hr) in Figure 8, and it can be observed that the air bubbles have been completely removed. From these results, it was confirmed that when a gas-permeable material is used as the outer packaging part of the liquid-filled culture bag of this embodiment, it takes longer to remove the air bubbles than when a gas-barrier material is used, but the air bubbles are removed.

[0066] (Test 4) A test was conducted to confirm whether a liquid-filled culture bag with air bubbles removed could be obtained when the culture bag was stored without using the liquid-filled culture bag of this embodiment. Specifically, the same culture bag as in Test 1 was first produced. Polyethylene film (manufactured by Toyo Seikan Group Holdings, Ltd.) was used as the material for the culture bag, and two sheets of film measuring 100 mm x 70 mm x 0.10 mm were produced.

[0067] Multiple hemispherical recesses were formed as a microstructure on the culture surface of the culture bag. The opening of the recesses had a diameter of 0.5 mm and a depth of 0.25 mm, and approximately 18,000 of these were formed. Hemispherical protrusions, 250 μm high and spaced approximately 500 μm apart, were formed on the outside of the culture area of ​​the culture bag. The oxygen permeability of the film of the culture bag was 8,500 ml / m. 2 ・day・atm (37℃-50%RH).

[0068] The film with the culture surface formed on it was then attached to the other film by heat sealing, forming one port, to which a needleless connector (manufactured by Qosina) was connected via a tube.

[0069] Next, the prepared culture bag was filled with 15 mL of medium (StemFit AK02N (Ajinomoto Co., Inc., product number RCAK02N)), and only large air bubbles were removed by suction with a syringe. The culture bag filled with medium was then sealed in an outer packaging section (Co-pack (R) ST1525, Asahi Kasei Pax Co., Ltd.) and packaged without degassing using a commercially available vacuum sealer (V301 Fuji Impulse Co., Ltd.) to produce a liquid-filled culture bag as a comparative example. Next, the obtained liquid-filled culture bag of the comparative example was stored in a refrigerator at 4 ° C., and micrographs of the recesses on the culture surface of the liquid-filled culture bag were taken at the start of storage, and after 2.5 hours, 6 hours, and 70 hours. The results are shown in Figure 9.

[0070] The black circle in the recessed portion of the photograph taken at the start of storage (0 hr) in Fig. 9 shows air bubbles trapped within the black circle. The black circle in the recessed portion of the photograph taken 2.5 hours after storage (2.5 hr) in Fig. 9 also shows air bubbles trapped within the black circle.

[0071] Furthermore, the black circles in the recesses in the photographs taken 6 hours (6 hr) and 70 hours (70 hr) in Figure 9 also show bubbles trapped within them. As such, it was found that simply packaging and storing the culture bag without providing a space that can be depressurized between the outside of the culture portion of the culture bag and the outer packaging portion does not remove the bubbles in the microstructure formed in the culture portion of the culture bag.

[0072] The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the present invention. For example, a culture bag having a size capable of forming 500,000 to 1,000,000 spheres can be selected and sealed in an outer packaging section to produce a liquid-filled culture bag.

[0073] The present invention can be suitably used when using a liquid-filled culture bag in which a culture medium or the like has been filled in advance and air bubbles have been removed from the culture section.

[0074] The contents of the documents cited in this specification and the Japanese application of this application are incorporated herein in their entirety by reference.

[0075] REFERENCE SIGNS LIST 10 culture bag 11 culture section 111 microstructure 112 first protrusion 12 bag sealing section 13 port 14 tube 15 cap 20 exterior packaging section 21 exterior sealing section 22 second protrusion 30 spacer section S reduced pressure space L liquid

Claims

1. A liquid-filled culture bag in which a culture bag having a microstructure formed in at least a part of the culture section is contained in an outer packaging section, characterized in that a space that can be decompressed exists between the outside of the culture section and the outer packaging section, and the liquid-filled culture bag is degassed and packaged.

2. The liquid-filled culture bag according to claim 1, characterized in that a plurality of first protrusions are provided on the outside of the culture section.

3. A liquid-filled culture bag according to claim 1 or 2, characterized in that a plurality of second protrusions are provided in the area of ​​the outer packaging portion facing the outside of the culture portion.

4. A liquid-filled culture bag according to claim 1 or 2, characterized in that a spacer section is provided between the outside of the culture section and the outer packaging section to form a space that can be depressurized.

5. The liquid-filled culture bag according to claim 1 or 2, characterized in that the microstructure is a plurality of recesses or a plurality of grooves.

6. The oxygen permeability of the material in the region of the culture bag where the culture section is provided is 3,000 ml / m 2 3. The liquid-filled culture bag according to claim 1, characterized in that the culture medium has a temperature of 100° C. or more.

7. A liquid-filled culture bag as described in claim 1 or 2, characterized in that the oxygen permeability of the material in the area of ​​the outer packaging portion facing the outside of the culture portion is lower than the oxygen permeability of the culture portion.

8. The oxygen permeability of the material of the region of the outer packaging part facing the outside of the culture part is 1,000 ml / m 2 8. The liquid-filled culture bag according to claim 7, characterized in that it has a gas barrier property of less than 1.0 .times.day.atm.

9. A method for manufacturing a liquid-filled culture bag in which a culture bag having a microstructure formed in at least a portion of a culture section is contained in an outer packaging section, characterized in that a space that can be depressurized exists between the outside of the culture section and the outer packaging section, the culture bag is filled with liquid, and the culture bag is degassed and packaged.

10. A method for manufacturing a liquid-filled culture bag as described in claim 9, characterized in that a plurality of first protrusions are provided on the outside of the culture section, and / or a plurality of second protrusions are provided in an area of ​​the outer packaging section facing the outside of the culture section, and / or a spacer section is provided that forms a space that can be reduced in pressure between the outside of the culture section and the outer packaging section.

11. A method for using the liquid-filled culture bag according to claim 1 or 2, characterized in that the liquid-filled culture bag is stored or transported at room temperature, refrigerated or frozen.

12. A method for using the liquid-filled culture bag according to claim 1 or 2, characterized in that the liquid-filled culture bag is used for culturing by injecting cells, culture medium, cell adhesion factors, cell growth factors and other cytokines into the liquid-filled culture bag.

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