Cell- or cell product-containing composition and method for producing cell or cell aggregate and / or cell product

Abstract

Provided is a method for producing cells or cell aggregates and / or cell products, the method facilitating removal of hydrogel-derived substances. The present invention provides a method for producing cells or cell aggregates and / or cell products, the method including: employing a capsule containing a core base material and a shell that covers the core base material to culture cells or cell aggregates in the core base material, thus obtaining cells or cell aggregates and / or cell products; and physically destroying or cutting at least the shell to obtain the cells or the cell aggregates and / or the cell products.

Description

Method for producing cell or cell product-containing compositions, cells or cell aggregates and / or cell products

[0001] The present invention relates to compositions containing cells or cell products, and methods for producing cells or cell aggregates and / or cell products.

[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 a culture plate. This type of culture is not limited to cell proliferation, but is also performed for purposes such as antibody production, viral vector production, and physiologically active substance production. However, culturing a large number of cells using this two-dimensional culture method requires a large number of culture plates.

[0003] Meanwhile, techniques for culturing cells in a three-dimensional or pseudo-three-dimensional culture environment using scaffolds such as porous membranes or hydrogels are known. The following Patent Documents 1 to 6 disclose culturing cells inside a hydrogel such as a tubular alginate gel. Patent Document 7 also discloses culturing cells inside a hydrogel such as a roughly spherical alginate gel. Cells housed in this hydrogel can be cultured in a reactor containing a liquid medium while still encased in the hydrogel.

[0004] International Publication No. WO 2011 / 046105 JP 2017-99303 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 2018 / 096277

[0005] When a hydrogel such as an alginate gel is used, the alginate gel is dissolved using a chelating agent such as EDTA when recovering cells from within the hydrogel.

[0006] However, dissolving a hydrogel results in the presence of hydrolysates (e.g., alginate) in the solution. Removal of the hydrolysates from the cell- or cell-product-containing composition recovered from the hydrogel may require complex processing. Therefore, a method for producing cells or cell aggregates and / or cell products that allows for easy removal of hydrogel-derived substances (e.g., hydrogel lysates), as well as a cell- or cell-product-containing composition from which a relatively high degree of hydrogel-derived substances has been removed, are desired.

[0007] In one embodiment, a method for producing cells or cell aggregates and / or cell products includes culturing cells or cell aggregates within a capsule comprising a core substrate and a shell covering the core substrate to obtain cells, cell aggregates and / or cell products, and physically destroying or cutting at least the shell to recover the cells or cell aggregates and / or cell products.

[0008] In one embodiment, the cell or cell product-containing composition may be produced by using a capsule comprising a core substrate and a shell covering the core substrate, culturing cells or cell aggregates within the core substrate to obtain cells, cell aggregates and / or cell products, and then physically destroying or cutting at least the shell and recovering them.

[0009] In one embodiment, the cell population or cell product is a cell or cell product-containing composition produced by recovering cells or cell aggregates or cell products coated with a hydrogel, and has a solution containing the cells or cell aggregates and / or the cell products, the solution containing hydrogel-derived components, and the concentration of the hydrogel-derived components in the solution is 0.5% or less.

[0010] 1 is a schematic diagram of a string-shaped capsule according to one embodiment. FIG. 2 is a schematic cross-sectional view of a string-shaped capsule according to one embodiment. FIG. 3 is a schematic diagram for explaining a manufacturing method of a string-shaped capsule according to one embodiment. FIG. 4 is a graph showing the number of K562 cells collected in Examples 1 to 3 and Reference Example 1. FIG. 5 is a graph showing the survival rate of K562 cells in Examples 1 to 3 and Reference Example 1. FIG. 6 is a micrograph showing the state of NHDF cells cultured in a capsule in Example 4. FIG. 7 is a micrograph showing the state of NHDF cells when the capsule is cut in Example 4. FIG. 8 is a micrograph showing the state of NHDF cells after shaking of the cut capsule in Example 4. FIG. 9 is a micrograph showing the state of NHDF cells cultured in a capsule in Example 5. FIG. 10 is a micrograph showing the state of NHDF cells after shaking of the cut capsule in Example 5. FIG. 11 is a graph showing the amount of viral vector recovered in Examples 6 and 7 and Reference Examples 2 and 3. FIG. 12 is a graph showing the results of evaluation of cell recovery loss in the presence or absence of alginic acid.

[0011] It should be noted that the present disclosure is not limited to the particular devices and methods described. The terms used in this disclosure are for the purpose of describing particular embodiments or examples and are not intended to limit the scope of the claims. As used herein, the term "about" means plus or minus 5% of the numerical value with which it is used.

[0012] [Capsule] The capsule may have a core substrate containing cells or cell aggregates and a shell covering the core substrate. The shape of the capsule is not particularly limited. The capsule may be, for example, approximately spherical or approximately string-like.

[0013] Preferably, the shape of the capsule 10 may be a substantially string-like shape that extends continuously (see also Figures 1 and 2). In this case, the core substrate 12 has a continuously extending string-like shape, and the shell 14 has a continuously extending cylindrical shape that covers the core substrate. The cross-sectional shape of the capsule 10 is not particularly limited, and may be a substantially circular or elliptical shape. When the shape of the capsule 10 is a substantially string-like shape that extends continuously, a large number of cells or cell aggregates can be handled while being contained within the capsule, which may facilitate handling during the cultivation and recovery of cells or cell aggregates, and the production and recovery of cell products.

[0014] The core substrate may contain one or more cells or cell aggregates. When the capsule has a shape similar to a continuously extending string, the core substrate may contain a large number of cells or cell aggregates. The cell aggregate may be, for example, a cell aggregate (spheroid). A spheroid may be formed by aggregation of dispersed cells within the capsule. It should be noted that, hereinafter, the term "cell" may refer not only to a single cell itself, but also to a cell aggregate formed by an aggregation of multiple cells or a cell aggregate (spheroid).

[0015] The core substrate and the shell may each be a single layer or may have a multi-layer structure. For example, the core substrate may be composed of a multi-layer substrate covered with a shell. In this case, the core substrates of the respective layers may be composed of the same material or different materials. Similarly, the shell covering the core substrate may be composed of a multi-layer material. In this case, the shells of the respective layers may be composed of the same material or different materials.

[0016] The length of each capsule (maximum length in one direction) may be, for example, 1 cm or more, preferably 5 cm or more, more preferably 10 cm or more, and even more preferably 20 cm or more. When the capsule has a continuously extending, roughly string-like shape, the length of the core substrate and shell 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 capsule length, the more cells or cell aggregates can be accommodated in one capsule. From the viewpoint of ease of handling, the capsule length may be, for example, 200 cm or less.

[0017] The outer diameter of the shell is not particularly limited, but may be, for example, in the range of 0.2 μm to 5000 μm. The outer diameter of the shell is preferably 1 μm to 2000 μm, and more preferably 10 μm to 1000 μm. Here, the outer diameter may be defined as the average value of outer diameters measured at multiple positions, for example, 10 positions.

[0018] If the capsule is substantially spherical, the outer diameter of the shell may be defined by the diameter of the shell. If the capsule is string-shaped, the outer diameter of the shell may be defined by the diameter in the radial direction of the shell (reference symbol R1 in FIG. 2).

[0019] The inner diameter of the shell is not particularly limited, but may be, for example, in the range of 0.1 μm to 4000 μm. The inner diameter of the shell is preferably in the range of 0.8 μm to 1000 μm, and more preferably 5 μm to 500 μm. Here, the inner diameter may be defined by the average value of the outer diameters measured at multiple positions, for example, 10 positions. If the capsule is string-shaped, the inner diameter of the shell may be defined by the inner diameter in the radial direction of the shell (reference symbol R2 in FIG. 2).

[0020] The thickness of the shell, i.e., the difference between the outer diameter and the inner diameter of the shell, may be preferably 10 μm to 800 μm, more preferably 100 μm to 600 μm, and even more preferably 200 μm to 500 μm. Here, the difference between the outer diameter and the inner diameter may be defined by the difference between the average value of the outer diameter and the average value of the inner diameter.

[0021] The capsule may also be formed by molding the core-shell structure described above. For example, the capsule may be formed into a desired shape by bonding a plurality of approximately spherical core-shell structures together. The capsule may also be formed by molding a roughly string-like core-shell structure into a predetermined shape. For example, the capsule may be formed into a desired shape by bundling or folding roughly string-like core-shell structures. Furthermore, the capsule according to this embodiment may be formed by coating the core-shell structure described above with another hydrogel such as alginate gel.

[0022] The core substrate may be, for example, a medium, a culture solution, a culture supernatant, a buffer solution, physiological saline, an extracellular matrix, an extracellular matrix derived from a living organism or a cell, a collagen solution, laminin, agarose, nanocellulose, methylcellulose, sucrose, polyvinyl alcohol, serum, plasma, a material processed from serum or plasma, hyaluronic acid, proteoglycan, elastin, pullulan, dextran, pectin, gellan gum, xanthan gum, guar gum, carrageenan, glucomannan, an alginate solution, a hydrogel, a chitosan gel, Matrigel, a collagen gel, a gelatin, a peptide gel, a fibrin gel, an alginate gel, or a mixture thereof.

[0023] The core substrate may contain various growth factors suitable for cell culture, cell maintenance and proliferation, cell function expression, cell production of various substances, etc. The growth factors may be, for example, 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 / or hepatocyte growth factor (HGF).

[0024] When the cells contained in the core substrate are adherent cells, the core substrate may optionally contain a solid scaffold material, which may be composed of a material such as a microcarrier.

[0025] The shell may be made of a polymer that is sufficiently permeable to, for example, cell culture medium components. The polymer may include, for example, at least one selected from the group consisting of hydrogels, polysaccharides, synthetic polymers, polyamino acids, ornithine, lysine, arginine, histidine, guanidine, polyallylamine, polyvinylamine, polyethyleneimine, allylamine-diallylamine copolymers, and allylamine-maleic acid copolymers.

[0026] Preferably, at least one of the core substrate and the shell contains a hydrogel. The hydrogel is not particularly limited as long as it is a gel produced by gelling a hydrogel preparation (hydrogel precursor). Such hydrogels may be selected from the group consisting of, for example, alginate gel, chitosan gel, collagen gel, gelatin, peptide gel, laminin gel, carrageenan, pectin, gellan gum, and fibrin gel, as well as mixtures thereof. The hydrogel precursor may be one that forms these hydrogels upon gelation.

[0027] Chitosan gels, collagen gels, gelatin, peptide gels, and laminin gels can be gelled, for example, by altering temperature, pH, and / or salt concentration. Fibrin gels are gelled by the action of the monomer fibrinogen with the enzyme thrombin. Alginate gels, carrageenan, pectin, and gellan gum can be gelled by the addition of cations.

[0028] When at least one of the core substrate and the shell contains a hydrogel, the hydrogel preferably contains an alginate gel. The alginate gel can be formed by partially crosslinking an alginate solution (precursor) with divalent metal ions. The alginate solution may be, for example, sodium alginate, potassium alginate, ammonium alginate, or a combination thereof. The alginate may also be a natural extract or a chemically modified alginate. Examples of chemically modified alginates include methacrylate-modified alginate. Alginate gel has low toxicity to cells and is therefore suitable as a container for encasing cells.

[0029] The hydrogel may also be a mixture of the above-mentioned alginate with agar, agarose, polyethylene glycol (PEG), polylactic acid (PLA), nanocellulose, etc. The weight of the alginate relative to the weight of the solvent in the alginate solution is not particularly limited, but is preferably 0.2 wt %, for example, preferably 0.25 wt %, and more preferably 0.5 wt %.

[0030] Examples of divalent metal ions used to obtain alginate gel include calcium ions, magnesium ions, barium ions, strontium ions, zinc ions, and iron ions. Preferably, the divalent metal ions are calcium ions or barium ions. The divalent metal ions are preferably provided to alginic acid in the form of a solution. Examples of solutions containing divalent metal ions include solutions containing calcium ions. Examples of such solutions include aqueous solutions such as calcium chloride, calcium carbonate, and calcium gluconate. Preferably, such solutions are aqueous solutions of calcium chloride or barium chloride.

[0031] The concentration of the divalent metal ion in the solution containing the divalent metal ion is, for example, 1 mM to 1 M, preferably 20 to 500 mM, and more preferably 100 mM.

[0032] The raw material for the alginate gel used as the hydrogel is preferably sodium alginate. In this case, the M / G ratio of sodium alginate may be preferably 0.1 to 1.8. The M / G ratio is determined by the ratio of D-mannuronic acid to L-guluronic acid in the alginic acid group. The M / G ratio varies depending on the type and composition of alginic acid.

[0033] The molecular weight (GPC) of sodium alginate is preferably 700,000 to 1,000,000, and more preferably 800,000 to 1,000,000.

[0034] Preferably, the shell contains a hydrogel and the core material contains a liquid or a sol. Such core-shell capsules can be produced by the methods described in any of the above-mentioned Patent Documents 1 to 7.

[0035] The cells or cell aggregates contained in the core substrate may include non-adherent cells (suspension cells) or adhesive cells. When the cells or cell aggregates are adhesive cells, the shell may include a material that is adhesive to the adhesive cells. Alternatively, a material that is adhesive to the adhesive cells may be attached to the inner surface of the shell. For example, the shell may be a hydrogel such as alginate gel, and fibrin may be attached to the inner surface of the hydrogel.

[0036] The type of cell is not particularly limited. Such cells may be, for example, pluripotent ES cells or iPS cells, various pluripotent stem cells (hematopoietic stem cells, neural stem cells, mesenchymal stem cells, etc.), or unipotent stem cells (hepatic stem cells, germline stem cells, etc.). Furthermore, the cells may be various differentiated cells, such as muscle cells such as skeletal muscle cells and cardiac muscle cells, nerve cells such as cerebral cortical cells, fibroblasts, epithelial cells, hepatocytes, pancreatic beta cells, skin cells, etc.

[0037] The cells may be virus-producing cells, viral vector-producing cells, antibody-producing cells, biopharmaceutical substance-producing cells, physiologically active substance-producing cells, immune cells, and / or cells for food applications. Cells for food applications may include at least one cell selected from, for example, adipocytes, blood cells, fibroblasts, nerve cells, skin cells, vascular cells, and pluripotent stem cells.

[0038] The viral vector-producing cells may be, for example, AAV viral vector-producing cells, adenoviral vector-producing cells, retroviral vector-producing cells, lentiviral vector-producing cells, Sendai viral vector-producing cells, or herpes simplex viral vector-producing cells. The viral producing cells may also be, for example, oncolytic viral cells, such as herpes simplex viral cells, adenoviral cells, or vaccinia viral cells.

[0039] A "viral vector producing cell" or "vector producing cell" may be a cell capable of producing a viral vector or viral vector particle. A retroviral vector producing cell may also be a "producer cell" or a "packaging cell." One or more DNA constructs of the viral vector system may be either stably integrated or episomally maintained in the viral vector producing cell. Alternatively, all DNA components of the viral vector system may be transiently transfected into a viral vector producing cell. In yet another alternative, a producer cell stably expressing some of the components may be transiently transfected with the remaining components required for vector production.

[0040] If the cell or cell mass is a viral vector-producing cell, the cellular product may be a viral vector, and if the cell or cell mass is a virus-producing cell, the cellular product may be a virus.

[0041] The antibody-producing cell is not particularly limited, and may be, for example, a genetically modified animal cell that produces an antibody. The host cell may be, for example, a cell selected from the group consisting of CHO cells, CHO cell sublines, COS cells, Sp2 / 0 cells, NS0 cells, SP2 cells, PERC6 cells, YB2 / 0 cells, YE2 / 0 cells, 1R983F cells, Namalwa cells, Wil-2 cells, Jurkat cells, Vero cells, Molt-4 cells, HEK293 cells, BHK cells, HT-1080 cells, KGH6 cells, P3X63Ag8.653 cells, C127 cells, JC cells, LA7 cells, ZR-45-30 cells, hTERT cells, NM2C5 cells, and UACC-812 cells.

[0042] When the cell or cell aggregate is a biopharmaceutical substance-producing cell, the cell product may be a biopharmaceutical substance. The biopharmaceutical substance may be, for example, any substance that can be used as a biopharmaceutical. The biopharmaceutical substance may be a virus, a viral vector, an antibody, or the like.

[0043] The physiologically active substance-producing cells are not particularly limited, and may be, for example, cells selected from the group consisting of insulin-secreting cells, pancreatic islets, pancreatic islet cells, dopamine-secreting cells, pituitary cells, growth hormone-secreting cells, parathyroid cells, nerve growth factor-secreting cells, blood coagulation factor-secreting cells, hepatocytes, parathyroid cells, and physiologically active substance expression vectors (genetically modified cells).

[0044] Furthermore, the cell or cell mass may include a tissue or organ formed by a cell mass, a microorganism such as bacteria, fungi such as oomycetes, myxomycetes and fungi, and algae. Examples of fungi include koji mold and yeast.

[0045] When the cell or cell aggregate is an antibody-producing cell, the cell product may be an antibody.

[0046] When the cell or cell aggregate is a physiologically active substance-producing cell, the cell product is a physiologically active substance, which may be, for example, a substance selected from the group consisting of insulin, dopamine, growth hormone, nerve growth factor, blood coagulation factor, and a physiologically active substance expression vector.

[0047] The diameter of the cells or cell aggregates may be smaller than the inner diameter of the shell. Furthermore, the diameter of the cells or cell aggregates and / or cell products is not particularly limited, but may be, for example, 10 nm or greater.

[0048] [Method for manufacturing capsules] An example of a method for manufacturing capsules including a core substrate containing cells or cell aggregates and a shell covering the core substrate will be described. The method for manufacturing the capsules and the method for containing cells or cell aggregates in the capsules are not particularly limited, but may be the same as the methods described in any of Patent Documents 1 to 7 mentioned above.

[0049] When the capsules are in the form of a continuous string, they can be produced, for example, as follows (see also Figure 3). First, a core substrate (suspension) 1 containing cells is flowed, while a shell precursor 3 is flowed around the suspension 1. The material of the core substrate is as described above. When the shell is a hydrogel, the shell precursor 3 may be a hydrogel precursor. The cells, core substrate, and hydrogel precursor are as described above.

[0050] Preferably, the suspension 1 is caused to flow as a laminar flow. This laminar flow is formed in the first inlet pipe 2. The shell precursor 3 is caused to flow so as to surround the outer periphery of the flow of suspension 1. In other words, the shell precursor 3 is coaxial with the suspension 1 and flows in the same direction. Preferably, the shell precursor 3 is caused to flow as a laminar flow. As a result, a flow of the shell precursor 3 is formed at the second inlet pipe 4 that surrounds the flow of suspension 1.

[0051] The shell precursor 3 is then gelled or solidified to form a tubular shell that covers the suspension 1. The gelling or solidification of the shell precursor 3 can be achieved by contacting the outer periphery of the flow of the shell precursor 3 with a sheath solution 5 containing a material that gels or solidifies the shell precursor 3. In the embodiment shown in Fig. 3, the sheath solution 5 surrounds the shell precursor (second laminar flow) 3 at the third inlet tube 6. In other words, the sheath solution 5 flows coaxially with the suspension 1 and the shell precursor 3 and in the same direction.

[0052] The flow of the suspension 1, the shell precursor 3, and the sheath solution 5 may be started in any order at the start of production, and may be stopped in any order at the end of production. However, from the viewpoint of confining cells without leakage, it is preferable that the flow of the suspension 1 be started last at the start of production, and that the flow of the suspension 1 be stopped first at the end of production.

[0053] The suspension 1, shell precursor 3, and sheath solution 5 flow out of the third introduction tube 6 and are immersed in a liquid or suspension 20, such as physiological saline or a liquid medium. Here, the shell precursor 3 flows out of the third introduction tube 6 while being gelled by the addition of a gelling agent. This forms a capsule 10 containing a tubular hydrogel that covers the suspension.

[0054] 3, capsules were formed by forming a flow of cell suspension 1, a flow of shell precursor 3, and a flow of sheath solution 5, which then flowed out from the third inlet tube 6. Alternatively, capsules of a similar structure can be formed by forming a flow of suspension 1, forming a flow of shell precursor 3 that covers the outer periphery of the first laminar flow, and discharging these flows into a container containing sheath solution 5 that contains a material that gels or solidifies the shell precursor 3. In this case, if the flow of suspension 1 and the flow of shell precursor 3 are intermittently discharged, a large number of spherical capsules can also be produced.

[0055] When the core substrate contains adherent cells, the core substrate may optionally contain a solid scaffold or a precursor of an adhesive material. The precursor of the adhesive material may contain, for example, fibrinogen, a component derived from bovine platelet lysate, and / or a component derived from human platelet lysate (hPL). Such components may be, for example, insoluble components in bovine platelet lysate or human platelet lysate, or may be a fibrin-containing substance. Here, the fibrin-containing substance may contain insoluble components derived from plasma, platelets, or platelet lysate. In this case, the adhesive material may be precipitated from the precursor of the adhesive material in the core substrate. This allows the adhesive material to be coated on the inner surface of the shell. For example, when the precursor of the adhesive material is fibrinogen, a component derived from bovine platelet lysate, and / or a component derived from human platelet lysate (hPL), fibrin can be attached to the inner surface of the shell.

[0056] [Method for producing cells or cell aggregates] First, cells are cultured using capsules comprising a core substrate containing cells or cell aggregates and a shell covering the core substrate (culture step). The cell culture is carried out while the cells are encapsulated. Therefore, the cells may be immersed in a solution such as a liquid medium together with the capsule in a encapsulated state. The cell culture conditions are determined appropriately depending on the type of cells to be cultured and the purpose of the culture.

[0057] Next, at least the shell of the capsule is physically broken or cut. The physical breaking or cutting of the shell may be by partially breaking the shell or by cutting the shell into multiple parts. The breaking or cutting of the shell can be performed by piercing the shell with a needle or by cutting the shell with a cutter or scissors.

[0058] When the capsules are in the form of a continuous string, the physical disruption or cutting of the shell is preferably accomplished by cutting the capsule string into multiple pieces.

[0059] The capsules containing the physically disrupted or cut shells are then placed in a solution, such as a culture medium, or allowed to move in the solution, causing the cells or cell aggregates and / or cell products to be expelled from the capsule interior. The solution may typically be a liquid culture medium.

[0060] In this step, the capsules containing the physically broken or cut shells are preferably moved in a solution such as a liquid medium. This operation may be performed by repeatedly aspirating and discharging the medium containing the capsules containing the physically broken or cut shells, or by rotating or shaking the container containing the medium containing the capsules. This allows the cells or cell aggregates to be more effectively removed from the capsules.

[0061] When the shell is physically cut, the shell is preferably cut, for example, 10 or more, 30 or more, or 50 or more times using a cutting means such as scissors or a cutter. Furthermore, it is preferable that the majority of the cut shells (small pieces), for example, 80% or more of the multiple small pieces, are divided into pieces having a diameter of, for example, 5 cm or less, 3 cm or less, or 1 cm or less. Here, the diameter of the small pieces may be defined by the maximum length of the small pieces.

[0062] Because the shell is physically destroyed or cut, this process can be performed without chemical treatment. Therefore, cells or cell aggregates inside the capsules can be recovered without using chelating agents such as EDTA (ethylenediaminetetraacetic acid), which are commonly used to dissolve hydrogels such as alginate gel. This allows the shell, such as alginate gel, to be easily removed from the solution. Furthermore, damage to the cells or cell aggregates caused by chelating agents is also avoided.

[0063] If the shell contains an adhesive material that is adhesive to adherent cells, or if the inner surface of the shell is coated with an adhesive material that is adhesive to adherent cells, a process for dissolving the adhesive material may be performed when recovering cells or cell aggregates. Dissolving the adhesive material can be achieved by adding a reagent for dissolving the adhesive material to the solution. For example, if fibrin is used as the adhesive material, a reagent for selectively dissolving fibrin can be added to the solution containing the capsules. The reagent for selectively dissolving fibrin may be, for example, a proteolytic enzyme such as nattokinase, urokinase, plasmin, fibrin / fibrinogen lysing enzyme, or trypsin. This facilitates the release of adherent cells from within the physically disrupted or cut capsules.

[0064] The treatment for dissolving the adhesive material may be carried out before or after physically destroying or cutting the shell, and even in this case, the treatment for dissolving the adhesive material is preferably carried out with a reagent that barely dissolves the shell.

[0065] Next, after physically destroying or cutting at least the shell, the capsules with the physically destroyed or cut shell are separated from the cells or cell aggregates. This separation can be performed, for example, by filtering the solution containing the capsules through a mesh. Alternatively or in addition, centrifugation may be used, which allows the capsules to be removed from a solution such as a liquid medium.

[0066] By the above process, cells or cell aggregates can be collected from inside the capsules to produce a cell-containing composition (cell population).

[0067] When cells or cell aggregates in a shell form spheroids, the diameter of the cells or cell aggregates is preferably smaller than the inner diameter of the shell. More preferably, the diameter of the cells or cell aggregates is preferably 90% or less of the inner diameter of the shell. This makes it easier for the cells or cell aggregates to escape from the capsule containing the physically destroyed or cut shell.

[0068] The length of each capsule (maximum length in one direction) may be, for example, 1 cm or more, preferably 5 cm or more, more preferably 10 cm or more, and even more preferably 20 cm or more. When the capsule has a continuously extending, roughly string-like shape, the length of the core substrate and shell may be, for example, 5 cm or more, more preferably 10 cm or more, and even more preferably 20 cm or more. When the capsule has such a macroscopic size, the shell becomes easier to physically destroy or cut. Furthermore, from the viewpoint of releasing cells from the physically destroyed or cut shell, it is preferable that the core substrate contains a liquid or a sol. In other words, it is preferable that the core substrate is not a solid or a gel.

[0069] When the capsule has a generally continuous string-like shape, it is preferable that the capsule be cut, for example, 10 or more times, 30 or more times, or 50 or more times, using a cutting means such as scissors or a cutter. Furthermore, it is preferable that the majority of the cut capsule (small pieces), for example, 80% or more of the multiple small pieces, be broken into pieces with lengths of, for example, 5 cm or less, 3 cm or less, or 1 cm or less. This allows cells or cell aggregates and / or cell products to be efficiently released from the inside of the capsule.

[0070] [Method for producing a cell product] First, cells are cultured using a capsule comprising a core substrate containing cells or cell aggregates and a shell covering the core substrate (culture step). Details of the culture step are as described above. However, in this culture step, the cells or cell aggregates produce a cell product. That is, the cells produce the cell product while contained in the capsule. Here, at least a portion of the cell product is retained within the capsule. The cell product may be, for example, a virus, a viral vector, an antibody, a biopharmaceutical substance, or a physiologically active substance.

[0071] Next, at least the shell of the capsule is physically broken or cut (shell breaking step). Details of the shell breaking step are as described above.

[0072] The capsules containing the physically broken or cut shells are then placed in a solution, such as a culture medium, or allowed to move in the solution, causing the cell products trapped within the capsules to be released from the capsules. The solution may typically be a liquid culture medium.

[0073] In this step, the capsules containing the physically broken or cut shells are preferably moved in a solution such as a liquid medium. This operation may be performed by shaking the capsules containing the physically broken or cut shells in the medium, or by repeatedly aspirating and discharging the medium containing the capsules using a pipette or needle. This allows the cell products to be more effectively removed from the capsules.

[0074] Because the shell is physically destroyed or cut, this process can be performed without chemical treatment. Therefore, cell products within the capsules can be recovered without dissolving the hydrogel, such as alginate gel, with a chelating agent, such as EDTA (ethylenediaminetetraacetic acid) or citric acid. This allows cells or cell aggregates to be easily recovered from the capsules while suppressing the residue of hydrogel-derived substances.

[0075] Next, after physically breaking or severing at least the shell, the capsules with the physically broken or severed shells are separated from the cell products. This separation can be performed, for example, by filtering the solution containing the capsules, thereby removing the capsules from a solution such as a liquid medium.

[0076] After removing capsules with physically broken or cut shells from the cell product by filtration as described above, the cells may be removed by extracting the culture supernatant, which is then centrifuged and purified to produce a cell product-containing composition.

[0077] If the cell products remain intracellularly, they may be extracted into the culture medium by disrupting the cells before removing them, which can prevent some of the cell products from being lost when the cells are removed.

[0078] In the above-described embodiment, the cells are removed after the capsules with physically broken or cut shells are removed by filtration. Alternatively, the cells may be removed by filtration together with the capsules with physically broken or cut shells.

[0079] The above process allows for the production of cellular products. The inventors of the present application have newly discovered that a portion of the cellular product remains within a shell, for example, a shell having a hydrogel such as alginate gel. In particular, the inventors have newly discovered that a portion of an antibody, virus, or viral vector remains within a shell having a hydrogel such as alginate gel. In the above embodiment, as described above, the shell is physically destroyed or cut, so that the cellular product remaining within the capsule can also be recovered. Therefore, the recovery rate of the cellular product is improved.

[0080] Preferably, the shell comprises a hydrogel, and the core substrate comprises a liquid or sol. When the core substrate is a liquid or sol, cells or cell aggregates and / or cell products can be easily released from capsules with a broken or cut shell by physically destroying or cutting the hydrogel shell. Therefore, the recovery rate of cell products can be improved.

[0081] The diameter of the cell product is not particularly limited, but may be, for example, 10 nm or more, preferably 20 nm or more, and more preferably 30 nm or more. Some of the cell products of such sizes may remain within a shell containing a hydrogel such as an alginate gel. Therefore, the method according to the above embodiment may be more preferably applied.

[0082] [Cell or cell product-containing composition] The cell or cell product-containing composition may be produced by the above-mentioned cell production method or cell product production method. Here, in the above-mentioned production method, the shell constituting the capsule is removed by breaking or cutting and filtering. Therefore, most of the shell-derived components can be removed from the produced cell population or cell product. From this perspective, the following cell or cell product-containing composition can be obtained.

[0083] That is, a cell- or cell product-containing composition produced by recovering cells or cell aggregates or cell products coated with a hydrogel may comprise a solution containing cells or the cell aggregates and / or cell products. The solution comprises hydrogel-derived components. The hydrogel-derived components may be obtained by dissolving a portion of the hydrogel that constitutes the core and / or shell.

[0084] The concentration of the hydrogel-derived component in the solution containing the cells or cell aggregates and / or cell products may be 0.5 wt% or less, preferably 0.25 wt% or less, and more preferably 0.1 wt% or less.

[0085] The concentration of the hydrogel-derived component in the solution may be, for example, 0.000015 w / w % or more, 0.000010 w / w % or more, or 0.000005 w / w % or more.

[0086] Here, the hydrogel may be an alginate gel as described above. In this case, the hydrogel-derived component is (ungelled) alginate. A small amount of alginate may remain in the solution containing the cell population or cell products.

[0087] The method for measuring the concentration of the hydrogel-derived component is not particularly limited, and may be any known method. For example, the concentration of alginic acid can be measured by the carbazole method. In the carbazole method, the concentration of alginic acid in a solution is measured by quantifying the amount of uronic acid contained in the alginic acid.

[0088] Example 1 Example 1 will be described. The following cells and materials were prepared. Cells: Human chronic myeloid leukemia-derived cells (K562 cells) Core substrate: 0.3 w / v% methylcellulose solution (R&D Scientific) Alginate precursor: Sodium alginate ("High-G ALG300" manufactured by Kimika Co., Ltd.) Culture medium: IMDM + 10% FBS + 0.1% gentamicin First, a cell suspension, alginate precursor (hydrogel precursor), and sheath solution were prepared. The sheath solution was an aqueous solution containing 100 mM calcium chloride and 3 w / v% sucrose.

[0089] The alginic acid precursor (hydrogel precursor) was a sodium alginate solution. The sodium alginate solution was prepared by adding the above sodium alginate to physiological saline and stirring. The concentration of sodium alginate relative to the physiological saline was 1 w / v%.

[0090] The cell suspension was prepared by introducing the above-mentioned K562 cells into a 0.3 w / v% methylcellulose solution (solvent: the above-mentioned medium) as a core substrate. The K562 cells are non-adherent cells in a suspension system. The initial density of the K562 cells in the cell suspension was 2 × 10 ７ The cells introduced into the methylcellulose solution were in the form of single cells.

[0091] Using the cell suspension, alginate precursor (hydrogel precursor), and sheath solution, a cell population was produced based on the capsule production method described above (see also Figure 3). Specifically, a flow of the cell suspension, a flow of the alginate precursor surrounding the cell suspension, and a flow of the sheath solution surrounding the alginate precursor were formed, and these flows were ejected into physiological saline. The alginate precursor was crosslinked by contact with the calcium chloride aqueous solution used as the sheath solution, forming a tubular alginate gel.

[0092] This produced string-like capsules in the saline solution, each having a cell-containing core and a shell surrounding the core. The cell-covered capsules were allowed to stand in the saline solution for a desired period of time.

[0093] Next, the cells together with the capsules were transferred to a liquid medium and cultured inside the capsules (culture step). The cells were cultured in the liquid medium together with the capsules in a encapsulated state. The cell culture was carried out at a temperature of 37°C for 3 days. The liquid medium used for the culture was as described above.

[0094] Next, the capsule shell was physically cut with scissors. For bent capsules, multiple cuts can be made at once with a cutter. This process was repeated 30 times, resulting in multiple cuts on the capsule.

[0095] Next, the solution containing the capsules with the physically cut shells was pipetted 30 times. Pipetting was performed by aspirating and discharging the medium containing the capsules with a pipette. It was confirmed that the cells were sufficiently removed from the cut shells by pipetting.

[0096] The capsules were then removed from the medium by filtration, producing a cell population containing a large number of K562 cells.

[0097] After 4 days of culture, the cell suspension containing the K562 cells was recovered from the capsules, and the capsules and cells were centrifuged to remove the solution, after which the amount of alginic acid was quantified. The quantification of alginic acid was performed using the carbazole method. The resulting alginic acid concentration was 12.4 μg / mL. In this case, the alginic acid concentration was 2.0 wt% or less. By washing the solution containing alginic acid and K562 cells, a solution containing 0.5 wt% or less alginic acid and K562 cells (cell-containing composition) was easily obtained.

[0098] Example 2: Instead of pipetting the solution containing capsules with physically broken shells, the container containing the solution containing capsules with physically broken shells was rotated in a mix rotor at 60 rpm for 15 minutes. A cell population containing a large number of K562 cells was produced in the same manner as in Example 1, except for this procedure.

[0099] Example 3: Instead of pipetting the solution containing capsules with physically broken shells, the container containing the solution containing capsules with physically broken shells was swung (shaken) at 125 rpm for 5 minutes. A cell population containing a large number of K562 cells was produced in the same manner as in Example 1, except for this procedure.

[0100] [Reference Example 1] Capsules were produced and cells were cultured in the same manner as in Example 1. After that, the capsules were dissolved without cutting them by placing them in a 1 mM EDTA-PBS solution and leaving them for 5 minutes. Then, the cells were recovered by pipetting with a P1000.

[0101] In Reference Example 1, the amount of alginic acid in the cell suspension containing the recovered K562 cells was quantified. The quantification of alginic acid was performed using the carbazole method. The resulting alginic acid concentration was 290 μg / mL. This was more than 20 times the alginic acid concentration in the solution recovered in Example 1.

[0102] [Measurement of viable cell count] The viable cell count and viability of the cell populations produced in Examples 1 to 3 and Reference Example 1 were measured. The viable cell count and viability were measured by trypan blue dye exclusion test.

[0103] 4 is a graph showing the number of K562 cells collected in Examples 1 to 3 and Reference Example 1. FIG. 5 is a graph showing the survival rate of K562 cells in Examples 1 to 3 and Reference Example 1.

[0104] Referring to FIG. 4, the number of K562 cells recovered by cutting the capsule shell was higher than that by dissolving the hydrogel capsule with EDTA-PBS solution.

[0105] Furthermore, referring to FIG. 5, the viability of K562 cells prepared by cutting the capsule shell was higher than that of K562 cells prepared by dissolving the hydrogel capsule with EDTA-PBS solution.

[0106] These results are believed to be due to the fact that in Examples 1 to 3, the shells were physically destroyed or cut and no chelating agent such as EDTA was used, which prevented damage to the K562 cells by the chelating agent during recovery from the capsules, resulting in a higher survival rate of the K562 cells.

[0107] Example 4 Example 4 will be described. The following cells and materials were prepared. Cells: Human neonatal dermal fibroblasts (NHDF cells) Core substrate: 0.3 w / v% methylcellulose solution (R&D Scientific) Alginate precursor: Sodium alginate ("High-G ALG300" manufactured by Kimika Co., Ltd.) Culture medium: DMEM low glucose + 10% FBS + 0.1% gentamicin First, a cell suspension, alginate precursor (hydrogel precursor), and sheath solution were prepared. The sheath solution was an aqueous solution containing 100 mM calcium chloride and 3 w / v% sucrose.

[0108] The alginic acid precursor (hydrogel precursor) was a sodium alginate solution. The sodium alginate solution was prepared by adding the above sodium alginate to physiological saline and stirring. The concentration of sodium alginate relative to the physiological saline was 1 w / v%.

[0109] The cell suspension was prepared by introducing the above-mentioned NHDF cells into a 0.3 w / v% methylcellulose solution (solvent: the above-mentioned medium) as a core substrate. The NHDF cells are adherent cells. The initial density of the NHDF cells in the cell suspension was 4 × 10 ６ The cells introduced into the methylcellulose solution were in the form of single cells.

[0110] Using the cell suspension, alginate precursor (hydrogel precursor), and sheath solution, a cell population was produced based on the capsule production method described above (see also Figure 3). Specifically, a flow of the cell suspension, a flow of the alginate precursor surrounding the cell suspension, and a flow of the sheath solution surrounding the alginate precursor were formed, and these flows were ejected into physiological saline. The alginate precursor was crosslinked by contact with the calcium chloride aqueous solution used as the sheath solution, forming a tubular alginate gel.

[0111] This resulted in the production of string-like capsules in the saline solution, each having a core containing NHDF cells and a shell surrounding the core. The capsules surrounding the NHDF cells were allowed to stand in the saline solution for a desired period of time.

[0112] Next, the NHDF cells together with the capsules were transferred to a liquid medium, and the NHDF cells were cultured inside the capsules (culture step). The NHDF cells were cultured in the liquid medium together with the capsules in a encapsulated state. The cells were cultured at a temperature of 37°C for 2 days. The liquid medium used for the culture was as described above.

[0113] Figure 6 is a micrograph showing the state of K562 cells cultured in a capsule in Example 4. Figure 6 shows the state of a capsule containing NHDF cells cultured for two days. As shown in Figure 6, the NHDF cells form spheroids within the capsule. Specifically, the string-like capsule contains a large number of NHDF cell spheroids.

[0114] Hydrogels such as alginate gels do not substantially function as scaffolds for adherent cells such as NHDF cells, and therefore, in Example 4, it is believed that the NHDF cells formed spheroids without adhering to a shell such as alginate gel.

[0115] Next, the capsule shell was physically cut using a cutter. For bent capsules, the cutter can cut multiple locations at once. This process was repeated 30 times, resulting in multiple cuts on the capsule (see Figure 7).

[0116] Next, the container containing the solution containing the capsules with the physically cut shells was shaken at 125 rpm for 5 minutes, which confirmed that the NHDF cells had been sufficiently released from the cut shells (see Figure 8).

[0117] The capsules were then removed from the medium by filtration, producing a cell population containing numerous NHDF cell spheroids.

[0118] Example 5 Example 5 will be described. The following cells and materials were prepared. Cells: Human neonatal dermal fibroblasts (NHDF cells) Core substrate: A solution prepared by mixing the following medium and hPL (AventaCell, UltraGRO-PURE:HPCHXCRL50) in a 1:1 ratio Alginic acid precursor: Sodium alginate ("High-G ALG300" manufactured by Kimika Co., Ltd.) Medium: DMEM low glucose + 10% FBS + 0.1% gentamicin

[0119] First, a cell suspension, an alginate precursor (hydrogel precursor), and a sheath solution were prepared. The sheath solution was an aqueous solution containing 100 mM calcium chloride and 3 w / v % sucrose.

[0120] The alginic acid precursor (hydrogel precursor) was a sodium alginate solution. The sodium alginate solution was prepared by adding the above sodium alginate to physiological saline and stirring. The concentration of sodium alginate relative to the physiological saline was 1 w / v%.

[0121] The cell suspension was prepared by introducing the NHDF cells into the core substrate. The NHDF cells are adhesive cells. The initial density of the NHDF cells in the cell suspension was 2 x 10 ６ The cells introduced into the core substrate were single-cell cells.

[0122] Using the cell suspension, alginate precursor (hydrogel precursor), and sheath solution, a cell population was produced based on the capsule production method described above (see also Figure 3). Specifically, a flow of the cell suspension, a flow of the alginate precursor surrounding the cell suspension, and a flow of the sheath solution surrounding the alginate precursor were formed, and these flows were ejected into physiological saline. The alginate precursor was crosslinked by contact with the calcium chloride aqueous solution used as the sheath solution, forming a tubular alginate gel.

[0123] The string-like capsules were left standing at 37°C for a desired period of time. The hPL contained fibrinogen. The fibrinogen precipitated as fibrin (an adhesive material) on the inner surface of the alginate gel shell. This resulted in the production of string-like capsules having a core containing NHDF cells, a shell covering the core, and fibrin attached to the inner surface of the shell.

[0124] Next, the NHDF cells together with the capsules were transferred to a liquid medium, and the NHDF cells were cultured inside the capsules (culture step). The NHDF cells were cultured in the liquid medium together with the capsules in a encapsulated state. The cells were cultured at a temperature of 37°C for 2 days. The liquid medium used for the culture was as described above.

[0125] Figure 9 is a micrograph showing the state of NHDF cells cultured in a capsule in Example 5. Figure 9 is a micrograph showing the state of NHDF cells cultured in a capsule in Example 5. Figure 9 shows the state of a capsule containing NHDF cells cultured for two days. As shown in Figure 9, the NHDF cells are adhered to the shell inside the capsule via fibrin.

[0126] In Example 5, fibrin is attached to the inner surface of the shell containing a hydrogel such as alginate gel. It is believed that this fibrin acts to allow NHDF cells, which are adhesive cells, to adhere to the alginate gel.

[0127] Next, the capsule shell was physically cut using a cutter. For bent capsules, the cutter can cut multiple locations at once. This cutting process was performed 30 times, resulting in multiple cuts on the capsule.

[0128] Next, the container containing the solution containing capsules with physically cut shells was shook at 125 rpm for 5 minutes. After this, the adhesive material, fibrin, was dissolved. Fibrin dissolution was achieved by adding nattokinase. Five minutes after the addition of nattokinase, the container containing the solution containing capsules with physically cut shells was further shook at 125 rpm for 15 minutes. This confirmed that the NHDF cells had sufficiently escaped from within the cut shells (see FIG. 10 ).

[0129] As shown in Example 5, even if the cultured cells adhere to the adhesive material that makes up the capsule, the adhesive cells can be sufficiently released from within the shell by at least physically destroying or cutting the shell and dissolving the adhesive material.

[0130] The capsules were then removed from the medium by filtration, producing a cell population containing numerous NHDF cell spheroids.

[0131] Example 6 Example 6 will be described. The following cells and materials were prepared. Cells: AAV-293 (Agelent, 240073-41) Alginate precursor: Sodium alginate (Kimika Co., Ltd., "High-G ALG300") Core substrate: 0.3 w / v% methylcellulose solution (R&D Scientific) 00) Culture medium: DMEM + 10% FBS + 1% L-Glutamine (Hereinafter, unless otherwise specified, the left side will be referred to as "culture medium") DMEM: high glucose, pyruvate (Gibco, 11995040) Fetal Bovine Serum (CORNING, 35-010-CV, lot. 19321001) L-Glutamine (200 mM) (Gibco, 25030081) TrypLE select (Gibco, 12563-011) 10 cm dish: Cell culture dish 100mm (Φ96x21mm) (TPP, 93100) ・1xPBS: 10xPBS (Fujifilm, 163-25265) diluted with MQ solution and autoclaved before use ・pEI-pro (PPU, 115-0015) ・6-well plate: Multi-dish round (6 well) (ThermoFisher, 140675) ・P1000 tip: Tips HRC UNV 1000μL 768C / 8 (RC research grade tip, general purpose, 1000uL) (Mettler-Toledo Rainin, 30374656) ・G18 needle: Terumon bevel needle 18G (Terumo, NN-1838N) ・10mL syringe: Terumo syringe 10mL lock (Terumo, SS-10LZ) AAVpro: Titration Kit (for Real Time PCR) Ver.2 (Takara, 6233) First, GFP, AAV1, and Helper plasmids were added to the pEI-pro solution to create the transfection solution. Next, the cell supernatant was removed, and the transfection solution was added to the cells (AAV-293) along with fresh medium. The cells were transfected by incubating overnight.

[0132] Next, we prepared string-like capsules by preparing a cell suspension, an alginate precursor (hydrogel precursor), and a sheath solution. The sheath solution was an aqueous solution containing 100 mM calcium chloride and 3 w / v% sucrose.

[0133] The alginic acid precursor (hydrogel precursor) was a sodium alginate solution. The sodium alginate solution was prepared by adding the above sodium alginate to physiological saline and stirring. The concentration of sodium alginate relative to the physiological saline was 0.5 w / v%.

[0134] The cell suspension was prepared by introducing the transfected cells into a 0.6 w / v% methylcellulose solution (solvent: the above-mentioned medium) as a core substrate. The transfected cells were cells that produce a viral vector (AAV). The initial density of the transfected cells in the cell suspension was 3 x 10 ６ The concentration was 100 μL / 100 μL.

[0135] Using the cell suspension, alginate precursor (hydrogel precursor), and sheath solution, capsules were produced according to the capsule production method described above (see also Figure 3). That is, a flow of cell suspension, a flow of alginate precursor surrounding the flow of cell suspension, and a flow of sheath solution surrounding the flow of alginate precursor were formed, and these flows were ejected into physiological saline. The alginate precursor was crosslinked by contact with the calcium chloride aqueous solution used as the sheath solution, forming a tubular alginate gel.

[0136] This produced string-like capsules in the saline solution, each having a cell-containing core and a shell surrounding the core. The cell-covered capsules were allowed to stand in the saline solution for a desired period of time.

[0137] Next, the cells together with the capsules were transferred to a liquid medium and cultured within the capsules (culture step). The cells were cultured in the liquid medium together with the capsules in a encapsulated state. The cells were cultured at a temperature of 37°C for 3 days (culture step). The liquid medium used for the culture was as described above. During this culture, the viral vector was produced from the cells.

[0138] Next, the capsule shell was physically cut using a cutter. For bent capsules, multiple cuts can be made at once using scissors. This process was repeated 10 times, resulting in multiple cuts on the capsule.

[0139] Next, the medium containing the cut capsules was pipetted 10 times using a P1000 tip. 0.5 mL of the pipetted solution was collected in a 1.5 mL tube. The culture supernatant (cell product) was used as Example 6.

[0140] Example 7 Example 7 will be described. In Example 7, additional steps were performed compared to Example 6. Preparation of cells, production of capsules, cell culture, cutting of capsules, and pipetting of the medium containing the cut capsules were performed in exactly the same manner as in Example 6. In Example 7, the solution pipetted in Example 6 was suspended 10 times using a G18 needle attached to a 10 mL capacity. This suspension of the solution was performed by sucking up and expelling the entire amount of the solution with the G18 needle. This process was repeated 10 times. The solution (cell product) obtained after this process was designated Example 7.

[0141] [Reference Example 2] In Example 6, the cells together with the capsules were transferred to a liquid medium, and 0.5 mL of the culture supernatant obtained when the cells were cultured in the capsules (culture step) was collected in a 1.5 mL tube. This culture supernatant (cell product) was designated Reference Example 2.

[0142] [Reference Example 3] The cells transfected in Example 6 were seeded on a culture dish without being placed in a capsule and cultured for 3 days in the same liquid medium under the same conditions. After the culture, 0.5 mL of the culture supernatant was collected in a 1.5 mL tube. This culture supernatant (cell product) was used as Reference Example 3.

[0143] [Titer Evaluation] DNase I was added to the culture supernatants obtained in Examples 6 and 7 and Reference Examples 2 and 3 to remove free genome and plasmid DNA from the culture supernatants.

[0144] Next, the mixture was heat-treated to inactivate DNase I. Then, lysis buffer was added to the mixture to elute the vector genome.

[0145] The amount (titer) of the viral vector was determined by real-time PCR using a solution in which the vector genome was eluted.

[0146] 11 is a graph showing the amount (titer) of viral vectors in Examples 6 and 7 and Reference Examples 2 and 3. In Examples 6 and 7, viral vectors were produced by cells in a encapsulated state, but viral vectors were recovered at the same level as in Reference Example 3, where cells were cultured without being encapsulated.

[0147] In Reference Example 2, the culture supernatant was collected without destroying or cutting the capsules. In Reference Example 2, the titer was lower than in Examples 6 and 7. This is thought to be due to a decrease in the amount of viral vector collected. In other words, by destroying or cutting the capsules for collection, the viral vector could be collected more effectively.

[0148] The inventors of the present application discovered that some cell products may be trapped within capsules, as described above, and came up with the idea of ​​a process of breaking or cutting the shell that constitutes the capsule. This allows for more efficient recovery of cell products, as described above. In addition, because cell products can be recovered without dissolving the shell, which is made of a gel such as alginate gel, alginate can be easily removed from the recovered product (cell product-containing composition).

[0149] [Evaluation of cell recovery loss] Next, a suspension was prepared by adding alginic acid (ALG300) to physiological saline containing T cells at the ratio shown in Table 1 below, and the suspension was subjected to the following operations, after which the number of remaining cells was counted.

[0150] The suspension was subjected to the following two operations: Operation 1: The suspension was placed in a 15 mL tube and the tube was rolled. Operation 2: The suspension was pipetted.

[0151] [Table 1] After the above-mentioned procedure 1 or 2, the number of viable cells was counted for each sample. The viable cell count was performed using a hemocytometer with trypan blue staining. Figure 12 is a graph showing the evaluation results of cell recovery loss in the presence or absence of alginate.

[0152] Referring to Figure 12, regardless of physical operation methods 1 and 2, the higher the alginate concentration, the greater the loss of T cell recovery. This is thought to be due to the fact that the viscosity of the cell-containing solution increases with the alginate concentration. In other words, this may be due to the cells adhering to the container along with the alginate as the viscosity of the solution increases, or being unable to be effectively separated by centrifugation. Therefore, when recovering cells from a solution, it is desirable to keep the concentration of alginate (hydrogel) in the solution as low as possible.

[0153] In the above-described embodiments and examples, most of the hydrogel, such as alginate gel, that constitutes the capsules is not dissolved by the chelating agent and is removed by filtration. Therefore, the amount of alginate remaining in the recovered cell population and cell products can be extremely small. This is believed to reduce the loss of cells or cell products during recovery.

[0154] It should be noted that at least the following inventions are clearly stated in this specification based on the above-mentioned description of the embodiments and / or examples.

[0155] [Appendix 1] A method for producing cells, cell aggregates, and / or cell products, comprising: culturing cells or cell aggregates within a core substrate using a capsule comprising a core substrate and a shell covering the core substrate, thereby obtaining cells, cell aggregates, and / or cell products; and physically destroying or cutting at least the shell, and recovering the cells, cell aggregates, and / or cell products.

[0156] [Appendix 2] The manufacturing method of Appendix 1, wherein at least one of the core substrate and the shell comprises a hydrogel.

[0157] [Appendix 3] The manufacturing method according to Appendix 2, wherein the hydrogel comprises an alginate gel.

[0158] [Appendix 4] The manufacturing method according to any one of Appendices 1 to 3, wherein the shell comprises a hydrogel, and the core substrate comprises a liquid or a sol.

[0159] [Appendix 5] The manufacturing method according to any one of Appendices 1 to 4, further comprising leaving or moving capsules containing the shell that have been physically broken or cut in a solution.

[0160] [Appendix 6] The manufacturing method according to any one of Appendices 1 to 5, comprising removing the capsules from the solution by filtering the solution containing the capsules after physically destroying or cutting at least the shells.

[0161] [Supplementary Note 7] The manufacturing method according to any one of Supplementary Notes 1 to 6, wherein the cells or cell aggregates include non-adherent cells.

[0162] [Appendix 8] The manufacturing method according to any one of Appendices 1 to 6, wherein the cells or cell aggregates include adhesive cells.

[0163] [Appendix 9] The capsules contain an adhesive material that is adhesive to the adherent cells, and the manufacturing method described in Appendix 8 further comprises dissolving the adhesive material before or after physically destroying or cutting at least the shell.

[0164] [Appendix 10] The manufacturing method according to Appendix 9, wherein the adhesive material is fibrin, and further comprising dissolving the fibrin at least before or after physically disrupting or cutting the shell.

[0165] [Appendix 11] The production method according to any one of Appendices 1 to 10, wherein the cells or cell aggregates include at least one of virus-producing cells, viral vector-producing cells, antibody-producing cells, biopharmaceutical substance-producing cells, physiologically active substance-producing cells, stem cells, cells differentiated from stem cells, immune cells, and cells for food use.

[0166] [Appendix 12] The production method according to any one of Appendices 1 to 11, wherein the cells or cell aggregates to be cultured include at least one selected from the group consisting of single cells, isolated cells, spheroids, fungi, and microorganisms.

[0167] [Appendix 13] A cell or cell product-containing composition produced by using a capsule comprising a core substrate and a shell covering the core substrate to culture cells or cell aggregates within the core substrate to obtain cells, cell aggregates, and / or cell products, and then physically destroying or cutting at least the shell and recovering them.

[0168] [Appendix 14] A cell or cell product-containing composition produced by recovering cells or cell aggregates or cell products coated with a hydrogel, the cell or cell product-containing composition having a solution containing the cells or cell aggregates, and / or the cell products, the solution containing a hydrogel-derived component, and the concentration of the hydrogel-derived component in the solution is 0.5 w / w% or less.

[0169] [Appendix 15] The cell or cell product-containing composition according to Appendix 14, wherein the hydrogel is an alginate gel, and the hydrogel-derived component is alginate.

[0170] [Appendix 16] The cell or cell product-containing composition according to appendix 14 or 15, wherein the concentration of the hydrogel-derived component in the solution is 0.000005 w / w% or more.

[0171] As mentioned above, the cells to be cultured in the above appendix are not limited to single cells, i.e., isolated cells or single cells, but may include cell aggregates such as spheroids, tissues, or organs. Furthermore, the cells or cell aggregates obtained by culture are not limited to isolated cells or single cells, but may include cell aggregates such as spheroids, tissues, or organs. However, it is preferable that the cells or cell aggregates to be cultured and / or the cells or cell aggregates obtained by culture are isolated cells, spheroids, fungi, and / or microorganisms. Examples of cell types that may constitute isolated cells or spheroids are as mentioned above.

[0172] As described above, the contents of the present invention have been disclosed through embodiments and examples, 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.

[0173] This application claims priority based on Japanese Patent Application No. 2022-190850, filed on November 29, 2022, the entire contents of which are incorporated herein by reference.

Claims

1. Culturing cells or cell aggregates in a capsule comprising a core substrate and a shell covering the core substrate to obtain cells, cell aggregates, and / or cell products; physically destroying or cutting at least the shell and recovering the cells or cell aggregates and / or cell products.

2. The method of claim 1 , wherein at least one of the core substrate and the shell comprises a hydrogel.

3. The method of claim 2 , wherein the hydrogel comprises an alginate gel.

4. the shell comprises a hydrogel; The method of claim 1 , wherein the core substrate comprises a liquid or a sol.

5. The method of any one of claims 1 to 4, further comprising placing or moving capsules containing the physically broken or cut shell in a solution.

6. 6. The method of claim 1, further comprising removing the capsules from a solution containing the capsules by filtering the solution after physically breaking or cutting at least the shells.

7. The method of claim 1 , wherein the cells or cell aggregates comprise non-adherent cells.

8. The method of claim 1 , wherein the cells or cell aggregates comprise adherent cells.

9. the capsule contains an adhesive material that is adhesive to the adherent cells; The method of claim 8 further comprising dissolving the adhesive material at least before or after physically breaking or cutting the shell.

10. the adhesive material is fibrin; The method of claim 9, further comprising dissolving the fibrin at least before or after physically disrupting or cutting the shell.

11. 11. The production method according to any one of claims 1 to 10, wherein the cells or cell aggregates comprise at least one of virus-producing cells, viral vector-producing cells, antibody-producing cells, biopharmaceutical substance-producing cells, physiologically active substance-producing cells, stem cells, cells differentiated from stem cells, immune cells, and cells for food use.

12. The method according to any one of claims 1 to 10, wherein the cells or cell aggregates to be cultured comprise at least one selected from the group consisting of single cells, isolated cells, spheroids, fungi, microorganisms, tissues, and organs.

13. A cell or cell product-containing composition produced by culturing cells or cell aggregates within a capsule comprising a core substrate and a shell covering the core substrate to obtain cells, cell aggregates and / or cell products, and then physically destroying or cutting at least the shell and recovering the cells or cell products.

14. A cell or cell product-containing composition produced by harvesting cells or cell aggregates or cell products coated with a hydrogel, comprising: a solution containing the cells or cell aggregates and / or cell products; the solution has a hydrogel-derived component; A cell or cell product-containing composition, wherein the concentration of hydrogel-derived components in the solution is 0.5 wt% or less.

15. the hydrogel is an alginate gel; The cell or cell product-containing composition of claim 14 , wherein the hydrogel-derived component is alginate.

16. 16. The cell or cell product-containing composition according to claim 14 or 15, wherein the concentration of the hydrogel-derived component in the solution is 0.000005 w / w% or more.

17. The method according to claim 1 , wherein physically destroying or cutting at least the shell is performed with a needle, a cutter, or scissors.

18. 18. The method of claim 1, wherein at least the step of physically destroying or cutting the shell is performed without using an agent that dissolves the shell.

19. 19. The method of any one of claims 1 to 12, 17 and 18, wherein physically breaking or cutting at least the shell comprises cutting the shell into multiple parts.