Method for producing cell capsule, method for producing cell population or cell product, solution, and cell population

Abstract

Provided is a method for producing a cell capsule capable of alleviating an adverse effect on a cell. This method for producing a cell capsule includes continuously or intermittently causing a hydrogel or a hydrogel precursor covering a cell to flow into a collection solution. The collection solution contains a pH buffer.

Description

Method for producing cell capsules, method for producing cell populations or cell products, solution and cell population

[0001] The present invention relates to a method for producing cell capsules, a method for producing a cell population or a cell product, and a solution and a cell population used in the method for producing cell capsules.

[0002] Currently, attempts are being made to culture a variety of cells in various fields, including developmental biology, drug discovery, and regenerative medicine. These cells are typically cultured two-dimensionally on the surface of plastic tissue culture vessels. Techniques for culturing cells in a three-dimensional or pseudo-three-dimensional culture environment using scaffolds such as porous membranes and hydrogels are known. The following Patent Documents 1 to 6 disclose culturing cells inside tubular or spherical hydrogels.

[0003] In recent years, spheroid culture, in which cells are cultured and aggregated in three dimensions, has attracted attention as an alternative to monolayer culture, which cultures cells in two dimensions. Compared to monolayer culture, spheroid culture can create a state closer to that of cells in vivo and is known to be able to bring out the specific functions that cells possess in vivo. For this reason, cell aggregates are expected to be useful tools in, for example, drug discovery research.

[0004] International Publication No. WO 2011 / 046105, International Publication No. WO 2017 / 091662, International Publication No. WO 2018 / 098295, International Publication No. WO 2019 / 178549, International Publication No. WO 2020 / 032221, International Publication No. WO 2009 / 108138, JP 2017-99303

[0005] The inventors of the present application have discovered the following new problem in producing tubular or spherical hydrogels as described in Patent Documents 1 to 6. In Patent Documents 1 to 6, a hydrogel precursor is continuously or intermittently discharged from a nozzle into a solution to produce a tubular or spherical hydrogel in the solution. If the hydrogel precursor is discharged for a long period of time for the purpose of encapsulating a large number of cells in the hydrogel, such as for mass cell culture, the pH (hydrogen ion exponent) of the solution will fluctuate, which may have an adverse effect on the cells encapsulated in the hydrogel.

[0006] Therefore, there is a need for a method for producing cell capsules and a method for producing cell populations or cell products that can mitigate the adverse effects on cells.

[0007] A method for producing cell capsules according to one embodiment includes continuously or intermittently flowing a hydrogel or a hydrogel precursor that covers cells into a capture solution, the capture solution including a pH buffer.

[0008] A method for producing a cell population or cell product according to one embodiment includes culturing cells inside a hydrogel using cell capsules produced by the above-described method for producing cell capsules.

[0009] The cell population according to one embodiment may be a cell population produced by the above-described method for producing a cell population or cell product.

[0010] The solution according to one embodiment is a solution used for producing cell capsules having a hydrogel that encases cells, and includes a pH buffer.

[0011] FIG. 1 is a schematic diagram showing the structure of a cell capsule according to one embodiment. FIG. 2 is a schematic cross-sectional view of the cell capsule shown in FIG. 1. FIG. 3 is a schematic diagram showing the structure of a cell capsule according to another embodiment. FIG. 4 is a schematic diagram illustrating a method for manufacturing a cell capsule according to the first embodiment. FIG. 5 is a schematic diagram illustrating a method for manufacturing a cell capsule according to the second embodiment. FIG. 6 is a schematic diagram illustrating a method for manufacturing a cell capsule according to the third embodiment. FIG. 7 is a graph showing the results of measuring the amount of lactate in the culture medium of human iPS cells on Day 7 in Reference Examples 1 to 6 and Examples 1 to 6. FIG. 8 is a graph showing the results of measuring the proliferation rate of human iPS cells on Day 7 in Reference Examples 1 to 6 and Examples 1 to 6. FIG. 9 is a graph showing the results of measuring the amount of lactate in the culture medium of human iPS cells on Day 7 in Examples 7 to 10. FIG. 10 is a graph showing the results of measuring the amount of lactate in the culture medium of human iPS cells on Day 7 in Reference Examples 7 to 10. Figure 11 is an enlarged photograph showing cell capsules on Day 2 in Examples 11 to 22. Figure 12 is a graph showing the results of measuring the amount of lactate in the medium of K562 cells on Day 2 in Examples 11 to 22. Figure 13 is a graph showing the results of measuring the amount of lactate in the medium of K562 cells on Day 2 in Reference Examples 11 to 22.

[0012] Hereinafter, embodiments will be described with reference to the drawings. In the following drawings, the same or similar parts are denoted by the same or similar reference numerals. However, it should be noted that the drawings are schematic and the ratios of the dimensions may differ from those of the actual parts.

[0013] The cell capsule has cells encased in a hydrogel. One or more cells may be encased in the hydrogel. The cells may be dispersed or in the form of cell aggregates. The hydrogel may encase the cells, and may have, for example, a generally tubular, string-like, spherical, or spherical shape.

[0014] FIG. 1 is a schematic diagram showing the configuration of a cell capsule according to one embodiment. FIG. 2 is a schematic cross-sectional view of the cell capsule shown in FIG. 1. In the embodiment shown in FIGS. 1 and 2, the hydrogel 14 has a generally tubular shape. Specifically, the cell capsule may include a tubular hydrogel 14 and a core 12 covered by the hydrogel 14. Cells are provided in the core 12. The hydrogel 14 surrounds the periphery of the core 12. More specifically, the hydrogel 14 surrounds the core 12 in a cross section perpendicular to the direction in which the hydrogel 14 extends. The tubular hydrogel 14 extends long, and the core 12 extends long like a string inside the tubular hydrogel 14. One or more cells (including spheroids) may be contained in one cell capsule.

[0015] The length of the hydrogel 14 may be, for example, 5 cm or more, more preferably 10 cm or more, and even more preferably 20 cm or more. The longer the hydrogel, the more cells can be contained inside one hydrogel. The length of the hydrogel 14 is not particularly limited, but may be, for example, 10 m or less.

[0016] FIG. 3 is a schematic diagram showing the configuration of a cell capsule according to another embodiment. In the embodiment shown in FIG. 3, the cell capsule has a substantially spherical shape. The hydrogel 14 has a substantially spherical shell shape. Specifically, the cell capsule may include a spherical hydrogel 14 and a core 12 covered by the hydrogel 14. The core 12 is substantially spherical and is provided inside the spherical hydrogel 14. Cells are provided within the core 12. The hydrogel 14 surrounds the entire core 12. The cell capsule according to the embodiment shown in FIG. 3 can also be considered to be a cell capsule according to the embodiment shown in FIGS. 1 and 2 with a shorter length. One or more cells (including spheroids) may be contained in a single cell capsule.

[0017] The outer diameter of the hydrogel 14 is not particularly limited, but may be, for example, in the range of 40 μm to 4000 μm, preferably in the range of 80 μm to 2000 μm, and more preferably in the range of 100 μm to 1000 μm. When the hydrogel has a tubular shape, the outer diameter of the hydrogel may be determined by the outer diameter of the hydrogel in a cross section perpendicular to the direction in which the cell capsules extend (see symbol R1 in FIG. 2).

[0018] The inner diameter of the hydrogel is not particularly limited, but may be, for example, in the range of 10 μm to 3000 μm, preferably in the range of 20 μm to 1000 μm, and more preferably in the range of 40 μm to 500 μm. When the hydrogel has a tubular shape, the inner diameter of the hydrogel may be determined by the inner diameter of the hydrogel in a cross section perpendicular to the direction in which the cell capsule extends (see symbol R2 in Figure 2). The aforementioned inner and outer diameters can be measured, for example, using a phase-contrast optical microscope.

[0019] The hydrogel 14 is obtained by gelling a hydrogel precursor. The hydrogel 14 is preferably one that can be sufficiently permeable to cell culture medium components.

[0020] The hydrogel 14 may include at least one selected from the group consisting of, for example, alginate gel, Matrigel, collagen gel, chitosan gel, gelatin, peptide gel, laminin gel, agarose gel, nanocellulose, methylcellulose, xytran, pectin, gellan gum, xanthan gum, guar gum, carrageenan, glucomannan, and fibrin gel.

[0021] Preferably, the hydrogel 14 is a gel whose main component is alginate, i.e., an alginate gel. In this case, the hydrogel precursor may be a solution whose main component is an alginate solution. The alginate gel can be formed by crosslinking an alginate solution, which is an alginate derivative, with a divalent metal ion. The alginate gel can be suitably used as a hydrogel for culturing cells or cell clusters in a state in which the cells or cell clusters are enclosed.

[0022] [First embodiment] In a method for manufacturing a cell capsule, first, a core, a hydrogel precursor, a gelling solution, and a collection solution are prepared. The core may be a cell suspension. The hydrogel precursor may contain a material that forms a hydrogel by gelation. The gelling solution may contain a material that gels the hydrogel precursor.

[0023] The method for producing cell capsules involves sequentially flowing a hydrogel or hydrogel precursor that encases the cells into a collection solution, the compositions of which are described in detail below: core, hydrogel precursor, gelling solution, and collection solution.

[0024] The method for producing a cell capsule according to the first embodiment will be described in more detail with reference to FIG. 4 . FIG. 4 is a schematic diagram for explaining the method for producing a cell capsule according to the first embodiment. The device for producing a cell capsule may have a first inlet 210, a second inlet 220, a third inlet 230, and an injection port 240. The first inlet 210 is an inlet for introducing a core. The second inlet 220 is an inlet for introducing a hydrogel precursor. The third inlet 230 is an inlet for introducing a solution (gelling solution) that gels the hydrogel precursor.

[0025] The hydrogel precursor flows in from the second inlet 220 and merges around the core. As a result, the hydrogel precursor flows around the core and in the direction of the core flow. That is, the hydrogel precursor surrounds the core in a cross section perpendicular to the core flow. More specifically, it is preferable that the hydrogel precursor and the core flow to form a laminar flow.

[0026] The gelling solution is a solution for gelling the hydrogel precursor to form a hydrogel. The gelling solution is joined to the core and the hydrogel precursor downstream of the joining point of the core and the hydrogel precursor. The gelling solution flows along with the flow of the hydrogel precursor around the hydrogel precursor surrounding the core. That is, the gelling solution surrounds the hydrogel precursor in a cross section perpendicular to the flow of the hydrogel precursor. It is preferable that the gelling solution forms a laminar flow together with the core and the hydrogel precursor.

[0027] In the first embodiment, the gelling solution merges with the hydrogel precursor, causing the hydrogel precursor to become a hydrogel. The hydrogel precursor may be completely in a hydrogel state when it flows out of the injection port 240, or may flow out in precursor form from the injection port 240. Therefore, the hydrogel or hydrogel precursor covers the core containing the cells and flows out into the collection solution 20 together with the core.

[0028] Furthermore, in the first embodiment, the gelling solution that covers the hydrogel or hydrogel precursor flows out into the collection solution together with the hydrogel or hydrogel precursor.

[0029] As shown in Figure 4, the hydrogel or hydrogel precursor is continuously drained into the collection solution together with the core. From the start of draining the hydrogel or hydrogel precursor, the hydrogel formed from the hydrogel or hydrogel precursor is maintained in the collection solution for, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more. The time for draining the core, hydrogel or hydrogel precursor, and gelling solution may be, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more.

[0030] The time for which the hydrogel or the hydrogel formed from the hydrogel precursor is maintained in the collection solution is not particularly limited, and may be, for example, 24 hours or less, 20 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, or 6 hours or less from the start of outflow of the hydrogel or hydrogel precursor. The time for which the core, hydrogel or hydrogel precursor, or gelling solution is allowed to outflow is also not particularly limited, and may be, for example, 24 hours or less, 20 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, or 6 hours or less.

[0031] In Figure 4, the hydrogel or hydrogel precursor is continuously discharged into the collection solution together with the core. However, the hydrogel or hydrogel precursor may be discharged intermittently into the collection solution together with the core.

[0032] In order to produce a large amount of cell population, the fluid with the fastest flow rate among the core, the hydrogel precursor or hydrogel, and the gelling solution may be flowed into the collection solution at a rate of, for example, 1 mL or more. Preferably, the cell-containing core may be flowed into the collection solution at a rate of, for example, 1 mL or more.

[0033] The average temperature of the trapping solution may be, for example, 40°C or less, 30°C or less, preferably 25°C or less, and more preferably 20°C or less. By lowering the average temperature of the trapping solution, adverse effects on cells can be mitigated. The average temperature can be calculated by measuring the temperatures of multiple portions of the trapping solution and dividing the measured temperature by the number of portions. If the temperature of the trapping solution is generally uniform throughout the trapping solution, the average temperature of the trapping solution can also be determined from the temperature at one point in the trapping solution.

[0034] The average temperature of the trapping solution may be, for example, 0°C or higher, 2°C or higher, or 4°C or higher. The temperature of the trapping solution may be within a range that arbitrarily combines the above upper and lower limits. The average temperature of the trapping solution is desirably maintained within the above range while the hydrogel or hydrogel precursor is being poured into the trapping solution. Alternatively, the average temperature of the trapping solution may be set within the above range immediately after the hydrogel or hydrogel precursor has been poured into the trapping solution. When the average temperature of the trapping solution is set within the above range immediately after the hydrogel or hydrogel precursor has been poured into the trapping solution, the cell capsules may be maintained in the trapping solution for, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more. The time for which the cell capsules are maintained in the trapping solution is not particularly limited, but may be, for example, 24 hours or less.

[0035] The core may be a cell suspension, and may contain at least one selected from the group consisting of physiological saline, an aqueous solution of inorganic salts, an aqueous solution of sugars, a culture medium, a culture supernatant, a buffer solution, an extracellular matrix, a thickener, and a hydrophilic resin (such as polyvinyl alcohol, polyacrylamide, or poly(N-isopropylacrylamide)).

[0036] The thickener may include at least one selected from the group consisting of, for example, chitosan gel, collagen solution, Matrigel, collagen, gelatin, alginate solution, alginate gel, peptide gel, laminin, agarose, nanocellulose, methylcellulose, hyaluronic acid, proteoglycan, elastin, pullulan, dextran, pectin, gellan gum, xanthan gum, guar gum, carrageenan, and glucomannan.

[0037] The core may contain various growth factors suitable for cell culture, cell maintenance and proliferation, or cell function expression, etc. Such growth factors may be, for example, at least one selected from the group consisting of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF).

[0038] The type of cell is not particularly limited. The cell may be, for example, an animal cell, preferably a human cell. The cell may be, for example, various types of pluripotent stem cells, human ES cells or human iPS cells, pluripotent stem cells, oligopotent or unipotent progenitor cells, etc. Examples of various types of pluripotent and pluripotent stem cells include iPS cells, ES cells, mesenchymal stem cells, neural stem cells, hepatic stem cells, skin stem cells, pancreatic stem cells, etc. Examples of stem cells and progenitor cells with oligopotent or unipotent properties include muscle stem cells, germ stem cells, respiratory progenitor cells, digestive progenitor cells, cardiac progenitor cells, etc. The adhesive cells may also be various differentiated cells, such as muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and cardiac muscle cells), neurons (e.g., cerebral cortical cells), fibroblasts, epithelial cells, endothelial cells, adipocytes, osteoblasts, chondrocytes, macrophages, dendritic cells, hepatocytes, hepatic stellate cells, pancreatic beta cells, keratinocytes, kidney cells, renal tubular cells, hair matrix cells, corneal endothelial cells, photoreceptors, pigment epithelial cells, goblet cells, and respiratory cells. Cells produced by inducing differentiation of these pluripotent or multipotent cells may also be used. Furthermore, the cells may be CHO cells or human hepatoma-derived cell lines. Furthermore, the cells include tissues or organs formed by aggregation of the aforementioned cells, as well as fungi (e.g., bacteria, oomycetes, myxomycetes, and fungi), and microorganisms (e.g., algae). Examples of fungi include koji mold and yeast.

[0039] The material constituting the hydrogel precursor is appropriately selected depending on the material of the hydrogel described above. That is, the hydrogel precursor may contain at least one precursor selected from the group consisting of alginate gel, Matrigel, collagen gel, chitosan gel, gelatin, peptide gel, laminin gel, agarose gel, nanocellulose, methylcellulose, dextran, pectin, gellan gum, xanthan gum, guar gum, carrageenan, glucomannan, fibrin gel, tetraPEG gel, polyacrylamide gel, polyrotaxane, and poly(N-isopropylacrylamide) polymer.

[0040] Preferably, the hydrogel precursor may be a solution (alginic acid derivative) whose main component is an alginic acid solution. The alginic acid solution may be, for example, sodium alginate, potassium alginate, ammonium alginate, or a combination thereof. The alginic acid derivative may be a natural extract or a chemically modified alginic acid. Examples of chemically modified alginic acid include methacrylate-modified alginic acid. Alginic acid solutions are easily crosslinked by divalent metal ions in a short time at or near room temperature, and tend to form alginic acid gels.

[0041] The capture solution contains a pH buffer, which stably maintains the pH of the capture solution. In the first embodiment, during the production of cell capsules, the hydrogel, hydrogel precursor, and / or gelling solution flows into the capture solution. In particular, when producing long cell capsules, the hydrogel, hydrogel precursor, and / or gelling solution flows into the capture solution for a long period of time. Even in such cases, if the capture solution contains a pH buffer, the pH of the capture solution is stably maintained for a long period of time. This can mitigate adverse effects on the cells in the cell capsules in the capture solution.

[0042] The capture solution may be, for example, acetate buffer, phosphate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, phosphate-buffered saline, or a so-called Good's buffer. Preferably, the pH buffer in the capture solution is a Good's buffer. Some Good's buffers are unlikely to form complexes with metal ions, and therefore may be suitable for use even when mixed with the gelling solution.

[0043] Examples of Good's buffers include ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), ADA (N-(2-acetamido)iminodiacetic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Bis-Tris (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), CAPS (3-cyclohexylaminopropanesulfonic acid), CAPSO (N-cyclohexylaminopropanesulfonic acid), and the like. N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), CHES (2-cyclohexylaminoethanesulfonic acid), DIPSO (3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), EPPS (4-(2-hydroxyethyl)-1-piperazine-1-propanesulfonic acid), HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), HEPES-Na (2-[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid), Sodium sulfonate), HEPPSO (4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid)), MES (2-morpholinoethanesulfonic acid monohydrate), MOPS (3-morpholinopropanesulfonic acid), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), PIPES (piperazine-1,4-bis(2-ethanesulfonic acid)), PIPES sodium salt (sodium piperazine-1,4-bis(2-ethanesulfonate)), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid), TAPS (N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), and Tricine (N-[tris(hydroxymethyl)methyl]glycine).

[0044] The capture solution may contain nutrients such as a carbon source, vitamins, and inorganic salts. For example, the capture solution may contain a medium with buffering capacity. The type of medium may be selected appropriately depending on the type of cells to be accommodated inside the tubular hydrogel.

[0045] The collection solution preferably contains a gelling agent that gels the hydrogel precursor. The gelling agent may be any material capable of forming a hydrogel by crosslinking the hydrogel precursor. For example, when the hydrogel precursor is alginic acid, the gelling agent may be a material capable of providing divalent cations. Examples of such gelling agents include salts of magnesium, calcium, strontium, or barium. Preferably, the gelling agent may be, for example, calcium chloride, calcium carbonate, calcium hydroxide, barium chloride, barium carbonate, or barium hydroxide. That is, the collection solution may contain divalent cations such as calcium ions or barium ions.

[0046] The concentration of the gelling agent, for example, the concentration of calcium chloride or barium chloride, may be, for example, 0 to 500 mmol / L, preferably 5 to 200 mmol / L.

[0047] When the collection solution contains a gelling agent, the buffer is preferably one that is not substantially precipitated by the gelling agent. When the collection solution contains a gelling agent, even if the hydrogel precursor flows out into the collection solution before becoming a hydrogel, the hydrogel precursor can become a hydrogel in the collection solution.

[0048] The pH of the capture solution may be, for example, in the range of 6.5 to 8.0, preferably 6.8 to 7.5. By keeping the pH in this range, adverse effects on cells can be more effectively suppressed. When the capture solution contains a pH buffer, the pH of the capture solution is likely to be maintained in the range of, for example, 6.5 to 8.0, preferably 6.8 to 7.5, during the time period during which the hydrogel precursor or hydrogel is released and / or during the time period during which the cells are incubated in the cell capsules.

[0049] The osmotic pressure of the capture solution may be, for example, in the range of 100 to 1500 mOsm, preferably 200 to 400 mOsm. When the osmotic pressure is in this range, adverse effects on cells can be further suppressed.

[0050] The gelling solution may be a solution containing a gelling agent. The gelling agent may be any material capable of forming a hydrogel by crosslinking the hydrogel precursor. For example, when the hydrogel precursor is alginic acid, the gelling agent may be any material capable of providing divalent cations. Examples of such gelling agents include salts of magnesium, calcium, strontium, or barium. Preferably, the gelling agent may be, for example, calcium chloride, calcium carbonate, calcium hydroxide, barium chloride, barium carbonate, or barium hydroxide. That is, the gelling solution may contain divalent cations such as calcium ions or barium ions.

[0051] The concentration of the divalent metal ion in the gelling solution, such as the concentration of calcium chloride or barium chloride, may be, for example, 10 to 500 mmol / L, more preferably 20 to 200 mmol / L.

[0052] The gelling solution preferably contains a pH buffer. In this case, it is more preferable that the pH buffer is a Good's buffer. As the pH buffer, those listed above can be used. In this case, even if a large amount of gelling solution flows into the collection solution, the hydrogen ion exponent in the collection solution is likely to be maintained stably. Therefore, the adverse effects on the cells in the cell capsules in the collection solution can be more mitigated.

[0053] The gelling solution may contain nutrients such as a carbon source, vitamins, and inorganic salts. For example, the gelling solution may contain a culture medium having buffering capacity. The type of culture medium may be appropriately selected depending on the type of cells to be accommodated inside the tubular hydrogel.

[0054] The pH of the gelling solution may be, for example, in the range of 6.5 to 8.0, preferably 6.8 to 7.5. When the pH is in this range, adverse effects on cells can be further suppressed.

[0055] The osmotic pressure of the gelling solution may be, for example, in the range of 100 to 1500 mOsm, preferably 200 to 400 mOsm. When the osmotic pressure is in this range, adverse effects on cells can be further suppressed.

[0056] The gelling solution preferably contains the same pH buffer as the collecting solution. In this case, even if a large amount of the gelling solution flows into the collecting solution, the pH of the collecting solution is likely to be maintained stable. The gelling solution more preferably has the same composition as the collecting solution. In this case, even if a large amount of the gelling solution flows into the collecting solution, the pH of the collecting solution is likely to be maintained stable.

[0057] [Second embodiment] A method for manufacturing a cell capsule according to the second embodiment will be described. Fig. 5 is a schematic diagram for explaining the method for manufacturing a cell capsule according to the second embodiment. In the second embodiment, the description of the same configuration and / or process as in the first embodiment may be omitted.

[0058] First, a core, a hydrogel precursor, a gelling solution, and a collection solution are prepared. The materials and cells constituting the core, the hydrogel precursor, and the collection solution are the same as those in the first embodiment.

[0059] 5, the apparatus for producing cell capsules may have a first inlet 210, a second inlet 220, and an injection port 240. The first inlet 210 is an inlet for introducing the cores. The second inlet 220 is an inlet for introducing the hydrogel precursor. In the second embodiment, the injection port 240 is immersed in the collection solution.

[0060] The hydrogel precursor flows in from the second inlet 220 and merges around the core. As a result, the hydrogel precursor flows around the core and in the direction of the core flow. That is, the hydrogel precursor surrounds the core in a cross section perpendicular to the core flow. More specifically, it is preferable that the hydrogel precursor and the core flow to form a laminar flow.

[0061] In a second embodiment, the collection solution contains a gelling agent that gels the hydrogel precursor. In this second embodiment, the hydrogel precursor coats the cell-laden core and flows continuously with the core into the collection solution. The hydrogel precursor gels in the collection solution to form a hydrogel. This forms a long tubular hydrogel that coats the cell-laden core.

[0062] As shown in Figure 5, the hydrogel precursor is continuously released into the collection solution together with the core. The core released together with the hydrogel precursor is maintained in the collection solution for, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more from the start of the release of the hydrogel precursor. The time for releasing the core and hydrogel precursor may be, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more.

[0063] The time for which the cores that have flowed out together with the hydrogel precursors are maintained in the collection solution is not particularly limited, and may be, for example, 24 hours or less, 20 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, or 6 hours or less from the start of outflow of the cores and hydrogel precursors. The time for which the cores and hydrogel precursors are allowed to flow out is also not particularly limited, and may be, for example, 24 hours or less, 20 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, or 6 hours or less.

[0064] The average temperature of the capture solution may be, for example, 40° C. or less, 30° C. or less, preferably 25° C. or less, and more preferably 20° C. or less. By lowering the average temperature of the capture solution, adverse effects on cells can be alleviated.

[0065] The average temperature of the trapping solution may be, for example, 0°C or higher, 2°C or higher, or 4°C or higher. The average temperature of the trapping solution may be within a range that arbitrarily combines the above upper and lower limits. The average temperature of the trapping solution is desirably maintained within the above range while the hydrogel precursor is being drained into the trapping solution. Alternatively, the average temperature of the trapping solution may be set within the above range immediately after the hydrogel precursor has been drained into the trapping solution. When the average temperature of the trapping solution is set within the above range immediately after the hydrogel precursor has been drained into the trapping solution, the cell capsules may be maintained in the trapping solution for, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more. The time for which the cell capsules are maintained in the trapping solution is not particularly limited, but may be, for example, 24 hours or less.

[0066] In order to produce a large amount of cell population, the fluid having the faster flow rate, either the core or the hydrogel precursor, may be flowed into the collection solution at a rate of, for example, 1 mL or more. Preferably, the cell-containing core may be flowed into the collection solution at a rate of, for example, 1 mL or more.

[0067] In the second embodiment, it is also possible to produce long cell capsules as explained in FIGS.

[0068] [Third embodiment] A method for manufacturing a cell capsule according to a third embodiment will be described. Fig. 6 is a schematic diagram for explaining a method for manufacturing a cell capsule according to the third embodiment. In the third embodiment, the description of the same configuration and / or process as in the first and second embodiments may be omitted.

[0069] First, a core, a hydrogel precursor, a gelling solution, and a collection solution are prepared. The materials and cells constituting the core, the hydrogel precursor, and the collection solution are the same as those in the first and second embodiments.

[0070] 6, the device for producing cell capsules may have a first inlet 210, a second inlet 220, and an ejection port 240, similar to the second embodiment. The first inlet 210 is an inlet for introducing the cores. The second inlet 220 is an inlet for introducing the hydrogel precursor. In the third embodiment, the ejection port 240 is located above the collection solution.

[0071] In the third embodiment, the collection solution contains a gelling agent that gels the hydrogel precursor. In the third embodiment, the hydrogel precursor covers the cell-containing core and intermittently flows out into the collection solution together with the core. At this time, the hydrogel precursor and core become approximately spherical droplets before reaching the collection solution from the injection port 240. The hydrogel precursor and core are introduced into the collection solution in the form of approximately spherical droplets. The hydrogel precursor gels in the collection solution to form a hydrogel. This produces a cell capsule that includes an approximately spherical core containing cells and an approximately spherical shell-shaped hydrogel covering the core.

[0072] As shown in Figure 6, the hydrogel precursor is intermittently released into the collection solution together with the cores. The cores released together with the hydrogel precursor are maintained in the collection solution for, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more from the start of the release of the hydrogel precursor. The time for releasing the cores and hydrogel precursor may be, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more. This allows for the production of a large number of spherical cell capsules.

[0073] The time for which the cores that have flowed out together with the hydrogel precursors are maintained in the collection solution is not particularly limited, and may be, for example, 24 hours or less, 20 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, or 6 hours or less from the start of outflow of the hydrogel precursors. The time for which the cores and hydrogel precursors are allowed to flow out is also not particularly limited, and may be, for example, 24 hours or less, 20 hours or less, 16 hours or less, 12 hours or less, 8 hours or less, or 6 hours or less.

[0074] The average temperature of the capture solution may be, for example, 40° C. or less, 30° C. or less, preferably 25° C. or less, and more preferably 20° C. or less. By lowering the average temperature of the capture solution, adverse effects on cells can be alleviated.

[0075] The average temperature of the trapping solution may be, for example, 0°C or higher, 2°C or higher, or 4°C or higher. The average temperature of the trapping solution may be within a range that arbitrarily combines the above upper and lower limits. The average temperature of the trapping solution is desirably maintained within the above range while the hydrogel precursor is being drained into the trapping solution. Alternatively, the average temperature of the trapping solution may be set within the above range immediately after the hydrogel precursor has been drained into the trapping solution. When the average temperature of the trapping solution is set within the above range immediately after the hydrogel precursor has been drained into the trapping solution, the cell capsules may be maintained in the trapping solution for, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or 4 hours or more. The time for which the cell capsules are maintained in the trapping solution is not particularly limited, but may be, for example, 24 hours or less.

[0076] In order to produce a large amount of cell population, the fluid having the faster flow rate, either the core or the hydrogel precursor, may be flowed into the collection solution at a rate of, for example, 1 mL or more. Preferably, the cell-containing core may be flowed into the collection solution at a rate of, for example, 1 mL or more.

[0077] In the above embodiments, a method for producing a cell capsule having a cell-containing core and a hydrogel covering the core has been described. However, it should be noted that the cell capsule produced by the production method of the present invention is not limited to this. For example, the cell capsule may have a structure containing a hydrogel in which cells are embedded. This can be achieved, for example, by having the core contain both a hydrogel precursor and cells. Even in this case, if the capture solution used to capture the hydrogel precursor or hydrogel during cell capsule production contains a pH buffer, adverse effects on the cells can be mitigated.

[0078] The cell capsules produced by the above-described cell capsule production method are used, for example, for cell culture. In this case, the cells are cultured by immersing the cell capsules in a medium. This allows the cells to be cultured inside the hydrogel.

[0079] The cell population of the present invention may be a cell population produced by the above-mentioned cell culture method. In one specific example, the cell population is formed by recovering cells from inside a hydrogel. Cell recovery can be achieved, for example, by dissolving and removing the hydrogel. The hydrogel can be removed by chemical reaction, enzymatic reaction, or the like. Specifically, the hydrogel can be removed by, for example, an EDTA / PBS solution, alginate lyase, or a combination thereof.

[0080] The cells or cell populations can produce a cell product while encapsulated in the cell capsule. This cell product can be produced, for example, by recovering the cell product from the culture medium in which the cells or cell populations are cultured. The cell product may be, but is not limited to, a protein, an extracellular vesicle, an antibody, or the like.

[0081] Example 1 Example 1 is described below. First, a core, a hydrogel precursor, a gelling solution, and a collection solution were prepared. The core was a cell suspension. The cell suspension was prepared by suspending human iPS cells (QHJI01s04) in Iscove's modified Dulbecco's medium containing 3% methylcellulose (R&D systems HSC001) and adding 0.6 w / v% methylcellulose. The initial cell density in the cell suspension was 1 x 10 ７ cells / mL.

[0082] The hydrogel precursor was a sodium alginate solution prepared by adding the above-mentioned sodium alginate ("High-G ALG300" manufactured by Kimika Co., Ltd.) to saline and stirring. The concentration of sodium alginate relative to the saline was 1.0 w / v%.

[0083] The gelling solution was an aqueous solution containing 100 mM calcium chloride and 3% w / v sucrose.

[0084] Using these cores, hydrogel precursor, and gelling solution, a cell mass was produced based on the cell capsule production method according to the first embodiment (see FIG. 4). That is, a core flow, a hydrogel precursor flow around the core flow, and a gelling solution flow around the hydrogel precursor flow were formed, and these flows were ejected into the collection solution from the ejection port 240.

[0085] The hydrogel precursor was crosslinked upon contact with the gelling solution to form a tubular alginate gel, producing a hydrogel covering the cell-containing core in the collection solution.

[0086] In Example 1, the collection solution was a 100 mM aqueous calcium chloride solution to which 25 mM HEPES buffer and a ROCK inhibitor (Y-27632) were added. The ROCK inhibitor was added to the final solvent to a concentration of 10 μM.

[0087] The core flow rate was approximately 50 μL / min, and the core was allowed to flow for approximately 1 minute. Therefore, the amount of core contained inside the tubular alginate gel was approximately 50 μL. After the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated for a predetermined time and temperature. In Example 1, the incubation temperature was 4° C., and the incubation time was 1 hour (see Table 1 below). During this incubation, the average temperature of the collection solution was maintained at the incubation temperature (4° C. in Example 1).

[0088] After incubating the cell capsules in the capture solution, they were removed from the capture solution and immersed in culture medium to initiate cell culture. In other words, cells were cultured inside the tubular hydrogel. The culture medium was "mTeSR-plus (STEMCELL Technologies)" supplemented with 0.1 v / v% gentamicin sulfate solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and 10 μM ROCK inhibitor (Y-27632).

[0089] The day when the cell culture was started was designated "Day 0," and the medium was completely replaced on Days 2, 5, and 6. The new medium was the same as the medium used on Day 0.

[0090] Cells were recovered from the cell capsules on Day 7. The viable cell count of the recovered cells was measured using a cell counter NC-200 (automatic cell counter; manufactured by MS Techno Systems), and the proliferation rate was calculated by dividing the total viable cell count by the initial cell number on Day 0.

[0091] The supernatant of the medium was collected on Day 7, and the amount of lactate in the supernatant was measured using a cell culture medium analyzer (Prime, manufactured by Nova Biomedical).

[0092] Example 2 Example 2 will be described below. The cell capsules according to Example 2 were produced in the same manner as in Example 1, except for the incubation temperature before the start of culture. Specifically, in Example 2, after the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 1 hour (see Table 1 below). Cell culture after incubation was performed in the same manner as in Example 1.

[0093] Example 3 Example 3 will be described below. The cell capsules according to Example 3 were produced in the same manner as in Example 1, except for the incubation time before the start of culture. Specifically, in Example 3, after the core, alginate gel, and gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 4°C for 2 hours (see Table 1 below). Cell culture after incubation was performed in the same manner as in Example 1.

[0094] Example 4 Example 4 will be described below. The cell capsules according to Example 4 were produced in the same manner as in Example 1, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 4, after the core, alginate gel, and gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 2 hours (see Table 1 below). Cell culture after incubation was performed in the same manner as in Example 1.

[0095] Example 5 Example 5 will be described below. The cell capsules of Example 5 were produced in the same manner as in Example 1, except for the incubation time before the start of culture. Specifically, in Example 5, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 4°C for 3 hours (see Table 1 below). Cell culture after incubation was performed in the same manner as in Example 1.

[0096] Example 6 Example 6 will be described below. The cell capsules according to Example 6 were produced in the same manner as in Example 1, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 6, after the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 3 hours (see Table 1 below). Cell culture after incubation was performed in the same manner as in Example 1.

[0097] Reference Examples 1 to 6 are described below. The cell capsules of Reference Examples 1 to 6 were produced in the same manner as in Examples 1 to 6, respectively, except for the composition of the capture solution. The capture solution of Reference Examples 1 to 6 was prepared by adding 10 μM of a ROCK inhibitor (Y-27632) to a 100 mM aqueous calcium chloride solution. Incubation of the cell capsules in the capture solution and culturing of the cells after incubation were carried out in the same manner as in Examples 1 to 6, respectively (see Table 1 below).

[0098] (Table 1)

[0099] Figure 7 is a graph showing the results of measuring the amount of lactate in the medium on Day 7 in Reference Examples 1 to 6 and Examples 1 to 6. In Figure 7, the amounts of lactate measured in Reference Example 1, Reference Example 2, Reference Example 3, Reference Example 4, Reference Example 5, Reference Example 6, Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6 are shown in order from left to right.

[0100] 7, it can be seen that in Reference Examples 1 to 6, the amount of lactate in the medium on Day 7 decreased as the incubation time increased. In particular, when the incubation temperature was the relatively high 20°C, the amount of lactate in the medium on Day 7 decreased significantly as the incubation time increased. It is believed that if the cell capsules were immersed in the collection solution for a long time before cell culture, this would have a negative impact on the cell culture, inhibiting cell growth and resulting in a decrease in the amount of lactate, an intracellular metabolic product.

[0101] When the average temperature of the trapping solution is 4°C as in Examples 1, 3, and 5, the amount of lactate in the medium on Day 7 is maintained at a high value even if the incubation time is long. Furthermore, when the average temperature of the trapping solution is 20°C as in Examples 2, 4, and 6, the amount of lactate in the medium on Day 7 is maintained at a high value so long as the incubation time is 2 hours or less. Comparing Reference Examples 3 to 6 with Examples 3 to 6, the amount of lactate in Examples 3 to 6 is higher when the incubation time is 2 to 3 hours.

[0102] Fig. 8 is a graph showing the measurement results of cell proliferation rates on Day 7 in Reference Examples 1 to 6 and Examples 1 to 6. In Fig. 8, the cell proliferation rates measured in Reference Example 1, Reference Example 2, Reference Example 3, Reference Example 4, Reference Example 5, Reference Example 6, Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6 are shown in order from the left.

[0103] Referring to Figure 8, it can be seen that in Reference Examples 1 to 6, the cell proliferation rate generally decreased as the incubation time increased. In particular, when the incubation temperature was the relatively high 20°C, the cell proliferation rate significantly decreased as the incubation time increased. This result is generally consistent with the lactate content measurement results described above. In other words, it can be seen that if the cell capsules are immersed in the collection solution for a long time before cell cultivation, it has a negative impact on the cell culture.

[0104] When the average temperature of the collection solution is 4°C as in Examples 1, 3, and 5, the cell culture rate is maintained at a high value even when the incubation time is long. Furthermore, when the average temperature of the collection solution is 20°C as in Examples 2, 4, and 6, the cell culture rate is maintained at a high value as long as the incubation time is 2 hours or less. Comparing Reference Examples 3 to 6 with Examples 3 to 6, the cell culture rate in Examples 3 to 6 is higher when the incubation time is 2 to 3 hours.

[0105] The difference between Examples 1 to 6 and Reference Examples 1 to 6 lies in the composition of the respective trapping solutions. In Examples 1 to 6, the trapping solutions contain HEPES, a Good's buffer. This is thought to have kept the pH in the trapping solutions relatively stable and alleviated adverse effects on cell culture.

[0106] Example 7 Example 7 is described below. The cell capsules of Example 7 were produced in the same manner as in Example 1, except for the composition of the capture solution. The capture solution of Example 7 was prepared by adding calcium chloride and a ROCK inhibitor (Y-27632) to a medium with pH buffering capacity. The medium was Dulbecco's modified Eagle's medium (DMEM / F-12). Calcium chloride was dissolved in the medium to a concentration of 100 mM. The ROCK inhibitor (Y-27632) was added to the final solvent to a concentration of 10 μM. Incubation of the cell capsules in the capture solution and cell culture after incubation were performed in the same manner as in Example 1 (see Table 2 below).

[0107] Example 8 Example 8 will be described below. The cell capsules of Example 8 were produced in the same manner as in Example 7, except for the incubation temperature before the start of culture. Specifically, in Example 8, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 1 hour (see Table 2 below). Cell culture after incubation was performed in the same manner as in Example 7.

[0108] Example 9 Example 9 will be described below. The cell capsules of Example 9 were produced in the same manner as in Example 7, except for the incubation time before the start of culture. Specifically, in Example 9, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 4°C for 3 hours (see Table 2 below). Cell culture after incubation was performed in the same manner as in Example 7.

[0109] Example 10 is described below. The cell capsules of Example 10 were produced in the same manner as in Example 7, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 10, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 3 hours (see Table 2 below). Cell culture after incubation was performed in the same manner as in Example 7.

[0110] Reference Examples 7 to 10 are described below. The cell capsules of Reference Examples 7 to 10 were produced in the same manner as in Examples 7 to 10, respectively, except for the composition of the capture solution. The capture solution of Reference Examples 7 to 10 was prepared by adding a ROCK inhibitor (Y-27632) to a 100 mM aqueous calcium chloride solution. The ROCK inhibitor (Y-27632) was added to a final solvent concentration of 10 μM. Incubation of the cell capsules in the capture solution and cell culture after incubation were carried out in the same manner as in Examples 7 to 10, respectively (see Table 2 below).

[0111] (Table 2)

[0112] Figure 9 is a graph showing the results of measuring the amount of lactate in the medium on Day 7 in Examples 7 to 10. The amounts of lactate measured in Examples 7, 8, 9, and 10 are shown in order from the left. Figure 10 is a graph showing the results of measuring the amount of lactate in the medium on Day 7 in Reference Examples 7 to 10. In Figure 10, the amounts of lactate measured in Reference Examples 7, 8, 9, and 10 are shown in order from the left.

[0113] 9, when the average temperature of the trapping solution is 4°C as in Examples 7 and 9, the amount of lactate in the medium on Day 7 is maintained at a high value even if the incubation time is long. Furthermore, when the average temperature of the trapping solution is 20°C as in Examples 8 and 10, the amount of lactate in the medium on Day 7 is maintained at a high value if the incubation time is 1 hour.

[0114] 10, it can be seen that in Reference Examples 7 to 10, as the incubation time increased, the amount of lactate in the medium on Day 7 decreased significantly. When the incubation temperature was a relatively high 20°C and the incubation time was 3 hours, the viable cell count could not be maintained.

[0115] Comparing Reference Examples 7 to 10 with Examples 7 to 10, it can be seen that the decrease in lactate amount was mitigated and the adverse effects on cells tended to be mitigated in Examples 7 to 10. This is thought to be because the collection solution in Examples 7 to 10 was a medium with buffering capacity, and the pH in the collection solution was maintained relatively stable.

[0116] Example 11 Example 11 is described below. First, a core, a hydrogel precursor, a gelling solution, and a collection solution were prepared. The core was a cell suspension. The cell suspension was prepared by suspending cells (human chronic myeloid leukemia-derived K562 cells) in Iscove's modified Dulbecco's medium containing 3% methylcellulose (R&D systems HSC001) to which 0.3 w / v% methylcellulose had been added. The initial cell density in the cell suspension was 1 x 10 ７ cells / mL.

[0117] The hydrogel precursor was a sodium alginate solution prepared by adding sodium alginate (High-G ALG300, manufactured by Kimica Co., Ltd.) to saline and stirring. The concentration of sodium alginate relative to the saline was 1.0 w / v%.

[0118] The gelling solution was an aqueous solution containing 100 mM calcium chloride and 3% w / v sucrose.

[0119] Using these cores, hydrogel precursor, and gelling solution, a cell mass was produced based on the cell capsule production method according to the first embodiment (see FIG. 4). That is, a core flow, a hydrogel precursor flow around the core flow, and a gelling solution flow around the hydrogel precursor flow were formed, and these flows were ejected into the collection solution from the ejection port 240.

[0120] The hydrogel precursor was crosslinked upon contact with the gelling solution to form a tubular alginate gel, producing a hydrogel covering the cell-containing core in the collection solution.

[0121] In Example 11, the collection solution was a medium with pH buffering capacity. The medium was Iscove's modified Dulbecco's medium containing 3% methylcellulose (R&D Systems HSC001), supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The core flow rate was approximately 50 μL / min, and the core was allowed to flow for approximately 1 minute. Therefore, the amount of core contained inside the tubular alginate gel was approximately 50 μL.

[0122] After the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated for a predetermined time and temperature. In Example 11, the incubation temperature was 4°C, and the incubation time was 1 hour (see Table 3 below).

[0123] After incubation in the capture solution, the cell capsules were removed from the capture solution and immersed in culture medium to initiate cell culture. In other words, cells were cultured inside the tubular hydrogel. The culture medium was Iscove's modified Dulbecco's medium containing 3% methylcellulose (R&D Systems HSC001), supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate solution (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0124] The day when the cell culture was started was designated "Day 0," and the cells were collected from the cell capsules on Day 2. The supernatant of the culture medium on Day 2 was also collected, and the amount of lactate in the supernatant was measured in the same manner as in Example 1. In this way, the amount of lactate produced from Day 0 to Day 2 was measured.

[0125] Example 12 is described below. The cell capsules of Example 12 were produced in the same manner as in Example 11, except for the incubation temperature before the start of culture. Specifically, in Example 12, after the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 1 hour (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0126] Example 13 Example 13 is described below. The cell capsules of Example 13 were produced in the same manner as in Example 11, except for the incubation temperature before the start of culture. Specifically, in Example 13, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 37°C for 1 hour (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0127] Example 14 Example 14 is described below. The cell capsules of Example 14 were produced in the same manner as in Example 11, except for the incubation time before the start of culture. Specifically, in Example 14, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 4°C for 2 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0128] Example 15 Example 15 is described below. The cell capsules of Example 15 were produced in the same manner as in Example 11, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 15, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 2 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0129] Example 16 Example 16 is described below. The cell capsules of Example 16 were produced in the same manner as in Example 11, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 16, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 37°C for 2 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0130] Example 17 Example 17 is described below. The cell capsules of Example 17 were produced in the same manner as in Example 11, except for the incubation time before the start of culture. Specifically, in Example 17, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 4°C for 4 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0131] Example 18 Example 18 is described below. The cell capsules of Example 18 were produced in the same manner as in Example 11, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 18, after the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 4 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0132] Example 19 Example 19 is described below. The cell capsules of Example 19 were produced in the same manner as in Example 11, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 19, after the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 37°C for 4 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0133] Example 20 is described below. The cell capsules of Example 20 were produced in the same manner as in Example 11, except for the incubation time before the start of culture. Specifically, in Example 20, after the core, alginate gel, and gelling solution were ejected into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 4°C for 6 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0134] Example 21 is described below. The cell capsules of Example 21 were produced in the same manner as in Example 11, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 21, after the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 20°C for 6 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0135] Example 22 is described below. The cell capsules of Example 22 were produced in the same manner as in Example 11, except for the incubation time and incubation temperature before the start of culture. Specifically, in Example 22, after the core, alginate gel, and gelling solution were dispensed into the collection solution, the lid of the container containing the collection solution was immediately closed, and the container was incubated at 37°C for 6 hours (see Table 3 below). Cell culture after incubation was performed in the same manner as in Example 11.

[0136] Figure 11 is an enlarged photograph showing the cell capsules on Day 2 in Examples 11 to 22. As shown in Figure 11, it can be seen that K562 cells are housed inside the tubular alginate.

[0137] Reference Examples 11 to 22 are described below. The cell capsules of Reference Examples 11 to 22 were produced in the same manner as in Examples 11 to 22, respectively, except for the composition of the capture solution. The capture solution of Reference Examples 11 to 22 was a 100 mM aqueous calcium chloride solution. Incubation of the cell capsules in the capture solution and cell culture after incubation were carried out in the same manner as in Examples 11 to 22, respectively (see Table 3 below).

[0138] (Table 3)

[0139] Figure 12 is a graph showing the results of measuring the amount of lactate in the medium of K562 cells on Day 2 in Examples 11 to 22. In Figure 12, the amounts of lactate measured in Examples 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 are shown in order from left to right.

[0140] Figure 13 is a graph showing the results of measuring the amount of lactate in the medium of K562 cells on Day 2 in Reference Examples 11 to 22. In Figure 13, the amounts of lactate measured in Reference Examples 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 are shown in order from the left.

[0141] 13, it can be seen that in the Reference Example, when the incubation time was 6 hours, the amount of lactate in the medium decreased regardless of the incubation temperature. On the other hand, in the Examples, the amount of lactate in the medium was maintained even when the incubation time was 6 hours. This is thought to be because the collection solution in Examples 11 to 22 was a medium with buffering capacity, and the pH in the collection solution was maintained relatively stable.

[0142] The above examples demonstrate that when cells encased in a tubular hydrogel need to be soaked in a capture solution for a long period of time, the capture solution preferably contains a pH buffer. In this case, the capture solution may be a culture medium with pH buffering capacity. In particular, the above examples demonstrate that such a capture solution is preferably maintained at a relatively low temperature during cell capsule production, for example, 40°C or less, 30°C or less, preferably 25°C or less, and more preferably 20°C or less.

[0143] Such a capture solution can be suitably used when producing very long cell capsules and / or large quantities of cell capsules. When producing very long cell capsules and / or large quantities of cell capsules, the time required to drain the core, hydrogel or hydrogel precursor, and gelling solution may reach, for example, 0.25 hours or more, 0.5 hours or more, or even 1 hour or more. In this case, the portion of the hydrogel or hydrogel precursor that is immersed in the capture solution at the start of the draining process will be immersed in the capture solution for a long period of time. Even in such cases, using a capture solution containing a pH buffer can mitigate adverse effects on the cells in the cell capsules.

[0144] As described above, the contents of the present invention have been disclosed through the embodiments, but the descriptions and drawings that form part of this disclosure should not be understood to limit the present invention. Various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art from this disclosure. Therefore, the technical scope of the present invention is defined only by the inventive features of the claims that can be reasonably understood from the above description.

[0145] This application claims priority based on Japanese Patent Application No. 2023-104592, filed on June 26, 2023, the entire contents of which are incorporated herein by reference.

Claims

1. A method for producing cell capsules, comprising continuously or intermittently flowing a hydrogel or a hydrogel precursor that covers cells into a capture solution, the capture solution including a pH buffer.

2. The method for producing a cell capsule according to claim 1, wherein the pH buffer comprises a Good's buffer.

3. A method for producing a cell capsule as described in claim 1 or 2, which comprises maintaining the hydrogel or the hydrogel generated from the hydrogel precursor in the collection solution for 0.25 hours or more from the start of the outflow of the hydrogel or hydrogel precursor.

4. A method for producing a cell capsule as described in claim 1 or 2, comprising maintaining the hydrogel or the hydrogel generated from the hydrogel precursor in the collection solution for at least 1 hour and not more than 24 hours from the start of outflow of the hydrogel or hydrogel precursor.

5. A method for producing cell capsules described in any one of claims 1 to 4, wherein the average temperature of the collection solution is 0°C or higher and 40°C or lower.

6. A method for producing cell capsules described in any one of claims 1 to 4, wherein the average temperature of the collection solution is 0°C or higher and 20°C or lower.

7. A method for producing cell capsules according to any one of claims 1 to 6, wherein the pH of the capture solution is in the range of 6.5 to 8.

0.

8. A method for producing a cell capsule described in any one of claims 1 to 7, wherein the hydrogel or hydrogel precursor covers a core containing the cells and flows out into the capture solution together with the core.

9. A method for producing a cell capsule according to any one of claims 1 to 8, wherein the collection solution contains a gelling agent that gels the hydrogel precursor.

10. A method for producing a cell capsule described in any one of claims 1 to 9, wherein a gelling solution covering the hydrogel or hydrogel precursor is allowed to flow into the capture solution together with the hydrogel or hydrogel precursor.

11. The method for producing a cell capsule according to claim 10, wherein the gelling solution contains a pH buffer.

12. The method for producing a cell capsule according to claim 10 or 11, wherein the pH buffer comprises a Good's buffer.

13. A method for producing a cell capsule according to any one of claims 10 to 12, wherein the gelling solution has the same pH buffer as the capture solution.

14. A method for producing a cell population or a cell product, comprising culturing cells inside a hydrogel using a cell capsule produced by the method for producing a cell capsule according to any one of claims 1 to 13.

15. A cell population produced by the method for producing a cell population or cell product according to claim 14.

16. A solution used in the manufacture of cell capsules having a hydrogel surrounding cells, the solution comprising a pH buffer.

17. The solution of claim 16, wherein the pH buffer is a Good's buffer.

18. The solution according to claim 16 or 17, further comprising a gelling agent for gelling the hydrogel precursor.

19. The solution of any one of claims 16 to 18, wherein the pH of the solution is in the range of 6.5 to 8.0.