A method for mass production of cell aggregates of uniform size.

Abstract

This provides a novel method for easily and mass-producing cell aggregates of substantially uniform size. [Solution] A method for producing cell aggregates using a cell culture bag, wherein the cell culture bag has a bottom surface with a plurality of recesses, and the method includes (1) adding cells and culture medium to the cell culture bag and stirring, and culturing while applying pressure to the cell culture bag, and (2) collecting the formed cell aggregates after the culturing is complete.

Description

[Technical Field]

[0001] The present invention relates to a method for easily and in large quantities producing cell aggregates of substantially uniform size.

[0002] [Background of the Invention] 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 plate with one or more recesses (commonly called a "well plate") to introduce cells and culture medium into each recess and then culturing them. However, this method carries the risk of contamination by foreign matter because the recesses are open to the atmosphere, and the process is cumbersome as cells and culture medium must be injected and retrieved from each recess, making large-scale mass culture a significant burden on workers.

[0003] In contrast, a method has been developed and reported for performing closed-system large-scale culture using a gas-permeable bag-shaped container (also called a "bag") with multiple recesses formed on its bottom surface (for example, Patent Document 3 described below). This method has the advantage of improving airtightness compared to the method using the well plates mentioned above, thereby reducing the risk of contamination by foreign matter. On the other hand, in closed cultures using such bags, it is not possible to control the number or position of cells in each recess, which can lead to variations in the size and number of cultures (more specifically, cell aggregates) formed between recesses. Therefore, it is necessary to restrict movement between recesses during culture, detach cells from each recess, and place them in the appropriate positions, which is a cumbersome process and places a significant burden on workers when culturing on a large scale. Therefore, in this field, there has been a strong demand for a closed culture method that can easily and mass-produce substantially uniform cell cultures.

[0004] Conventionally, the following methods have been developed and reported for closed culture using gas-permeable bag-like containers. Patent Document 1 discloses a method for culturing adherent cells, which includes adding adherent cells and culture medium to a bag-shaped container having a container wall that serves as a cell culture surface, and then pressing the bag-shaped container to increase the internal pressure, thereby suppressing the flow of the contents and promoting cell adhesion to the cell culture surface, or suppressing the detachment of adhered cells.

[0005] Patent Document 2 discloses a cell culture method that includes placing a flexible culture vessel containing cells and culture medium on a mounting surface, applying pressure to the flexible culture vessel to deform the outer surface of the flexible culture vessel in contact with the mounting surface to form multiple recesses, continuing the culture in this state, and after cell aggregates are formed in the recesses, releasing the pressure on the flexible culture vessel to flatten the recesses formed on the outer surface of the vessel and continuing the culture. Patent Document 2 states that if the recesses are maintained during culture, there is a risk of inconvenience in the circulation of the culture medium and the diffusion of gases, so the pressure applied during culture is released to flatten the recesses, and it is also stated that flattening the recesses can increase the area used for cell culture.

[0006] Patent Document 3 discloses a method for culturing cell aggregates (spheroids) using a cell culture bag having multiple recesses formed on its lower surface. In this method, it is necessary to peel off spheroids adhering to the inner surface of the recesses in order to form one spheroid in each recess and to improve the efficiency of cell proliferation and differentiation induction. During this peeling process, the multiple recesses are closed to prevent the spheroids from moving between the recesses. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2016-86774 [Patent Document 2] WO2016 / 208526 [Patent Document 3] Japanese Patent Publication No. 2019-118319

Summary of the Invention

Problems to be Solved by the Invention

[0008] None of the above prior arts discloses or suggests anything about simply and massively producing cell aggregates having substantially uniform sizes.

[0009] Therefore, an object of the present invention is to provide a new means capable of simply and massively producing cell aggregates having substantially uniform sizes.

Means for Solving the Problems

[0010] As a result of intensive studies to solve the above problems, the present inventors have found that by adding cells and a culture medium to a gas-permeable flexible cell culture bag having a plurality of recesses on the lower surface and culturing while applying pressure to the cell culture bag, cell aggregates having substantially uniform sizes are formed in each recess. Further, it has been found that after the completion of culturing, a large number of formed cell aggregates can be taken out from each recess by a simple operation and can be recovered more simply from the cell culture bag. The present invention is based on these new findings and includes the following inventions.

[0011] [1] A method for producing cell aggregates using a cell culture bag, comprising: The cell culture bag has an upper surface and a lower surface, and the lower surface is provided with a plurality of recesses; (1) A step of adding cells and a medium to the cell culture bag, stirring, and culturing while applying pressure to the cell culture bag from at least one direction; (2) A step of recovering the formed cell aggregates after the completion of culturing. A method including these steps. [2] The method according to [1], wherein 100,000 to 1,000,000 cell aggregates are recovered. [3] The method according to [1] or [2], wherein the recovered cell aggregates have substantially uniform particle diameters. [4] Any of the methods of [1] to [3], where 70% or more of the recovered cell aggregates have a particle diameter within ±10% of the median particle diameter of the cell aggregates. [5] Any of the methods of [1] to [4], where 70% or more of the recovered cell aggregates have a particle diameter of 140 μm to 160 μm. [6] Any of the methods of [1] to [5], where each of the recovered cell aggregates contains 200 to 2000 cells. [7] Any of the methods of [1] to [6], where the cells are cells that have been cryopreserved and thawed. [8] Any of the methods of [1] to [7], where the cells are insulin-secreting cells. [8a] The method of [8], where the insulin-secreting cells are insulin-producing cells. [9] Any of the methods of [1] to [8], where the cell culture bag is inverted top to bottom to recover the cell aggregates.

[10] Any of the methods of [1] to [9], where air is injected into the cell culture bag to recover the cell aggregates.

[0012]

[11] Any of the methods of [1] to

[10] , where the step of culturing while applying pressure to the cell culture bag from at least one direction is a step of culturing while applying pressure to the cell culture bag from above, below, or both above and below.

[12] The method of

[11] , where the step of culturing while applying pressure to the cell culture bag from at least one direction is a step of culturing while applying pressure to the cell culture bag from above.

[13] Any of the methods of [1] to

[12] , where the ratio of the depth to the diameter of the concave portion is 1:1.5 to 2.5.

[14] Any of the methods of [1] to

[13] , where the concave portion is spherical crown-shaped.

[15] Any of the methods of [1] to

[14] , where the pitch of the concave portion is 0.35 mm to 0.49 mm.

[16] Any of the methods of [1] to

[15] , where the depth of the concave portion is 150 μm to 220 μm.

[17] Any of the methods of [1] to

[16] , where the liquid depth in the cell culture bag is 1.5 mm to 6 mm.

[18] Place cells in a cell culture bag at a rate of 1 × 10 5 ~1 × 10 7 Add in a volume of cells / mL, using one of the methods [1] to

[17] .

[19] Place cells in a cell culture bag at a rate of 1 × 10 4 ~5×10 6 cells / cm 2 Add in the amount specified by one of the following methods [1] to

[18] .

[20] Add culture medium after cell aggregate formation, or any of the methods [1] to

[19] .

[0013]

[21] Any method [1] to

[20] , wherein the lower surface comprises 100,000 to 1,000,000 recesses.

[22] 1 to 1000 recesses per unit area of ​​the bottom surface / cm 2 Prepare by any of the following methods [1] to

[21] .

[23] A population of cell aggregates for transplantation, wherein more than 70% of the cell aggregates have a particle size of 140 μm to 160 μm.

[24] A group of cell aggregates

[23] containing 100,000 to 1,000,000 cell aggregates.

[25] A population of cell aggregates of

[23] or

[24] , including insulin-secreting cells. [25a] A group of cell aggregates of

[25] in which insulin-secreting cells are insulin-producing cells.

[26] A cell culture bag containing 100,000 to 1,000,000 cell aggregates having substantially uniform particle size.

[27] A cell culture bag of

[26] in which more than 70% of the cell aggregates have a particle size of 140 μm to 160 μm.

[28] A cell culture bag of

[26] or

[27] containing cell aggregates of insulin-secreting cells. [28a] A cell culture bag of

[28] in which insulin-secreting cells are insulin-producing cells. This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2020-93447, filed on 28 May 2020, which forms the basis of the priority claim of this application. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Effects of the Invention]

[0014] According to the present invention, a novel method is available that enables the simple and large-scale production of cell aggregates of substantially uniform size. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 is a schematic diagram showing an example of a cell culture bag usable in the present invention, where (a) is an oblique view and (b) is a perspective view. [Figure 2] Figure 2 is a schematic diagram showing another example of a cell culture bag usable in the present invention, where (a) is an oblique view and (b) is a perspective view. [Figure 3] Figure 3(1) is a perspective view showing an example of a mounting platform for placing a cell culture bag usable in the present invention, and Figure 3(2) is a schematic diagram showing that a recess is formed on the lower surface of the cell culture bag by placing the cell culture bag on the mounting platform and applying pressure by sandwiching it between the pressing member. [Figure 4] Figure 4 is a schematic cross-sectional view of a culture apparatus usable in the present invention, where (a) shows an example with a mounting surface having an opening for receiving the recess of a cell culture bag, and (b) shows an example with a flat mounting surface. [Figure 5] Figure 5 is a perspective view showing another example of a culture apparatus that can be used in the present invention. [Figure 6] Figure 6 is a photograph of cell aggregates produced by the method of the present invention. [Figure 7] Figure 7 is a graph showing the diameter distribution of cell aggregates produced from recovered insulin-producing cells. [Figure 8] Figure 8 is a graph showing the diameter distribution of cell aggregates produced from recovered human iPS cells, with (A) those produced without applying pressure during culture (control) and (B) those produced with applying pressure during culture. [Modes for carrying out the invention]

[0016] 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.

[0017] "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 greater than or equal to day / atm.

[0018] Examples of flexible film materials that can be used for cell culture bags include, but are not limited to, resin films such as polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyester, silicone-based elastomer, polystyrene-based elastomer, tetrafluoroethylene-hexafluoropropylene copolymer (FEP). These can be used in the form of a single-layer film or in the form of a multilayer (e.g., two-layer or three-layer) film by laminating the same or different materials. When forming a cell culture bag by heat-sealing two films together, it is preferable to have a layer that functions as a sealant layer considering heat-sealability. For example, the 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 the inner layer are preferably composed of materials having high gas permeability, heat-sealability, and transparency. In addition, the inner layer is preferably composed of a material having low cytotoxicity in addition to the above characteristics. As such materials, for example, linear low-density polyethylene (LLDPE), very low-density polyethylene (VLDPE / ULDPE), low-density polyethylene (LDPE), or blends of these polyethylene-based resins can be preferably used. The outer layer is preferably a polyethylene-based resin with a density of 0.886 g / cm 3 to 0.93 g / cm 3 . The outer layer may be omitted as appropriate. Also, the surface (at least the bottom surface) that becomes the inner surface of the cell culture bag is preferably subjected to a low cell adhesion treatment in order to facilitate cell aggregation at the center of the bottom of the recess. Examples of the low cell adhesion treatment include coating with a phospholipid polymer, a polyvinyl alcohol derivative, a surfactant, albumin, etc. The thickness of the film can be 50 to 300 μm, preferably about 80 to 160 μm, and the thickness can be the same or different on the upper and lower surfaces of the cell culture bag.

[0019] 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. For example, it can be appropriately selected from the range of 10 to 1 million, 100 to 1 million, 1,000 to 1 million, or 10,000 to 1 million, preferably 100,000 to 1 million, or 300,000 to 1 million.

[0020] 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 depth-to-diameter ratio of the recess is preferably 1:1 to 4, more preferably 1:1.5 to 2.5, and even more preferably 1:1.8 to 2.2. For example, in one embodiment, the depth of the recess can be 100 μm to 1000 μm, for example 150 μm to 220 μm, and the opening diameter can be 300 μm to 1500 μm, for example 225 μm to 550 μm.

[0021] The arrangement of the recesses is preferably staggered to maximize the area occupied by the recesses on the bottom surface, but they may be arranged in a grid pattern if necessary. The distance (pitch) between the centers of adjacent recesses 4 can be 0.35 mm to 0.49 mm, preferably 0.38 mm to 0.45 mm. Alternatively, the distance between the contours of adjacent recesses can be set to greater than 0 and ~1 mm, preferably 0.05 to 0.1 mm. There are 1 to 1000 recesses per unit area of ​​the bottom surface / cm². 2 For example, 30-800 pieces / cm 2 , or 60-700 pieces / cm 2 It can be established.

[0022] The size of the cell culture bag is not particularly limited, but for example, it can be 10 to 1000 mm in length, for example, 50 to 500 mm in length and 10 to 1000 mm in width, for example, 50 to 500 mm in width. 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 about 2 mm to 8 mm, when pressure is applied to the cell culture bag containing the culture medium.

[0023] 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).

[0024] 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).

[0025] 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.

[0026] 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'.

[0027] 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.

[0028] 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', causing the lower film 22' to be pressed against the mounting surface 5a, and a portion of it to protrude from the recess 5b', thereby forming a recess 4'.

[0029] 2.Culture equipment In the present invention, a culture apparatus can be used that has a configuration capable of applying pressure to a cell culture bag filled with cells and a predetermined culture medium. The culture apparatus comprises at least a mounting platform on which the cell culture bag is placed, a pressing member that holds down the upper surface of the cell culture bag, a support mechanism that supports the vertical movement of one or both of these, a pressure applying means that presses one or both of these toward the cell culture bag, and 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 culture apparatus may be portable and moved into an incubator for use, or it may be installed inside an incubator.

[0030] In this invention, "applying pressure" means making the liquid depth uniform in a cell culture bag filled with cells and a predetermined culture medium. In a cell culture bag filled with cells and a predetermined culture medium without applied pressure, the liquid depth is not uniform due to "twists," "wrinkles," "folds," "bends," etc., formed in the flexible upper and / or lower film, resulting in a mixture of areas with relatively high and relatively low liquid depths. By "applying pressure," these "twists," "wrinkles," "folds," "bends," etc., formed in the upper and / or lower film can be straightened, making the liquid depth uniform in the cell culture bag. The step of "applying pressure" can be performed by sandwiching the cell culture bag filled with cells and a predetermined culture medium between the upper surface (mounting surface) of the mounting stand and the lower surface of the pressing member. In other words, this can be done by pressing the pressing member downwards toward the cell culture bag using a support mechanism and pressure application means, thereby applying pressure from above the cell culture bag, and / or by pushing the mounting platform upwards toward the cell culture bag using a support mechanism and pressure application means, thereby applying pressure from below the cell culture bag. Alternatively, the pressure application can be omitted, and pressure can be applied to the cell culture bag by the weight of the pressing member itself. These means can straighten out any "twists," "wrinkles," "folds," "bends," etc., formed on the upper and / or lower film in the area in contact with the lower surface of the pressing member, thereby making the liquid depth in the cell culture bag uniform.

[0031] Figure 4 shows a schematic cross-sectional view of an example of a culture apparatus usable in the present invention. The culture apparatus 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 stage 5 to apply pressure.

[0032] The culture apparatus 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 against the top surface portion 21a of the upper 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 accept 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, so crushing and deformation of the recesses 4 are avoided, preventing cells from flowing out of the recesses 4, and furthermore, gas permeability from the bottom film 22 is increased. The shape of the openings 5b is not limited to openings, for example, it may be a recess, or it may be a wire mesh, and it may be a shape that can accept not only each of the recesses 4, but also a shape that can accept a plurality of recesses 4, as long as crushing and deformation of the recesses 4 are avoided.

[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 culture device having the same configuration as the culture 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, mesenchymal stem cells, adipose stem cells, fibroblasts, myoblasts, chondrocytes, vascular endothelial cells, and the like. Alternatively, multiple cells may be mixed and used to form cell aggregates as organoids.

[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 that possess 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). Currently, there are various types of "induced pluripotent stem cells." These include iPS cells established by Yamanaka et al. by introducing four factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126:663-676), human cell-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131:861-872), Nanog-iPS cells established by selecting cells based on Nanog expression after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPS cells produced by methods that do not include c-Myc (Nakagawa M, Yamanaka iPS cells established by introducing six virus-free factors (S., et al. Nature Biotechnology, (2008) 26, 101-106) can also be used.In addition, induced pluripotent stem cells established by introducing the four factors OCT3 / 4, SOX2, NANOG, and LIN28, as created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), and induced pluripotent stem cells created by Sakurada et al. (Japanese Patent Publication No. 2008-307007) can also be used. In addition, all published papers (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No. Any induced pluripotent stem cells known in the art described in (7,795-797) or patents (e.g., JP 2008-307007, JP 2008-283972, US2008 / 2336610, US2009 / 047263, WO2007 / 069666, WO2008 / 118220, WO2008 / 124133, WO2008 / 151058, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852) may be used.

[0042] Furthermore, the "differentiation-inducing cells" of pluripotent stem cells mentioned above refer to cells characterized by a predetermined phenotype or expression of a specific marker obtained by differentiating pluripotent stem cells. A "marker" refers to a cell antigen or gene that is specifically expressed by a predetermined cell type, such as a "marker protein" or "marker gene." Preferably, the marker is a cell surface marker, in which case enrichment, isolation, and / or detection of viable cells can be performed. The marker may be a positive selection marker or a negative selection marker.

[0043] The detection of marker proteins can be performed using immunological assays (ELISA, immunostaining, flow cytometry, etc.) that utilize antibodies specific to the marker protein. The detection of marker genes can be performed using nucleic acid amplification methods and / or nucleic acid detection methods (RT-PCR, microarrays, biochips, etc.) known in the field. For example, a "positive" result for a marker protein means that it is detected positively by flow cytometry, while a "negative" result means that it is below the detection limit by flow cytometry. Similarly, a "positive" result for a marker gene means that it is detected by RT-PCR, while a "negative" result means that it is below the detection limit by RT-PCR.

[0044] Examples of differentiated cells usable in the present invention include insulin-secreting cells obtained by differentiation induction from pluripotent stem cells. Insulin-secreting cells include insulin-producing cells and / or pancreatic β-cells.

[0045] "Insulin-producing cells" and "pancreatic β-cells" are cells differentiated from pluripotent stem cells according to known methods (WO2009 / 012428, WO2016 / 021734, Stem Cell Research (2015) 14, 185-197). Insulin-producing cells are characterized by the expression of at least one marker, insulin and NK6 homeobox 1 (NKX6.1). Pancreatic β-cells are more mature than insulin-producing cells and are characterized by the expression of at least one marker, MAFA, UCN3, and IAPP.

[0046] 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.

[0047] 4. Cell culture and recovery of cell aggregates The cells are added to the culture medium and cell culture bag, mixed, and then cultured in the bag while pressure is applied to the bag.

[0048] The culture medium can be appropriately selected and used according to the type of cells being used. The cells and culture medium are added through the port on the cell culture bag using a pump such as a peristaltic pump or a syringe, and then mixed. Mixing helps to ensure that the number of cells entering each recess on the bottom of the cell culture bag is approximately uniform in subsequent processes. Mixing can be performed once or multiple times at any point before pressure is applied to the cell culture bag. For example, mixing can be performed before or after placing the cell culture bag on the mounting platform of the culture device, or immediately before applying pressure to the cell culture bag in the culture device. Mixing may be performed manually or by a robotic arm, and / or using a vibrator.

[0049] The number of cells added to the cell culture bag can be adjusted as appropriate depending on factors such as the size of the cell culture bag, the size and number of recesses on the bottom of the cell culture bag, the type of cells used, and the desired size of the cell aggregates. It is not particularly limited, but preferably 1 × 10 5 ~1 × 10 7 cells / mL, more preferably 5 × 10 5 ~5×10 6 The range of cells / mL, and / or 1 × 10 4 ~5×10 6 cells / cm 2 , more preferably 1 × 10 5 ~2×10 6 cells / cm 2 The amount can be selected from a range. If the number of cells added is too little or too much outside this range, it may not be possible to produce large quantities of cell aggregates of a uniform size.

[0050] The amount of culture medium added to the cell culture bag can be adjusted as appropriate depending on factors such as the size of the cell culture bag, the number of cells added to the bag, the culture period, and the desired size of the cell aggregates, and is not particularly limited. However, the amount should preferably be such that the liquid depth of the culture medium added to the cell culture bag when pressure is applied to the cell culture bag (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 the inner surface of the top film) is about 1 mm to 20 mm, more preferably about 2 mm to 8 mm. If too much culture medium is added to the cell culture bag when seeding the cells, the liquid depth will be high, and the higher the liquid depth, the longer it will take for the cells to settle. During this time, the cells may move horizontally due to thermal convection, etc., and may not settle at a uniform concentration, making it difficult to ensure that the number of cells in each recess is approximately uniform. In such cases, the amount of culture medium added when seeding the cells may be reduced, and additional culture medium may be added after the formation of cell aggregates.

[0051] Pressure can be applied to the cell culture bag using the culture apparatus described above, 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.

[0052] The amount of pressure applied to the cell culture bag in the culture apparatus should be sufficient to smooth out any "twists," "wrinkles," "folds," or "bends" formed on the flexible upper and / or lower film facing the pressing member, and to ensure a uniform liquid depth in the cell culture bag. It can be appropriately adjusted depending on factors such as the size and depth of the cell culture bag, the material of the cell culture bag, and the amount of culture medium added to the cell culture bag. While not particularly limited, it is preferably 0.001 to 0.1 kgf / cm². 2 , more preferably 0.002~0.05 kgf / cm² 2The value can be selected from the range. If the applied pressure is too low, it may not be possible to straighten out the "twists," "wrinkles," "folds," "bends," etc., formed on the top and / or bottom films, which may prevent the liquid depth in the cell culture bag from becoming uniform, and ultimately prevent the particle size of the resulting cell aggregates from becoming substantially uniform. On the other hand, if the applied pressure is too high, it may damage the cell culture bag or affect the survival and proliferation of the cells.

[0053] Cell culture is performed in a culture device while maintaining pressure on the cell culture bag. This ensures a uniform liquid depth, which in turn allows for a roughly uniform number of cells entering each recess and / or a roughly uniform distribution of culture medium components. Once cells enter the recess, cell aggregation occurs, forming cell aggregates. The culture conditions can be adjusted according to the cells used, in an incubator set 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%). When moving the culture device containing the cell culture bag into the incubator, this may be done manually or by a robotic arm. The culture period is not particularly limited and can be appropriately determined depending on factors such as the desired cell aggregate size and cell type, but is for example, 1-10 days, preferably 2-7 days. The culture medium may also be changed as needed.

[0054] Next, after the culture is complete, the applied pressure is released and the formed cell aggregates are collected. The collection of the formed cell aggregates can be carried out by any means that allows the cell aggregates to float out of the recess into the culture medium. Such means can be physical, and may include inverting the cell culture bag, applying vibration to the cell culture bag, using a protruding member from the outside of the cell culture bag to push up the recess (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 by a robotic arm, and / or by 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. The cell aggregates floating in the culture medium can be collected together with the culture medium, etc., through the port provided on the cell culture bag.

[0055] 5.Cell aggregates According to the method of the present invention, cell aggregates having substantially uniform particle sizes can be produced simply and in large quantities.

[0056] The number of cell aggregates produced can be adjusted based on the number of recesses on the bottom surface of the cell culture bag, and is not particularly limited, but is preferably between 100,000 and 1,000,000, or between 300,000 and 1,000,000.

[0057] "Having substantially uniform particle size" means that 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more of the manufactured cell aggregates have a particle size within ±10%, preferably within ±5%, of the median particle size of the cell aggregates. The particle size and particle size distribution of the cell aggregates can be measured by combining one or more of the conventionally known methods, such as measurement using microscopic observation, dynamic light scattering, laser diffraction, centrifugal sedimentation, FFF method, and electrical detection method.

[0058] The size of the cell aggregates produced may vary depending on factors such as the type and number of cells used, the size of the cell culture bag, the size and number of recesses on the bottom of the cell culture bag, and the culture period. For example, in one embodiment, 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more of the produced cell aggregates may have a particle size of 100 μm to 200 μm, preferably 120 μm to 180 μm, and more preferably 140 μm to 160 μm. If the size of the cell aggregates is larger than this range, it may cause significant stress to the cells or increase the number of apoptosis, which may hinder the formation of cell aggregates.

[0059] The number of cells contained in a cell aggregate can vary depending on factors such as the type and number of cells used, the size of the cell culture bag, the size and number of recesses on the bottom of the cell culture bag, and the culture period, but it is generally around 200 to 2000 cells per cell aggregate.

[0060] The manufactured cell aggregates can be transplanted into living animals for cell transplantation therapy. The manufactured cell aggregates are useful as cell-based therapies for treating diseases, either as is, encapsulated, or gelled with biodegradable hydrogels and then transplanted to the affected area.

[0061] Furthermore, the manufactured cell aggregates can also be used as prodrugs. A "prodrug" is a group of cells that, after being transplanted into a living organism, differentiate and transform into cells that have the function of treating diseases.

[0062] Furthermore, the manufactured cell aggregates can be administered to the target patient as is, or mixed with a pharmacologically acceptable carrier, as a pharmaceutical composition.

[0063] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0064] Example 1: Production of cell aggregates using insulin-producing cells 1. Preparation of insulin-producing cells Insulin-producing cells were prepared by differentiating and inducing differentiation from human iPS cells according to conventionally known methods (WO2009 / 012428, WO2016 / 021734, Stem Cell Research (2015) 14, 185-197).

[0065] The human iPS cells used were the Ff-I14s04 strain established by Kyoto University.

[0066] For differentiation induction, undifferentiated iPS cells were first seeded into a bioreactor. The iPS cells maintained on a culture dish were treated with EDTA solution and dissociated until they became single cells. Subsequently, the iPS cells dispersed in culture medium were placed into the bioreactor at a rate of 6 × 10⁶ per unit. 6 The seeds were seeded at a density of one cell and cultured in suspension with stirring at 37°C. Stem FitAK03N medium supplemented with 10 μM Y-27632 was used as the culture medium at the time of seeding, and the cells were cultured for 1 day to form cell aggregates.

[0067] Next, differentiation from iPS cells to endodermal cells was induced. The cells were cultured for 1 day in Dulbecco's modified Eagle medium (Thermo Fisher Scientific) containing activin A (10 ng / mL) (PeproTech), the GSK3β inhibitor CHIR99021 (3 μM) (Axon Medchem), 1% dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries) 1% B-27 (registered trademark) (Thermo Fisher Scientific), and 0.1% Pluronic (registered trademark) F68 (Sigma). Subsequently, the cells were cultured for 2 days in Dulbecco's modified Eagle medium containing activin A (10 ng / mL) (PeproTech), 1% dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries) insulin-free B-27 (registered trademark) (Thermo Fisher Scientific), and 0.1% Pluronic (registered trademark) F68 (Sigma).

[0068] Next, differentiation induction from endodermal cells to endocrine progenitor cells was carried out according to conventionally known methods (WO2009 / 012428, WO2016 / 021734, Stem Cell Research (2015) 14, 185-197), (Nature Biotechnology 2014;32:1121-1133)).

[0069] Next, differentiation induction was performed from endocrine progenitor cells to insulin-producing cells. Endocrine progenitor cells were cultured in Iscob's modified Eagle medium-option Zn containing 1% B-27 (registered trademark), magnesium L-ascorbic acid phosphate (58 mg / L) (Fujifilm Wako Pure Chemical Industries), activin receptor-like kinase 5 inhibitor II (10 μM) (Santa Curz), LDN-193189 (100 nM) (Medchemexpress), triiodothyronine (1 μM) (Sigma), RO4929097 (1 μM) (Selleck), and 0.1% Pluronic F68 (Sigma). ++ Insulin-producing cells were obtained by culturing them for 4 days in a medium supplemented with PD-166866 (1 μM) (Sigma) for an additional 3 days. The obtained insulin-producing cells were dispersed into single cells using TrypLE (registered trademark) Select (Thermo Fisher Scientific).

[0070] 2. Production of cell aggregates The cell culture bag used was one with a structure similar to that of the cell culture bag shown in Figure 1. Specifically, it consisted of two flexible films made of 100 μm thick, gas-permeable polyethylene layered and fused around the edges, and had a port. The bottom film of the cell culture bag was 700 cm². 2 A recess with a depth of 150 μm and a diameter of 350 μm is made on the lower surface of 4.5 × 10 5 Individually equipped (642 recesses per unit area of ​​the bottom surface / cm²) 2 The top film has a bulging shape with a height of 4 mm. The inner surface of the film is coated with a low-cell adhesion coating made of phospholipid polymer. The culture apparatus used was the one shown in Figure 5.

[0071] In a cell culture bag, insulin-producing cells (1.25 × 10¹⁶) prepared as described above are placed together with a culture medium containing differentiation factors (triiodothyronine, activin receptor-like kinase 5 inhibitor II, zinc sulfate, heparin, N-acetylcysteine, Trolox, R428, etc.) from a conventionally known method (Nature Biotechnology 2014;32:1121-1133), lock inhibitors (Y27632, etc.), fibroblast growth factor receptor inhibitors (PD-166866, etc.) (MCDB131 / 20mM glucose / sodium bicarbonate / fatty acid-free bovine serum-derived albumin / ITS-X / Glutamax / ascorbic acid / penicillin / streptomycin) according to a conventional method (Nature Biotechnology 2014;32:1121-1133). 6 The cell culture bag (cells / mL) was sealed and the port was closed. The bag was then placed upside down on a flat surface with the recessed side facing upwards, and the contents were agitated by pressing down alternately with both hands from above. Immediately after agitation, the cell culture bag was inverted so that the recessed side was facing downwards and placed on the mounting platform of the culture device. A pressure of 2 kgf was applied from the top surface of the bag using a pressing device. The culture device in this configuration was then moved into the incubator and cultured for 4 days at 37°C and 5% CO2.

[0072] After the culture was complete, the cell culture bag was removed from the culture device and turned inside out, allowing the cell aggregates formed in each recess to suspend from the recesses into the culture medium. A syringe was connected to the port, and the cell aggregates were collected along with the culture medium through the port. Only a small amount of cell aggregates remained in the cell culture bag, indicating that almost all of the cell aggregates were recovered. A photograph of the recovered cell aggregate is shown in Figure 6. Flow cytometry analysis of the cells in the recovered cell aggregate revealed that 41.6% of the cells expressed both insulin and NKX6.1, confirming the production of insulin-producing cells.

[0073] The culture medium containing the recovered cell aggregates was divided into 175 cm³. 2 The cells were transferred to a flask, and the cell aggregates were photographed using a camera attached to a phase-contrast microscope. The particle size of each cell aggregate was then measured from the images. The results are shown in Figure 7.

[0074] More than 70% of the recovered cell aggregates had particle sizes within ±10% of the median particle size of the cell aggregates, with particle sizes ranging from 140 μm to 160 μm.

[0075] These results confirm that, according to the method of the present invention, cell aggregates having substantially uniform particle sizes can be produced in large quantities and easily.

[0076] Example 2: Production of cell aggregates using iPS cells 1. Preparation of iPS cells Human iPS cells (1231A3 strain) were subcultured according to the protocol published by the Center for iPS Cell Research and Application (CiRA) of Kyoto University, detached with TrypLE + EDTA, and suspended in StemFit medium (Ajinomoto) supplemented with 1% lock inhibitor (Y27632).

[0077] 2. Production of cell aggregates The cell culture bag used was one with a similar structure to the cell culture bag shown in Figure 1. Specifically, it consisted of two flexible films made of 100 μm thick, gas-permeable polyethylene layered and fused around the edges, and had a port. The bottom film of the cell culture bag was 50 cm 2 A recess with a depth of 200 μm and a diameter of 500 μm is made on the lower surface of 1.8 × 10 4 Individually equipped (360 recesses per unit area of ​​the bottom surface / cm²) 2 The top film has a bulging shape with a height of 4 mm. The inner surface of the film is coated with a low-cell adhesion coating made of phospholipid polymer.

[0078] Place 20 mL of the aforementioned cell suspension (2.25 × 10) into the cell culture bag. 5The cell culture bag (1 cell / mL) was sealed, the port was closed, and the bag was placed upside down with the recess facing upwards. The suspension inside the bag was agitated by pressing down alternately with both hands from above. Immediately after agitation, the cell culture bag was inverted so that the recess was facing downwards and placed on the mounting platform of the culture apparatus. A pressure of 2 kgf was applied to the top surface of the bag using a pressing device. For the control group, the procedure was the same except that the 2 kgf pressure was not applied. The culture apparatus in this configuration was then moved into an incubator and cultured for 2 days under conditions of 37°C and 5% CO2.

[0079] After the culturing was complete, the cell culture bag was removed from the culture device and turned inside out, allowing the cell aggregates formed in each recess to suspend from the recesses into the culture medium. A syringe was connected to the port, and the culture medium containing the cell aggregates was collected through the port.

[0080] The culture medium containing the recovered cell aggregates was transferred entirely to a 10 cm petri dish. The cell aggregates were photographed using a camera attached to a phase-contrast microscope, and the particle size of each cell aggregate was measured from the images. The results are shown in Figure 8.

[0081] When comparing the particle size deviations of the recovered cell aggregates, those produced by culturing without applying pressure showed a deviation of approximately ±10% of the center particle size, while those obtained by culturing with applied pressure showed a deviation of approximately ±5% of the center particle size.

[0082] These results confirm that, according to the method of the present invention, cell aggregates having substantially uniform particle sizes can be produced in large quantities and easily. [Explanation of Symbols]

[0083] 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'' culture equipment 11 Cell aggregates

Claims

1. A method for producing cell aggregates using a cell culture bag, The cell culture bag has an upper surface and a lower surface, and the lower surface has multiple recesses. (1) Add cells and culture medium to a cell culture bag and mix, and culture the cell culture bag while applying pressure from at least one direction. (2) After the culturing is complete, a step to collect the formed cell aggregates, A method that includes this.

2. The method according to claim 1, wherein 100,000 to 1,000,000 cell aggregates are recovered.

3. The method according to claim 1 or 2, wherein the recovered cell aggregates have substantially uniform particle sizes.

4. The method according to any one of claims 1 to 3, wherein 70% or more of the recovered cell aggregates have a particle size within ±10% of the median particle size of the cell aggregates.

5. The method according to any one of claims 1 to 4, wherein 70% or more of the recovered cell aggregates have a particle size of 140 μm to 160 μm.

6. The method according to any one of claims 1 to 5, wherein each recovered cell aggregate contains 200 to 2000 cells.

7. The method according to any one of claims 1 to 6, wherein the cells are freeze-thawed cells.

8. The method according to any one of claims 1 to 7, wherein the cells are insulin-secreting cells.

9. The method according to any one of claims 1 to 8, wherein the cell culture bag is inverted upside down to collect the cell aggregates.

10. The method according to any one of claims 1 to 9, wherein air is injected into a cell culture bag to collect cell aggregates.

11. The method according to any one of claims 1 to 10, wherein the step of culturing cells in a cell culture bag while applying pressure from at least one direction is the step of culturing cells in a cell culture bag while applying pressure from above, below, or both above and below.

12. The method according to claim 11, wherein the step of culturing the cell culture bag while applying pressure from at least one direction is the step of culturing while applying pressure from above.

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

5.

14. The method according to any one of claims 1 to 13, wherein the recess is spherical in shape.

15. The method according to any one of claims 1 to 14, wherein the pitch of the recesses is 0.35 mm to 0.49 mm.

16. The method according to any one of claims 1 to 15, wherein the depth of the recess is 150 μm to 220 μm.

17. The method according to any one of claims 1 to 16, wherein the liquid depth in the cell culture bag is 1.5 mm to 6 mm.

18. Place cells in a cell culture bag at a rate of 1 x 10 5 ~1 x 10 7 The method according to any one of claims 1 to 17, wherein the amount is added is cells / mL.

19. Place cells in a cell culture bag at a rate of 1 x 10 4 ~5 x 10 6 cells / cm 2 The method according to any one of claims 1 to 18, wherein the amount is added.

20. The method according to any one of claims 1 to 19, wherein culture medium is added after cell aggregate formation.

21. The method according to any one of claims 1 to 20, wherein the lower surface is provided with 100,000 to 1,000,000 recesses.

22. 1 to 1000 recesses per unit area of ​​the bottom surface / cm² 2 The method according to any one of claims 1 to 21, comprising:

23. A population of cell aggregates for transplantation, wherein more than 70% of the cell aggregates have a particle size of 140 μm to 160 μm.

24. A population of cell aggregates according to claim 23, comprising 100,000 to 1,000,000 cell aggregates.

25. A population of cell aggregates according to claim 23 or 24, comprising insulin-secreting cells.

26. A cell culture bag containing 100,000 to 1,000,000 cell aggregates with substantially uniform particle size.

27. The cell culture bag according to claim 26, wherein more than 70% of the cell aggregates have a particle size of 140 μm to 160 μm.

28. The cell culture bag according to claim 26 or 27, wherein the cell aggregate contains insulin-secreting cells.

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