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

Abstract

The invention provides a culture bag filled with liquid, which can efficiently remove bubbles accumulated in tiny concave parts in the culture bag without a large amount of labor and special equipment, and can reduce tedious work and pollution risks on a manufacturing site. A liquid-filled culture bag in which a culture bag, in which a fine structure is formed in at least a portion of a culture part, is housed in an outer packaging part, the liquid-filled culture bag having a depressurizable space between the outside of the culture part and the outer packaging part, and the liquid-filled culture bag being packaged by degassing. It is preferable that a plurality of first protrusions are provided on the outside of the culture part. Furthermore, it is preferable that a plurality of second protrusions are provided in a region facing the outside of the culture part in the outer packaging part. Furthermore, it is preferable that the culture device is provided with a partition part that forms a pressure-reducing space between the outer packaging part and the outside of the culture part.

Description

Technical Field

[0001] This invention relates to cell culture technology, and in particular to culture bags having minute recesses or the like in the culture section. Background Technology

[0002] In culturing spheroids or organoids of adhesion cells such as iPS cells and ES cells, culture bags with multiple tiny indentations (pores) in the culture section are sometimes used. When such culture bags are filled with culture medium, air bubbles can become trapped and accumulate in these tiny indentations, hindering cell culture. In other words, if air bubbles remain in the tiny indentations of the culture bag, they can impede the formation of spheroids (cell aggregates) composed of iPS cells, etc., but removing all air bubbles from the tiny indentations is extremely difficult.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: International Publication No. 2021 / 241665

[0006] Patent Document 2: Japanese Patent Application Publication No. 2020-80670 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] One method for removing air bubbles accumulated in tiny depressions is to use water pressure to remove the bubbles by wiping them with liquid using a micropipette. However, in such operations using a micropipette, especially when the depressions are small, it is sometimes impossible to remove the air bubbles completely.

[0009] Furthermore, this method is only applicable to open systems and is not suitable for removing air bubbles accumulated in the culture section of the culture bag.

[0010] Here, Patent Document 1 discloses a method of applying pressure to a culture bag after filling it with culture medium into a culture bag having tiny recesses in the culture section, thereby removing air from the culture bag.

[0011] Furthermore, Patent Document 2 discloses filling a culture bag with tiny recesses in its culture section with degassed culture medium to remove air bubbles accumulated in the tiny recesses. Moreover, this document also discloses placing the culture bag with tiny recesses in its culture section in a vacuum exhaust device and exposing it to a vacuum, thereby removing air from the culture bag.

[0012] However, these methods suffer from drawbacks such as the time-consuming process of bubble removal and the need for specialized equipment. Furthermore, in regenerative medicine and cell therapy manufacturing, on-site adjustments are required to remove bubbles from the culture bags, which complicates the manufacturing process and may lead to contamination.

[0013] Therefore, the inventors conducted in-depth research and developed a liquid-filled culture bag that can eliminate these problems, thus completing this invention.

[0014] That is, the present invention was made in view of the above circumstances, and its object is 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, which can efficiently remove air bubbles accumulated in tiny depressions in the culture bag without a lot of effort or specialized equipment, and can reduce tedious operations and the risk of contamination at the manufacturing site.

[0015] Solution for solving the problem

[0016] To achieve the above objectives, the liquid-filled culture bag of the present invention is configured such that at least a portion of the culture section has a microstructure formed in the culture bag, which is contained in the outer packaging section of the liquid-filled culture bag, wherein there is a pressure-reducing space between the outer side of the culture section and the outer packaging section, and the liquid-filled culture bag is degassed and packaged.

[0017] Furthermore, preferably, the liquid-filled culture bag of the present invention is configured to have a plurality of first protrusions on the outer side of the culture section, and preferably, it is configured to have a plurality of second protrusions in the area of ​​the outer packaging section facing the outer side of the culture section, and preferably, it is configured to have a spacer that forms a pressure-reducing space between the outer side of the culture section and the outer packaging section.

[0018] Furthermore, preferably, the liquid-filled culture bag of the present invention is configured such that the microstructure consists of multiple recesses or multiple grooves.

[0019] Furthermore, preferably, the oxygen permeability of the component comprising the culture section in the liquid-filled culture bag of the present invention is 3000 ml / m². 2 ·day·atm and above.

[0020] Furthermore, preferably, the oxygen permeability of the component of the liquid-filled culture bag of the present invention, which is configured in the outer packaging portion facing the outer side of the culture portion, is lower than or equal to the oxygen permeability of the culture portion. More preferably, the oxygen permeability of the component of the outer packaging portion facing the outer side of the culture portion has 1000 ml / m³. 2 Air resistance below day·atm.

[0021] Furthermore, it is also preferred that the liquid-filled culture bag of the present invention is configured to be composed of various combinations of the above-mentioned liquid-filled culture bags.

[0022] The method for manufacturing a liquid-filled culture bag of the present invention is as follows: a method for manufacturing a liquid-filled culture bag in which at least a portion of the culture section has a microstructure is contained in an outer packaging section, wherein a pressure-reducing space exists between the outer side of the culture section and the outer packaging section, liquid is filled into the culture bag, and the culture bag is degassed and packaged.

[0023] Furthermore, preferably, the method for manufacturing the liquid-filled culture bag of the present invention is configured as follows: having a plurality of first protrusions on the outer side of the culture section, and / or having a plurality of second protrusions in the outer packaging section in the area facing the outer side of the culture section, and / or having a spacer that forms a pressure-reducing space between the outer side of the culture section and the outer packaging section.

[0024] The method of using the liquid-filled culture bag of the present invention is as follows: the above-mentioned method of using the liquid-filled culture bag, wherein it is stored or transported at room temperature, refrigerated or frozen.

[0025] Furthermore, the method of using the liquid-filled culture bag of the present invention is as follows: the above-mentioned method of using the liquid-filled culture bag, wherein cells, culture medium, cell adhesion factors, cell growth factors and other cytokines are injected into the liquid-filled culture bag for culture.

[0026] Invention Effects

[0027] According to the present invention, a liquid-filled culture bag, a method for manufacturing the liquid-filled culture bag, and a method for using the liquid-filled culture bag are provided, which can efficiently remove air bubbles accumulated in tiny depressions in the culture bag without requiring a lot of effort or specialized equipment, and can reduce tedious operations and the risk of contamination at the manufacturing site. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the structure of a liquid-filled culture bag according to an embodiment of the present invention.

[0029] Figure 2 This is a schematic cross-sectional view showing the structure of a liquid-filled culture bag according to an embodiment of the present invention.

[0030] Figure 3 This is a schematic cross-sectional view showing the configuration of a modified example 1 of the liquid-filled culture bag according to an embodiment of the present invention.

[0031] Figure 4 This is a schematic cross-sectional view showing the configuration of a modified example 2 of the liquid-filled culture bag according to an embodiment of the present invention.

[0032] Figure 5 This is a schematic cross-sectional view showing the configuration of a modified example 3 of the liquid-filled culture bag according to an embodiment of the present invention.

[0033] Figure 6 This is a photograph showing the results of Experiment 1.

[0034] Figure 7 This is a photograph showing the results of Experiment 2.

[0035] Figure 8 This is a photograph showing the results of experiment 3.

[0036] Figure 9 This is a photograph showing the results of experiment 4. Detailed Implementation

[0037] The following provides a detailed description of 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. However, the present invention is not limited to the specific content of the following embodiments and examples.

[0038] First, the liquid-filled culture bag of the present invention will be described in detail with reference to the accompanying drawings.

[0039] The liquid-filled culture bag of this embodiment is characterized in that at least a portion of the culture section is formed with a microstructure and the culture bag is contained in the outer packaging section of the liquid-filled culture bag, wherein there is a pressure-reducing space between the outer side of the culture section and the outer packaging section, and the liquid-filled culture bag is degassed and packaged.

[0040] That is, the culture bag with a microstructure formed in at least a portion of the culture section is filled with liquid and is contained in the outer packaging section.

[0041] Specifically, such as Figure 1 and Figure 2 As shown, the liquid-filled culture bag of this embodiment consists of a culture bag 10 and an outer packaging section 20, with the culture bag 10 sealed inside the outer packaging section 20.

[0042] The culture bag 10 is a bag-shaped container made of soft packaging material for culturing cells, etc., and has a culture section 11 inside, which has a culture surface as a region for culturing cells.

[0043] The outer side of the culture section 11 refers to the bag wall portion containing the culture section 11. Figure 2 The side opposite to the culture surface in the bottom part of the bag (the middle part is the bottom part of the bag). Figure 2(The middle part is the side with the first protrusion 112). That is, the outer side of the culture part 11 refers to the area on the outer side of the culture bag 10 that corresponds to the culture surface.

[0044] It should be noted that, in Figure 2 In this case, the culture section 11 is only provided on the bottom side of the culture bag 10, but it may also be provided only on the top plate side of the culture bag 10, or it may be provided on both the bottom and top plate sides. In these cases, the outer side of the culture section 11 on the top plate side refers to the area on the outer side of the culture bag 10 corresponding to the culture surface on the top plate side, and the outer side of the culture section 11 on the bottom plate side refers to the area on the outer side of the culture bag 10 corresponding to the culture surface on the bottom plate side. Figures 3-5 The same applies to the variant examples shown.

[0045] The culture bag 10 can be manufactured, for example, by sandwiching one or more ports between two membranes and heat-sealing the periphery. Figure 1 The culture bag 10 includes a bag sealing part 12 formed by heat sealing, a port 13, a tube 14 communicating with the port 13, and a cap 15 sealing the tube 14. It should be noted that the method of manufacturing the culture bag 10 is not limited to this; for example, the culture bag 10 can also be manufactured by other methods such as using a vacuum-formed membrane or a blow-formed membrane.

[0046] Furthermore, the number of ports on the culture bag 10 is not limited to one; it can also have two or more ports.

[0047] Furthermore, the sealing of tube 14 is not limited to the method of using cap 15. For example, a connector with a cap function can be used to seal tube 14, or a cap can be provided to connect a common connector to tube 14 and seal the connector.

[0048] Microstructures 111 are formed in the culture section 11 of the culture bag 10. Sometimes, these microstructures 111 are, for example, multiple recesses (pores) or multiple grooves. In addition, cell low adhesion treatment, cell adhesion treatment, etc. can be applied to the culture section 11 having these microstructures 111.

[0049] The shape of the multiple recesses is not particularly limited; for example, they can be hemispherical, conical, pyramidal, composed of cylinders and cones, or composed of prisms and pyramids. Furthermore, the shape of the multiple grooves is not particularly limited; for example, they can be V-shaped with a cross-section perpendicular to the long dimension of the groove, and can be straight or curved.

[0050] The shape of the opening in the multiple recesses is preferably a circle, a quadrilateral, etc., and the diameter of the circle or the inscribed circle can be set to less than 2 mm, less than 1.5 mm, or less than 1.0 mm.

[0051] In addition, the depth of the multiple recesses can be set to less than 1.5mm, less than 1.0mm, less than 0.5mm, or less than 0.35mm.

[0052] Furthermore, the width and depth of the multiple slots can also be set to less than 1.5mm, less than 1.0mm, less than 0.5mm, or less than 0.35mm respectively.

[0053] By setting the microstructure 111 in the culture section 11 as multiple recesses, a sphere composed of multiple iPS cells, other adhesion cells, etc. can be appropriately formed using the culture bag 10.

[0054] Furthermore, by setting the microstructure 111 in the culture section 11 as multiple grooves, the adhering cells can be properly cultured by adhering to the culture section 11 while increasing the area of ​​the culture surface of the culture bag 10.

[0055] The culture bag 10 is filled with liquid L. Preferably, liquid L is culture medium, water for injection, phosphate buffer, physiological saline, etc.

[0056] For the culture bag with liquid filling completed in this embodiment, when air bubbles are temporarily removed from the microstructures 111 in the culture section 11 to wet the surface of the microstructures 111, air bubbles will generally not be generated again in the microstructures 111. Therefore, it is also preferable to prefill not only the culture medium but also other liquids such as water for injection as liquid L.

[0057] Preferably, the components of the culture bag 10, at least the region having the culture section 11, are made of breathable components so that air bubbles accumulated in the microstructure 111 can be removed to the outside of the culture bag 10. For example, the oxygen permeability of the components in the region of the culture section 11 at 37°C is preferably 3000 ml / m³. 2 • day • atm or more, preferably 5000ml / m 2 •day·atm or above, further preferably 8000ml / m 2 ·day·atm and above.

[0058] In this embodiment, the culture bag after liquid filling has a pressure-reducing space (pressure-reducing space) S between the outside of the culture section 11 and the outer packaging section 20.

[0059] As described above, the outer side of the culture section 11 refers to the area on the outer side of the culture bag 10 that corresponds to the culture surface.

[0060] like Figure 2As shown, the liquid-filled culture bag of this embodiment has a plurality of first protrusions 112 on the outside of the culture section 11, which can appropriately form a decompression space S between the outside of the culture section 11 and the outer packaging section 20.

[0061] In addition, it is also preferred that, such as Figure 3 As shown in Modification 1, the culture bag of this embodiment is designed such that the bottom part of the culture bag 10 is a U-shaped continuous body and has the shape of both the concave part (microstructure 111) inside the bag and the convex part (first protrusion 112) outside the bag.

[0062] Moreover, such as Figure 4 As shown in Modification 2, the liquid-filled culture bag of this embodiment can also form a decompression space S between the outer side of the culture section 11 and the outer packaging section 20 by having a plurality of second protrusions 22 in the area of ​​the outer packaging section 20 facing the outer side of the culture section 11.

[0063] The first protrusion 112 and the second protrusion 22 only need to form a decompression space S between the outer side of the culture section 11 and the outer packaging section 20, and their shape, size and number are not particularly limited.

[0064] Moreover, in addition, such as Figure 5 As shown in Modification 3, the liquid-filled culture bag of this embodiment can also appropriately form a decompression space S between the outer side of the culture section 11 and the outer packaging section 20 by having a spacer 30 that forms a space for decompression between the outer side of the culture section 11 and the outer packaging section 20.

[0065] The spacer 30 only needs to form a space that can reduce pressure between the outer side of the culture section 11 and the outer packaging section 20. Its specific structure is not particularly limited. For example, a plate-shaped member with multiple through holes or a porous member can be used appropriately.

[0066] It should be noted that, in this embodiment, the culture bag 10 can also be sealed into another outer packaging section, and this outer packaging section can be sealed into the outer packaging section 20, thereby making the culture bag 10 a double package.

[0067] Even if the liquid-filled culture bag of this embodiment is set up in this way, for example, if the spacer 30 functions as described above in other outer packaging parts, and thus a decompression space S is formed between the outside of the culture part 11 and the outer packaging part 20, air bubbles can be properly removed from the culture part 11.

[0068] Furthermore, in the liquid-filled culture bag of this embodiment, it is preferable that a plurality of first protrusions 112 are provided on the outer side of the culture section 11, and a plurality of second protrusions 22 are provided in the area of ​​the outer packaging section 20 facing the outer side of the culture section 11. It is also preferable that a plurality of first protrusions 112 are provided on the outer side of the culture section 11, and a spacer 30 is provided to form a pressure-reducing space between the outer side of the culture section 11 and the outer packaging section 20. It is also preferable that a plurality of second protrusions 22 are provided in the area of ​​the outer packaging section 20 facing the outer side of the culture section 11, and a spacer 30 is provided to form a pressure-reducing space between the outer side of the culture section 11 and the outer packaging section 20. It is also preferable that all of these configurations are provided simultaneously.

[0069] According to the liquid-filled culture bag of this embodiment, by forming such a decompression space S, the outer side of the culture section 11 and the outer packaging section 20 will not be completely in contact, so that a vacuum to decompression state space is generated in the outer packaging section 20, which can efficiently remove the bubbles accumulated in the microstructure 111 into the decompression space S.

[0070] In this embodiment, the culture bag after filling is degassed and packaged while the culture bag 10 is sealed inside the outer packaging section 20.

[0071] That is, in the culture bag after liquid filling in this embodiment, the outer packaging section 20 is in a state of vacuum to depressurization, becoming a state of approximately vacuum.

[0072] This degassed packaging can be performed using commercially available heat-sealing equipment. For example, commercial degassed sealing machines manufactured by FUJIIMPULSE Co., Ltd. can be used appropriately.

[0073] In the liquid-filled culture bag of this embodiment, an outer sealing part 21 is formed on the periphery of the outer packaging part 20.

[0074] In the liquid-filled culture bag of this embodiment, the outer packaging part 20 may not have gas barrier properties, but it is preferable to have gas barrier properties.

[0075] This is because, as described later in the embodiments, by using a gas-barrier material in the outer packaging section 20, the time required to remove bubbles accumulated in the microstructure 111 can be shortened compared to using a non-gas-barrier material.

[0076] Specifically, in the liquid-filled culture bag of this embodiment, the oxygen permeability of the component in the area of ​​the outer packaging section 20 facing the outside of the culture section 11 is preferably below the oxygen permeability of the culture section 11, and more preferably 8000 ml / m 2 • Day • atm below, preferably 1000 ml / m 2• Day • atm below, preferably 100ml / m 2 •day •atm and below.

[0077] Here, in Experiment 3 described later, it is shown that even if the oxygen permeability of the membrane in the culture section is the same as that in the outer packaging section, bubbles can be removed from the culture section, although it takes time. Furthermore, by setting the oxygen permeability of the outer packaging section to be lower than that of the membrane in the culture section, and by setting the difference between them to be greater, the amount of gas discharged from the culture section to the depressurization space S is greater than the amount of gas entering the depressurization space S from the outside of the outer packaging section, thus shortening the time for removing bubbles accumulated in the culture section.

[0078] In the liquid-filled culture bag of this embodiment, the material of the culture bag 10 can be a resin film, and polyolefin resins such as polyethylene, polypropylene, and polymethylpentene can be used. For example, copolymers of polyethylene, ethylene and α-olefins, copolymers of ethylene and vinyl acetate, and ionomers of ethylene and acrylic acid, methacrylic acid copolymers and metal ions can be used. In addition, styrene-based elastomers and polyester-based thermoplastic elastomers can also be used. Moreover, soft vinyl chloride resin, polybutadiene resin, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomers, silicone-based thermoplastic elastomers, and styrene-based elastomers, such as SBS (styrene / butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butene / styrene), SEPS (styrene / ethylene / propylene / styrene), and fluorinated resins can also be used.

[0079] In the liquid-filled culture bag of this embodiment, the material used as the outer packaging part 20 can be, for example, polyethylene, polypropylene, laminated film having a sealant layer and a barrier layer, vapor-deposited film (barrier layer examples: ethylene-vinyl alcohol copolymer (EVOH), nylon, polyamide, vapor deposition examples: aluminum vapor deposition, alumina vapor deposition, silicon dioxide vapor deposition, etc.).

[0080] According to this embodiment of the liquid-filled culture bag, air bubbles accumulated in the tiny depressions inside the culture bag can be efficiently removed without a lot of effort or specialized equipment, reducing tedious operations and the risk of contamination at the manufacturing site.

[0081] The method for manufacturing a liquid-filled culture bag according to this embodiment is characterized in that a culture bag with a microstructure formed in at least a part of the culture section is contained in an outer packaging section. In this method, there is a space for depressurization between the outer side of the culture section and the outer packaging section, liquid is filled into the culture bag, and the culture bag is degassed and packaged.

[0082] Furthermore, the manufacturing method of the liquid-filled culture bag in this embodiment is preferably configured as follows: having a plurality of first protrusions on the outside of the culture section, and / or having a plurality of second protrusions in the outer packaging section in the area facing the outside of the culture section, and / or having a spacer that forms a pressure-reducing space between the outside of the culture section and the outer packaging section.

[0083] According to the method for manufacturing a liquid-filled culture bag according to this embodiment, air bubbles present in the microstructure of the culture section formed in the culture bag can be removed to the space between the outer side of the culture section and the outer packaging section, and a liquid-filled culture bag in which air bubbles are removed from the microstructure can be obtained.

[0084] In addition, a pressure-reducing space is provided between the outside of the culture section and the outer packaging section, so that the culture bag can be degassed and packaged while it is filled with liquid, thereby enabling the inspection of leakage from the culture bag.

[0085] The characteristic of the method of using the liquid-filled culture bag of this embodiment is that it is the same as the method of using the liquid-filled culture bag of this embodiment, and is stored or transported at room temperature, refrigerated or frozen.

[0086] Furthermore, it is also preferred that the method of using the liquid-filled culture bag of this embodiment is as follows: injecting target cells, culture medium, cell adhesion factors, cell growth factors and other cytokines into the liquid-filled culture bag of this embodiment for culture.

[0087] Furthermore, it is also preferable to design a method in which the target cells and culture medium are injected after the filled liquid has been temporarily removed from the culture bag for culturing.

[0088] According to the method of using the liquid-filled culture bag of this embodiment, the culture bag can be stored and transported while it is filled with liquid, and air bubbles can be removed during storage and transportation.

[0089] Furthermore, in this embodiment of the method of use, since air bubbles are removed beforehand, the filling and culture bag does not require the removal of air bubbles from the recess before cell culture, thus enabling proper cell culture.

[0090] Example

[0091] The following describes the experiments conducted to confirm the bubble removal effect achieved by the liquid-filled culture bag, the method for manufacturing the liquid-filled culture bag, and the method for using the liquid-filled culture bag according to the embodiments of the present invention.

[0092] (Experiment 1)

[0093] An experiment was conducted to confirm that a culture bag with bubble removal could be obtained through this embodiment.

[0094] Specifically, firstly, make Figure 1 and Figure 3 The culture bag shown. As the material for the culture bag, two films of 100mm × 70mm × 0.10mm were made using polyethylene film (manufactured by Toyo Seikan Group Holding Co., Ltd.).

[0095] In addition, multiple hemispherical recesses are formed in the culture section of the culture bag. The diameter of the opening of each recess is 0.5 mm and the depth is 0.25 mm, with approximately 18,000 recesses formed.

[0096] Hemispherical protrusions, each 250 μm high and spaced approximately 500 μm apart, are formed on the outer side of the culture section of the culture bag. Furthermore, the oxygen permeability of the membrane in the culture bag is 8500 ml / m³. 2 ·day·atm(37℃-50%RH).

[0097] Then, the membrane with the culture surface is bonded to the other membrane by heat sealing. At this point, a port is formed, and a needleless connector (manufactured by Qosina) is connected to the port via a tube.

[0098] Next, fill the prepared culture bag with 15 mL of culture medium (StemFit AK02N (Ajinomoto Co., Ltd., product number RCAK02N)) and remove only the large air bubbles by aspirating them with a syringe.

[0099] Then, the culture bag filled with culture medium is sealed into the outer packaging unit (Corpak(R)ST1525, Asahi Kasei Pax Co., Ltd.), and degassed using a commercially available degassing sealing machine (V301, FUJIIMPULSE Co., Ltd.) to produce the liquid-filled culture bag of this embodiment.

[0100] The membrane in the outer packaging section is composed of ONy#15 / LDPE20 / L-LDPE#40 (total 75μm), and its oxygen permeability is 23ml / m. 2 ·day·atm(23℃-50%RH).

[0101] Next, the culture bags with completed liquid filling were stored in a refrigerator at 4°C, and microscopic images of the concave areas of the culture surface were taken at the start of storage, 2.5 hours later, and 5.5 hours later. The results are presented below. Figure 6 .

[0102] exist Figure 6The black circle in the recessed area of ​​the photo taken at the start time (0hr) shows the presence of air bubbles. It should be noted that the white circle in the center is a reflection of light, but air bubbles are present in that area.

[0103] Next, in Figure 6 In the image taken 2.5 hours later (2.5hr), roughly crescent-shaped particles were observed in the concave area, but these were smaller air bubbles, indicating that the bubbles had been partially removed. It should be noted that external water droplets caused by condensation are also reflected in this image.

[0104] Furthermore, in Figure 6 The black circle was not captured in the indentation of the photo taken 5.5 hours later (5.5hr), indicating that the bubble was completely removed.

[0105] Based on the results, it can be seen that, according to this embodiment, a culture bag with properly removed air bubbles can be obtained after filling.

[0106] (Experiment 2)

[0107] Using the culture bag filled with liquid obtained through this embodiment, an experiment was conducted to confirm that cell culture could be performed.

[0108] Specifically, cell culture was carried out using culture bags that had been filled with liquid and stored for three months after being prepared in Experiment 1.

[0109] After three months of storage, drain all the culture medium from the culture bag that has been filled with liquid. Fill the culture bag with 20 ml of StemFit AK02N (Ajinomoto Co., Ltd., product number RCAK02N) containing 10 mM Y-27632 (Wako Pure Chemical Industries, Ltd.) and seed the cells by placing approximately 150 human induced pluripotent stem (iPS) cells (strain 1231A3) in each recess.

[0110] Then, microscopic images of the concave areas of the culture surface of the culture bag after filling with liquid were taken 24 hours after the start of culture. The results are shown below. Figure 7 .

[0111] Figure 7 The photo on the left shows the state of the culture surface before the culture bag was removed after three months of storage, once the culture had been filled. As shown in the photo, there are no air bubbles in the recesses of the culture surface.

[0112] Figure 7 The top and bottom images in the center show the state of the culture surface at the start time (0hr), while the small particles in the recesses in the magnified image below are cells.

[0113] Figure 7The top and bottom photos on the right show the state of the culture surface 24 hours after the start of the culture, and observe that spherical bodies are formed in the center of each recess.

[0114] According to the results, since the air bubbles are removed before filling the culture bag according to this embodiment, cell culture can be carried out properly without removing air bubbles from the recess before cell culture.

[0115] (Experiment 3)

[0116] When using a breathable material as the outer packaging material of the liquid-filled culture bag in this embodiment, a test is conducted to confirm whether air bubbles have been removed.

[0117] Specifically, the same culture bag as in Experiment 1 was first produced. Two films measuring 100mm × 70mm × 0.10mm were made using polyethylene film (manufactured by Toyo Seikan Group Holdings Co., Ltd.) as the material for the culture bag.

[0118] In addition, multiple hemispherical recesses are formed on the culture surface of the culture bag. The diameter of the opening of each recess is 0.5 mm and the depth is 0.25 mm, forming approximately 18,000 recesses.

[0119] Hemispherical protrusions, each 250 μm high and spaced approximately 500 μm apart, are formed on the outer side of the culture section of the culture bag. Furthermore, the oxygen permeability of the membrane in the culture bag is 8500 ml / m³. 2 ·day·atm(37℃-50%RH).

[0120] Then, the membrane with the culture surface is bonded to the other membrane by heat sealing. At this point, a port is formed, and a needleless connector (manufactured by Qosina) is connected to the port via a tube.

[0121] Next, fill the prepared culture bag with 15 mL of culture medium (StemFit AK02N (Ajinomoto Co., Ltd., product number RCAK02N)) and remove only the large air bubbles by aspirating them with a syringe.

[0122] Then, the culture bag filled with culture medium was sealed into an air-permeable outer packaging section, and degassed using a commercially available degassing sealing machine (V301, FUJIIMPULSE Co., Ltd.) to produce the liquid-filled culture bag of this embodiment. As the outer packaging section, a bag of the same size as the Corpac® ST1525 used in Experiment 1 was produced by heat-sealing a polyethylene film (manufactured by Toyo Seikan Group Holdings Co., Ltd.) made from the same polyethylene film used for the culture bag. The oxygen permeability of the film in this outer packaging section was 8500 ml / m³.2 ·day·atm(37℃-50%RH).

[0123] Next, the obtained culture bags with completed liquid filling were stored in a refrigerator at 4°C, and microscopic images of the concave areas of the culture surface were taken at the start of storage, 2.5 hours, 6 hours, and 70 hours. The results are presented below. Figure 8 .

[0124] exist Figure 8 The black circle in the recess of the photo taken at the start time (0hr) indicates the presence of accumulated air bubbles.

[0125] In addition, Figure 8 The black circles in the recesses of the photos taken 2.5 hours (2.5hr) and 6 hours (6hr) show that air bubbles still remain.

[0126] On the other hand, Figure 8 The black circles representing bubbles were not captured in the indentation of the photograph taken 70 hours later (70hr), indicating that the bubbles were completely removed.

[0127] Based on the results, it was confirmed that when using a breathable material as the outer packaging material of the liquid-filled culture bag of this embodiment, the bubbles were removed, although it took longer to remove them, compared to using a gas-barrier material.

[0128] (Experiment 4)

[0129] An experiment was conducted to confirm whether a 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.

[0130] Specifically, the same culture bag as in Experiment 1 was first produced. Two films measuring 100mm × 70mm × 0.10mm were made using polyethylene film (manufactured by Toyo Seikan Group Holdings Co., Ltd.) as the material for the culture bag.

[0131] Multiple hemispherical recesses are formed on the culture surface of the culture bag as microstructures. The diameter of the opening of each recess is 0.5 mm and the depth is 0.25 mm, forming approximately 18,000 recesses.

[0132] Hemispherical protrusions, each 250 μm high and spaced approximately 500 μm apart, are formed on the outer side of the culture section of the culture bag. Furthermore, the oxygen permeability of the membrane in the culture bag is 8500 ml / m³. 2 ·day·atm(37℃-50%RH).

[0133] Then, the membrane with the culture surface is bonded to the other membrane by heat sealing. At this point, a port is formed, and a needleless connector (manufactured by Qosina) is connected to the port via a tube.

[0134] Next, fill the prepared culture bag with 15 mL of culture medium (StemFit AK02N (Ajinomoto Co., Ltd., product number RCAK02N)) and remove only the large air bubbles by aspirating them with a syringe.

[0135] Then, the culture bags filled with culture medium were sealed into the outer packaging department (Corpak(R)ST1525, Asahi KaseiPax Co., Ltd.) and packaged without degassing using a commercially available degassing sealing machine (V301FUJIIMPULSE Co., Ltd.) to produce liquid-filled culture bags as a comparative example.

[0136] Next, the completed culture bags of the comparative examples were stored in a refrigerator at 4°C, and microscopic images of the concave areas of the culture surface were taken at the start of storage, 2.5 hours, 6 hours, and 70 hours. The results are presented below. Figure 9 .

[0137] exist Figure 9 The black circle in the recess of the photo taken at the start time (0hr) indicates the presence of accumulated air bubbles.

[0138] In addition, Figure 9 The image taken 2.5 hours later (2.5hr) also shows the presence of air bubbles in the concave area within the black circle.

[0139] Moreover, in Figure 9 In the photos taken 6 hours (6hr) and 70 hours (70hr), bubbles were also observed to have accumulated in the recesses within the black circles.

[0140] As such, it can be seen that without providing a pressure-reducing space between the outer side of the culture section of the culture bag and the outer packaging section, it is impossible to remove air bubbles formed in the microstructure of the culture section of the culture bag simply by packaging and storing the culture bag.

[0141] This invention is not limited to the above-described embodiments and examples. Within the scope of this invention, various modifications and implementations are of course possible.

[0142] For example, as a culture bag, a culture bag of a size that can form 500,000 to 1,000,000 spheres can be selected, and it can be sealed in the outer packaging to manufacture a liquid-filled culture bag, etc., which can be modified appropriately.

[0143] Industrial availability

[0144] The present invention can be appropriately used in cases where a culture bag is filled with a pre-filled culture medium or the like and air bubbles are removed from the culture section.

[0145] The entire contents of the documents described in this specification and the contents of the Japanese application specification for this application are incorporated herein by reference.

[0146] Explanation of reference numerals in the attached figures

[0147] 10: Culture bag;

[0148] 11: Training Department;

[0149] 111: Microstructure;

[0150] 112: First protrusion;

[0151] 12: Bag sealing section;

[0152] 13: Port;

[0153] 14: pipe;

[0154] 15: Hat;

[0155] 20: Outer Packaging Department;

[0156] 21: Exterior sealing part;

[0157] 22: Second protrusion;

[0158] 30: Spacing section;

[0159] S: Decompression space;

[0160] L: Liquid.

Claims

1. A culture bag with liquid filling, characterized in that, A culture bag with a microstructure formed in at least a portion of the culture section is contained in a liquid-filled culture bag in an outer packaging section, wherein there is a pressure-reducing space between the outer side of the culture section and the outer packaging section, and the liquid-filled culture bag is degassed and packaged.

2. The culture bag with liquid filling completed according to claim 1, characterized in that, The outer side of the culture section has multiple first protrusions.

3. The culture bag with liquid filling completed according to claim 1 or 2, characterized in that, The outer packaging section has a plurality of second protrusions in the area facing the outer side of the culture section.

4. The culture bag with liquid filling completed according to claim 1 or 2, characterized in that, The liquid-filled culture bag has a spacer that forms a pressure-reducing space between the outer side of the culture section and the outer packaging section.

5. The culture bag with liquid filling completed according to claim 1 or 2, characterized in that, The microstructure consists of multiple recesses or multiple grooves.

6. The culture bag with liquid filling completed according to claim 1 or 2, characterized in that, The oxygen permeability of the component in the culture bag containing the culture section is 3000 ml / m³. 2 ·day·atm and above.

7. The culture bag with liquid filling completed according to claim 1 or 2, characterized in that, The oxygen permeability of the component in the outer packaging section facing the outside of the culture section is lower than that of the culture section.

8. The culture bag with liquid filling completed according to claim 7, characterized in that, The oxygen permeability of the component in the outer packaging section facing the outer side of the culture section is 1000 ml / m³. 2 Air resistance below day·atm.

9. A method for manufacturing a culture bag with liquid filling, characterized in that, A method for manufacturing a culture bag in which at least a portion of the culture section has a fine structure is contained within an outer packaging section and the culture bag is filled with liquid to complete the culture bag manufacturing process. There is a pressure-reducing space between the outer side of the culture section and the outer packaging section. Fill the culture bag with liquid. The culture bags are degassed and packaged.

10. The method for manufacturing a culture bag with liquid filling according to claim 9, characterized in that, The outer side of the culture section has a plurality of first protrusions, and / or the outer packaging section has a plurality of second protrusions in the area facing the outer side of the culture section, and / or has a spacer that forms a pressure-reducing space between the outer side of the culture section and the outer packaging section.

11. A method for using a culture bag that has been filled with liquid, characterized in that, The method of using the culture bag after filling with liquid according to claim 1 or 2, wherein, Store or transport at room temperature, refrigerated, or frozen.

12. A method for using a culture bag that has been filled with liquid, characterized in that, The method of using the culture bag after filling with liquid according to claim 1 or 2, wherein, Cells, culture medium, cell adhesion factors, cell growth factors, and other cytokines are injected into the culture bag after filling with liquid for culture.

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