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 with which it is possible to alleviate adverse effects on cells. A method for producing a cell capsule includes continuously or intermittently discharging a hydrogel or a hydrogel precursor covering cells into a collection solution. The collection solution comprises a pH buffer.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for producing a cell capsule, a method for producing a cell group or a cell product, a solution used in the method for producing a cell capsule, and a cell group. BACKGROUND

[0002] At present, various cells are being attempted to be cultured in various fields of biogenesis, innovative drugs, regenerative medicine, and the like. Typically, these cells are two-dimensionally cultured on the surface of a plastic container for tissue culture. There are known techniques of culturing cells in a three-dimensional or quasi-three-dimensional culture environment using a scaffold such as a porous membrane or a hydrogel. Patent Documents 1 to 6 below disclose culturing cells on the inside of a tubular or spherically shaped hydrogel.

[0003] In recent years, attention is being paid to spheroid culture of culturing cells to be three-dimensionally aggregated, instead of two-dimensionally culturing cells in monolayer culture. It is known that spheroid culture can construct a state close to that of cells in vivo, and exert specific functions that cells have in vivo, compared to monolayer culture. Therefore, for example, in innovative drug research and the like, cell blocks are expected as a useful tool.

[0004] PRIOR ART DOCUMENTS

[0005] PATENT DOCUMENTS

[0006] Patent Document 1: International Publication No. 2011 / 046105

[0007] Patent Document 2: International Publication No. 2017 / 091662

[0008] Patent Document 3: International Publication No. 2018 / 098295

[0009] Patent Document 4: International Publication No. 2019 / 178549

[0010] Patent Document 5: International Publication No. 2020 / 032221

[0011] Patent Document 6: International Publication No. 2009 / 108138

[0012] Patent Document 7: Japanese Patent Application Publication No. 2017-99303 SUMMARY

[0013] PROBLEMS TO BE SOLVED BY THE INVENTION

[0014] With respect to the production of a tubular or spheroid hydrogel like that described in Patent Documents 1 to 6, the inventors of the present application have found a new problem as described below. In Patent Documents 1 to 6, a hydrogel precursor is continuously or intermittently discharged from a nozzle into a solution in which a tubular or spheroid hydrogel is produced. In order to encapsulate a large number of cells in the hydrogel, for example, for the purpose of mass culture of cells or the like, the discharge of the hydrogel precursor is performed for a long time, and as a result, the pH (hydrogen ion index) in the solution changes, which can adversely affect the cells encapsulated in the hydrogel.

[0015] Therefore, a method for producing a cell capsule, a method for producing a cell population or a cell product, or the like, which can alleviate adverse effects on cells, is desired.

[0016] A method for producing a cell capsule of one embodiment includes continuously or intermittently discharging a cell-encapsulating hydrogel or a hydrogel precursor into a collection solution, the collection solution containing a pH buffering agent.

[0017] A method for producing a cell population or a cell product of one embodiment includes culturing cells inside a hydrogel using a cell capsule produced by the method for producing a cell capsule described above.

[0018] A cell population of one embodiment can be a cell population produced by the method for producing a cell population or a cell product described above.

[0019] A solution of one embodiment is a solution used in the production of a cell capsule having a cell-encapsulating hydrogel, and contains a pH buffering agent. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic view illustrating the structure of a cell capsule of one embodiment.

[0021] Figure 2 is Figure 1 is a schematic cross-sectional view of a cell capsule.

[0022] Figure 3 is a schematic view illustrating the structure of a cell capsule of another embodiment.

[0023] Figure 4 is a schematic view for explaining a method for producing a cell capsule of the first embodiment.

[0024] Figure 5 is a schematic view for explaining a method for producing a cell capsule of the second embodiment.

[0025] Figure 6 is a schematic view for explaining a method for producing a cell capsule of the third embodiment.

[0026] Figure 7is a graph showing the measurement results of the lactate amount in the medium of human iPS cells on Day 7 (Day 7) in Reference Examples 1 to 6 and Examples 1 to 6.

[0027] Figure 8 is a graph showing the measurement results of the proliferation rate of human iPS cells at the time point of Day 7 (Day 7) in Reference Examples 1 to 6 and Examples 1 to 6.

[0028] Figure 9 is a graph showing the measurement results of the lactate amount in the medium of human iPS cells on Day 7 (Day 7) in Examples 7 to 10.

[0029] Figure 10 is a graph showing the measurement results of the lactate amount in the medium of human iPS cells on Day 7 (Day 7) in Reference Examples 7 to 10.

[0030] Figure 11 is an enlarged photograph showing the cell capsules on Day 2 (Day 2) in Examples 11 to 22.

[0031] Figure 12 is a graph showing the measurement results of the lactate amount in the medium of K562 cells on Day 2 (Day 2) in Examples 11 to 22.

[0032] Figure 13 is a graph showing the measurement results of the lactate amount in the medium of K562 cells on Day 2 (Day 2) in Reference Examples 11 to 22. DETAILED DESCRIPTION

[0033] Hereinafter, the embodiments will be described with reference to the drawings. In the drawings below, the same or similar signs are attached to the same or similar portions. However, it should be noted that the drawings are schematic, and the ratio of each dimension and the like is different from reality.

[0034] The cell capsule has cells covered with a hydrogel. One or a plurality of cells can be covered with a hydrogel. The cells can be dispersed cells, or can be in the form of a cell aggregate. The hydrogel, as long as it covers the cells, can have a shape such as an approximately tubular shape, an approximately band-like shape, an approximately spherical shape, or an approximately spherical shape.

[0035] Figure 1 is a schematic diagram showing the structure of a cell capsule of one embodiment. Figure 2 is Figure 1 a schematic cross-sectional view of the cell capsule shown in Figure 1 and Figure 2In the manner shown, the hydrogel 14 has an approximately tubular shape. Specifically, the cell capsule may have a tubular hydrogel 14 and a nucleus 12 encapsulated by the hydrogel 14. The cell is disposed within the nucleus 12. The hydrogel 14 surrounds the nucleus 12. More specifically, the hydrogel 14 surrounds the nucleus 12 in a cross-section orthogonal to the direction in which the hydrogel 14 extends. The tubular hydrogel 14 extends to a longer horizontal level, and the nucleus 12 extends to a longer horizontal level in a band-like manner inside the tubular hydrogel 14. One or more cells (including spheroids) may be contained within a single cell capsule.

[0036] The length of hydrogel 14 is, 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 within a single hydrogel. The length of hydrogel 14 is not particularly limited and can be, for example, less than 10 m.

[0037] Figure 3 This is a schematic diagram illustrating the structure of a cell capsule in another manner. Figure 3 In the illustrated configuration, the cell capsule has an approximately spherical shape. The hydrogel 14 has an approximately shell-like shape. Specifically, the cell capsule may have a shell-like hydrogel 14 and a nucleus 12 encapsulated by the hydrogel 14. The nucleus 12 is approximately spherical and is disposed inside the shell-like hydrogel 14. The cell is disposed within the nucleus 12. The hydrogel 14 completely surrounds the nucleus 12. It can be considered that... Figure 3 The cell capsule shown is shortened. Figure 1 and Figure 2 The cell capsule is of the length shown. One or more cells (including spheroids) may be contained in one cell capsule.

[0038] The outer diameter of the hydrogel 14 is not particularly limited, for example, it is 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 can be specified by the outer diameter of the hydrogel in a cross-section orthogonal to the direction of extension of the cell capsule (see reference). Figure 2 The symbol R1).

[0039] The inner diameter of the hydrogel is not particularly limited, 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 can be defined by the inner diameter of the hydrogel in a cross-section orthogonal to the direction of extension of the cell capsule (see reference). Figure 2 The symbol R2). The aforementioned inner and outer diameters can be determined, for example, by a phase-difference optical microscope.

[0040] The hydrogel 14 can be obtained by gelating a hydrogel precursor. The hydrogel 14 is preferably a hydrogel that can have sufficient permeability to a cell culture medium component.

[0041] The hydrogel 14 can include at least one selected from, for example, an alginate gel, Matrigel, a collagen gel, a chitosan gel, gelatin, a peptide gel, a laminin gel, an agarose gel, nanocellulose, methylcellulose, dextrin, pectin, gellan gum, xanthan gum, guar gum, carrageenan, glucomannan, and a fibrin gel.

[0042] The hydrogel 14 is preferably an alginate gel that uses an alginate gel as a main component. In this case, the hydrogel precursor can be a solution that uses an alginate solution as a main component. The alginate gel can be formed by cross-linking an alginate solution that is an alginate derivative with a divalent metal ion. The alginate gel can be suitably used as a hydrogel for culturing cells or cell blocks in a state of being wrapped in cells or cell blocks.

[0043] [1st Embodiment]

[0044] In the method of manufacturing the cell capsule, first, a core, a hydrogel precursor, a gelation solution, and a collection solution are prepared. The core can be a cell suspension. The hydrogel precursor can include a material that forms a hydrogel by gelation. The gelation solution can include a material that gelates the hydrogel precursor.

[0045] The method of manufacturing the cell capsule includes continuously flowing the hydrogel or the hydrogel precursor that coats the cells into the collection solution. The composition of the core, the hydrogel precursor, the gelation solution, and the collection solution will be described in detail below.

[0046] For the method of manufacturing the cell capsule of the 1st embodiment, a device for manufacturing the cell capsule is used. Figure 4 A more detailed description will be given. Figure 4 is a schematic diagram for explaining the method of manufacturing the cell capsule of the 1st embodiment. The device for manufacturing the cell capsule can have a 1st inlet 210, a 2nd inlet 220, a 3rd inlet 230, and a discharge port 240. The 1st inlet 210 is a flow inlet through which the core flows in. The 2nd inlet 220 is a flow inlet through which the hydrogel precursor flows in. The 3rd inlet 230 is a flow inlet through which a solution that gelates the hydrogel precursor (a gelation solution) flows in.

[0047] The hydrogel precursor flows in from the 2nd inlet 220 and converges around the core. Thereby, the hydrogel precursor flows around the core flow in the direction of the core flow. That is, the hydrogel precursor surrounds the core in a cross section orthogonal to the core flow. More specifically, it is preferable that the hydrogel precursor and the core flow in a manner to form a laminar flow.

[0048] The gelling solution is used to gelle a hydrogel precursor to form a hydrogel. The gelling solution merges with the core and hydrogel precursor downstream of the confluence point. The gelling solution flows along the hydrogel precursor flow, surrounding the core and the hydrogel precursor. That is, the gelling solution surrounds the hydrogel precursor in a cross-section orthogonal to the hydrogel precursor flow. Preferably, the gelling solution, core, and hydrogel precursor form a laminar flow.

[0049] In the first embodiment, the gelling solution merges with the hydrogel precursor, transforming the hydrogel precursor into a hydrogel. The hydrogel precursor may be completely transformed into a hydrogel state or may flow out of the injection port 240 in its original state. Therefore, the hydrogel or hydrogel precursor encapsulates the nucleus containing the cell and flows out together with the nucleus into the collection solution 20.

[0050] In addition, in the first embodiment, the gelation solution coating the hydrogel or hydrogel precursor flows out together with the hydrogel or hydrogel precursor into the collection solution.

[0051] like Figure 4 As shown, the hydrogel or hydrogel precursor, along with the core, continuously flows out into the collection solution. From the start of the flow-out of the hydrogel or hydrogel precursor, the hydrogel or hydrogel formed from the hydrogel precursor is held in the collection solution for, for example, more than 0.25 hours, more than 0.5 hours, more than 1 hour, more than 2 hours, or more than 4 hours. The time for the core, hydrogel or hydrogel precursor, and gelation solution to flow out can be, for example, more than 0.25 hours, more than 0.5 hours, more than 1 hour, more than 2 hours, or more than 4 hours.

[0052] There is no particular limitation on the time during which the hydrogel or hydrogel precursor is retained in the collection solution, and it can be, for example, less than 24 hours, less than 20 hours, less than 16 hours, less than 12 hours, less than 8 hours, or less than 6 hours from the time the hydrogel or hydrogel precursor is eluted. Furthermore, there is no particular limitation on the time during which the core, hydrogel or hydrogel precursor, or gelling solution is eluted, and it can be, for example, less than 24 hours, less than 20 hours, less than 16 hours, less than 12 hours, less than 8 hours, or less than 6 hours.

[0053] exist Figure 4 In this process, the hydrogel or hydrogel precursor flows continuously along with the core into the collecting solution. However, it is not limited to this; the hydrogel or hydrogel precursor may also flow intermittently along with the core into the collecting solution.

[0054] From the viewpoint of producing a large number of cell populations, the fluid with the fastest flow rate in the nucleus, hydrogel precursor, or hydrogel and gelation solution can flow into the collection solution, for example, more than 1 mL. Preferably, the nucleus containing the cells flows into the collection solution, for example, more than 1 mL.

[0055] The average temperature of the collection solution can be, for example, 40°C or lower, 30°C or lower, preferably 25°C or lower, and more preferably 20°C or lower. By lowering the average temperature of the collection solution, the adverse effects on the cells can be mitigated. The average temperature can be calculated by dividing the temperature of a plurality of portions of the collection solution by the number of portions for which the temperature was measured. In the case where the temperature of the collection solution is substantially uniform throughout the collection solution, the average temperature of the collection solution can also be determined by the temperature at one location in the collection solution.

[0056] In addition, the average temperature of the collection solution can be, for example, 0°C or higher, 2°C or higher, or 4°C or higher. The temperature of the collection solution can be within a range that is any combination of the above upper limit and the above lower limit. It is desirable that the average temperature of the collection solution be maintained within the above range during the period in which the hydrogel or hydrogel precursor is flowing into the collection solution. Alternatively, the average temperature of the collection solution can be set to the above range immediately after the hydrogel or hydrogel precursor has just finished flowing into the collection solution. In the case where the average temperature of the collection solution is set to the above range immediately after the hydrogel or hydrogel precursor has just finished flowing into the collection solution, the cell capsules can be 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 which the cell capsules are maintained in the collection solution is not particularly limited and can be, for example, 24 hours or less.

[0057] The core can be a cell suspension. The core can contain at least one selected from, for example, physiological saline, an aqueous solution of an inorganic salt, an aqueous solution of a saccharide, a culture medium, a culture supernatant, a buffer, an extracellular matrix, a thickening agent, and a hydrophilic resin (polyvinyl alcohol, polyacrylamide, poly N-isopropyl acrylamide, etc.).

[0058] The thickening agent can contain at least one selected from, for example, a chitosan gel, a collagen solution, Matrigel, collagen, gelatin, an alginate solution, an alginate gel, a peptide gel, laminin, agarose, nanocellulose, methylcellulose, hyaluronic acid, proteoglycans, elastin, pullulan, dextran, pectin, gellan gum, xanthan gum, guar gum, carrageenan, and glucomannan.

[0059] The core can contain various growth factors suitable for the culture, maintenance, proliferation, or functional expression of cells, and the like. Such growth factors can be at least one selected from, for example, epithelial 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 cell proliferation factor (VEGF), and hepatocyte proliferation factor (HGF).

[0060] The kind of the cell is not particularly limited. The cell can be, for example, an animal cell, and preferably can be a human cell. The cell can be various kinds of stem cells having, for example, differentiation pluripotency, human ES cells having differentiation totipotency, human iPS cells, stem cells having differentiation multipotency, progenitor cells having differentiation oligopotent or monopotent, and the like. As the various kinds of stem cells having differentiation totipotency and pluripotency, for example, iPS cells, ES cells, mesenchymal stem cells, neural stem cells, hepatic stem cells, skin stem cells, pancreatic stem cells, and the like can be given. As the stem cells, progenitor cells having differentiation oligopotent or monopotent, for example, muscle stem cells, germ stem cells, respiratory tract progenitor cells, digestive tract progenitor cells, cardiac progenitor cells, and the like can be given. In addition, the adherent cell can be various kinds of cells after differentiation, such as muscle cells such as skeletal muscle cells, smooth muscle cells, cardiac muscle cells, neural cells such as cerebral cortex cells, fibroblasts, epithelial cells, endothelial cells, adipocytes, osteoblasts, chondrocytes, macrophages, dendritic cells, hepatocytes, hepatic stellate cells, pancreatic β cells, keratinocytes, renal cells, renal tubular cells, hair follicle cells, corneal endothelial cells, retinal cells, pigment epithelial cells, cup cells, respiratory tract cells, and the like. These cells can use cells produced by differentiation induction from cells having differentiation totipotency or pluripotency. In addition, the cell can be a CHO cell, a cell strain from human hepatocarcinoma, or the like. Furthermore, the cell also includes tissues or internal organs composed of a collection of the above-described cells, and bacteria, oomycetes, mycelia, and fungi, microorganisms such as algae. As examples of the fungi, for example, Aspergillus, Saccharomyces can be given.

[0061] The material constituting the hydrogel precursor is appropriately selected depending on the material of the above-described hydrogel. That is, the hydrogel precursor can include at least one precursor selected from, for example, 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, Tetra-PEG gel, polyacrylamide gel, polyrotaxane, poly(N-isopropylacrylamide) polymer.

[0062] The hydrogel precursor can preferably be a solution in which an alginate solution is a main component (alginate derivative). The alginate solution can be, for example, sodium alginate, potassium alginate, or ammonium alginate, or a combination thereof. The alginate derivative can be a natural extract or a chemically modified product. As the chemically modified alginate, for example, methacrylate-modified alginate and the like can be given. At normal temperature or in the vicinity thereof, the alginate solution is easily crosslinked by divalent metal ions in a short time, and is easily formed into an alginate gel.

[0063] The collection solution contains a pH buffer. Thus, the hydrogen ion index of the collection solution is stably maintained. In the first embodiment, in the production of the cell capsule, the hydrogel, the hydrogel precursor, and / or the gelling solution flows into the collection solution. In particular, in the case of production of a long cell capsule, the hydrogel, the hydrogel precursor, and / or the gelling solution flows into the collection solution for a long time. Even in such a case, as long as the collection solution contains a pH buffer, the hydrogen ion index of the collection solution can be stably maintained for a long time. Thus, the adverse effects on the cells in the cell capsule in the collection solution can be mitigated.

[0064] The collection solution can be, for example, an acetate buffer, a phosphate buffer, a citrate buffer, a citrate phosphate buffer, a borate buffer, a tartrate buffer, a phosphate buffered saline, or a so-called Good's buffer. It is preferable that the pH buffer in the collection solution be a Good's buffer. Some Good's buffers are not apt to form complexes with metal ions, and thus can be considered to be suitably used even when mixed with the gelling solution.

[0065] The Good's buffer can contain at least one selected from, for example, 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-cyclohexyl-2-hydroxy-3-aminopropanesulfonic 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-piperazino]ethanesulfonic acid sodium), 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-ethanesulfonic acid)), POPS0 (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).

[0066] The collection solution can contain nutrients such as, for example, a carbon source, vitamins, inorganic salts, and the like. As such a substance, the collection solution can contain a culture medium having a buffering capacity. The kind of the culture medium can be appropriately selected depending on the kind of the cells accommodated in the inner side of the tubular hydrogel.

[0067] The collection solution preferably contains a gelation agent that gelates the hydrogel precursor. The gelation agent can be any material that can form a hydrogel by cross-linking the hydrogel precursor. For example, in the case where the hydrogel precursor is alginic acid, the gelation agent can be a material capable of providing a divalent cation. As such a gelation agent, there can be mentioned, for example, a salt of magnesium, calcium, strontium, or barium. Preferably, the gelation agent can be, for example, calcium chloride, calcium carbonate, calcium hydroxide, barium chloride, barium carbonate, or barium hydroxide. That is, the collection solution can contain a divalent cation such as, for example, a calcium ion or a barium ion.

[0068] The concentration of the gelling agent, for example, the concentration of calcium chloride or barium chloride, is, for example, 0 to 500 mmol / L, and preferably can be 5 to 200 mmol / L.

[0069] In the case where the collection solution contains a gelling agent, the buffer is preferably composed of a substance that does not substantially precipitate due to the action of the gelling agent. Even in the case where the hydrogel precursor has flowed out into the collection solution before the hydrogel precursor becomes a hydrogel when the collection solution contains a gelling agent, the hydrogel precursor can become a hydrogel in the collection solution.

[0070] The pH of the collection solution is, for example, in the range of 6.5 to 8.0, and preferably can be 6.8 to 7.5. By making the pH in this range, adverse effects on the cells can be further inhibited. In the case where the collection solution contains a pH buffer, the pH of the collection solution is easily maintained in the range of, for example, 6.5 to 8.0, and preferably 6.8 to 7.5 during the period in which the hydrogel precursor or the hydrogel flows out, and / or during the period in which the cells in the cell capsules are cultured.

[0071] The osmotic pressure of the collection solution is, for example, in the range of 100 to 1500 mOsm, and preferably can be 200 to 400 mOsm. By making the osmotic pressure in this range, adverse effects on the cells can be further inhibited.

[0072] The gelling solution can be a solution containing a gelling agent. The gelling agent can be any material that can form a hydrogel by cross-linking a hydrogel precursor. For example, in the case where the hydrogel precursor is alginic acid, the gelling agent can be a material that can provide a divalent cation. As such a gelling agent, for example, a salt of magnesium, calcium, strontium, or barium can be given. Preferably, the gelling agent can be, for example, calcium chloride, calcium carbonate, calcium hydroxide, barium chloride, barium carbonate, or barium hydroxide. That is, the gelling solution can contain a divalent cation such as, for example, a calcium ion or a barium ion.

[0073] The concentration of the divalent metal ion in the gelling solution, for example, the concentration of calcium chloride or barium chloride, is, for example, 10 to 500 mmol / L, and more preferably can be 20 to 200 mmol / L.

[0074] The gelling solution preferably contains a pH buffer. At this time, the pH buffer is more preferably a Good's buffer. As the pH buffer, the above-mentioned buffers can be used. At this time, even if a large amount of the gelling solution flows into the collection solution, the hydrogen ion index in the collection solution is easily stably maintained. Thus, adverse effects on the cells in the cell capsules in the collection solution can be further alleviated.

[0075] The gelling solution can contain, for example, nutrients such as a carbon source, vitamins, inorganic salts, and the like. As such a substance, the gelling solution can contain a culture medium having a buffering capacity. The kind of culture medium can be appropriately selected depending on the kind of cells accommodated inside the tubular hydrogel.

[0076] The pH of the gelling solution is, for example, in the range of 6.5 to 8.0, and preferably can be in the range of 6.8 to 7.5. By making the pH in this range, the adverse effects on cells can be further suppressed.

[0077] The osmotic pressure of the gelling solution is, for example, in the range of 100 to 1500 mOsm, and preferably can be in the range of 200 to 400 mOsm. By making the osmotic pressure in this range, the adverse effects on cells can be further suppressed.

[0078] The gelling solution preferably has the same pH buffer as the collection solution. At this time, even if a large amount of the gelling solution flows into the collection solution, the hydrogen ion index in the collection solution is easily stably maintained. The gelling solution more preferably has the same composition as the collection solution. At this time, even if a large amount of the gelling solution flows into the collection solution, the hydrogen ion index in the collection solution is more easily stably maintained.

[0079] [2nd Embodiment]

[0080] The method for producing the cell capsule of the 2nd embodiment will be described. Figure 5 is a schematic view for explaining the method for producing the cell capsule of the 2nd embodiment. In the 2nd embodiment, for the same constitution and / or process as the 1st embodiment, the explanation thereof is sometimes omitted.

[0081] First, a core, a hydrogel precursor, a gelling solution, and a collection solution are prepared. As for the core, the hydrogel precursor, and materials constituting the collection solution, cells, and the like, the same as the 1st embodiment.

[0082] In the 2nd embodiment, as shown in Figure 5 , the device for producing the cell capsule can have a 1st inlet 210, a 2nd inlet 220, and a discharge port 240. The 1st inlet 210 is a flow inlet through which the core flows. The 2nd inlet 220 is a flow inlet through which the hydrogel precursor flows. In the 2nd embodiment, the discharge port 240 is immersed in the collection solution.

[0083] The hydrogel precursor flows from the 2nd inlet 220 and merges around the core flow. Thereby, the hydrogel precursor flows around the core flow in the direction of the core flow. That is, the hydrogel precursor surrounds the core in a cross section orthogonal to the core flow. More specifically, it is preferable that the hydrogel precursor and the core flow in a manner to form a laminar flow.

[0084] In the second embodiment, the collecting solution contains a gelling agent that gels the hydrogel precursor. In the second embodiment, the hydrogel precursor, encapsulating a cell nucleus, flows continuously into the collecting solution along with the nucleus. The hydrogel precursor gels in the collecting solution to form a hydrogel. Thus, an elongated tubular hydrogel encapsulating a cell nucleus is formed.

[0085] like Figure 5 As shown, the hydrogel precursor and the core continuously flow out together into the collection solution. The core, flowing out along with the hydrogel precursor, remains 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, starting from the flow-out of the hydrogel precursor. The time for the core and hydrogel precursor to flow out is, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, or possibly 4 hours or more.

[0086] There is no particular limitation on the time the nucleus, which flows out along with the hydrogel precursor, can remain in the collection solution. From the start of the flow-out of the nucleus and hydrogel precursor, it can be, for example, less than 24 hours, less than 20 hours, less than 16 hours, less than 12 hours, less than 8 hours, or less than 6 hours. Furthermore, there is no particular limitation on the time for the nucleus and hydrogel precursor to flow out; it can be, for example, less than 24 hours, less than 20 hours, less than 16 hours, less than 12 hours, less than 8 hours, or less than 6 hours.

[0087] The average temperature of the collected solution is, for example, below 40°C, below 30°C, preferably below 25°C, and more preferably below 20°C. By lowering the average temperature of the collected solution, the adverse effects on the cells can be mitigated.

[0088] Furthermore, the average temperature of the collection solution can be, for example, above 0°C, above 2°C, or above 4°C. The average temperature of the collection solution can be any combination of the aforementioned upper and lower limits. It is desirable that the average temperature of the collection solution remain within the aforementioned range during the flow of the hydrogel precursor into the collection solution. Alternatively, the average temperature of the collection solution can be set to the aforementioned range immediately after the hydrogel precursor has finished flowing into the collection solution. When the average temperature of the collection solution is set to the aforementioned range immediately after the hydrogel precursor has flowed into the collection solution, the cell capsule can be held 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. There is no particular limitation on the time the cell capsule is held in the collection solution, and it can be, for example, less than 24 hours.

[0089] From the viewpoint of producing a large number of cell populations, a faster-flowing fluid in the nucleus and hydrogel precursor can flow into the collection solution, for example, more than 1 mL. Preferably, the nucleus containing the cells flows into the collection solution, for example, more than 1 mL.

[0090] In the second embodiment, a long cell capsule as described in Figure 1 and Figure 2 is manufactured.

[0091] [Third Embodiment]

[0092] A method of manufacturing a cell capsule of the third embodiment will be described. Figure 6 is a schematic view for explaining the method of manufacturing a cell capsule of the third embodiment. In the third embodiment, for the same constitution and / or process as the first and second embodiments, the explanation thereof is sometimes omitted.

[0093] First, a core, a hydrogel precursor, a gelation solution, and a collection solution are prepared. As for the materials, cells, and the like constituting the core, the hydrogel precursor, and the collection solution, the same as the first and second embodiments is used.

[0094] In the third embodiment, as shown in Figure 6 , the device for manufacturing a cell capsule is the same as the second embodiment, and can have a first inlet 210, a second inlet 220, and a discharge outlet 240. The first inlet 210 is a flow inlet through which the core flows in. The second inlet 220 is a flow inlet through which the hydrogel precursor flows in. In the third embodiment, the discharge outlet 240 is located above the collection solution.

[0095] In the third embodiment, the collection solution has a gelation agent that causes the hydrogel precursor to gel. In the third embodiment, the hydrogel precursor coats the core containing the cells and intermittently flows out into the collection solution together with the core. At this time, the hydrogel precursor and the core become droplets of approximately spherical shape before reaching the collection solution from the discharge outlet 240. The hydrogel precursor and the core are dropped into the collection solution in the form of the droplets of approximately spherical shape. The hydrogel precursor gels in the collection solution, forming a hydrogel. Thus, a cell capsule containing a core of approximately spherical shape containing cells, and a hydrogel of approximately spherical shell shape coating the core is manufactured.

[0096] As shown in Figure 6 , the hydrogel precursor intermittently flows into the collection solution together with the core. From the start of the flow of the hydrogel precursor, the core flowing out together with the hydrogel precursor is kept 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, 4 hours or more. The time for which the core and the hydrogel precursor flow out can be, for example, 0.25 hours or more, 0.5 hours or more, 1 hour or more, 2 hours or more, 4 hours or more. Thus, a large number of spherical cell capsules can be obtained.

[0097] The time for which the core is kept in the collection solution together with the hydrogel precursor is not particularly limited, and can 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 the effusion of the hydrogel precursor. In addition, the time for which the core and the hydrogel precursor are effused is not particularly limited, and can 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.

[0098] The average temperature of the collection solution is, for example, 40°C or less, 30°C or less, and preferably 25°C or less, and more preferably can be 20°C or less. By lowering the average temperature of the collection solution, the adverse effects on the cells can be mitigated.

[0099] In addition, the average temperature of the collection solution can be, for example, 0°C or more, 2°C or more, or 4°C or more. The average temperature of the collection solution can be a range obtained by arbitrarily combining the above-mentioned upper limit value and the above-mentioned lower limit value. It is desirable that the average temperature of the collection solution be maintained within the above-mentioned range during the period in which the hydrogel precursor is effused into the collection solution. Alternatively, the average temperature of the collection solution can be set to the above-mentioned range immediately after the hydrogel precursor has finished being effused into the collection solution. In the case in which the average temperature of the collection solution is set to the above-mentioned range immediately after the hydrogel precursor has finished being effused into the collection solution, the cell capsule can be kept 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 which the cell capsule is kept in the collection solution is not particularly limited, and can be, for example, 24 hours or less.

[0100] From the viewpoint of manufacturing a large number of cell groups, the fluid that flows faster in the core and the hydrogel precursor can flow into the collection solution, for example, 1 mL or more. It is preferable that the core containing the cells flow into the collection solution, for example, 1 mL or more.

[0101] In the above-mentioned embodiments, the manufacturing method of the cell capsule having a core containing cells and a hydrogel that coats the core was described. However, it is to be noted that the cell capsule manufactured by the manufacturing method of the present application is not limited to these. For example, the cell capsule can have a structure in which a hydrogel containing buried cells. This can be achieved, for example, by causing the above-mentioned core to contain both the hydrogel precursor and the cells. Even in this case, as long as the collection solution in which the hydrogel precursor or the hydrogel is collected contains a pH buffering agent at the time of manufacturing the cell capsule, the adverse effects on the cells can be mitigated.

[0102] The cell capsule manufactured by the above-mentioned manufacturing method of the cell capsule can be used, for example, for the culture of cells. At this time, the cells are cultured by immersing each cell capsule in a culture medium. Thereby, the cells can be cultured inside the hydrogel.

[0103] The cell population of the present application can be a cell population produced by the above-described cell culture method. In a specific example, the cell population is formed by recovering the cells from the inside of the hydrogel. The recovery of the cells can be achieved by, for example, dissolving and removing the hydrogel. The hydrogel can be removed by chemical reactions and enzyme reactions, and the like. Specifically, the hydrogel can be removed by, for example, an EDTA / PBS solution or an alginate lyase, or a combination thereof.

[0104] The cells or cell population can produce a cell product in a state of being encapsulated by the cell capsules. The cell product can be produced by recovering from the culture medium in which, for example, the cells or cell population have been cultured. The cell product is not particularly limited and can be, for example, a protein, an extracellular vesicle, an antibody, and the like.

[0105] [Example 1]

[0106] Example 1 is described below. First, a core, a hydrogel precursor, a gelation solution, and a collection solution were prepared. The core was a cell suspension. The cell suspension was adjusted by suspending human iPS cells (QHJI01s04) in a medium containing 3% methylcellulose (R&D systems HSC001) added to Iscove's Modified Dulbecco's Medium with 0.6 w / v% methylcellulose. The initial cell density in the cell suspension was 1 x 10 7 cells / mL.

[0107] The hydrogel precursor was a sodium alginate solution. The sodium alginate solution was generated by adding the above-described sodium alginate ("High-G ALG300" manufactured by Kimica Corporation) to physiological saline and stirring. The concentration of the sodium alginate with respect to the physiological saline was 1.0 w / v% percent concentration.

[0108] The gelation solution was an aqueous solution containing 100 mM calcium chloride and 3 w / v% sucrose.

[0109] Using these core, hydrogel precursor, and gelation solution, a cell population was produced based on the production method of the cell capsules of the first embodiment (see FIG. 1). That is, a core stream, a hydrogel precursor stream around the core stream, and a gelation solution stream around the hydrogel precursor stream were formed, and these streams were discharged from the discharge port 240 into the collection solution. Figure 4 ). That is, a core stream, a hydrogel precursor stream around the core stream, and a gelation solution stream around the hydrogel precursor stream were formed, and these streams were discharged from the discharge port 240 into the collection solution.

[0110] The hydrogel precursor was crosslinked by contact with the gelation solution, and a tubular alginate gel was formed. Thus, a hydrogel encapsulating the core containing the cells was produced in the collection solution.

[0111] In Example 1, the collection solution was a solution in which 25 mM of HEPES buffer and a ROCK inhibitor (Y-27632) were added to 100 mM of an aqueous calcium chloride solution. The ROCK inhibitor was added so as to have a concentration of 10 μM with respect to the final solvent.

[0112] The flow rate of the core flow was about 50 μL / min, and the core flow was performed for about 1 minute. Thus, the amount of the core accommodated inside the tubular alginate gel was about 50 μL. Immediately after the core, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container accommodating the collection solution was closed, and the container was incubated for a given time and at a given temperature. In Example 1, the incubation temperature was 4°C, and the incubation time was 1 hour (see Table 1 below). During the incubation, the average temperature of the collection solution was maintained at the incubation temperature (4°C in Example 1).

[0113] After the incubation of the cell capsule in the collection solution, the cell capsule was taken out of the collection solution, immersed in a culture medium, and the culture of the cells was started. That is, the cells were cultured in a state of being accommodated inside the tubular hydrogel. The culture medium was a medium in which 0.1 v / v% of a gentamicin sulfate solution (manufactured by FUJIFILM Wako Pure Chemical Corporation) and 10 μM of a ROCK inhibitor (Y-27632) were added to "mTeSR-plus" (manufactured by STEMCELL Technologies Inc.).

[0114] When the date of the start of the culture of the cells was set as "Day 0", the culture medium was exchanged in its entirety on Day 2, Day 5, and Day 6. The new culture medium was the same as the culture medium used on Day 0.

[0115] The cells were recovered from the cell capsule on Day 7. Using a cell counting device NC-200 (automatic cell counter; manufactured by MS Techno Systems), the number of viable cells of the recovered cells was measured, and the proliferation rate was calculated by dividing the total number of viable cells by the initial number of cells on Day 0.

[0116] In addition, the supernatant of the culture medium on Day 7 was recovered, and the amount of lactate in the supernatant was measured. The amount of lactate was measured by a cell culture medium analysis device (manufactured by Nova Biomedical; Prime).

[0117] [Example 2]

[0118] Example 2 is described below. The cell capsules of Example 2 were manufactured in the same manner as Example 1 except for the culture temperature before the start of the culture. Specifically, in Example 2, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was cultured at 20°C for 1 hour (see Table 1 below). The culture of the cells after the culture was performed by the same method as Example 1.

[0119] [Example 3]

[0120] Example 3 is described below. The cell capsules of Example 3 were manufactured in the same manner as Example 1 except for the culture time before the start of the culture. Specifically, in Example 3, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was cultured at 4°C for 2 hours (see Table 1 below). The culture of the cells after the culture was performed by the same method as Example 1.

[0121] [Example 4]

[0122] Example 4 is described below. The cell capsules of Example 4 were manufactured in the same manner as Example 1 except for the culture time and the culture temperature before the start of the culture. Specifically, in Example 4, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was cultured at 20°C for 2 hours (see Table 1 below). The culture of the cells after the culture was performed by the same method as Example 1.

[0123] [Example 5]

[0124] Example 5 is described below. The cell capsules of Example 5 were manufactured in the same manner as Example 1 except for the culture time before the start of the culture. Specifically, in Example 5, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was cultured at 4°C for 3 hours (see Table 1 below). The culture of the cells after the culture was performed by the same method as Example 1.

[0125] [Example 6]

[0126] Example 6 is described below. The cell capsules of Example 6 were manufactured identically to Example 1 except for the culture time before the start of culture and the culture temperature. Specifically, in Example 6, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container that housed the collection solution was covered, and the container was cultured at 20°C for 3 hours (see Table 1 below). The culture of the cells after culture was performed by the same method as Example 1.

[0127] [Reference Examples 1 to 6]

[0128] Reference Examples 1 to 6 are described below. The cell capsules of Reference Examples 1 to 6 were manufactured identically to Examples 1 to 6, respectively, except for the composition of the collection solution. The collection solution of Reference Examples 1 to 6 was a solution in which 10 μM of a ROCK inhibitor (Y-27632) was added to a 100 mM calcium chloride aqueous solution. The culture of the cell capsules in the collection solution, the culture of the cells after culture were performed by the same method as Examples 1 to 6, respectively (see Table 1 below).

[0129]

[0130] Figure 7 is a graph showing the results of measurement of the amount of lactate in the culture medium at Day 7 in Reference Examples 1 to 6 and Examples 1 to 6. In Figure 7 , 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.

[0131] Referring to Figure 7 , it is seen that in Reference Examples 1 to 6, the amount of lactate in the culture medium at Day 7 decreased as the culture time became longer. In particular, in the case where the culture temperature was relatively high at 20°C, the amount of lactate in the culture medium at Day 7 decreased significantly as the culture time became longer. It is considered that if the cell capsules are immersed in the collection solution for a long time before the culture of the cells, adverse effects on the culture of the cells occur, the proliferation of the cells is inhibited, and thus the amount of lactic acid as a metabolic product in the cells decreases.

[0132] In the case where the average temperature of the collection solution was 4°C as in Examples 1, 3, and 5, even if the culture time was longer, the lactate amount in the medium on Day 7 remained at a high value. In addition, in the case where the average temperature of the collection solution was 20°C as in Examples 2, 4, and 6, if the culture time was 2 hours or less, the lactate amount in the medium on Day 7 remained at a high value. When Reference Examples 3 to 6 and Examples 3 to 6 were compared, in the case where the culture time was 2 to 3 hours, the lactate amount in Examples 3 to 6 became higher.

[0133] Figure 8 is a graph showing the measurement results of the proliferation rate of the cells at the time point of Day 7 in Reference Examples 1 to 6 and Examples 1 to 6. In Figure 8 , 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.

[0134] Referring to Figure 8 , it was found that in Reference Examples 1 to 6, as the culture time was longer, the proliferation rate of the cells generally showed a decreasing tendency. In particular, in the case where the culture temperature was relatively high at 20°C, as the culture time was longer, the proliferation rate of the cells greatly decreased. This result substantially matched the measurement results of the lactate amount described above. That is, it was found that if the cell capsules were immersed in the collection solution for a long time before the culture of the cells, adverse effects on the culture of the cells were caused.

[0135] In the case where the average temperature of the collection solution was 4°C as in Examples 1, 3, and 5, even if the culture time was longer, the culture rate of the cells remained at a high value. In addition, in the case where the average temperature of the collection solution was 20°C as in Examples 2, 4, and 6, if the culture time was 2 hours or less, the culture rate of the cells remained at a high value. When Reference Examples 3 to 6 and Examples 3 to 6 were compared, in the case where the culture time was 2 to 3 hours, the culture rate of the cells in Examples 3 to 6 became higher.

[0136] The difference between Examples 1 to 6 and Reference Examples 1 to 6 was present in the composition of the collection solution. In Examples 1 to 6, the collection solution contained HEPES as a Good's buffer. Thereby, the hydrogen ion index in the collection solution was relatively stably maintained, and it was considered that adverse effects on the culture of the cells were alleviated.

[0137] [Example 7]

[0138] Example 7 is explained below. The cell capsules of Example 7 were manufactured in the same manner as Example 1 except for the composition of the collection solution. The collection solution of Example 7 was a solution to which calcium chloride and a ROCK inhibitor (Y-27632) were added to a medium having a pH buffering capacity. The medium was Dulbecco's Modified Eagle Medium (DMEM / F-12). The calcium chloride was dissolved in the medium in a concentration of 100 mM. The ROCK inhibitor (Y-27632) was added in a concentration of 10 μM relative to the final solvent. For the culture of the cell capsules in the collection solution, the culture of the cells after the culture, the same method as Example 1 was performed (see Table 2 below).

[0139] [Example 8]

[0140] Example 8 is explained below. The cell capsules of Example 8 were manufactured in the same manner as Example 7 except for the culture temperature before the start of the culture. Specifically, in Example 8, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was covered, and the container was cultured at 20°C for 1 hour (see Table 2 below). For the culture of the cells after the culture, the same method as Example 7 was performed.

[0141] [Example 9]

[0142] Example 9 is explained below. The cell capsules of Example 9 were manufactured in the same manner as Example 7 except for the culture time before the start of the culture. Specifically, in Example 9, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was covered, and the container was cultured at 4°C for 3 hours (see Table 2 below). For the culture of the cells after the culture, the same method as Example 7 was performed.

[0143] [Example 10]

[0144] Example 10 is explained below. The cell capsules of Example 10 were manufactured in the same manner as Example 7 except for the culture time and the culture temperature before the start of the culture. Specifically, in Example 10, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was covered, and the container was cultured at 20°C for 3 hours (see Table 2 below). For the culture of the cells after the culture, the same method as Example 7 was performed.

[0145] [Reference Examples 7 to 10]

[0146] The following describes Reference Examples 7 to 10. The cell capsules of Reference Examples 7 to 10 were manufactured identically to Examples 7 to 10, except for the composition of the collection solution. The collection solution of Reference Examples 7 to 10 was a solution in which a ROCK inhibitor (Y-27632) was added to a 100 mM calcium chloride aqueous solution. The ROCK inhibitor (Y-27632) was added at a concentration of 10 μM relative to the final solvent. The culture of the cell capsules in the collection solution, and the culture of the cells after the culture, were performed by the same methods as in Examples 7 to 10, respectively (see Table 2 below).

[0147]

[0148] Figure 9 is a graph showing the measurement results of the amount of lactate in the medium at Day 7 in Examples 7 to 10. The amount of lactate measured in Example 7, Example 8, Example 9, and Example 10 is shown in order from the left. Figure 10 is a graph showing the measurement results of the amount of lactate in the medium at Day 7 in Reference Examples 7 to 10. In Figure 10 , the amount of lactate measured in Reference Example 7, Reference Example 8, Reference Example 9, and Reference Example 10 is shown in order from the left.

[0149] Referring to Figure 9 , in the case where the average temperature of the collection solution was 4°C as in Example 7, 9, the amount of lactate in the medium at Day 7 remained at a high value even if the culture time was lengthened. In addition, in the case where the average temperature of the collection solution was 20°C as in Example 8, 10, the amount of lactate in the medium at Day 7 remained at a high value if the culture time was 1 hour.

[0150] Referring to Figure 10 , in Reference Examples 7 to 10, it was found that the amount of lactate in the medium at Day 7 decreased significantly as the culture time was lengthened. In the case where the culture temperature was relatively high at 20°C and the culture time was 3 hours, the number of viable cells could not be maintained.

[0151] When Reference Examples 7 to 10 and Examples 7 to 10 were compared, it was found that there was a tendency that the decrease in the amount of lactate was moderated, and the adverse effects on the cells were moderated, in Examples 7 to 10. It is considered that this is because the collection solution was a medium having a buffering capacity in Examples 7 to 10, and the hydrogen ion index in the collection solution was maintained relatively stable.

[0152] [Example 11]

[0153] Example 11 is explained below. First, the core, the hydrogel precursor, the gelation solution, and the collection solution were prepared. The core was a cell suspension. The cell suspension was adjusted by the following method: cells (K562 cells from human chronic myelogenous leukemia) were suspended in a medium to which 0.3 w / v% methylcellulose was added to Iscove's Modified Dulbecco's Medium containing 3% methylcellulose (R&D systems HSC001). The initial cell density in the cell suspension was 1 x 10 7 cells / mL.

[0154] The hydrogel precursor was a sodium alginate solution. The sodium alginate solution was prepared by adding sodium alginate ("High-G ALG300" manufactured by Kimica Corporation) to physiological saline and stirring. The concentration of the sodium alginate with respect to the physiological saline was 1.0 w / v% percent concentration.

[0155] The gelation solution was an aqueous solution containing 100 mM calcium chloride and 3 w / v% sucrose.

[0156] Using these core, hydrogel precursor, and gelation solution, a cell cluster was manufactured based on the method for manufacturing a cell capsule of Embodiment 1 (refer to Figure 4 ). That is, a core stream, a hydrogel precursor stream around the core stream, and a gelation solution stream around the hydrogel precursor stream were formed, and these streams were discharged from the discharge port 240 into the collection solution.

[0157] The hydrogel precursor was crosslinked by contact with the gelation solution, and a tubular alginate gel was formed. Thus, a hydrogel that coats the core containing the cells was manufactured in the collection solution.

[0158] In Example 11, the collection solution was a medium having a pH buffering capacity. The medium was a medium to which 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate solution (manufactured by FUJIFILM Wako Pure Chemical Corporation) were added to Iscove's Modified Dulbecco's Medium containing 3% methylcellulose (R&D systems HSC001). The speed of the core stream was about 50 μL / min, and the core stream flowed for about 1 minute. Thus, the amount of the core accommodated inside the tubular alginate gel was about 50 μL.

[0159] Immediately after the core, alginate gel, and gelation solution were discharged into the collection solution, the lid of the container that accommodates the collection solution was covered, and the container was incubated at a given temperature for a given time. In Example 11, the incubation temperature was 4°C, and the incubation time was 1 hour (refer to Table 3 below).

[0160] After the cell capsules were incubated in the collection solution, the cell capsules were taken out of the collection solution and immersed in the culture medium, and the culture of the cells was started. That is, the cells were cultured in a state of being accommodated inside the tubular hydrogel. The culture medium was a culture medium to which 10% of fetal bovine serum (FBS) and 0.1% of a gentamicin sulfate solution (manufactured by FUJIFILM Wako Pure Chemical Corporation) were added to Iscove's Modified Dulbecco's Medium containing 3% of methylcellulose (R&D systems HSC001).

[0161] When the date on which the culture of the cells was started was set as "Day 0", the cells were recovered from the cell capsules on Day 2. In addition, the supernatant of the culture medium on Day 2 was recovered, and the amount of lactate in the supernatant was measured by the same method as in Example 1. Thus, the amount of lactate produced from Day 0 to Day 2 was measured.

[0162] [Example 12]

[0163] Example 12 is described below. The cell capsules of Example 12 were manufactured in the same manner as in Example 11 except for the culture temperature before the start of the culture. Specifically, in Example 12, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container accommodating the collection solution was closed, and the container was incubated at 20°C for 1 hour (see Table 3 below). The culture of the cells after the incubation was performed by the same method as in Example 11.

[0164] [Example 13]

[0165] Example 13 is described below. The cell capsules of Example 13 were manufactured in the same manner as in Example 11 except for the culture temperature before the start of the culture. Specifically, in Example 13, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container accommodating the collection solution was closed, and the container was incubated at 37°C for 1 hour (see Table 3 below). The culture of the cells after the incubation was performed by the same method as in Example 11.

[0166] [Example 14]

[0167] Example 14 is described below. The cell capsules of Example 14 were manufactured in the same manner as in Example 11 except for the culture time before the start of the culture. Specifically, in Example 14, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container accommodating the collection solution was closed, and the container was incubated at 4°C for 2 hours (see Table 3 below). The culture of the cells after the incubation was performed by the same method as in Example 11.

[0168] [Example 15]

[0169] Example 15 is explained below. The cell capsules of Example 15 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation and the incubation temperature. Specifically, in Example 15, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 20°C for 2 hours (see Table 3 below). The incubation of the cells after incubation was performed by the same method as Example 11.

[0170] [Example 16]

[0171] Example 16 is explained below. The cell capsules of Example 16 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation and the incubation temperature. Specifically, in Example 16, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 37°C for 2 hours (see Table 3 below). The incubation of the cells after incubation was performed by the same method as Example 11.

[0172] [Example 17]

[0173] Example 17 is explained below. The cell capsules of Example 17 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation. Specifically, in Example 17, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 4°C for 4 hours (see Table 3 below). The incubation of the cells after incubation was performed by the same method as Example 11.

[0174] [Example 18]

[0175] Example 18 is explained below. The cell capsules of Example 18 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation and the incubation temperature. Specifically, in Example 18, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 20°C for 4 hours (see Table 3 below). The incubation of the cells after incubation was performed by the same method as Example 11.

[0176] [Example 19]

[0177] Example 19 is explained below. The cell capsules of Example 19 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation and the incubation temperature. Specifically, in Example 19, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 37°C for 4 hours (see Table 3 below). The culture of the cells after incubation was performed by the same method as Example 11.

[0178] [Example 20]

[0179] Example 20 is explained below. The cell capsules of Example 20 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation. Specifically, in Example 20, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 4°C for 6 hours (see Table 3 below). The culture of the cells after incubation was performed by the same method as Example 11.

[0180] [Example 21]

[0181] Example 21 is explained below. The cell capsules of Example 21 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation and the incubation temperature. Specifically, in Example 21, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 20°C for 6 hours (see Table 3 below). The culture of the cells after incubation was performed by the same method as Example 11.

[0182] [Example 22]

[0183] Example 22 is explained below. The cell capsules of Example 22 were manufactured in the same manner as Example 11 except for the incubation time before the start of incubation and the incubation temperature. Specifically, in Example 22, immediately after the nucleus, the alginate gel, and the gelling solution were discharged into the collection solution, the lid of the container containing the collection solution was closed, and the container was incubated at 37°C for 6 hours (see Table 3 below). The culture of the cells after incubation was performed by the same method as Example 11.

[0184] Figure 11 are enlarged photographs showing the cell capsules on Day 2 in Examples 11 to 22. As shown in Figure 11 , it was observed that the inside of the tubular alginate contained K562 cells.

[0185] [Reference Examples 11 to 22]

[0186] The following describes Reference Examples 11 to 22. The cell capsules of Reference Examples 11 to 22 were manufactured in the same manner as Examples 11 to 22, except for the composition of the collection solution. The collection solution of Reference Examples 11 to 22 was a 100 mM aqueous calcium chloride solution. The cultivation of the cell capsules in the collection solution, and the cultivation of the cells after cultivation, were performed by the same methods as in Examples 11 to 22, respectively (see Table 3 below).

[0187]

[0188] Figure 12 is a graph showing the results of measurement of the amount of lactate in the culture medium of K562 cells on Day 2 in Examples 11 to 22. In Figure 12 , the amount of lactate measured in Example 11, Example 12, Example 13, Example 14, Example 15, Example 16, Example 17, Example 18, Example 19, Example 20, Example 21, and Example 22 is shown in order from the left.

[0189] Figure 13 is a graph showing the results of measurement of the amount of lactate in the culture medium of K562 cells on Day 2 in Reference Examples 11 to 22. In Figure 13 , the amount of lactate measured in Reference Example 11, Reference Example 12, Reference Example 13, Reference Example 14, Reference Example 15, Reference Example 16, Reference Example 17, Reference Example 18, Reference Example 19, Reference Example 20, Reference Example 21, and Reference Example 22 is shown in order from the left.

[0190] Referring to Figure 13 , in the Reference Examples, it was found that the amount of lactate in the culture medium decreased regardless of the cultivation temperature when the cultivation time was 6 hours. On the other hand, in the Examples, the amount of lactate in the culture medium was maintained even when the cultivation time was 6 hours. It is considered that this is because the collection solution was a culture medium having a buffering capacity in Examples 11 to 22, and the hydrogen ion index in the collection solution was maintained relatively stable.

[0191] According to the above Examples, it was found that the collection solution preferably contains a pH buffering agent when the cells to be covered with the tubular hydrogel are left in the collection solution for a long period of time. At this time, the collection solution can be a culture medium having a pH buffering capacity. In particular, according to the above Examples, it was found that such a collection solution is preferably maintained at a relatively low temperature, for example, 40°C or lower, 30°C or lower, preferably 25°C or lower, and more preferably 20°C or lower, at the time of manufacture of the cell capsule.

[0192] Such a collection solution can be suitably used when manufacturing very long cell capsules and / or a large number of cell capsules. In the case of manufacturing very long cell capsules and / or a large number of cell capsules, the time for which the core, hydrogel or hydrogel precursor, gelling solution is allowed to flow out can be, for example, 0.25 hours or more, 0.5 hours or more, or 1 hour or more. At this time, in the hydrogel or hydrogel precursor, the portion that is immersed in the collection solution at the start of the flow out is immersed in the collection solution for a long time. Even in such a case, by using a collection solution that contains a pH buffering agent, the adverse effects on the cells in the cell capsules can be mitigated.

[0193] The present application has been disclosed by embodiments as described above, but the discussion and drawings that form a part of this disclosure should not be construed as limiting the present application. Various alternative embodiments, examples, and applications of the present application can be apparent to those skilled in the art from the disclosure. Therefore, the technical scope of the present application is determined only by the specific matters of the invention in the patent claims reasonably derived from the above description.

[0194] This application claims priority based on Japanese Patent Application No. 2023-104592 filed on June 26, 2023, the entire contents of which are hereby incorporated by reference.

Claims

1. A method for producing a cell capsule, comprising: continuously or intermittently discharging a cell-coated hydrogel or a hydrogel precursor into a collection solution, the collection solution containing a pH buffering agent.

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

3. The method for producing a cell capsule according to claim 1 or 2, comprising: maintaining the hydrogel or a hydrogel generated from the hydrogel precursor in the collection solution for 0.25 hours or more from the start of the discharge of the hydrogel or the hydrogel precursor.

4. The method for producing a cell capsule according to claim 1 or 2, comprising: maintaining the hydrogel or a hydrogel generated from the hydrogel precursor in the collection solution for 1 hour or more and 24 hours or less from the start of the discharge of the hydrogel or the hydrogel precursor.

5. The method for producing a cell capsule according to 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. The method for producing a cell capsule according to 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. The method for producing a cell capsule according to any one of claims 1 to 6, wherein the pH of the collection solution is in the range of 6.5 to 8.

0.

8. The method for producing a cell capsule according to any one of claims 1 to 7, wherein the hydrogel or the hydrogel precursor coats a nucleus containing the cell and is discharged together with the nucleus into the collection solution.

9. The method for producing a cell capsule according to any one of claims 1 to 8, wherein the collection solution contains a gelation agent that causes the hydrogel precursor to gel.

10. The method for producing a cell capsule according to any one of claims 1 to 9, wherein a gelation solution that coats the hydrogel or the hydrogel precursor is discharged together with the hydrogel or the hydrogel precursor into the collection solution.

11. The method for producing a cell capsule according to claim 10, wherein the gelation solution contains a pH buffering agent.

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

13. The method for producing a cell capsule according to any one of claims 10 to 12, wherein the gelation solution has the same pH buffering agent as the collection solution.

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

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

16. A solution for producing a cell capsule having a cell-coated hydrogel, the solution containing a pH buffering agent.

17. The solution according to claim 16, wherein the pH buffering agent is a Good's buffer.

18. The solution according to claim 16 or 17, comprising a gelation agent that gelates the hydrogel precursor.

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

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