Method for freezing cell aggregates

Abstract

Provided is a method for freezing cell aggregates including nervous cells. Provided is a method for freezing cell aggregates including nervous cells and having a three-dimensional structure, the method comprising the following step (1) and step (2): step (1) for immersing, in a cryopreservation solution, cell aggregates including nervous cells at a temperature of 0 °C to 30 °C before freezing, and preparing the cell aggregates immersed in the cryopreservation solution; and step (2) for freezing the cell aggregates including nervous cells under a gaseous phase condition of a liquid nitrogen container having a temperature of at most -150 °C.

Description

Technical Field

[0001] The present application relates to a method for freezing cell aggregates containing nervous system cells.

Background Art

[0002] In recent years, the development of pharmaceuticals containing cells as active ingredients has been underway. For example, a cell therapy in which pluripotent stem cells (PSCs) are induced to differentiate into dopamine-producing neurons or their progenitor cells and transplanted into the midbrain of a patient is considered a promising treatment method for Parkinson's disease (Non-Patent Documents 1 to 3). In cell pharmaceuticals, achieving cryopreservation of the final product is an essential factor for spreading cell therapy (Patent Documents 1 to 4). Different from cell biology research, when used clinically, cryopreserved cells are transplanted immediately without performing recovery culture after thawing. Therefore, it is important that the engraftability, function / activity, and cell viability are maintained after thawing for cryopreserved cells.

[0003] It has been suggested that in the transplantation of solid tissues, the presence of donor blood vessels and antigen-presenting cells as they are induces a stronger immune response than the transplantation of cell suspensions (Non-Patent Document 4). On the other hand, if the immune response is suppressed by syngeneic transplantation or immunosuppressive agents, this problem is solved, and the transplantation of ventral midbrain (VM) tissue shows higher survival rates of dopamine-producing neurons and behavioral recovery than the transplantation of cell suspensions (Non-Patent Document 5). In addition, mechanical dissociation processes and enzymatic dissociation processes for obtaining cell suspensions can change cell characteristics and cause cell damage. Therefore, in clinical applications, it is desirable to formulate the final product as a cell mass rather than a cell suspension. However, there is a problem that cell masses are more difficult to cryopreserve than single cells.

[0004] As methods for freezing cells, a slow method and a vitrification method are known (Non-Patent Documents 6 to 8). The slow freezing method is a method of freezing cells at about 1°C / min together with a low concentration of cryoprotective agent (CPA) (such as 10% dimethyl sulfoxide (DMSO)) (Patent Document 5, Non-Patent Documents 9 and 10). In the slow freezing method, ice formation first starts in the extracellular space, resulting in the concentration of the extracellular fluid. As a result, water is withdrawn from the cells due to the osmotic pressure gradient across the cell membrane. This dehydration of the cells avoids intracellular ice formation. However, if the cells are dehydrated excessively, the cells are damaged by the concentrated intracellular fluid and CPA in the cryopreservation solution. On the other hand, the vitrification method is a rapid cooling method in which a high concentration of cryoprotective agent (for example, DMSO, acetamide or ethylene glycol) is added and then the cells are immediately transferred into liquid nitrogen and frozen all at once in an amorphous state (Patent Document 6, Non-Patent Document 11). That is, it is a method of minimizing the growth of ice crystals by solidifying the solvents inside and outside the cells in a glassy state. The vitrification method requires strict time control. However, in the case of cell aggregates, unlike the case of single cells dispersed in the solvent, since it takes time for the cryopreservation solution to penetrate into the cell aggregates, there is concern that the vitrification effect of the cryopreservation solution cannot be obtained sufficiently. Therefore, its application to clinical cell production is technically difficult (Non-Patent Document 12). As described above, since precise control of ice formation and cell dehydration is required, a cryopreservation method for cell masses for clinical use has not been established. In addition, Non-Patent Document 13 discloses that by using a gas-phase liquid nitrogen storage container that can be stored at -190°C and freezing cultured cells in the gas phase, mycoplasma contamination can be prevented. However, nothing was known about freezing cell aggregates under liquid nitrogen gas phase.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

[0006] [Non-Patent Document 1] Doi et al., Stem Cell Reports, 2(3), 337 - 350, 2014 [Non-Patent Document 2] Sundberg et al., Stem Cells 31, 1548 - 1562, 2013 [Non-Patent Document 3] Nolbrant et al., Nature Protocols, 12(9), 1962 - 1979, 2017 [Non-Patent Document 4] Redmond et al., Neurobiology of Disease, 29(1), 103 - 116, 2008 [Non-Patent Document 5] Fricker et al., PLoS ONE, 7(10), e47169, 2012 [Non-Patent Document 6] Chong et al., Stem Cells, 27(1), 29 - 39, 2009 [Non-Patent Document 7] Smith et al., Fertility and Sterility, 94(6), 2088 - 2095, 2010 [Non-Patent Document 8] Jang et al., Integrative Medicine Research, 6(1), 12 - 18, 2017 [Non-Patent Document 9] Schwartz et al., Journal of Neuroscience Research, 74(6), 838-851, 2003

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present application aims to provide a method for freezing cell aggregates containing nervous system cells.

Means for Solving the Problems

[0008] As a result of intensive studies, the present inventors have found a method for freezing cell aggregates containing nervous system cells and have completed the present invention. That is, the present invention is as follows: [1] A method for freezing a cell aggregate having a three-dimensional structure and containing nervous system cells, comprising the following steps (1) and (2): (1) A step of immersing a cell aggregate containing nervous system cells in a cryopreservation solution at 0°C or higher and 30°C or lower before freezing to prepare a cell aggregate immersed in the cryopreservation solution, and (2) A step of freezing the cell aggregate immersed in the cryopreservation solution under the gas phase of a liquid nitrogen container at -150°C or lower. 〔2〕The method according to 〔1〕, wherein in step (1), the cell aggregates are immersed in a cryopreservation solution for 15 minutes to 360 minutes. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the ratio of the volume of the cell aggregates and the cryopreservation solution contained in the container to the volume of the gas phase in the liquid nitrogen container is 5% or less. 〔4〕The method according to any one of 〔1〕 to 〔3〕, wherein the packing density of the cell aggregates with respect to the preservation solution is 50 to 500 cells / ml. 〔5〕The method according to 〔4〕, wherein the size of the container containing the cell aggregates and the cryopreservation solution is 0.5 to 5 ml. 〔6〕The method according to any one of 〔1〕 to 〔5〕, wherein the cell aggregates containing nervous system cells are cell aggregates containing nervous system cells derived from pluripotent stem cells. 〔7〕The method according to any one of 〔1〕 to 〔6〕, wherein the cell aggregates containing nervous system cells contain cells that are at least one of FOXA2, TH, and NURR1 positive. 〔8〕The method according to 〔7〕, wherein the cell aggregates containing nervous system cells contain FOXA2-positive and LMX1A-positive cells. 〔9〕The method according to 〔7〕, wherein the cell aggregates containing nervous system cells contain FOXA2-positive, TH-positive, and NURR1-positive cells. 〔10〕The method according to any one of 〔1〕 to 〔9〕, wherein the nervous system cells are dopamine-producing nerve cells or their precursor cells. 〔11〕The method according to any one of 〔1〕 to 〔10〕, wherein the cell aggregates contain 60% or more dopamine-producing neural precursor cells. 〔12〕The method according to any one of 〔1〕 to 〔11〕, wherein the cell aggregates containing nervous system cells are cell aggregates having an equivalent circle diameter of 150 μm to 1000 μm. 〔13〕The method according to 〔12〕, wherein the cell aggregates contain 500 to 150,000 cells. 〔14〕The method according to any one of 〔1〕 to 〔13〕, wherein the number of cells contained in the cryopreservation solution is 80,000 to 5,000,000 cells / mL. A method for long-term preservation of cell aggregates containing nervous system cells, comprising storing a container containing cell aggregates obtained by the method according to any one of [1] to

[14] in the gas phase or the liquid phase of a liquid nitrogen container. 〔16〕A transplantation composition containing, as an active ingredient, cell aggregates obtained by the method according to any one of [1] to

[14] . 〔17〕The transplantation composition according to

[16] above, characterized by containing cell aggregates having a circular equivalent diameter of 150 μm to 1000 μm and a cryopreservation solution, containing 60% or more of dopamine-producing neural progenitor cells, and not requiring culturing for recovery after thawing. 〔18〕The transplantation composition according to

[16] or

[17] above, wherein the number of cells is 80,000 to 5,000,000 cells / mL. 〔19〕The transplantation composition according to any one of

[16] to

[18] above, containing 10 to 500 cell aggregates / ml. 〔20〕The transplantation composition according to any one of

[16] to

[19] above, wherein the cell aggregates and the cryopreservation solution are filled in a container of 0.5 mL to 15 mL. 〔21〕A method for producing a transplantation composition containing, as an active ingredient, dopamine-producing neural progenitor cells, comprising freezing cell aggregates having a circular equivalent diameter of 150 μm to 1000 μm, containing 60% or more of dopamine-producing neural progenitor cells and having a cell number of 80,000 to 5,000,000 cells / mL, by the method according to any one of [1] to

[14] . 〔22〕The method for producing a transplantation composition according to

[21] above, wherein the cell aggregates and the cryopreservation solution are filled in a container of 0.5 mL to 15 mL. 〔23〕A method for treating a disease requiring regeneration of dopamine neurons, comprising the following steps: (1) A step of thawing the transplantation composition according to any one of

[16] to

[20] at 30°C to 40°C, and (2) A step of transplanting the transplantation composition obtained in (1) into the striatal region of a patient. 〔24〕The method according to

[23] above, characterized in that after thawing in (1), the cryopreservation solution is replaced with a medium for administration without culturing, and step (2) is performed. Regarding.

Advantages of the Invention

[0009] This application provides a method for freezing cell aggregates containing nervous system cells. The cell aggregates of nervous system cells frozen by the method of this application show a high cell survival rate and maintain functional properties such as neurite outgrowth ability. Further, by using the present invention, since the cell aggregates after freezing can be stored as they are without being transferred to a storage device, a temporary temperature rise due to transfer to the storage device can be avoided, and damage to unnecessary cell aggregates can be prevented. Even when storing at a temperature lower than the liquid nitrogen gas phase, since it can be directly immersed in liquid nitrogen without being taken out of the container, the number of steps can be reduced as an industrial manufacturing method, and workability is improved.

Brief Description of the Drawings

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Figure 12

Mode for Carrying Out the Invention

[0022] In this specification and the claims, when a numerical value is accompanied by the term "about", it is intended to include the range of ±10% of that value. For example, "about 20" shall include "18 to 22". A numerical range includes all numerical values between the two endpoints and the numerical values of the two endpoints. "About" regarding a range is applied to both endpoints of that range. Therefore, for example, "about 20 to 30" shall include "18 to 33".

[0023] 〔Nervous System Cells〕 The present application provides a method for freezing a cell aggregate containing neural cells having a three-dimensional structure.

[0024] Neural cells include neuronal cells (neuronal cell or neuron), and progenitor cells of the neuronal cells, i.e., neural progenitor cells (neural progenitor cell or neural precursor cell), etc. Neural cells may be neural cells derived from any site, such as neural cells of the central nervous system, or somatic nervous system neural cells of the motor nerves and sensory organs or neural cells of the peripheral nervous system of the autonomic nervous system, and include nerves (neurons), neural crest-derived cells, glial cells such as oligodendrocytes or astrocytes, and their stem cells or progenitor cells, etc. Examples of neural cells include cells that express a neural cell marker. Examples of neural cell markers include, but are not limited to, NCAM, βIII Tubulin (TUJ1), tyrosine hydroxylase (TH), serotonin, nestin, MAP2, MAP2AB, NEUN, GABA, glutamate, CHAT, SOX1, BF1, EMX1, VGLUT1, PAX, NKX, GSH, Telencephalin, GLUR1, CAMKII, CTIP2, TBR1, Reelin, TBR1, BRN2, OTX2, LMX1A, LMX1B, EN1, NURR1, PITX3, DAT, GIRK2, and TH, etc. The fact that a cell is a neural cell can be confirmed by the expression of one or more neural cell markers. In the present specification, examples of neural cells include cells that express one or more, two or more, or three or more of the above neural cell markers.

[0025] Neural cells of the central nervous system can be classified according to the difference in the site where the neural cells are present. That is, neurons and their progenitor cells derived from the forebrain, telencephalon, diencephalon, cerebrum, hypothalamus, midbrain, hindbrain, midbrain-hindbrain boundary region, cerebellum, retina, pituitary gland, or spinal cord are included.

[0026] Neurons derived from the forebrain are neurons present in forebrain tissues (i.e., telencephalon, cerebrum, hippocampus or choroid plexus, diencephalon, hypothalamus, etc.). Neurons of the forebrain can be confirmed by the expression of forebrain neuron markers. Examples of forebrain neuron markers include OTX1 (forebrain), BF1 (also called FOXG1), or SIX3 (which is also a marker for the telencephalon or cerebrum). In this specification, as nervous system cells, cells that express one or more, two or more, or three or more of the above forebrain neuron markers, telencephalon or cerebrum markers are included.

[0027] As neurons derived from the cerebrum, dorsal cells (e.g., cerebral cortex cells, Cajal-Retzius cells, hippocampal neurons, etc.) or ventral cells (e.g., basal ganglia cells of the cerebrum, etc.) can be mentioned. Examples of ventral cerebral neuron markers include basal ganglia neuron markers (e.g., GSH2, MASH1, NKX2.1, NOZ1). Examples of dorsal cerebral neuron markers include cerebral cortex neuron markers (e.g., PAX6, EMX1, TBR1). In this specification, as nervous system cells, cells that express one or more, two or more, or three or more of the above cerebral neuron markers, basal ganglia neuron markers, or cerebral cortex neuron markers are included.

[0028] As nervous system cells derived from the midbrain, neural progenitor cells derived from the ventral part of the midbrain, dopamine-producing neurons (also called dopaminergic neurons or Dopaminergic neuron), or dopamine-producing neural progenitor cells (also called dopaminergic progenitor cells or Dopaminergic progenitor) can be mentioned. Examples of markers for nervous system cells derived from the midbrain include FOXA2, EN2, TUJ1, etc. Examples of FOXA2-positive and TUJ1-positive nervous system cells include dopamine-producing neural progenitor cells and dopamine-producing neurons. In addition, dopamine-producing neurons can be identified using the fact that they are FOXA2-positive, NURR1-positive, and TH-positive as indicators.

[0029] In addition, dopamine-producing neural progenitor cells can be identified using the indicators that they are positive for FOXA2 and LMX1A. More preferably, they contain cells that are positive for one or more of OTX2, LMX1A, LMX1B, CORIN, SHH, AADC, βIII-Tubulin, EN1, NURR1, PITX3, DAT, GIRK2, and TH. In this specification, unless otherwise specified, cell aggregates containing dopamine-producing neural progenitor cells may also contain dopamine-producing neurons or dopaminergic neurons, etc.

[0030] In this specification, examples of nervous system cells include cells that express one or more, two or more, or three or more of markers of nervous system cells derived from the midbrain, markers of dopamine-producing neural progenitor cells, or markers of dopamine-producing neurons.

[0031] In this specification, examples of dopamine-producing neural progenitor cells include cells that express FOXA2 and / or LMX1A (FOXA2-positive and / or LMX1A-positive cells), preferably cells that express one or more, two or more, or three or more selected from the group consisting of OTX2, LMX1B, EN1, CORIN, SHH, AADC, and βIII-Tubulin in addition to FOXA2 and LMX1A.

[0032] In this specification, examples of dopamine-producing neurons (dopamine neurons) include cells that express TH and / or NURR1 (TH-positive and / or NURR1-positive cells), preferably cells that express one or more, two or more, or three or more selected from the group consisting of FOXA2, AADC, DAT, and GIRK2 in addition to TH and NURR1.

[0033] Examples of neurons derived from the midbrain-hindbrain boundary region include neurons present in the cerebellum, cerebellar plate tissue, ventricular zone, rhombic lip, etc. Examples of midbrain-hindbrain boundary region markers include EN2 (midbrain), GBX2 (hindbrain), and N-Cadherin (neural progenitor cells in the midbrain-hindbrain boundary region). Examples of cerebellar neural progenitor cell markers include KIRREL2, PTF1A, or SOX2, which are GABAergic neural progenitor cell markers, and ATOH1 or BARHL1, which are cerebellar granule cell progenitor cell markers. In this specification, examples of nervous system cells include cells that express one or more, two or more, or three or more of the above midbrain-hindbrain boundary region markers, cerebellar neural progenitor cell markers, GABAergic neural progenitor cell markers, or cerebellar granule cell progenitor cell markers. Examples of neurons derived from the retina include photoreceptor cells, photoreceptor cell progenitors, retinal pigment epithelial cells, corneal cells, etc.

[0034] In addition, nervous system cells can also be classified according to the differences in neurotransmitters they produce (secrete). For example, dopamine-producing neurons, dopamine-producing neural progenitor cells, GABA neurons, GABA neural progenitor cells, cholinergic neurons, cholinergic neural progenitor cells, serotonin neurons, serotonin neural progenitor cells, glutamic acid neurons, glutamic acid neural progenitor cells, noradrenergic neurons, noradrenergic neural progenitor cells, adrenergic neurons, adrenergic neural progenitor cells, etc.

[0035] Examples of nervous system cells of the motor nerves and sensory organs include cholinergic neurons or their progenitor cells, etc. Examples of nervous system cells of the autonomic nerves include cholinergic neurons, adrenergic neurons, or their progenitor cells, etc.

[0036] Preferred examples of the nervous system cells in this specification include dopamine-producing neurons (dopamine neurons) and dopamine-producing neural progenitor cells (dopamine neural progenitor cells).

[0037] Biologically-derived nervous system cells are cells isolated from mammals such as humans. For example, as cells isolated from human brain tissue, cells contained in fetal midbrain tissue as described in Nature Neuroscience, 2, 1137 (1999) or N. Engl. J. Med.; 344: 710-9 (2001) are exemplified.

[0038] Nervous system cells may also be cells obtained by inducing differentiation from pluripotent stem cells such as embryonic stem cells (ES cells) and iPS cells. Methods for inducing the differentiation of nervous system cells from pluripotent stem cells include, for example, the methods described in Non-Patent Documents 3 and 4 and WO2015 / 034012 above (dopaminergic neural progenitor cells), WO2009 / 148170 (nervous system cells such as the cerebrum), WO2013 / 065763, WO2016 / 013669 or WO2017 / 126551 (nervous system cells of the pituitary gland or hypothalamus), WO2016 / 039317 (nervous system cells of the cerebellum), WO2015 / 076388 (nervous system cells of the telencephalon), Numasawa-Kuroiwa, Y et al., Stem Cell Reports, 2: 648-661 (2014) (neural progenitor cells), Qiu, L et al., Stem Cells Transl Med. 6(9): 1803-1814 (2017) (dopamine-producing neural progenitor cells).

[0039] Furthermore, nervous system cells may be cells obtained by inducing differentiation from multipotent stem cells such as mesenchymal stem cells (MSCs). Examples of methods for inducing the differentiation of nervous system cells from mesenchymal stem cells include the methods described in J Chem Neuroanat. 96: 126-133 (2019).

[0040] 〔Pluripotent Stem Cells〕 A pluripotent stem cell refers to a stem cell that has the pluripotency to differentiate into almost all cells existing in a living body and also has the ability to proliferate. Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ stem cells, tissue stem cells, somatic cells, etc. Although not particularly limited, pluripotent stem cells include, for example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, pluripotent stem cells (Muse cells) derived from cultured fibroblasts and bone marrow stem cells, etc. The pluripotent stem cell may be an ES cell, an ntES cell, or an iPS cell. Considering ethical aspects, the pluripotent stem cell may be an iPS cell. Note that embryonic stem cells are established from embryos within 14 days after fertilization.

[0041] Embryonic stem cells were first established in 1981 and have been applied to the production of knockout mice since 1989. In 1998, human embryonic stem cells were established and are being used in regenerative medicine. Embryonic stem cells can be produced by culturing the inner cell mass on feeder cells or in a medium containing LIF (leukemia inhibitory factor). Methods for producing embryonic stem cells are described, for example, in WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from a predetermined institution or purchased as commercial products. For example, KhES-1, KhES-2, and KhES-3, which are human embryonic stem cells, are available from the Institute for Frontier Medical Sciences, Kyoto University. The Rx::GFP strain (derived from the KhES-1 strain), which is a human embryonic stem cell, is available from the National Institutes of Natural Sciences, RIKEN. The EB5 cell line and D3 cell line, which are mouse embryonic stem cells, are available from the National Institutes of Natural Sciences, RIKEN and ATCC, respectively.

[0042] One type of embryonic stem cell, nuclear transfer embryonic stem cells (ntES cells), can be established from cloned embryos created by transplanting the nucleus of a somatic cell into an enucleated egg.

[0043] EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF, and bFGF (Cell, 70:841-847, 1992).

[0044] As used herein, "induced pluripotent stem cells" refer to cells in which pluripotency has been induced by reprogramming somatic cells by known methods or the like. Specifically, differentiated somatic cells such as fibroblasts or peripheral blood mononuclear cells are reprogrammed by the expression of any combination of a plurality of genes selected from a group of reprogramming genes including OCT3 / 4, SOX2, KLF4, MYC (c-MYC, N-MYC, L-MYC), GLIS1, NANOG, SALL4, LIN28, ESRRB, etc., to induce pluripotency. Preferred combinations of reprogramming factors include (1) OCT3 / 4, SOX2, KLF4, and MYC (c-MYC or L-MYC), (2) OCT3 / 4, SOX2, KLF4, LIN28, and L-MYC (Stem Cells, 2013; 31:458-466), (3) OCT3 / 4, SOX2, NANOG, and LIN28 (Science 2007; 318: 1917-1920), etc.

[0045] Induced pluripotent stem cells were established in mouse cells by Yamanaka et al. in 2006 (Cell, 2006, 126(4), pp.663-676). Induced pluripotent stem cells were also established in human fibroblasts in 2007 and have pluripotency and self-renewal ability similar to embryonic stem cells (Cell, 2007, 131(5), pp.861-872; Science, 2007, 318(5858), pp.1917-1920; Nat. Biotechnol., 2008, 26(1), pp.101-106).

[0046] In addition to the method of producing induced pluripotent stem cells by direct reprogramming by gene expression, induced pluripotent stem cells can also be produced by a method of inducing induced pluripotent stem cells from somatic cells by the addition of compounds or the like (Science, 2013, 341, pp.651-654).

[0047] It is also possible to obtain established induced pluripotent stem cells. For example, human induced pluripotent stem cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University are available from Kyoto University. As established induced pluripotent stem cells, for example, Ff-I01 cells, Ff-I01s04 cells, QHJ-I01, and Ff-I14 cells established at Kyoto University are available from Kyoto University.

[0048] The somatic cells used in the production of induced pluripotent stem cells are not particularly limited, and examples include tissue-derived fibroblasts, blood cell line cells (e.g., peripheral blood mononuclear cells (PBMC), T cells), hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, and the like.

[0049] When reprogramming is carried out by the expression of several types of genes in the production of induced pluripotent stem cells, the means for expressing the genes are not particularly limited. Examples of such means include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, or adeno-associated virus vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors or episomal vectors) (e.g., calcium phosphate method, lipofection method, RetroNectin method, or electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, or electroporation method), direct injection methods of proteins (e.g., methods using needles, lipofection method, or electroporation method), and the like.

[0050] Induced pluripotent stem cells can be produced in the presence or absence of feeder cells (feeder-free). When producing induced pluripotent stem cells in the presence of feeder cells, induced pluripotent stem cells can be produced by a known method in the presence of undifferentiated maintenance factors. The medium used when producing induced pluripotent stem cells in the absence of feeder cells is not particularly limited, but a known embryonic stem cell and / or induced pluripotent stem cell maintenance medium, or a medium for establishing induced pluripotent stem cells feeder-free can be used. Examples of the medium for establishing induced pluripotent stem cells feeder-free include feeder-free media such as Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit medium. When producing induced pluripotent stem cells, for example, somatic cells can be used to produce induced pluripotent stem cells by introducing four factors, OCT3 / 4, SOX2, KLF4, and MYC (L-MYC or c-MYC), into the somatic cells using a Sendai virus vector feeder-free.

[0051] The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably pluripotent stem cells of rodents (e.g., mouse or rat) or primates (e.g., human or monkey), more preferably human or mouse pluripotent stem cells, and even more preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0052] [Cell aggregate] When the cell aggregate in this specification "has a three-dimensional structure", it means that the cultured cells form a cell aggregate (Cell aggregate or sphere), which is a three-dimensional cell population formed by the cells adhering to each other, for example, by suspension culture or three-dimensional culture. The cell aggregate of nervous system cells is also called a neurosphere. The shape of the cell aggregate is not particularly limited and may be spherical or non-spherical. The cell aggregate in this specification is preferably a cell aggregate having a three-dimensional shape close to spherical. The three-dimensional shape close to spherical is a shape having a three-dimensional structure and, when projected onto a two-dimensional plane, shows, for example, a circular or elliptical shape.

[0053] The size of the cell aggregate containing nervous system cells and having a three-dimensional structure is not particularly limited, but usually, the equivalent circle diameter is 150 μm to 1000 μm, and in one aspect, for example, 200 μm to 800 μm, or 300 μm to 500 μm. Also, the cell aggregate containing nervous system cells and having a three-dimensional structure usually contains 500 to 150,000 cells, and in one aspect, for example, 1000 to 100,000 cells, 1000 to 70,000 cells, or 3000 to 30,000 cells.

[0054] The cell aggregate containing nervous system cells may contain other cells together with the nervous system cells. Examples of cell aggregates containing 60% or more, 70% or more, 80% or more, and more preferably 90% or more of nervous system cells are given.

[0055] In one aspect, the cell aggregate containing nervous system cells may contain 60% or more, 70% or more, or 80% or more of dopamine-producing neural progenitor cells and / or dopamine-producing neurons. That is, the cell aggregate containing nervous system cells may contain 60% or more, 70% or more, or 80% or more of nervous system cells expressing one or more markers selected from FOXA2, LMX1A, LMX1B, NURR1, and TH.

[0056] In one aspect, the cell aggregate containing nervous system cells contains 40% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more of dopamine-producing neural progenitor cells.

[0057] In one aspect, the cell aggregate containing nervous system cells contains 40% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more of cells that express one or more, two or more, or three or more markers of dopamine-producing neural progenitor cells.

[0058] In one aspect, the cell aggregate containing nervous system cells contains 40% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more of FOXA2-positive and LMX1A-positive cells. In one aspect, the cell aggregate further contains 40% or less of TH-positive and NURR1-positive cells.

[0059] In one aspect, the cell aggregate containing nervous system cells may contain 0% or more, 10% or more, or 20% or more of FOXA2-positive, TH-positive, and NURR1-positive cells.

[0060] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells may contain 60% or less, 50% or less, 40% or less, 5 - 50%, 5 - 40%, or 5 - 20% of NURR1-positive cells.

[0061] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons may contain 30% or less, 20% or less, 1 - 30%, 5 - 30%, 1 - 20%, 5 - 20%, or 5 - 15% of TH-positive cells.

[0062] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons may contain 30% or less, 1 - 25%, 1 - 20%, or 5 - 20% of KI67-positive cells.

[0063] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons may contain 20% or less, 10% or less, 5% or less, or 1% or less of SOX1-positive cells.

[0064] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells may contain PAX6-positive cells at 5% or less, 2% or less, 1% or less, or 0.5% or less.

[0065] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells further contains TH-positive and NURR1-positive cells at 20% or less, specifically 1% - 20%, more specifically 5% - 15%.

[0066] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells contains FOXA2-positive and LMX1A-positive cells at 50% or more, preferably 60% or more, 70% or more, or 80% or more, and contains TH-positive and NURR1-positive cells at 20% or less, 1% - 20%, more specifically 5% - 15%.

[0067] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells further has SOX1-positive cells at 10% or less, preferably 7% or less, more preferably 3% or less, and PAX6-positive cells at 5% or less, preferably 4% or less, more preferably 2% or less.

[0068] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells contains FOXA2-positive and LMX1A-positive cells at 60% or more, and contains TH-positive and NURR1-positive cells at 1% - 20%, has SOX1-positive cells at 10% or less, preferably 7% or less, more preferably 3% or less, and has PAX6-positive cells at 5% or less, preferably 4% or less, more preferably 2% or less.

[0069] In one aspect, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells contains FOXA2-positive and LMX1A-positive cells at 60% or more of the total cell number, and may contain TH-positive and NURR1-positive cells at 20% or less, 1 - 20%, or 5 - 15% of the total cell number.

[0070] In one aspect, the above cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons is a cell aggregate having an equivalent circle diameter of 150 μm to 1000 μm.

[0071] In one aspect, the above cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons contains 60% or more of FOXA2-positive and LMX1A-positive cells, 1% to 20% of NURR1-positive and TH-positive cells, and is a cell aggregate having an equivalent circle diameter of 150 μm to 1000 μm.

[0072] 〔Freezing method〕 The method of the present application includes the step of (1) immersing a cell aggregate containing nervous system cells having a three-dimensional structure in a cryopreservation solution at 0°C or higher and 30°C or lower before freezing to prepare a cell aggregate immersed in the cryopreservation solution.

[0073] In the present application, the cryopreservation solution means an aqueous liquid containing a cryoprotective substance. The cryoprotective substance means a substance having a high affinity for water molecules and a high effect of suppressing the growth of ice crystals in the cryopreservation solution. For example, dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG), and glycerin are included. In the present application, the cryoprotective substance is preferably dimethyl sulfoxide, glycerin, and / or propylene glycol. When dimethyl sulfoxide, glycerin, and / or propylene glycol is used as the cryoprotective substance, the concentration of the cryoprotective substance in the cryopreservation solution is usually about 2 to 12%, preferably about 5 to 12%, more preferably about 7 to 12%, still more preferably about 7 to 10%, and even more preferably about 10%.

[0074] As the aqueous liquid, for example, buffers such as physiological saline, PBS, EBSS, and HBSS, culture solutions for culturing cells and tissues such as DMEM, GMEM, and RPMI, serum, serum substitutes, or mixtures thereof can be used.

[0075] In addition, as the cryopreservation solution, commercially available cryopreservation solutions mainly composed of dimethyl sulfoxide (DMSO), glycerin and / or propylene glycol can be used. Specifically, as the cryopreservation solution, there are commercially available cryopreservation solutions such as STEM-CELL BANKER (SCB; ZENOAQ), STEM-CELL BANKER DMSO free (SCB DMSO free; ZENOAQ), Bambanker hRM (BBK; NIPPON Genetics), Bambanker DMSO Free (BBK DMSO Free; NIPPON Genetics), CryoStor CS5 (CS5; BioLife Solutions), CryoStor CS10 (CS10; BioLife Solutions), and Synth-a-Freeze (SaF; Thermo Fisher Scientific). For example, it is desirable to use a cryopreservation solution containing 7 to 12%, preferably about 10% of dimethyl sulfoxide, glycerin and / or propylene glycol (such as STEM-CELL BANKER, Bambanker hRM, CryoStor CS10, and Synth-a-Freeze). More preferably, Bambanker hRM can be used.

[0076] In this specification, when freezing cell aggregates, the number of cells (cell packing density) relative to the cryopreservation solution is 80,000 to 5,000,000 cells / mL, 100,000 to 4,000,000 cells / mL, or 200,000 to 2,000,000 cells / mL, 300,000 to 1,000,000 cells / mL.

[0077] In this specification, when freezing cell aggregates, the equivalent circle diameter of the cell aggregates is 150 to 1000 μm, 150 μm to 600 μm, or 300 μm to 500 μm.

[0078] In this specification, the volumes of the cell aggregates and the preservation solution are 0.25 mL to 2 mL, 0.5 mL to 1.5 mL, or 0.5 mL to 1 mL.

[0079] In this specification, the cell aggregates and the preservation solution may be filled in a container of 0.5 mL to 15 mL, 1 mL to 5 mL, or 1 mL to 2 mL.

[0080] The freezing point of the cryopreservation solution in the present application is not particularly limited, but is usually about -1°C to -10°C, preferably -3°C to -10°C, more preferably -3°C to -6°C, and even more preferably about -5°C.

[0081] When immersing the cell aggregates containing nervous system cells in the cryopreservation solution, the temperature is usually 0°C or higher and 30°C or lower, preferably 0°C or higher and 20°C or lower, more preferably 0°C or higher and 10°C or lower, and even more preferably 0°C or higher and 5°C or lower.

[0082] In addition, the time for immersing the cell aggregates containing nervous system cells in the cryopreservation solution is usually 5 minutes to 360 minutes, 5 minutes to 240 minutes, 5 minutes to 180 minutes, preferably 5 minutes to 120 minutes, 5 minutes to 60 minutes, 15 minutes to 360 minutes, 15 minutes to 240 minutes, 15 minutes to 180 minutes, 15 minutes to 150 minutes, preferably 15 minutes to 120 minutes, and more preferably 15 minutes to 60 minutes.

[0083] The method of the present application also includes a step of freezing the cell aggregates immersed in the cryopreservation solution obtained in step (1) under the gas phase of a liquid nitrogen container at -150°C or lower.

[0084] In this specification, the liquid nitrogen container is a container filled with liquid nitrogen, and in the container, the gas phase space is maintained at -150°C or lower. The temperature of the gas phase space is not particularly limited as long as it is -150°C or lower. For example, it may be about -160°C, about -170°C, about -180°C, or about -190°C.

[0085] The ratio of the volume of liquid nitrogen to the volume of the gas phase space in the liquid nitrogen container is not particularly limited as long as the temperature of the gas phase space can be maintained at -150°C or lower. Specifically, a volume ratio of about 1:2 to 1:10 can be mentioned. As such liquid nitrogen containers, G48-6R (Taiyo Nippon Sanso) and CryoSystem 6000 (MVE) are commercially available.

[0086] Alternatively, the liquid nitrogen in the liquid nitrogen container can be impregnated into an absorbent such as glass fiber to form a liquid nitrogen gas phase atmosphere. As this type of liquid nitrogen container called a dry shipper type, DR-22DS (Taiyo Nippon Sanso) and CryoShipper (MVE) are commercially available.

[0087] In the method of the present application, the container filled with the cell aggregates immersed in the cryopreservation solution is placed in the gas phase space in the liquid nitrogen container.

[0088] The ratio of the volume of the cell aggregates and the cryopreservation solution filled in the container to the volume of the gas phase in the liquid nitrogen container is not particularly limited as long as the temperature of the gas phase space can be maintained at -150°C or lower, and it can be appropriately adjusted according to the capacity and volume of the liquid nitrogen gas phase storage used, but it is preferably 5% or less. For example, when the volume of the gas phase space is 1500 L, the number of containers (vials) that can be filled with 2 mL of cell aggregates and cryopreservation solution is about 1 to 35200.

[0089] In the method of the present invention, the packing density of the cell aggregates with respect to the cryopreservation solution is 5 to 500 cells / ml or 10 to 500 cells / ml, preferably 50 to 500 cells / ml, and more preferably 50 to 200 cells / ml. There are no particular limitations on the shape and material of the container for filling the cell aggregates, and a container that can seal the cell aggregates and the cryopreservation solution can be used.

[0090] A container filled with a composition containing cell aggregates frozen by the method of the present application and a cryopreservation solution can be stored in a liquid nitrogen container for a long period of time. That is, the method of the present application may further include the step of storing a container filled with the frozen cell aggregates obtained in step (2) in the gas phase or the liquid phase of a liquid nitrogen container, and can be stored for a long period at -150°C or lower.

[0091] Alternatively, a container filled with a composition containing cell aggregates frozen by the method of the present application and a cryopreservation solution can be transferred to a separate deep freezer, programmable freezer, proton freezer, rapid liquid freezer, electrostatic air rapid freezer, CellAlive System (CAS) rapid freezer, brine rapid freezer, or another liquid nitrogen container for storage, and stored for a long period at -80°C or lower, preferably -150°C or lower. For example, it may be about -160°C, about -170°C, about -180°C, or about -190°C.

[0092] The storage period of "long-term storage" is not limited, for example, it means storing for a period of 1 week or more, 1 month or more, half a year or more, 1 year or more, 3 years or more, 5 years or more, etc. There is no particular upper limit to the storage period of long-term storage, for example, it includes storing for a period of 10 years, 20 years, 30 years, 50 years, 100 years, or more.

[0093] The frozen cell aggregates can be thawed and used as appropriate. The thawing method is not particularly limited, but from the viewpoints of function, activity, and cell viability, it is desirable to thaw at a temperature around body temperature in a short time. Specifically, it is desirable to thaw at 30°C to 40°C, preferably 35°C to 38°C, and more preferably at a temperature near human body temperature, for example, about 37°C.

[0094] The cell aggregates frozen by the method of the present invention can maintain properties equivalent to those of unfrozen cell aggregates. For example, when the cell aggregates frozen by the method of the present invention are subjected to recovery culture for 7 days after thawing, the marker expression rate is equivalent to that of the cell aggregates before freezing. For example, in the case of cell aggregates containing dopamine-producing neural progenitor cells, examples of the marker include FOXA2, LMX1A, NURR1, or TH. Here, the equivalent marker expression rate means that the numerical difference in the ratio of marker-expressing cells to the total number of cells is 10% or less before freezing and after thawing or after culturing for 7 days after thawing.

[0095] [Pharmaceutical composition] Furthermore, the present application provides a pharmaceutical composition, that is, a composition for transplantation (preparation), containing, as an active ingredient, a cell aggregate frozen or long-term preserved by the above method.

[0096] The pharmaceutical composition (composition for transplantation) of the present invention is a concept including both the pharmaceutical composition frozen by the method of the present invention and the pharmaceutical composition obtained by thawing it. That is, examples of the pharmaceutical composition (composition for transplantation) of the present invention include a frozen or non-frozen composition containing a cell aggregate containing nervous system cells and a cryopreservation solution, and a composition containing a cell aggregate containing nervous system cells and an administration medium in which the cryopreservation solution has been replaced with an administration medium after thawing.

[0097] Examples of the pharmaceutical composition (composition for transplantation) of the present invention include the compositions for transplantation described in the above

[16] to

[20] .

[0098] In one aspect, the present invention encompasses a composition for transplantation in which the number of cells is 80,000 to 5,000,000 cells / mL, 100,000 to 4,000,000 cells / mL, or 200,000 to 2,000,000 cells / mL, 300,000 to 1,000,000 cells / mL, containing 40% or more, preferably 60% or more, 60% or more, 80% or more, 85% or more, or 90% or more of FOXA2-positive and LMX1A-positive cells in the total number of cells, and containing 40% or less, 1% to 20%, or 5% to 15% of TH-positive and NURR1-positive cells in the total number of cells.

[0099] In one aspect, the equivalent circle diameter of the cell aggregate is 150 to 1000 μm, 150 μm to 600 μm, or 300 μm to 500 μm.

[0100] In one aspect, the volume of the cell aggregate and the preservation solution is 0.25 mL to 2 mL, 0.5 mL to 1.5 mL, or 0.5 mL to 1 mL.

[0101] In one aspect, the cell aggregate and the preservation solution may be filled in a container of 0.5 mL to 15 mL, 0.5 mL to 5 mL, or 1 mL to 2 mL.

[0102] In one aspect, the present invention includes a transplantation composition characterized by not requiring culturing for recovery after thawing.

[0103] In one aspect, the transplantation composition according to any one of

[16] to

[20] above, which contains 8 to 192 cell aggregates per ml, has an average particle diameter of the cell aggregates of 150 μm to 1000 μm, and has 80,000 to 2,400,000 cells per container, is included.

[0104] The cell aggregate is useful as a pharmaceutical composition for transplantation for a patient suffering from a disease that requires transplantation of nervous system cells, and can be used as a medicine such as a therapeutic agent for a disease accompanied by degeneration, injury or dysfunction of nervous system cells. That is, a pharmaceutical composition containing the cell aggregate of the present invention and a pharmaceutically acceptable carrier is also within the scope of the present invention.

[0105] Examples of diseases that require transplantation of nervous system cells or diseases accompanied by damage or dysfunction of nervous system cells include, for example, spinal cord injury, motor neuron disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, Alzheimer's disease, retinitis pigmentosa, age-related macular degeneration, Parkinson's syndrome (including Parkinson's disease).

[0106] As one aspect of the present invention, there is provided a pharmaceutical composition for treating Parkinson's disease, which contains, as an active ingredient, a cell aggregate containing the dopamine-producing neural progenitor cells and / or dopamine-producing neurons of the present invention. The number of dopamine-producing neural progenitor cells and / or dopamine-producing neurons contained in the therapeutic agent for Parkinson's disease is not particularly limited as long as the graft can engraft after administration. For example, it may contain 1.0×10 4 or more cells per transplantation. Also, it may be appropriately adjusted according to symptoms and body size. Transplantation of dopamine-producing neural progenitor cells into the diseased site can be performed, for example, by the methods described in Nature Neuroscience, 2, 1137 (1999) or N Engl J Med. ; 344: 710-9 (2001).

[0107] As one aspect, the pharmaceutical composition of the present invention (also referred to as a transplantation composition) contains a cell aggregate containing nervous system cells to be transplanted into a human and a cryopreservation solution. The pharmaceutical composition of the present invention includes both a frozen solid form and a liquid form before or after thawing. The pharmaceutical composition may appropriately contain additives used to maintain cell viability within a range that does not affect the freezing rate and freezing temperature. Examples of the cryopreservation solution include those described above.

[0108] The pharmaceutical composition or transplantation composition of the present invention is used for transplantation after thawing, removing the cryopreservation solution, and replacing it with an administration medium that can be administered to a living body as described below. That is, a composition containing the thawed cell aggregate and the administration medium also falls within the scope of the pharmaceutical composition (also referred to as a transplantation composition) of the present invention.

[0109] [Method for manufacturing the transplantation composition] The pharmaceutical composition (transplantation composition) described in

[16] to

[20] above can be manufactured by the freezing method described in any one of [1] to

[14] above. That is, the present invention includes a method for manufacturing the above-mentioned pharmaceutical composition (transplantation composition).

[0110] [Treatment method] As one aspect of the present invention, there is provided a method for treating a disease requiring replenishment of nervous system cells, which includes a step of transplanting the cell aggregates of the present invention into a patient suffering from a disease requiring transplantation of nervous system cells.

[0111] As one aspect of the present invention, cell aggregates containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons obtained in the present invention can be administered to patients with Parkinson's disease as a pharmaceutical composition, specifically as a transplantation material.

[0112] Specifically, a frozen pharmaceutical composition containing cell aggregates containing the dopamine-producing neural progenitor cells and / or dopamine-producing neurons of the present invention and a cryopreservation solution is thawed, suspended in an appropriate transplantation medium such as physiological saline as appropriate, and transplanted into an area where the patient lacks dopamine nerves, such as the striatum. For example, after thawing the pharmaceutical composition, it may be washed with a medium containing an appropriate carrier and replaced with a transplantation medium for suspending the cell aggregates when transplanting the cryopreservation solution into a human. The thawing temperature is not particularly limited, but as described above, it is 30°C to 40°C, preferably 35°C to 38°C, and more preferably a temperature near the human body temperature, such as about 37°C. The cell aggregates contained in the pharmaceutical composition (transplantation composition) of the present invention can be transplanted into a living body by replacing the cryopreservation solution with an administration medium without performing culture for recovery after thawing.

[0113] Here, the carrier used in the transplantation medium (administration medium) for cell aggregates containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons is not particularly limited as long as it is a substance used to maintain the survival of cells, and substances well-known to those skilled in the art can be used. Specifically, a physiological aqueous solvent (physiological saline, buffer solution, serum-free medium, etc.) can be used. If necessary, in transplantation medicine, a preservative, stabilizer, reducing agent, isotonic agent, etc. usually used may be added to the pharmaceutical composition containing the tissue or cells to be transplanted.

[0114] Also, upon transplantation, the thawed cell aggregates may be stored in a medium necessary to maintain the viability of each cell aggregate. Examples of the "medium necessary to maintain the viability" include a culture medium, a physiological buffer solution, etc., and are not particularly limited as long as the cell population containing dopaminergic neural progenitor cells and / or dopaminergic neurons survives, and those skilled in the art can appropriately select it. As an example, a medium prepared using a medium commonly used for culturing animal cells as a basal medium can be mentioned. Examples of the basal medium include BME medium, BGJb medium, CMRL 1066 medium, GMEM medium, Improved MEM Zinc Option medium, Neurobasal medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, or a mixed medium thereof, etc., which can be used for culturing animal cells.

[0115] By transplanting the above cell aggregates, the transplanted dopaminergic neural progenitor cells and / or dopaminergic neurons, and the dopaminergic neural progenitor cells and / or dopaminergic neurons induced after transplantation functionally engraft in the administered patient.

[0116] Here, "engraftment" as used in this specification means that the transplanted cells survive in the living body for a long period (e.g., 30 days or more, 60 days or more, 90 days or more) and adhere and remain in the organ.

[0117] "Functional engraftment" as used in this specification means a state in which the transplanted cells have engrafted and are performing their original functions in the living body.

[0118] "Functional engraftment rate" as used in this specification means the ratio of the cells that have achieved functional engraftment among the transplanted cells. The functional engraftment rate of the transplanted dopaminergic neural progenitor cells can be determined, for example, by measuring the number of TH-positive cells in the transplant.

[0119] By transplanting the above cell aggregates, the functional engraftment rate of the transplanted cells and dopamine-producing neural progenitor cells and / or dopamine-producing neurons induced after transplantation is 0.1% or more, preferably 0.2% or more, more preferably 0.4% or more, even more preferably 0.5% or more, and even more preferably 0.6% or more.

[0120] As one aspect of the present invention, a method for treating a disease requiring regeneration of dopamine-producing nerves, which includes the following steps, is included. (1) A step of thawing the transplantation composition according to any one of the above

[16] to

[20] at 30°C to 40°C, preferably at 37°C ± 3°C. (2) A step of transplanting the transplantation composition obtained in (1) into the striatal region of a patient. As one aspect of the present invention, a treatment method is included, which is characterized in that after thawing, without performing culturing, replacing the cryopreservation solution with an administration medium, and performing step (2).

[0121] Examples of mammals to be transplanted in this specification include, for example, humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, etc. Preferably, rodents (e.g., mice, rats) or primates (e.g., humans, monkeys) are mentioned, and more preferably humans are mentioned.

Example

[0122] Examples are shown below for a more detailed explanation, but the present application is not limited by the examples in any way.

[0123] Example 1: Production of cell aggregates Maintenance and neural differentiation of human iPS cells Human iPSCs (1231A3) (Kyoto University) were maintained in StemFit medium (Ajinomoto) on a 6-well plate coated with iMatrix511 (Nippi). To initiate neural differentiation, the iPSCs were incubated with TrypLE select (Invitrogen) for 10 minutes, then dissociated into single cells and seeded onto a 6-well plate coated with iMatrix511 (Nippi) at a density of 5×10 6 cells / well together with the differentiation medium. The differentiation medium is a medium containing GMEM supplemented with 8% KSR, 0.1 mM MEM non-essential amino acids (all from Invitrogen), sodium pyruvate (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol. The differentiation medium was changed daily from the day after seeding until day 12. To efficiently induce neural differentiation, LDN193189 (STEMGENT) and A83-01 (Wako) were added. Also, to induce floor plate cells, 2 μM purmorphamine and 100 ng ml -1 FGF8 (Wako) were added on days 1 to 7, and 3 μM CHIR99021 (Wako) was added on days 3 to 12.

[0124] Culture On day 12 of culture, the cells were re-seeded onto a low-adhesion U-bottom 96-well plate (Sumitomo Bakelite) at a density of 1.5×10 -1 cells / well in a neural differentiation medium containing Neurobasal medium supplemented with B27 supplement, 2 μM Glutamax-I (all from Invitrogen), 10 ng ml -1 GDNF, 200 mM ascorbic acid, 20 ng ml 4 BDNF (all from Wako) and 400 μM dbcAMP (Sigma-Aldrich). The medium was changed every 3 days, and 30 μM of Y-27632 (Wako) was added to the first medium. For long-term culture, the floating cell aggregates were cultured in the neural differentiation medium until day 28 to obtain cell aggregates.

[0125] Test Example 1: Cell viability assay The cell aggregates before freezing obtained in Example 1 were immersed in a dispersion solution for nerve cells (Wako Pure Chemical Industries: Cat. No. 297-78101) in a water bath (37°C) for 10 minutes. After dispersing the cell mass into single cells by pipetting, the viable cell count was measured using a Countess™ automated cell counter (Thermo Fisher Scientific: Cat. No. C10281).

[0126] In addition, the frozen cell aggregates obtained in Examples 2 to 7 and Comparative Example 1 below were immersed in a water bath (37°C) and thawed in about 2 minutes, and then recovered and cultured in a nerve differentiation medium. After removing the supernatant 1 day after the recovery culture, the viable cell count was measured in the same manner as the cell aggregates before freezing.

[0127] The cell viability rate (%) after freeze-thawing in Examples 2 to 7 and Comparative Example 1 was calculated by dividing the viable cell count of the cell mass after freezing by the viable cell count of the cell mass before freezing.

[0128] Test Example 2: Neurite outgrowth assay One cell aggregate before freezing obtained in Example 1 was seeded per well on a 24-well plate coated with iMatrix511, cultured in a nerve differentiation medium for 5 days, and then fixed with 4% paraformaldehyde. The fixed cell mass was stained with a PE-labeled anti-PSA-NCAM antibody (Miltenyi Biotec) and visualized using a fluorescence microscope (BZ-9000; Keyence). The area of the neurites extended from the cell aggregates was measured using Photoshop (Adobe systems) and WinRoof (Mitani Corporation).

[0129] In addition, the frozen cell aggregates obtained in Examples 3 to 7 below were immersed in a water bath (37°C) and thawed in about 2 minutes, and then cultured in a nerve differentiation medium for 5 days. Using the cell aggregates obtained after culturing for 5 days, the area of the neurites was measured in the same manner as the above cell aggregates.

[0130] Example 2: Freezing of cell aggregates Sixty-four cell aggregates on the 28th day produced by the method of Example 1 were collected together with the culture medium into 15 mL centrifuge tubes (IWAKI: Cat. No. 2323-015), and after allowing the cell aggregates to sediment naturally, the supernatant was removed. Then, 1.0 mL of Bambanker (registered trademark) DMSO Free (Rinfotec) of the cryopreservation solution was added to obtain a cell aggregate suspension. The entire amount of the cell aggregate suspension was injected into a sample container (SARSTEDT: Cat. No. 72.687.028S, 1.5 mL screw cap microtube), allowed to stand on ice for 40 minutes, and then placed in a cryobox. In order to evaluate the effect of the freezing method of the cell aggregates, the cryoboxes were carried into a liquid nitrogen cryopreservation container (Taiyo Nippon Sanso: Cat. No. DR-430M, vapor phase storage type), a -80°C ultra-low temperature freezer (Panasonic: Cat. No. KM-DU73Y1), and a -150°C ultra-low temperature freezer (Panasonic: Cat. No. MDF-C2156VAN) respectively for freezing, and used as test preparations of the cell aggregates.

[0131] According to the method described in Test Example 1, the cell viability (Figure 1) of the test preparations taken out from the liquid nitrogen cryopreservation container, the -80°C ultra-low temperature freezer, and the -150°C ultra-low temperature freezer was evaluated.

[0132] As a result, when frozen in the vapor space of the liquid nitrogen cryopreservation container, the cell viability after thawing was 53.2 ± 1.5% (mean value ± standard deviation, n = 11). On the other hand, when frozen in the -80°C freezer (actual measured temperature in the storage -77°C) and the -150°C freezer (actual measured temperature in the storage -145°C), the cell viability after thawing was 13.7 ± 6.9% (mean value ± standard deviation, n = 11) and 36.8 ± 7.9% (mean value ± standard deviation, n = 11) respectively (Figure 1).

[0133] From this, it was found that when frozen in the liquid nitrogen vapor space, the cell viability after thawing was high, and furthermore, the variation between sample containers was significantly suppressed.

[0134] Example 3: Influence of immersion time of cell aggregates in cryopreservation solution On the 28th day, 64 cell aggregates produced by the method of Example 1 were collected together with the culture medium into a 15 mL centrifuge tube (IWAKI: Cat. No. 2323-015), and the cell aggregates were allowed to sediment naturally. After removing the supernatant, 1.0 mL of Bambanker® hRM (Lymphotec) of the cryopreservation solution was added to obtain a cell mass suspension. To evaluate the effect of the immersion time of the cell aggregates in the cryopreservation solution, the entire amount of the cell aggregate suspension was injected into a sample container (SARSTEDT: Cat. No. 72.687.028S, 1.5 mL screw-cap microtube), and allowed to stand on ice for 15 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, or 360 minutes. Then, they were placed in a cryobox and transferred to a liquid nitrogen cryopreservation container (Taiyo Nippon Sanso: Cat. No. DR-430M, vapor-phase storage type) for freezing to obtain test preparations.

[0135] According to the methods described in Test Example 1 and Test Example 2, the cell viability and neurite outgrowth ability of the test preparations taken out from the liquid nitrogen cryopreservation container were evaluated. The results are shown in FIGS. 2 and 3. From 15 minutes to 120 minutes of the immersion time in the cryopreservation solution, particularly high tendencies of cell viability and neurite outgrowth ability were recognized.

[0136] From this, it was considered that the immersion time of the cell aggregates in the cryopreservation solution was particularly preferably from 15 minutes to 120 minutes.

[0137] Example 4: Influence of the volume of the gas phase part of the liquid nitrogen container Sixty-four cell aggregates on the 28th day produced by the method of Example 1 were collected together with the culture medium in a 15-mL centrifuge tube (IWAKI: Cat. No. 2323-015), and after allowing the cell aggregates to sediment naturally, the supernatant was removed. Then, 2 mL of Bambanker (registered trademark) hRM (Rinfotec) of the cryopreservation solution was added to obtain a cell mass suspension. The entire amount of the cell aggregate suspension was transferred to a 2-mL sample container (Nalgene: Cat. No. NL5000-0020), sealed with a cap, and then allowed to stand on ice for 35 minutes. To evaluate the influence of the volume ratio of the cell aggregates and the cryopreservation solution in the sample container on the gas phase volume of the liquid nitrogen container, one box containing three 2-mL sample containers arranged vertically in a 25-well cryobox (the volume ratio of the cell aggregates and the cryopreservation solution filled in the 2-mL sample container to the gas phase volume of the liquid nitrogen container was 0.3%), or two boxes each containing twenty-five 2-mL sample containers arranged vertically in a 25-well cryobox (the volume ratio of the cell aggregates and the cryopreservation solution filled in the 2-mL sample container was 5%) were prepared. These were carried into a 2-L Dewar flask (ISOTHERM: Cat. No. 27B-E) filled with liquid nitrogen and having a liquid nitrogen gas phase atmosphere prepared in advance, frozen, and used as the test preparation.

[0138] According to the methods described in Test Example 1 and Test Example 2, the cell viability and neurite outgrowth ability of the test preparation taken out from the liquid nitrogen cryopreservation container (2-L Dewar flask) were evaluated.

[0139] As a result, when the volume ratio of the cell aggregates and the cryopreservation solution filled in the sample container to the gas phase volume of the liquid nitrogen container was 0.3% and 5%, the cell viability and neurite outgrowth ability were equivalent (Figs. 4 and 5).

[0140] From this, it was found that the volume ratio of the cell aggregates and the cryopreservation solution to the gas phase volume of the liquid nitrogen container did not affect the cell viability and neurite outgrowth ability.

[0141] Example 5: Influence of the number of cell aggregates filled

[0142] On the 28th day, the cell aggregates produced by the method of Example 1 were collected at 10, 25, 50, 100, 250, or 500 per 15 mL centrifuge tube (IWAKI: Cat. No. 2323-015) together with the medium, and the cell aggregates were allowed to sediment naturally. After removing the supernatant, 1.0 mL of Bambanker® hRM (Rinfotec) of the cryopreservation solution was added to obtain a cell aggregate suspension. The entire amount of the cell aggregate suspension was transferred to a sample container (1.5 mL Sumilon Super Quality Slim Tube: Sumitomo Bakelite: Cat. No. MS-4702WS), allowed to stand on ice for 35 minutes, and then placed in a cryobox. This was transferred to a liquid nitrogen cryopreservation container (Taiyo Nippon Sanso: Cat. No. DR-430M, vapor phase preservation type) and frozen to obtain a test preparation of the cell aggregates.

[0143] According to the methods described in Test Example 1 and Test Example 2, the cell viability and neurite outgrowth ability of the test preparation taken out from the liquid nitrogen cryopreservation container were evaluated.

[0144] As a result, in the range of cell aggregate packing density from 10 to 500 cells / mL, the cell viability and neurite outgrowth ability were equivalent, and it was found that the cell aggregate packing density did not affect the cell viability and neurite outgrowth ability (Figs. 6 and 7).

[0145] From this, it was found that the cell aggregate packing density in the sample container did not affect the cell viability and neurite outgrowth ability.

[0146] Example 6: Influence of the amount of filling liquid per cell aggregate (size of the sample container) Sixty-four cell aggregates on the 28th day produced by the method of Example 1 were collected together with the culture medium into 15-mL centrifuge tubes (IWAKI: Cat. No. 2323-015), and after allowing the cell aggregates to sediment naturally, the supernatant was removed. Then, 0.5 mL, 1.5 mL, or 5 mL of Bambanker (registered trademark) hRM (Lymphotec) of the cryopreservation solution was added to obtain a cell mass suspension. To evaluate the influence of the filling volume of the cell aggregates, the total amounts of 0.5 mL, 1.5 mL, and 5 mL of the cell aggregate suspensions were transferred into 0.5-mL sample containers (SARSTEDT: Cat. No. 72.733.001), 1.5-mL sample containers (SARSTEDT: Cat. No. 72.687.028S), and 5-mL sample containers (Sumitomo Bakelite: Cat. No. MS-4605), respectively. After allowing them to stand on ice for 35 minutes, they were placed in a cryobox, and these were carried into a liquid nitrogen cryopreservation container (Taiyo Nippon Sanso: Cat. No. DR-430M, vapor-phase storage type) and frozen to obtain test preparations.

[0147] According to the methods described in Test Example 1 and Test Example 2, the cell viability and neurite outgrowth ability of the test preparations taken out from the liquid nitrogen cryopreservation container were evaluated.

[0148] As a result, it was found that in 0.5-mL, 1.5-mL, and 5-mL sample containers, the cell viability and neurite outgrowth ability were equivalent, and the filling volume per cell aggregate did not affect the cell viability and neurite outgrowth ability (Figs. 8 and 9).

[0149] Example 7: Influence of the equivalent circle diameter of cell aggregates Among the methods of Example 1, 0.5×10 4 cells / well, 1.0×10 4 cells / well, 2.0×10 4 cells / well or 3.0×10 4The cells / wells were reseeded into the neural differentiation medium, and on the 28th day, four types of cell aggregates with different sizes (average equivalent circle diameter: 329 μm, 457 μm, 594 μm, or 676 μm) were obtained. These were collected by size into 15 mL centrifuge tubes (IWAKI: Cat. No. 2323-015) for each medium, and after allowing the cell aggregates to sediment naturally, the supernatant was removed. Then, 1.0 mL of Bambanker® hRM (Lymphotec), a cryopreservation solution, was added to obtain a cell aggregate suspension. These cell aggregate suspensions were each transferred in their entirety to sample containers (1.5 mL Sumilon Super Quality Slim Tube: Sumitomo Bakelite: Cat. No. MS-4702WS), allowed to stand on ice for 45 minutes, and then placed in a cryobox. This was placed into a liquid nitrogen cryopreservation container (Taiyo Nippon Sanso: Cat. No. DR-430M, vapor-phase storage type), frozen, and used as the test preparation of the cell aggregates.

[0150] According to the methods described in Test Example 1 and Test Example 2, the cell viability and neurite outgrowth of the test preparation removed from the liquid nitrogen cryopreservation container were evaluated.

[0151] As a result, the cell viabilities of the cell aggregates with average equivalent circle diameters of 329 μm, 457 μm, 594 μm, and 676 μm were equivalent, and a particularly high tendency of neurite outgrowth ability was observed at average equivalent circle diameters of 329 μm, 457 μm, and 594 μm (Figures 10 and 11).

[0152] From this, it was considered that an equivalent circle diameter of the cell aggregates from 329 μm to 594 μm was particularly preferable.

[0153] Comparative Example 1 Sixty-four cell aggregates on the 28th day produced by the method of Example 1 were collected together with the medium into a 15 mL centrifuge tube (IWAKI: Cat. No. 2323-015), and the cell aggregates were allowed to sediment naturally. After removing the supernatant, 1.0 mL of Bambanker® DMSO Free (Rinfotec), a cryopreservation solution, was added to obtain a cell aggregate suspension. The entire amount of the cell aggregate suspension was transferred to a sample container (SARSTEDT: Cat. No. 72.687.028S, 1.5 mL screw cap microtube), and the container was allowed to stand on ice for 30 minutes. Then, the container was placed in a cryobox, immersed in the liquid phase of liquid nitrogen, cooled to -190 °C and frozen, and then transferred to a liquid nitrogen cryopreservation container (Taiyo Nippon Sanso: Cat. No. DR-430M, vapor phase storage type) for storage to obtain a test preparation.

[0154] As a result, when frozen by cooling to -190 °C in the liquid phase of liquid nitrogen, the cell viability was significantly lower than that in freezing in the gas phase (see Example 2) (Fig. 12).

Claims

1. A method for freezing a cell aggregate containing nervous system cells having a three-dimensional structure, comprising the following steps (1) and (2): (1) A step of immersing a cell aggregate containing nervous system cells in a cryopreservation solution at 0°C or higher and 10°C or lower for 5 to 360 minutes before freezing to prepare a cell aggregate immersed in the cryopreservation solution, and (2) A step of freezing the cell aggregate immersed in the cryopreservation solution under the gas phase of a liquid nitrogen container at -150°C or lower.

2. The method according to claim 1, wherein the immersion in the cryopreservation solution is performed at 0°C or higher and 5°C or lower before freezing.

3. The method according to claim 1 or 2, wherein in step (1), the cell aggregate is immersed in the cryopreservation solution for 15 to 360 minutes.

4. The method according to any one of claims 1 to 3, wherein the ratio of the volume of the cell aggregate and the cryopreservation solution contained in the container to the volume of the gas phase in the liquid nitrogen container is 5% or less.

5. The method according to any one of claims 1 to 4, wherein the packing density of the cell aggregate with respect to the preservation solution is 10 to 500 cells / mL.

6. The method according to claim 5, wherein the packing density of the cell aggregate with respect to the preservation solution is 50 to 500 cells / mL.

7. The method according to claim 5 or 6, wherein the size of the container containing the cell aggregate and the cryopreservation solution is 0.5 to 5 mL.

8. The method according to any one of claims 1 to 7, wherein the cell aggregate containing nervous system cells is a cell aggregate containing nervous system cells derived from pluripotent stem cells.

9. The method according to any one of claims 1 to 8, wherein the cell aggregate containing nervous system cells contains 60% or more nervous system cells.

10. The method according to any one of claims 1 to 9, wherein the cell aggregate containing nervous system cells contains cells in which at least one of FOXA2, TH, and NURR1 is positive.

11. The method according to claim 10, wherein the cell aggregate containing nervous system cells contains FOXA2-positive and LMX1A-positive cells.

12. The method according to claim 10, wherein the cell aggregate containing nervous system cells contains FOXA2-positive, TH-positive, and NURR1-positive cells.

13. The method according to any one of claims 1 to 12, wherein the nervous system cells are dopamine-producing nerve cells or their precursor cells.

14. The method according to any one of claims 1 to 13, wherein the cell aggregate containing nervous system cells contains 60% or more dopamine-producing nerve precursor cells.

15. The method according to any one of claims 1 to 14, wherein the cell aggregate containing nervous system cells is a cell aggregate having an equivalent circle diameter of 150 μm to 1000 μm.

16. The method according to claim 15, wherein the cell aggregate contains 500 to 150,000 cells.

17. The method according to any one of claims 1 to 16, wherein the number of cells contained in the cryopreservation solution is 80,000 to 5,000,000 cells / mL.

18. A method for long-term preservation of a cell aggregate containing nervous system cells, comprising storing a container containing the cell aggregate obtained by the method according to any one of claims 1 to 17 in the gas phase or the liquid phase of a liquid nitrogen container.

19. A method for producing a transplantation composition containing dopamine-producing neural progenitor cells as an active ingredient, comprising freezing a cell aggregate having an equivalent circle diameter of 150 μm to 1000 μm, containing 80,000 to 5,000,000 cells / mL, and containing 60% or more of dopamine-producing neural progenitor cells, by the method according to any one of claims 1 to 17.

20. The method for producing a transplantation composition according to claim 19, wherein the cell aggregate and the cryopreservation solution are filled in a container of 0.5 mL to 15 mL.

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