Cell culture system including removal of air from cell culture bags

Abstract

The purpose of the present invention is to provide a new means that makes it possible to conveniently and efficiently remove air contained in a cell culturing bag when a culture medium is dispensed. Provided is a system for culturing cells or a cell aggregate using a cell culturing bag, the system including a mechanism for applying a pressure, before adding cells to the cell culturing bag, to the cell culturing bag into which a culture medium has been added, the cell culturing bag having a lower face including a plurality of recesses, so as to remove air in the cell culturing bag through the cell culturing bag.

Description

[Technical Field]

[0001] The present invention relates to a culture system for removing air from a cell culture bag having multiple recesses and for producing cultured cells or cell aggregates within the bag. [Background technology]

[0002] In recent years, in fields such as gene therapy and regenerative medicine, it has become common to culture target cells in large quantities under artificial conditions. For example, one method involves using a cell culture plate (also called a "well plate") with one or more recesses, where cells and culture medium are introduced into each recess for cultivation. However, this method carries the risk of contamination by foreign substances because the recesses are open to the atmosphere.

[0003] To obtain even larger quantities of cells, there is a need for larger containers and smaller, more numerous recesses. As a result, "micro-pattern plates," which have multiple small recesses arranged within a single well, have been developed and are being used. However, due to the small size of the recesses, micro-pattern plates often have air bubbles that clog the recesses when culture medium is added. In this case, a water stream created using a pipette is blown directly onto the air blocking the recesses to remove the air, but this can result in the culture medium becoming full of bubbles, making it difficult to see the cells. Furthermore, it is difficult to completely remove the air that clogs the recesses, and this becomes especially difficult as the holes become smaller and the number of recesses increases (as the micro-pattern plate becomes larger), which also places a significant burden on the operator.

[0004] In response to this, a method has been developed and reported for performing large-scale closed-system culture using gas-permeable bag-like containers (also called "cell culture bags") (for example, Patent Documents 1-4, etc.). This method has the advantage of improving airtightness compared to the method using the aforementioned well plates, thereby reducing the risk of contamination by foreign substances.

[0005] On the other hand, even in closed cultures using such cell culture bags, air can enter along with the culture medium when it is added, sometimes hindering cell culture and observation. In particular, in the case of cell culture bags with multiple minute recesses (for example, Patent Documents 3 and 4), air bubbles can adhere to the inside of the recesses and block them, which is a major obstacle to cell culture.

[0006] Traditionally, removing air from cell culture bags involved physically agitating the bag to dislodge air bubbles attached to the inner wall, causing them to float in the culture medium, collect them in one place, and then aspirate them with a syringe or similar tool. However, this process was time-consuming and difficult to completely remove the air. In particular, the process became more difficult as the cell culture bags became larger.

[0007] Therefore, in this field, there has been a strong demand for a new method that allows for the simple and efficient removal of air that enters the cell culture bag when culture medium is added, thereby enabling cell culture. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2012-239401 [Patent Document 2] Japanese Patent Publication No. 2016-86774 [Patent Document 3] WO2016 / 208526 [Patent Document 4] Japanese Patent Publication No. 2019-118319 [Overview of the project] [Problems that the invention aims to solve]

[0009] Patent Document 1 discloses that when using an incubator in a dry state rather than a normal humidified state, the partial pressure of the air component inside the cell culture bag containing the cells and culture medium can be made equal to or greater than the atmospheric pressure of the dry air inside the culture incubator, thereby preventing air from moving from the outside to the inside of the bag and preventing the generation of bubbles inside the cell culture bag. Furthermore, it states that if bubbles still occur, they can be eliminated by further pressurizing the cell culture bag. The air targeted in Patent Document 1 is not the air that entered the cell culture bag together with the culture medium when it was added. Also, in Patent Document 1, the cell culture bag is pressurized by being sandwiched between a pressure plate and a stacking platform, and at this time the surface of the bag is in close contact with the surface of the pressure plate and stacking platform. Bubbles generated inside the bag cannot escape from the position in close contact with the surface of the pressure plate and stacking platform and dissolve and disappear into the culture medium. Therefore, when the pressure on the cell culture bag is released, bubbles may reappear.

[0010] Therefore, none of the above-mentioned prior art discloses or suggests anything about completely removing air that enters the cell culture bag when culture medium is added. Thus, the present invention aims to provide a new means that enables simple and efficient removal of air that enters the cell culture bag when culture medium is added. [Means for solving the problem]

[0011] As a result of diligent research to solve the above problems, the inventors have found that by adding culture medium to a cell culture bag equipped with multiple recesses and then applying pressure to the bag, air can be removed from inside the bag. The present invention is based on this novel finding and encompasses the following inventions.

[0012] [1] A cell or cell aggregate culture system using a cell culture bag, The cell culture bag has a bottom surface with multiple recesses, A mechanism for removing air inside the cell culture bag through the cell culture bag by applying pressure to the cell culture bag with culture medium added, before adding cells. The culture system as described above. [2] The culture system according to [1], wherein the pressure is applied to the cell culture bag from above the cell culture bag. [3] The culture system according to [1] or [2], further comprising a mechanism for culturing after stirring the cell culture bag with cells added. [4] The culture system according to any one of [1] to [3], further comprising a mechanism for recovering the cultured cells or cell aggregates formed after culturing. [5] The culture system according to [4], comprising a mechanism for recovering the cultured cells or cell aggregates formed by inverting the cell culture bag upside down after culturing. [6] The culture system according to any one of [1] to [5], wherein the lower surface of the cell culture bag has 10 to 1,000,000 recesses. [7] The culture system according to any one of [1] to [6], wherein the recesses in the cell culture bag are spherical crown-shaped. [8] The culture system according to any one of [1] to [], wherein the ratio of the diameter to the depth of the recesses in the cell culture bag is 1:0.25 to 1. [9] The culture system according to any one of [1] to [8], wherein the diameter of the recesses is 300 μm to 1500 μm and the depth is 100 μm to 1000 μm.

[10] A method for removing air from a cell culture bag having a plurality of recesses, comprising: adding culture medium to the cell culture bag and then applying pressure to remove air inside the cell culture bag through the cell culture bag. [] The method according to

[10] , wherein the lower surface of the cell culture bag has 10 to 1,000,000 recesses.

[12] The method according to

[10] or

[11] , wherein the recesses in the cell culture bag are spherical crown-shaped.

[13] The method according to any one of

[10] to

[12] , wherein the ratio of the depth to the diameter of the recesses in the cell culture bag is 1:1.5 to 2.5.

[14] Any of the methods

[10] to

[13] where the ratio of the diameter to the depth of the recess is 1:0.25 to 1.

[15] Any of the methods

[10] to

[14] where the diameter of the recess is 300 μm to 1500 μm and the depth is 100 μm to 1000 μm.

[16] Any of the methods

[10] to

[15] where the pressure is applied to the cell culture bag from above the cell culture bag. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2020-93446, which is the basis of the priority of this application. All publications, patents, and patent applications cited in this specification are hereby incorporated herein by reference in their entirety.

Advantages of the Invention

[0013] According to the present invention, air contained in a cell culture bag having a plurality of recesses at the time of introducing a culture medium can be removed simply and efficiently.

Brief Description of the Drawings

[0014] [Figure 1] FIG. 1 is a schematic diagram showing an example of a cell culture bag having a plurality of recesses that can be used in the present invention, (a) is a perspective view, and (b) is a perspective view. [Figure 2] FIG. 2 is a schematic diagram showing another example of a cell culture bag that can be used in the present invention, (a) is a perspective view, and (b) is a perspective view. [Figure 3] FIG. 3(1) is a perspective view showing an example of a mounting table for mounting a cell culture bag that can be used in the present invention, and FIG. 3(2) is a schematic diagram showing that a recess is formed on the lower surface of the cell culture bag by mounting the cell culture bag on the mounting table and applying pressure while sandwiching it with a pressing member. [Figure 4] FIG. 4 is a cross-sectional schematic diagram of a pressing device that can be used in the present invention, (a) shows an example having a mounting surface formed with an opening for receiving a recess of a cell culture bag, and (b) shows an example where the mounting surface is a flat surface. [Figure 5]Figure 5 is a perspective view showing another example of a pressing device that can be used in the present invention. [Figure 6] Figure 6 shows (A) a photograph and (B) a graph illustrating the relationship between the magnitude of the applied pressure and the storage time regarding the presence or absence of air bubbles in the recessed area. [Figure 7-1] Figure 7-1 is a photograph showing the results of observing the presence or absence of air bubbles in the recessed area of ​​a cell culture bag under pressure over time. [Figure 7-2] Figure 7-2 is a photograph showing the results of observing over time the presence or absence of air bubbles in a recess of a cell culture bag equipped with a gas-impermeable film on the bottom surface, under applied pressure. [Figure 8] Figure 8 is a photograph showing the results of observing the presence or absence of air bubbles in the recessed area of ​​a large cell culture bag under pressure over time. [Modes for carrying out the invention]

[0015] 1. Cell culture bag In the present invention, "cell culture bag" means a culture container formed in the shape of a bag using a gas-permeable flexible film material. Preferably, the cell culture bag has a shape that includes at least a bottom surface with a recess, and further has a shape that includes an upper surface facing the bottom surface.

[0016] "Gas permeability" refers to the property of allowing gas to pass through a flexible film material. In the cell culture bag of this invention, the oxygen permeability measured at a test temperature of 37°C according to the gas permeability test method of JIS K 7126 is 5000 mL / (m³). 2 It is preferable that it is 2000 days or more (atm).

[0017] Flexible film materials usable for cell culture bags include, but are not limited to, resin films such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone elastomer, polystyrene elastomer, and tetrafluoroethylene-hexafluoropropylene copolymer (FEP). These may be used in the form of a single-layer film or in the form of a multilayer (e.g., two or three layers) film by laminating the same or different materials. When forming a cell culture bag by overlapping and heat-sealing two films, it is preferable to have a layer that functions as a sealant layer, taking into consideration heat sealability. For example, a flexible film material can have a three-layer structure consisting of an inner layer, a base layer, and an outer layer from the inside of the cell culture bag. The base layer and inner layer are preferably made of materials that have high gas permeability, heat sealability, and transparency. Furthermore, in addition to the above properties, the inner layer is preferably made of a material that has low cytotoxicity. Suitable materials for this purpose include polyethylene resins such as linear low-density polyethylene (LLDPE), very low-density polyethylene (VLDPE / ULDPE), low-density polyethylene (LDPE), or blends thereof. The outer layer has a density of 0.886 g / cm³. 3 ~0.93g / cm 3 A polyethylene-based resin is preferred. The outer layer may be omitted as appropriate. Furthermore, the inner surface of the cell culture bag (at least the bottom surface) is preferably treated to reduce cell adhesion in order to encourage cells to gather in the center of the bottom of the recess. Examples of treatments to reduce cell adhesion include coating with phospholipid polymers, polyvinyl alcohol derivatives, surfactants, albumin, etc. The thickness of the film can be 50 to 300 μm, preferably about 80 to 160 μm, and the top and bottom surfaces of the cell culture bag may have the same thickness or different thicknesses.

[0018] The number of recesses on the underside of the cell culture bag can be determined according to the size of the cell culture bag and the desired number of cell aggregates, and can be appropriately selected from a range of, for example, 10 to 1 million.

[0019] The shape of the recess is not particularly limited, but a shape that facilitates cell accumulation at the bottom of the recess is preferred. For example, the shape of the recess can be a spherical or mortar-shaped (conical) depression. Furthermore, to facilitate cell accumulation at the bottom of the recess, the ratio of the diameter to depth of the recess is preferably 1:0.25 to 1, and more preferably 1:0.3 to 0.6. For example, in one embodiment, the recess can have an opening diameter of 300 μm to 1500 μm and a depth of 100 μm to 1000 μm.

[0020] The arrangement of the recesses is preferably in a staggered pattern so that the area occupied by the recesses on the lower surface is as large as possible, but they may be arranged in a grid pattern if necessary. The distance (pitch) between the centers of adjacent recesses 4 is not particularly limited, but it is preferable to make it as small as possible within the range where the contours of the recesses do not overlap, for example, the gap between contours can be set to be greater than 0, ~1 mm, preferably 0.05 ~ 0.1 mm.

[0021] The thickness of the cell culture bag is not particularly limited, but it should be thick enough to allow the liquid depth (the distance from the inner surface of the flat surface of the bottom film that comes into contact with the culture medium, excluding the recessed areas) to be 1 mm to 20 mm, preferably 2 mm to 8 mm.

[0022] Cell culture bags may be equipped with a tubular component (hereinafter sometimes referred to as a "port") through which culture medium, cells, etc., can be passed. The port can be molded from a thermoplastic resin such as polyethylene, polypropylene, polyvinyl chloride, polyethylene elastomer, or tetrafluoroethylene-hexafluoropropylene copolymer (FEP).

[0023] The form of the "cell culture bag" in the present invention can refer to the form of a bag-shaped culture container formed using a conventionally known gas-permeable flexible film material (e.g., Japanese Patent No. 5344094, WO2016 / 208526, Japanese Unexamined Patent Publication No. 2016-86774, Japanese Unexamined Patent Publication No. 2019-118319).

[0024] Figure 1 shows an example of a cell culture bag according to the present invention. The cell culture bag 1 has a bag-like structure comprising a bag body 2 formed by overlapping an upper film 21 constituting the upper surface and a lower film 22 constituting the lower surface and sealing the peripheral portion 20, and a port 3. Multiple recesses 4 arranged in a staggered pattern are formed and held in the lower film 22 by molding techniques such as thermo-press molding. The upper film 21 constituting the upper surface has a substantially flat top portion 21a that covers the entire upper portion of the multiple recesses 4, and a bulging shape with an inclined portion 21b around the top portion 21a. This allows it to seal with the lower film 22 to form a cell culture bag with thickness, and when the bag is filled with culture medium or when pressure is applied to the bag, deformation that causes the periphery of the lower film 22 to lift can be suppressed.

[0025] Figure 2 shows another example of a cell culture bag according to the present invention. In the cell culture bag 1', the recess is not formed directly on the bottom film 22', but is formed after the cell culture bag 1' is placed on the mounting platform, following the shape of the mounting surface of the mounting platform that contacts the bottom film 22'.

[0026] The mounting platform for cell culture bag 1' is a plate-shaped member having a flat mounting surface for placing the cell culture bag and a recess for forming a recess on the lower surface of the cell culture bag. The mounting platform can be made of metal, hard resin, or the like, and the recess on the mounting surface only needs to be shaped so that the lower surface of the pressed cell culture bag can form the aforementioned recess. The recess may be formed by providing corresponding recesses and / or protrusions on the mounting surface, or through holes appropriately placed on the mounting surface can be used as recesses.

[0027] Figure 3(1) shows an example of a mounting platform 5' that forms a recess in the cell culture bag 1'. The mounting platform 5' has a flat mounting surface 5a' for placing the cell culture bag 1' and a recess 5b' in the form of a through-hole for forming a recess in the lower surface of the cell culture bag 1'. As shown in Figure 3(2), after placing the cell culture bag 1' filled with cells and a predetermined culture medium on the mounting platform 5', pressure is applied by sandwiching the cell culture bag 1' between the pressing member 6 (described later) and the mounting platform 5'. As a result, the lower film 22' is pressed against the mounting surface 5a, and a portion of it protrudes from the recess 5b' without contacting the mounting surface 5a, thereby forming a recess 4'.

[0028] 2. Pressing device In the present invention, a pressing device can be used that is capable of applying pressure to a cell culture bag filled with a predetermined culture medium. The pressing device comprises at least a mounting base on which the cell culture bag is placed, a pressing member that presses against the upper surface of the cell culture bag, a support mechanism that supports the vertical movement of one or both of these, and a pressure applying means that presses one or both of these toward the cell culture bag. It may also further comprise a liquid delivery means for injecting and discharging cells and culture medium through a port in the cell culture bag. If pressure is applied to the cell culture bag by the weight of the pressing member, the pressure applying means may be omitted. Examples of liquid delivery means include, but are not limited to, pumps such as peristaltic pumps and syringes. The pressing device may be portable and moved into an incubator for use, or it may be installed inside the incubator.

[0029] In the present invention, the mounting platform has a shape in which its mounting surface supports the cell culture bag with low or no contact with the recess. As a result, unlike the cell culture bag in Patent Document 1 described above, when pressure is applied, the surface of the cell culture bag does not come into close contact with the surface of the mounting platform, maintaining gas permeability and allowing air to move from inside to outside the bag.

[0030] In this invention, "applying pressure" means increasing the internal pressure inside a cell culture bag filled with a predetermined culture medium, and this can be done by sandwiching the cell culture bag between the upper surface (mounting surface) of the mounting platform and the lower surface of the pressing member. Specifically, this can be done by using the support mechanism and pressure application means to push the pressing member downwards toward the cell culture bag and apply pressure from above the cell culture bag, and / or by using the support mechanism and pressure application means to push the mounting platform upwards toward the cell culture bag and apply pressure from below the cell culture bag.

[0031] Figure 4 shows a schematic cross-sectional view of an example of a pressing device usable in the present invention. The pressing devices 100 and 100'' in Figure 4 have a configuration in which a pressing member 6 is pressed down from above onto a cell culture bag 1 placed on a mounting base 5 to apply pressure.

[0032] The pressing device 100, 100'' comprises a mounting stage 5 on which the cell culture bag 1 is placed, a pressing member 6 having a bottom surface 6a that presses the top surface portion 21a of the top film 21, and a support mechanism 7 that supports the pressing member 6.

[0033] Note that in Figure 4, the illustration of the liquid delivery mechanism used to inject and discharge cells and culture medium from port 3 of the cell culture bag 1 is omitted.

[0034] The mounting platform 5 has a mounting surface 5a on which the cell culture bag 1 is placed horizontally. This mounting surface 5a may be flat (Figure 4(b)), or it may have openings 5b that receive a plurality of recesses 4 formed in the bottom film 22 of the cell culture bag 1 (Figure 4(a)). If there are openings 5b, the mounting surface 5a supports the bottom film 22 without contacting the recesses 4. The shape of the openings 5b is not limited to openings, and may be, for example, recesses, or mesh-like, and may be a shape that can receive not only each of the recesses 4, but also a shape that can receive a plurality of recesses 4.

[0035] The pressing member 6 is a plate-shaped member having a planar shape that matches the top surface portion 21a of the cell culture bag 1, and has a flat bottom surface 6a.

[0036] The support mechanism 7 consists of a frame 71 provided on the mounting surface 5, guide pins 72 extending upward from the four corners of the upper surface of the pressing member 6 and penetrating the frame 71 so as to be vertically movable, and a pressure applying means 73 that pushes the pressing member 6 downward. The pressure applying means 73 pushes the pressing member 6 toward the cell culture bag 1 and applies pressure to the cell culture bag 1. The pressure to be applied can be adjusted by adjusting the force or height to which the pressure applying means 73 pushes down the pressing member 6. The pressure applying means 73 only needs to be configured to enable pushing down the pressing member 6 with a predetermined force or to a predetermined height, and can consist of screws, springs, magnets, or a combination thereof.

[0037] Figure 5 shows a perspective view of another example of a culture apparatus usable in the present invention. The culture apparatus 100' in Figure 5 comprises a mounting base 5 having a mounting surface 5a, and a top lid 8' connected to the mounting base 5 via a hinge 9', the top lid 8' being configured to open and close on the hinge 9' as an axis. The top lid 8' consists of a pressing member 6 having a flat bottom surface 6a that matches the top surface portion 21a of the cell culture bag 1, guide pins (not shown) extending upward from the four corners of the upper surface of the pressing member 6 and penetrating the top lid 8' so as to be vertically movable, and a pressure applying means (not shown) for pushing the pressing member 6 downward toward the cell culture bag 1.

[0038] When placing the cell culture bag 1 into the culture apparatus 100', first the top lid 8' is opened via the hinge 9' (Figure 5(a)), and after the cell culture bag 1 is placed on the mounting surface 5a which has openings (not shown) that accept multiple recesses, the top lid 8' is closed and held in place by the locking mechanism 10', as shown in Figure 5(b). Pressure can be applied to the cell culture bag 1 by pressing the pressing member 6 downward with the pressure applying means until a predetermined pressure is applied.

[0039] Figures 4 and 5 above show examples using cell culture bag 1. However, when using cell culture bag 1', a mounting stand such as mounting stand 5', which has a mounting surface 5a' capable of forming a recess in the cell culture bag 1', can be used as the mounting stand. Otherwise, a pressing device having the same configuration as the pressing device described above can be used.

[0040] 3.Cells In the present invention, the cells used to produce cell aggregates are not particularly limited, but examples include pluripotent stem cells or their differentiated cells, neural stem cells, hepatocytes, corneal stem cells, pancreatic islet cells, cardiomyocytes, and the like.

[0041] In the present invention, "pluripotent stem cell" refers to embryonic stem cells (ES cells) and cells that possess similar pluripotency, that is, cells that potentially have the ability to differentiate into various tissues of the living organism (all of the endoderm, mesoderm, and ectoderm). Examples of cells with similar pluripotency to ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification). Preferably, in the present invention, pluripotent stem cells refer to human pluripotent stem cells. "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors) such as Oct3 / 4, Sox2, Klf4, and c-Myc. Furthermore, "differentiation-inducing cells" of the above-mentioned pluripotent stem cells refer to cells characterized by a predetermined phenotype or expression of a marker obtained by differentiating pluripotent stem cells. "Marker" refers to a cell antigen or its gene that is specifically expressed by a predetermined cell type, such as a "marker protein" or "marker gene."

[0042] The cells usable in this invention may be cells collected from living organisms, cultured cells, or cells obtained by freeze-thawing. The cells used are in a dissociated or dispersed state.

[0043] 4. A method for removing air from a cell culture bag filled with a cell or cell aggregate culture system and culture medium. (1) Mechanism for removing air from the cell culture bag The cell or cell aggregate culture system of the present invention is characterized by having a mechanism that applies pressure to a cell culture bag filled with culture medium to remove air inside the bag, particularly bubbles adhering to recesses, and the following is carried out in this mechanism.

[0044] The culture medium can be appropriately selected and used according to the type of cells being used. The culture medium is added through the port on the cell culture bag using a pump such as a peristaltic pump or a syringe. The amount of culture medium added to the cell culture bag can be appropriately adjusted according to factors such as the size of the cell culture bag, the number of cells to be added to the cell culture bag, the culture period, and the desired size of the cell aggregates, and is not particularly limited, but it is preferable to fill the cell culture bag to its full capacity with culture medium so that no air layer is formed inside the bag.

[0045] Pressure can be applied to the cell culture bag using the aforementioned pressing device, by sandwiching the cell culture bag between a mounting platform on which the cell culture bag is placed and a pressing member facing the top surface of the cell culture bag.

[0046] The magnitude and duration of pressure applied to the cell culture bag should be sufficient to remove air from inside the bag, especially bubbles adhering to the recesses. This can be adjusted as appropriate depending on factors such as the size of the cell culture bag and the size and depth of the recess openings, and is not particularly limited. For example, 0.001 kgf / cm² 2 ~0.2 kgf / cm² 2 Preferably 0.01 kgf / cm² 2 ~0.1 kgf / cm² 2It can be processed for 1 hour to 3 days, preferably 2 hours to 1 day, at a pressure selected from the range. In the present invention, the removal of air in the cell culture bag is carried out before cells are added to the cell culture bag. Thereby, it is not necessary to consider the influence of the applied pressure on the cells, and the optimum pressure magnitude and application time for removing the air in the bag can be applied. The application of pressure to the cell culture bag may be carried out in an incubator used for culturing, or may be carried out outside the incubator.

[0047] According to the present invention, by applying pressure to the cell culture bag, air in the cell culture bag filled with the culture medium, particularly air bubbles adhering in the recesses, can be removed to the outside of the bag through the gas-permeable flexible film material constituting the cell culture bag, and it does not dissolve air or air bubbles in the culture medium and cause them to disappear. Therefore, even when the subsequently applied pressure is released, the removed air and air bubbles do not reappear.

[0048] (2) Mechanism for culturing cells The cell or cell aggregate culture system of the present invention can be provided with a mechanism for culturing cells in the bag from which air in the cell culture bag filled with the above culture medium has been removed. The following is implemented in the mechanism. Cells are introduced through a port provided in the cell culture bag using a liquid feeding means such as a pump like a peristaltic pump or a syringe. The number of cells to be introduced into the cell culture bag can be appropriately adjusted according to factors such as the size of the cell culture bag, the culture period, the desired size of the cell aggregate, etc., and is not particularly limited. For example, it can be about 1×10 5 ~1×10 7 cells / mL. After the cells are added to the cell culture bag, they are stirred with the culture medium. By stirring, the number of cells entering each recess on the lower surface of the cell culture bag can be made approximately uniform in a subsequent step. Stirring may be carried out manually or by a robot arm, and / or can be carried out using a vibrator.

[0049] Cell culture can be carried out under appropriate conditions depending on the cells used, and can be done in an incubator adjusted to an appropriate temperature (30-40°C, e.g., 37°C), CO2 concentration (5-10%, e.g., 5%), and humidity (90-95%, e.g., 95%). The culture period can be appropriately determined depending on factors such as the desired cell aggregate size and cell type, and is not particularly limited, but is for example 1-10 days, preferably 2-7 days.

[0050] (3) Mechanism for collecting the formed cultured cells or cell aggregates The cell or cell aggregate culture system of the present invention may include a mechanism for recovering the cultured cells or cell aggregates formed in the recess after the completion of the culture. The following is carried out in this mechanism. Recovery of the cultured cells or cell aggregates formed in the recess can be carried out using any means that allows the cell aggregates to float out of the recess into the culture medium. Such means can be physical means, such as inverting the cell culture bag upside down, applying vibration to the cell culture bag, pushing up the recess from the outside of the cell culture bag using, for example, a protruding member (JP 2019-118319 A), or lifting the cell culture bag from the mounting surface and flattening the recess (if the recess is formed by a depression in the mounting surface). These means may be carried out by hand or a robotic arm, and / or using a vibrator. Alternatively, the cell aggregates can be floated out of the recess by a jet of air or a suitable buffer such as culture medium or physiological saline injected into the cell culture bag through the port of the cell culture bag. Cell aggregates suspended in the culture medium can be collected along with the culture medium and other materials through a port provided on the cell culture bag.

[0051] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0052] Experiment 1. Removal of air from medium-sized cell culture bags. (1-1) Medium cell culture bag A medium-sized cell culture bag with a similar structure to the cell culture bag shown in Figure 1 was prepared and used. Specifically, it consisted of two gas-permeable flexible films made of 100 μm thick polyethylene layered together and sealed around the edges (the area inside the seal was 50 cm²). 2 ), and has a configuration that includes a port. The bottom film of the cell culture bag has a recess with a diameter of 500 μm and a depth of 200 μm, 1.8 × 10 4 Each individual unit has a bulging top film that extends 4 mm in height. The inner surface of the film is coated with a phospholipid polymer to reduce cell adhesion.

[0053] (1-2) Experimental Method Add 10 mL of culture medium to the medium-sized cell culture bag through the port, close the port, and in a CO2 incubator (37°C, 95% humidity, 5% CO2), apply pressure using a pressure device at 0 kgf, 0.5 kgf, 1.0 kgf, 2.0 kgf, or 4.0 kgf (surface pressure: 0 kgf / cm²). 2 , 0.01 kgf / cm 2 , 0.02 kgf / cm 2 , 0.04 kgf / cm² 2 , or 0.08 kgf / cm² 2 They were stored under pressure in each of the following conditions. The presence or absence of air bubbles in the depressions was observed before storage (0 hours), and at 2.5 hours, 4.5 hours, 9 hours, 15 hours, and 72 hours after storage.

[0054] (1-3) Results Figure 6 shows the relationship between the magnitude of the applied pressure and the storage time regarding the presence or absence of air bubbles in the recesses. In the photographic diagram (A) of Figure 6, the black color indicates the presence of air bubbles in the recesses (the round areas), while the grayish-white color indicates the absence of air bubbles. Based on each photographic diagram, the relationship between the magnitude of the applied pressure and the time required for the air bubbles in the recesses to disappear is shown in the graph (B) of Figure 6. It was observed that the time required for the air bubbles to disappear decreases as the magnitude of the applied pressure increases. For example, when attempting to eliminate air bubbles in the recesses of a medium-sized cell culture bag overnight (approximately 12 hours), it was confirmed that an applied pressure of 0.5 kgf to 1.0 kgf was sufficient. Furthermore, since the volume of air present in a larger recess increases with the diameter of the recess, the size of the recess can also be a factor that affects the time required for the air bubbles to disappear. In the aforementioned medium-sized cell culture bag, when the recess diameter was changed from 0.5 μm to 1.26 μm, the time required for the air bubbles to disappear increased from 9 hours to 57 hours.

[0055] Experiment 2. Removal of air from a cell culture bag equipped with a gas-impermeable film on the bottom surface. (2-1) Medium-sized cell culture bag with a gas-impermeable film on the bottom surface A medium-sized cell culture bag with the same configuration as the cell culture bag used in Experiment 1 above was prepared and used, except that the gas-permeable film on the bottom surface (recessed side) was replaced with a multilayer gas-impermeable film made by bonding polyethylene and ethylene-vinyl alcohol copolymer (EVOH). The gas-impermeable film (thickness 118.5 μm) consists of polyethylene (thickness 67.7 μm), an adhesive layer (thickness 17.3 μm), and EVOH (thickness 30.5 μm) from the inside out. The gas permeability of the gas-permeable film and gas-impermeable film used in the experiment is shown below.

[0056] [Table 1]

[0057] (2-2) Experimental method A cell culture bag equipped with a gas-impermeable film on its bottom surface (hereinafter referred to as the "gas-impermeable cell culture bag") and the gas-permeable cell culture bag prepared in Experiment 1 above were each filled with 10 mL of culture medium through the port, and the ports were closed. In a CO2 incubator (37°C, 95% humidity, 5% CO2), a pressure device was used to apply 0.8 kgf (surface pressure: 0.016 kgf / cm²) of pressure. 2 They were stored under pressure in each of the following conditions. The presence or absence of air bubbles in the recesses was observed before storage (0 hours) and from 1 hour to 23 hours after storage.

[0058] (2-3) Results Figures 7-1 (gas-permeable cell culture bag) and 7-2 (gas-impermeable cell culture bag) show the results of observing the presence or absence of air bubbles in the recesses of each cell culture bag over time. In each photograph, a black color in the recess (the rounded area) indicates the presence of air bubbles, while a grayish-white color indicates the absence of air bubbles. In the gas-permeable cell culture bag (Figure 7-1), air bubbles disappeared after 15-16 hours of storage, whereas in the gas-impermeable cell culture bag (Figure 7-2), air bubbles remained even after 23 hours of storage. These results confirm that this method effectively removes air bubbles present in the recesses of the cell culture bag through the gas-permeable film.

[0059] Experiment 3. Removal of air from large cell culture bags (3-1) Large cell culture bag A large cell culture bag with a similar structure to the cell culture bag shown in Figure 1 was fabricated and used. It consisted of two layers of gas-permeable flexible polyethylene film, each 100 μm thick, with the edges sealed (the area inside the seal was 1,000 cm²). 2 ), and has a configuration that includes a port. The bottom film of the cell culture bag has a recess with a diameter of 350 μm and a depth of 150 μm, measuring 65 × 10 4 Each unit has a bulge shape on the top film, which is raised to a height of 4 mm. The inner surface of the film is coated with a low-cell adhesion coating made of phospholipid polymer.

[0060] (3-2) Experimental Method Add 200 mL of culture medium to the large cell culture bag through the port, close the port, and in a CO2 incubator (37°C, 95% humidity, 5% CO2), apply 1.0 kgf (surface pressure: 0.01 kgf / cm²) using a pressure device. 2 It was stored while applying pressure.

[0061] (3-3) Results Figure 8 shows the results of observing the presence or absence of air bubbles in the recesses of a large cell culture bag over time. In the photograph in Figure 8, the black areas in the recesses (the round areas) indicate the presence of air bubbles, while the grayish-white areas indicate the absence of air bubbles. The air bubbles in the recesses disappeared approximately 8 hours after storage. This confirmed that even in large cell culture bags, air bubbles can be removed by applying pressure.

[0062] The results above confirm that, according to the method of the present invention, air bubbles adhering to multiple recesses in a cell culture bag filled with culture medium can be removed simply and efficiently. The diameter of the recesses in the large cell culture bag used in this experiment (350 μm) is smaller than the diameter of the recesses in the medium cell culture bag described above (500 μm), and the size of the air bubbles in the recesses is also smaller, so they were removed in a short time. [Explanation of Symbols]

[0063] 1. 1' Cell culture bag 2. 2' Bag body 20, 20' Peripheral area 21, 21' Top film 21a, 21a' Top surface 21b, 21b' Sloped section 22, 22' Bottom film 3, 3' Port 4, 4' recess 5, 5' mounting platform 5a, 5a' Mounting surface 5b opening 5b' Indentation 6 Pressing member 6a Bottom 7 Support mechanism 71 frames 72 Guide pins 73 Pressure application means 8' top lid 9' Hinge 10' Locking Mechanism 100, 100', 100'' Pressing device 11 Air

Claims

1. A cell or cell aggregate culture system using a cell culture bag, The cell culture bag has a bottom surface with multiple recesses, A mechanism that applies pressure to remove air from inside the cell culture bag by passing it through the bag before adding cells to the cell culture bag containing the culture medium. The culture system comprising the above.

2. The culture system according to claim 1, wherein pressure is applied to the cell culture bag by applying pressure from above the cell culture bag.

3. Furthermore, the culture system according to claim 1 or 2, further comprising a mechanism for culturing the cell culture bag to which the cells have been added after agitation.

4. Furthermore, the culture system according to any one of claims 1 to 3, further comprising a mechanism for recovering the cultured cells or cell aggregates formed after the completion of culture.

5. The culture system according to claim 4, further comprising a mechanism for inverting the cell culture bag after the completion of culture to collect the formed cultured cells or cell aggregates.

6. The cell culture system according to any one of claims 1 to 5, wherein the lower surface of the cell culture bag is provided with 10 to 1 million recesses.

7. The culture system according to any one of claims 1 to 6, wherein the recess in the cell culture bag is spherical.

8. The culture system according to any one of claims 1 to 7, wherein the ratio of the diameter to the depth of the recess in the cell culture bag is 1:0.25 to 1.

9. A culture system according to any one of claims 1 to 8, wherein the diameter of the recess is 300 μm to 1500 μm and the depth is 100 μm to 1000 μm.

10. A method for removing air from a cell culture bag having multiple recesses on its bottom surface, A method comprising the steps of adding culture medium to a cell culture bag, and then applying pressure to remove air from inside the cell culture bag through the bag.

11. The method according to claim 10, wherein the lower surface of the cell culture bag is provided with 10 to 1 million recesses.

12. The method according to claim 10 or 11, wherein the recess in the cell culture bag is spherical.

13. The method according to any one of claims 10 to 12, wherein the ratio of depth to diameter of the recess in the cell culture bag is 1:1.5 to 2.

5.

14. The method according to any one of claims 10 to 13, wherein the ratio of the diameter to the depth of the recess is 1:0.25 to 1.

15. The method according to any one of claims 10 to 14, wherein the diameter of the recess is 300 μm to 1500 μm and the depth is 100 μm to 1000 μm.

16. The method according to any one of claims 10 to 15, wherein pressure is applied to the cell culture bag by applying pressure from above the cell culture bag.

Images