Method for freezing neural cells

Abstract

To provide a method for freezing a cell aggregate containing nervous system cells.SOLUTION: A method for freezing a cell aggregate containing neural cells having a three-dimensional structure, the method comprising the steps of: (1) contacting the cell aggregate containing the neural cells having a three-dimensional structure with a preservation solution at 0°C or higher and 30°C or lower before freezing; A step of preparing a cell aggregate immersed in a preservation solution, and (2) a step of cooling and freezing the cell aggregate immersed in the preservation solution obtained in step (1) at least from a temperature about 5 °C higher than the freezing point of the preservation solution to a temperature about 5 °C lower than the freezing point at an average temperature decrease rate of 2 to 7 °C / min.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present application relates to a method for freezing cell aggregates containing neural cells. [Background technology]

[0002] Based on reports of clinical trials using fetal midbrain cells, transplantation of dopamine-producing (DA) neurons is considered to be a promising treatment for Parkinson's disease (PD) (Non-Patent Document 1). Furthermore, methods have been reported for inducing differentiation of pluripotent stem cells (PSCs), such as embryonic stem cells (ES cells or ESCs) and induced pluripotent stem cells (iPS cells or iPSCs), into dopamine-producing neurons or their precursor cells. The present inventors' group has already reported a method for producing dopamine-producing neurons or dopamine-producing neuronal precursor cells from human induced pluripotent stem cells (Non-Patent Document 2). Other groups have also reported the production of PSC-derived dopamine-producing neurons (Non-Patent Documents 3 and 4).

[0003] For pharmaceuticals that use cells as active ingredients, achieving cryopreservation of the final product is an essential element for the widespread adoption of cell therapy (Patent Documents 1 to 4). Unlike cell biology research, when used clinically, cryopreserved cells are preferably transplanted immediately after thawing without undergoing recovery culture. Therefore, it is important that the frozen cells maintain their engraftment capacity, function, activity, and cell viability after thawing.

[0004] It has been suggested that solid tissue transplants induce stronger immune responses than cell suspension transplants due to the presence of donor blood vessels and antigen-presenting cells (Non-Patent Document 5). On the other hand, syngeneic transplants or suppression of the immune response with immunosuppressants eliminate this issue, and transplants of ventral midbrain (VM) tissue demonstrate higher dopaminergic neuronal survival and behavioral recovery than cell suspension transplants (Non-Patent Document 6). Furthermore, the mechanical and enzymatic dissociation processes used to obtain cell suspensions can alter cellular properties and cause cell damage. Therefore, for clinical applications, it is desirable to administer transplanted cells as cell clusters rather than cell suspensions. However, cell clusters are more difficult to cryopreserve than single cells.

[0005] When a single cell suspension of cryopreserved PSC-derived dopamine-producing neurons was transplanted into the rat striatum, viable TH neurons were observed. + The percentage of cells in frozen PSCs is reduced to approximately 60% compared to unfrozen cells (Non-Patent Document 7). On the other hand, in most studies in which human or rat ventral midbrain (VM) tissue has been cryopreserved, the survival rate of dopaminergic neurons in vivo has been reduced to less than 20% compared to unfrozen tissue (Non-Patent Documents 8-10). Therefore, there is a need to develop a freezing method that can maintain the survival of PSC-derived dopaminergic neuronal cell clusters.

[0006] Generally, there are two cell cryopreservation methods (Non-Patent Documents 11-13). Of these, the slow cryopreservation method involves freezing cells at approximately 1°C / min with a low concentration of cryoprotectant (CPA) (e.g., 10% dimethyl sulfoxide (DMSO)) (Patent Document 5, Non-Patent Documents 14 and 15). On the other hand, the vitrification method is a rapid cooling method in which cells are transferred to liquid nitrogen immediately after adding a high concentration of cryoprotectant (Patent Document 6, Non-Patent Document 16). Because the vitrification method requires strict time control, its application to clinical cell production is technically difficult (Non-Patent Document 17).

[0007] On the other hand, in the slow cryopreservation method, ice formation first begins in the extracellular space, leading to the concentration of extracellular fluid. As a result, water is drawn from the cell by the osmotic gradient across the cell membrane. This dehydration of the cells prevents intracellular ice formation. However, if cells are excessively dehydrated, they can be damaged by the concentrated intracellular fluid and CPA in the cryopreservation solution. Because precise control of ice formation and cell dehydration is required, a clinical cryopreservation method for cell clusters has not been established. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-104061 [Patent Document 2] WO2017 / 159862 specification [Patent Document 3] Special Publication No. 2015-521469 [Patent Document 4] Special Publication No. 2008-501320 [Patent Document 5] Japanese Patent Application Laid-Open No. 2011-103885 [Patent Document 6] Japanese Patent Application Laid-Open No. 2013-110988 [Non-patent literature]

[0009] [Non-Patent Document 1] Piccini et al; Nature Neuroscience, 2(12), 1137-1140, 1999 [Non-patent document 2] Doi et al., Stem Cell Reports, 2(3), 337-350, 2014 [Non-patent document 3] Sundberg et al., Stem Cells 31, 1548-1562, 2013 [Non-patent document 4] Nolbrant et al., Nature Protocols, 12(9), 1962-1979, .2017

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0010] The present application aims to provide a method for freezing cell aggregates containing neural cells. [Means for solving the problem]

[0011] As a result of extensive research, the present inventors have discovered a method for freezing cell aggregates containing nervous system cells, and have completed the present invention. [1] A method for freezing cell aggregates containing neural cells having a three-dimensional structure, comprising the following steps (1) and (2): (1) contacting a cell aggregate containing neural cells having a three-dimensional structure with a preservation solution at 0°C or higher and 30°C or lower before freezing to prepare a cell aggregate immersed in the preservation solution; and (2) A process of freezing the cell aggregates immersed in the preservation solution obtained in step (1) by cooling them from a temperature at least approximately 5°C higher than the freezing point of the preservation solution to a temperature at least approximately 5°C lower than the freezing point at an average rate of 2 to 7°C / min. [2] The method according to [1] above, wherein the temperature is lowered at an average rate of 3 to 7°C / min in step (2). [3] The method according to [1] or [2] above, wherein in step (1), the cell aggregates are contacted with the preservation solution for 15 to 90 minutes, preferably 15 to 60 minutes. [4] The method according to any one of [1] to [3] above, wherein the freezing point of the preservative solution is -1°C to -10°C. [5] The method according to any one of [1] to [4] above, wherein the preservation solution is an aqueous liquid containing 7% to 12% dimethyl sulfoxide and / or propylene glycol, and step (2) is a step of cooling from 0±5°C to -30±5°C at an average temperature decreasing rate of 2 to 5°C / min. [6] The method according to any one of [1] to [5] above, wherein the temperature is lowered at an average rate of 3 to 5°C / min in step (2). [7] The method according to any one of [1] to [6] above, further comprising the following step (3): (3) A step of cooling the frozen cell aggregates obtained in step (2) to −50° C. or below. [8] The method according to any one of [1] to [7] above, wherein the cell aggregate containing neural cells is a cell aggregate containing neural cells derived from pluripotent stem cells. [9] The method according to any one of [1] to [8] above, wherein the cell aggregate containing nervous system cells contains cells positive for at least one of FOXA2, TH, and NURR1.

[10] The method described in [9] above, wherein the cell aggregates containing nervous system cells contain FOXA2-positive and LMX1A-positive cells.

[11] The method described in [9] above, wherein the cell aggregates containing nervous system cells contain FOXA2-positive, TH-positive, and NURR1-positive cells.

[12] The method described in any one of [1] to [8], wherein the cell aggregates containing nervous system cells contain FOXA2-positive and LMX1A-positive cells at 40% or more of the total cell number, and TH-positive and NURR1-positive cells at 40% or less of the total cell number.

[13] The method according to any one of [1] to

[12] above, wherein the cell aggregate containing nervous system cells contains dopaminergic neural progenitor cells and / or dopaminergic neural cells.

[14] The method according to any one of [1] to

[13] above, wherein the cell aggregate contains 500 to 150,000 cells.

[15] The method according to any one of [1] to

[14] above, wherein the number of cells contained in the preservation solution is 80,000 to 5,000,000 cells / mL and the equivalent circle diameter of the cell aggregates is 150 to 1,000 μm.

[16] The method according to any one of [1] to

[15] above, wherein the volume of the cell aggregate and the preservation solution is 0.25 mL to 2 mL.

[17] The method according to any one of [1] to

[16] above, wherein the cell aggregate and the preservation solution are filled in a container of 0.5 ml to 15 ml.

[18] A method for long-term preservation of cell aggregates containing neural cells having a three-dimensional structure, comprising maintaining frozen cell aggregates obtained by the method described in any one of [1] to

[17] at -80°C or below.

[19] A method according to any one of [1] to

[18] , characterized in that frozen cell aggregates are obtained that do not require recovery culture after thawing.

[20] A composition for transplantation containing, as an active ingredient, a cell aggregate that has been frozen or long-term preserved by the method according to any one of [1] to

[19] above.

[21] A cell aggregate containing 500 to 150,000 cells, having a circle equivalent diameter of 150 μm to 1,000 μm, and containing 60% or more dopamine-producing neural progenitor cells and dopamine-producing neurons derived from pluripotent stem cells, and a cryopreservation solution containing 7% to 12% dimethyl sulfoxide or propylene glycol and having a freezing point of -1°C to -10°C, and having the following properties: (1) Approximately 60% or more of the total number of cells survives after thawing. (2) have neurite outgrowth activity of 50% or more compared to before freezing; (3) A frozen transplant composition showing that the change in the positivity rates of FOXA2, LMX1A, NURR1 and TH in cells surviving after thawing is within ±10%.

[22] The transplant composition according to

[20] or

[21] , wherein the cell count is 80,000 to 5,000,000 cells / mL, and the composition contains FOXA2-positive and LMX1A-positive cells at 40% or more of the total cell count, and TH-positive and NURR1-positive cells at 40% or less of the total cell count.

[23] The transplant composition according to any one of

[20] to

[22] above, wherein the cell aggregate has an equivalent circle diameter of 150 to 1000 μm.

[24] The transplant composition according to any one of

[20] to

[23] , which does not require culture for recovery after thawing.

[25] A transplant composition described in any one of

[20] to

[24] , which contains 8 to 192 cell aggregates / ml, the equivalent circle diameter of the cell aggregates is 150 μm to 1000 μm, and the number of cells per container is 80,000 to 2,400,000.

[26] The transplant composition described in any one of

[20] to

[25] above, wherein the volume of the cell aggregate and the preservation solution is 0.25 mL to 2 mL.

[27] The transplant composition according to any one of

[20] to

[26] above, which is filled in a container of 0.5 ml to 15 ml.

[28] A method for producing a composition for transplantation containing dopamine-producing neural progenitor cells as an active ingredient, comprising freezing cell aggregates with a circle equivalent diameter of 150 μm to 1000 μm, wherein the cell count is 80,000 to 5,000,000 cells / mL, and the aggregates contain FOXA2-positive and LMX1A-positive cells at 40% or more of the total cell count and TH-positive and NURR1-positive cells at 40% or less of the total cell count, using the method described in any one of [1] to

[17] above.

[29] A method for producing a transplant composition described in

[28] , which contains as an active ingredient an aggregate of nervous system cells, the aggregate comprising a population of cell aggregates having a circle equivalent diameter of 150 to 1000 μm, the number of cells per container being 80,000 to 2,400,000, and the volume of the cell aggregates and preservation solution being 0.25 mL to 2 mL, and is filled in a 0.5 mL to 15 mL container.

[30] A method for treating a disease requiring regeneration of dopamine-producing neurons, comprising the following steps: (1) thawing the transplant composition according to any one of

[20] to

[27] at 30°C to 40°C, preferably at 37°C ± 3°C; (2) A step of transplanting the transplant composition obtained in (1) into the striatum region of a patient.

[31] The method according to

[30] , wherein after thawing, the cryopreservation solution is replaced with an administration medium without culturing, and step (2) is carried out. Regarding. [Effects of the Invention]

[0012] The present application provides a method for cryopreserving cell aggregates containing neural cells. Neural cells cryopreserved by the method of the present application exhibit high cell viability and maintain functional properties. [Brief explanation of the drawings]

[0013] [Figure 1] A schematic diagram showing the protocol for inducing differentiation of iPSCs into dopamine-producing neural progenitor cells and the timing of the evaluation experiment. The abbreviations in the diagram indicate the components added to the medium: LDN: LDN193189, A: A83-01, Y: Y-27632, Pur: palmorphamin, CHIR: CHIR99021, AA: ascorbic acid.

[0014] [Figure 2A] Effect of cryopreservation solutions on iPSC-derived dopamine-producing neural progenitor cells. Viable cell recovery after thawing of unsorted cells (n=4) after 15 minutes of permeation in the cryopreservation solutions shown in Table 1 and cryopreservation at 0.5°C / min. [Figure 2B] Effect of cryopreservation solutions on iPSC-derived dopamine-producing neural progenitor cells. Neurite outgrowth of cell clusters derived from unsorted cells, which were permeated with the cryopreservation solutions shown in Table 1 for 15 minutes and then cryopreserved at 0.5°C / min (n=4). [Figure 2C] Effect of cryopreservation solutions on iPSC-derived dopamine-producing neural progenitor cells. Immunostained images of neurites from cell clusters derived from unsorted cells, which were permeated with the cryopreservation solutions listed in Table 1 for 15 minutes and then cryopreserved at 0.5°C / min, were stained with an early neural marker (PSA-NCAM). The scale bar indicates 1 mm.

[0015] [Figure 3]Time-temperature curves for the sample (straight line), the freezing chamber (dashed line), and the program (dotted line). A Bambanker hRM was used as the sample. The lower figure shows an enlarged view of the temperature change due to the release of latent heat in the upper figure.

[0016] [Figure 4A] Effect of freezing programs on iPSC-derived dopaminergic neural progenitor cells. Viable cell recovery after thawing of unsorted cells cryopreserved using various freezing programs after 15 minutes of permeabilization in Bambanker hRM. [Figure 4B] Effect of freezing programs on iPSC-derived dopaminergic neural progenitor cells. Neurite outgrowth of cell clusters derived from unsorted cells cryopreserved with various freezing programs after 15 minutes of permeation in Bambanker hRM.

[0017] [Figure 5A] Effect of prolonged exposure to cryopreservation media on iPSC-derived dopaminergic neural progenitor cells. Viable cell recovery after thawing of unsorted cells cryopreserved after 60 minutes of exposure to Bambanker hRM in various freezing programs. [Figure 5B] Effect of freezing after prolonged exposure to cryopreservation media on iPSC-derived dopaminergic neural progenitor cells. Neurite outgrowth of cell clusters derived from unsorted cells cryopreserved after 60 minutes of exposure to Bambanker hRM in various freezing programs.

[0018] [Figure 6A] In vitro characteristics of cryopreserved cell masses. Viable cell recovery rate after thawing. [Figure 6B] Characteristics of cryopreserved cell clusters in vitro. Neurite outgrowth of cell clusters. [Figure 6C]Characteristics of cryopreserved cell clusters in vitro. Immunostaining of cell clusters on day 35 and day 7 after thawing. The left image shows immunostaining for FOXA2 / DAPI, the center image shows immunostaining for NURR1 / TH, and the right image shows immunostaining for SOX1 / KI67 / PAX6 / DAPI. The scale bar indicates 100 μm. [Figure 6D] Characteristics of cryopreserved cell masses in vitro. Percentage of FOXA2+ cells, NURR1+ cells, and TH+ cells relative to total cells at 35 days and 7 days after thawing. [Figure 6E] Characteristics of cryopreserved cell masses in vitro. Percentage of SOX1+ cells, PAX6+ cells, and KI67+ cells relative to total cells at 35 days and 7 days after thawing. [Figure 6F] Characteristics of cryopreserved cell clusters in vitro. Gene expression of cell clusters relative to GAPDH measured by quantitative RT-PCR. Expression level of undifferentiated cells (day 0) was set to 1. [Figure 6G] Characteristics of cryopreserved cell clusters in vitro. Gene expression of cell clusters relative to GAPDH measured by quantitative RT-PCR. Expression level of undifferentiated cells (day 0) was set to 1. [Figure 6H] Characterization of cryopreserved cell masses in vitro. Principal component analysis of microarray data showing temporal changes in gene expression in unfrozen (circles) and frozen (triangles) cells. [Figure 6I] In vitro characteristics of cryopreserved cell masses. Scatter plots of microarray data from unfrozen and frozen cells from the same lot are shown on day 35 (X axis) or day 7 after thawing (Y axis). Black circles indicate genes with a signal intensity of 50 or higher in one sample, while open circles indicate genes with a signal intensity of 50 or lower in both samples. [Figure 6J] Characteristics of cryopreserved cell masses in vitro. Scatter plots of microarray data from unfrozen cells from different lots on day 35 are shown. Black circles indicate genes with a signal intensity of 50 or higher in one sample, and white circles indicate genes with a signal intensity of 50 or lower in both samples. [Figure 6K]Characterization of cryopreserved cell clusters in vitro. Immunostaining of TUBB3, TH, and DAPI in iPSC-derived dopaminergic neurons after thawing on day 28 + 21. Scale bar indicates 50 μm. [Figure 6L] Characterization of cryopreserved cell clumps in vitro. Representative evoked action potentials of iPSC-derived dopaminergic neurons after thawing on day 28+21. [Figure 6M] In vitro characteristics of cryopreserved cell masses. Results of dopamine release induced by high potassium stimulation on day 56 or 28+28 after thawing.

[0019] [Figure 7A] Time course of dopaminergic neural progenitor cell marker expression after thawing. Percentage of FOXA2+ cells to total cells on days 28, 29, 31, and 35, and on days 0, 1, 3, and 7 after thawing. [Figure 7B] Time course of dopamine-producing neural progenitor cell marker expression after thawing. Percentage of NURR1+ cells to total cells on days 28, 29, 31, and 35, and on days 0, 1, 3, and 7 after thawing. [Figure 7C] Time course of dopaminergic neural progenitor cell marker expression after thawing. TH gene expression in cell clusters relative to GAPDH was measured by quantitative RT-PCR. The expression level in undifferentiated cells (day 0) was set to 1.

[0020] [Figure 8A] Graft survival and function of cryopreserved cell masses. Methamphetamine-induced rotational movement in graft-bearing rats. Data are shown as mean ± SEM (n = 6-8). Two-way ANOVA with Tukey's multiple comparison test was performed, and significance levels are indicated by **p<0.01 and ****p<0.001 relative to the vehicle group. [Figure 8B] Graft survival and function of cryopreserved cell masses. HNA immunostaining in representative grafts derived from unfrozen (top) and frozen (bottom) cells. [Figure 8C] Graft survival and function of cryopreserved cell masses. Number of viable HNA+ cells in the grafts. [Figure 8D] Graft survival and function of cryopreserved cell masses. DAB staining of TH in a representative graft derived from unfrozen cells. [Figure 8E] Graft survival and function of cryopreserved cell mass. Enlarged image of the box in Figure 8D. The right panel is an enlarged image of the box in the left panel. The scale bar indicates 200 μm. [Figure 8F] Graft survival and function of cryopreserved cell masses. DAB staining of TH in a representative graft derived from frozen cells. [Figure 8G] Graft survival and function of cryopreserved cell mass. Enlarged image of the box in Figure 8F. The right panel is an enlarged image of the box in the left panel. The scale bar indicates 200 μm. [Figure 8H] Graft survival and function of cryopreserved cell masses. Number of viable TH+ cells in the grafts. [Figure 8I] Graft survival and function of cryopreserved cell masses. Immunostaining of frozen cell-derived grafts for FOXA2, TH, and HNA (top panel), and KI67 and HNA (bottom panel). Scale bar indicates 50 μm. [Figure 8J] Graft survival and function of cryopreserved cell masses. Ratio of FOXA2+ cells to HNA+ cells. [Figure 8K] Graft survival and function of cryopreserved cell masses. Ratio of KI67+ cells to HNA+ cells. [Figure 9] 1 shows the viability and neurite outgrowth activity of cryopreserved cell masses thawed under different conditions. [Figure 10A] Marker expression in cell clusters derived from cells not sorted with anti-CORIN antibody. Immunostained images of cell clusters for LMX1A, FOXA2, DAPI (top row), NURR1, TH, DAPI (middle row), SOX1, KI67, PAX6, DAPI (bottom row). The scale bar indicates 100 μm. [Figure 10B] Marker expression in cell clusters derived from cells not sorted with anti-CORIN antibody. Percentage of FOXA2+ / LMX1A+ cells, NURR1+ cells, and TH+ cells relative to total cells. [Figure 10C] Marker expression of cell clusters derived from cells not sorted with anti-CORIN antibody. Percentage of SOX1+ cells, PAX6+ cells, and KI67+ cells relative to total cells. [Figure 11] Marker expression in cell clusters from the same lot as those shown in Figure 6C, immediately before freezing (day 28). Immunostained images of cell clusters for LMX1A, FOXA2, and DAPI (top row), NURR1, FOXA2, TH, and DAPI (middle row), and SOX1, KI67, PAX6, and DAPI (bottom row). Scale bar indicates 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0021] In this specification and claims, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0022] [Nervous system cells] The present application provides a method for freezing cell aggregates containing neural cells having a three-dimensional structure.

[0023] Neural system cells include nerve cells (neuron) and precursor cells of the nerve cells, ie, neural progenitor cells or neural precursor cells. Nervous system cells may be cells of any site, such as central nervous system cells, or peripheral nervous system cells, such as motor nerve or sensory somatic nervous system cells or autonomic nervous system cells, and examples thereof include nerves (neurons), neural crest-derived cells, glial cells such as oligodendrocytes or astrocytes, and their stem or progenitor cells. Nervous system cells include cells that express nervous system cell markers. 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. Expression of one or more of these neural cell markers can confirm that a cell is a neural cell. As used herein, neural cells include cells that express one or more, two or more, or three or more of the above neural cell markers.

[0024] Neural cells in the central nervous system can be classified according to the location of the nervous cells, namely, neurons derived from the forebrain, telencephalon, diencephalon, cerebrum, hypothalamus, midbrain, hindbrain, midbrain-hindbrain junction, cerebellum, retina, pituitary gland, or spinal cord, and their progenitor cells.

[0025] Forebrain-derived neurons are neurons present in forebrain tissues (i.e., telencephalon, cerebrum, hippocampus or choroid, diencephalon, hypothalamus, etc.). Forebrain neurons can be identified by the expression of forebrain neuron markers. Examples of forebrain neuron markers include OTX1 (forebrain), BF1 (also known as FOXG1), and SIX3 (also a marker for the telencephalon or cerebrum). As used herein, nervous system cells include cells that express one or more, two or more, or three or more of the above forebrain neuron markers, telencephalon, or cerebrum markers.

[0026] Cerebral-derived neurons include dorsal cells (e.g., cerebral cortical cells, Cajal-Retzius cells, hippocampal neurons, etc.) and ventral cells (e.g., basal ganglia cells, etc.). Ventral cerebral neuron markers include basal ganglia neuron markers (e.g., GSH2, MASH1, NKX2.1, NOZ1). Dorsal cerebral neuron markers include cerebral cortical neuron markers (e.g., PAX6, EMX1, TBR1). As used herein, neural cells include cells that express one or more, two or more, or three or more of the above-mentioned cerebral neuron markers, basal ganglia neuron markers, or cerebral cortical neuron markers.

[0027] Examples of midbrain-derived neural cells include ventral midbrain-derived neural progenitor cells, dopaminergic neurons (also called dopaminergic neurons), and dopaminergic neural precursor cells (also called dopaminergic neural precursor cells or dopaminergic progenitors). Markers for midbrain-derived neural cells include FOXA2, EN2, and TUJ1. Examples of FOXA2- and TUJ1-positive neural cells include dopaminergic neural precursor cells and dopaminergic neurons. Dopaminergic neurons can be identified using FOXA2-, NURR1-, and TH-positive indicators.

[0028] Furthermore, dopamine-producing neural progenitor cells can be identified using FOXA2- and LMX1A-positive indicators. More preferably, the cell aggregate contains cells positive for one or more of OTX2, LMX1A, LMX1B, CORIN, SHH, AADC, βIII-Tubulin, EN1, NURR1, PITX3, DAT, GIRK2, and TH. As used herein, unless otherwise specified, a cell aggregate containing dopamine-producing neural progenitor cells may also contain dopamine-producing neurons or dopaminergic neurons.

[0029] As used herein, nervous system cells include cells that express one or more, two or more, or three or more of markers for midbrain-derived nervous system cells, markers for dopaminergic neural progenitor cells, or markers for dopaminergic neurons.

[0030] As used herein, dopamine-producing neural progenitor cells include cells that express FOXA2 and / or LMX1A (FOXA2-positive and / or LMX1A-positive), preferably cells that express one or more, two or more, or three or more selected from the group consisting of OTX2, LMX1B, CORIN, SHH, AADC, and βIII-Tubulin in addition to FOXA2 and LMXA1.

[0031] As used herein, dopamine-producing neurons (dopamine neurons) include cells that express TH and / or NURR1 (TH-positive and / or NURR1-positive), 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.

[0032] Neurons derived from the midbrain-hindbrain boundary region include neurons present in the cerebellum, cerebellar plate tissue, ventricular zone, rhombic lip, etc. Midbrain-hindbrain boundary region markers include EN2 (midbrain), GBX2 (hindbrain), and N-Cadherin (neuronal progenitor cells in the midbrain-hindbrain boundary region). Cerebellar neural progenitor cell markers include GABAergic neural progenitor cell markers KIRREL2, PTF1A, or SOX2, and cerebellar granule cell progenitor cell markers ATOH1 or BARHL1. As used herein, neural cells include cells expressing one or more, two or more, or three or more of the above-mentioned midbrain-hindbrain boundary region markers, cerebellar neural progenitor cell markers, GABAergic neural progenitor cell markers, or cerebellar granule cell progenitor cell markers. Examples of neural cells derived from the retina include photoreceptor cells, photoreceptor precursor cells, retinal pigment epithelial cells, and corneal cells.

[0033] Nervous system cells can also be classified according to the neurotransmitters they produce (secrete), and examples include dopaminergic neurons, dopaminergic neuronal precursor cells, GABAergic neurons, GABAergic neuronal precursor cells, cholinergic neurons, cholinergic neuronal precursor cells, serotonergic neurons, serotonergic neuronal precursor cells, glutamic acid neurons, glutamic acid neuronal precursor cells, noradrenergic neurons, noradrenergic neuronal precursor cells, adrenergic neurons, and adrenergic neuronal precursor cells.

[0034] Examples of nervous system cells of the motor nerves and sensory organs include cholinergic neurons and their precursor cells. Examples of autonomic nervous system cells include cholinergic neurons, adrenergic neurons, and their precursor cells.

[0035] As used herein, preferred examples of nervous system cells include dopamine-producing neurons (dopamine neurons) and dopamine-producing neural progenitor cells (dopamine neural progenitor cells).

[0036] Neural cells derived from living organisms are cells isolated from mammals such as humans. For example, cells isolated from human brain tissue include cells contained in fetal midbrain tissue as described in Nature Neuroscience, 2, 1137 (1999) or N. Engl. J. Med.; 344:710-9 (2001).

[0037] Neural 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 differentiation of neural cells from pluripotent stem cells include, for example, the methods described in the above-mentioned Non-Patent Documents 3 and 4 and WO2015 / 034012 (dopamine-producing neural progenitor cells), WO2009 / 148170 (nervous system cells of the cerebrum, etc.), 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) is an example.

[0038] Alternatively, the neural cells may be cells obtained by inducing differentiation from multipotent stem cells such as mesenchymal stem cells (MSCs). Examples of methods for inducing differentiation of neural cells from mesenchymal stem cells include the method described in J Chem Neuroanat. 96:126-133 (2019).

[0039] [Pluripotent stem cells] Pluripotent stem cells are stem cells that possess the pluripotency to differentiate into almost all cell types present in the body and also possess the ability to proliferate. Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES) cells, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, cultured fibroblasts, and pluripotent stem cells derived from bone marrow stem cells (Muse cells). Pluripotent stem cells may be ES cells, ntES cells, or iPS cells. From an ethical perspective, pluripotent stem cells may also be iPS cells. Embryonic stem cells are established from embryos within 14 days of fertilization.

[0040] Embryonic stem cells were first established in 1981 and have been used to generate knockout mice since 1989. Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell mass on feeder cells or in a medium containing LIF (leukemia inhibitory factor). Methods for producing embryonic stem cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, and US6,280,718. Embryonic stem cells are available from designated institutions or commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. The human embryonic stem cell Rx::GFP strain (derived from the KhES-1 strain) is available from RIKEN, a national research and development agency. The EB5 cell line and the D3 cell line, which are mouse embryonic stem cells, are available from the National Research and Development Agency, RIKEN, and ATCC, respectively.

[0041] Nuclear transfer embryonic stem cells (ntES cells), a type of embryonic stem cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg cell from which the nucleus has been removed.

[0042] EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF, and bFGF (Cell, 70:841-847, 1992).

[0043] As used herein, "induced pluripotent stem cells" refer to cells in which pluripotency has been induced by reprogramming somatic cells using known methods, etc. Specific examples include cells in which pluripotency has been induced by reprogramming differentiated somatic cells such as fibroblasts or peripheral blood mononuclear cells through the expression of any combination of multiple 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. 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), and (3) OCT3 / 4, SOX2, NANOG, and LIN28 (Science 2007; 318: 1917-1920).

[0044] In 2006, Yamanaka et al. established induced pluripotent stem cells using mouse cells (Cell, 2006, 126(4), pp. 663-676). In 2007, induced pluripotent stem cells were also established using human fibroblasts, and they possess the same pluripotency and self-renewal capabilities as 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).

[0045] In addition to methods for producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be produced by methods for inducing induced pluripotent stem cells from somatic cells by adding chemical compounds, etc. (Science, 2013, 341, pp. 651-654).

[0046] It is also possible to obtain established induced pluripotent stem cells, such as human induced pluripotent stem cell lines established at Kyoto University, such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells, which are available from Kyoto University. Examples of established induced pluripotent stem cells available from Kyoto University include Ff-I01 cells, Ff-I01s04 cells, QHJ-I01, and Ff-I14 cells.

[0047] Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells (PBMCs), T cells), hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.

[0048] When producing induced pluripotent stem cells, if reprogramming is performed by expressing several types of genes, the means for expressing the genes is 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 viral 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), and direct protein injection methods (e.g., needle method, lipofection method, or electroporation method).

[0049] 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, they can be produced in the presence of undifferentiated maintenance factors by known methods. The medium used to produce induced pluripotent stem cells in the absence of feeder cells is not particularly limited, and known maintenance media for embryonic stem cells and / or induced pluripotent stem cells, or media for establishing feeder-free induced pluripotent stem cells can be used. Examples of media for establishing feeder-free induced pluripotent stem cells 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, induced pluripotent stem cells can be produced by introducing the four factors OCT3 / 4, SOX2, KLF4, and MYC (L-MYC or C-MYC) into somatic cells in a feeder-free environment using a Sendai virus vector.

[0050] The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (e.g., mouse or rat) or primate (e.g., human or monkey) pluripotent stem cells, 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).

[0051] [Cell aggregate] As used herein, the term "cell aggregates having a three-dimensional structure" means that cultured cells form cell aggregates (or spheres), which are three-dimensional cell groups formed by cells adhering to one another, for example, through suspension culture or three-dimensional culture. Cell aggregates of nervous system cells are also called neurospheres. The shape of the cell aggregates is not particularly limited, and they may be spherical or non-spherical. The cell aggregates used herein are preferably cell aggregates having a three-dimensional shape close to a sphere. A three-dimensional shape close to a sphere is a shape having a three-dimensional structure, and when projected onto a two-dimensional surface, it is, for example, circular or elliptical.

[0052] The size of the cell aggregate containing neural cells having a three-dimensional structure is not particularly limited, but typically has a circle-equivalent diameter of 150 μm to 1,000 μm, for example, 200 μm to 800 μm, or 300 μm to 500 μm in one embodiment. Furthermore, the cell aggregate containing neural cells having a three-dimensional structure typically contains 500 to 150,000 cells, for example, 1,000 to 100,000 cells, 1,000 to 70,000 cells, or 3,000 to 30,000 cells in one embodiment.

[0053] The cell aggregate containing nervous system cells may contain other cells in addition to nervous system cells, and examples thereof include cell aggregates containing 60% or more, 70% or more, 80% or more, and more preferably 90% or more nervous system cells.

[0054] In one embodiment, the cell aggregate containing neural cells may contain 60% or more, 70% or more, or 80% or more dopaminergic neural progenitor cells and / or dopaminergic neurons. That is, the cell aggregate containing neural cells may contain 60% or more, 70% or more, or 80% or more neural cells expressing one or more markers selected from FOXA2, LMX1A, LMX1B, NURR1, and TH.

[0055] In one embodiment, the cell aggregate containing neural cells contains 40% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more dopamine-producing neural progenitor cells.

[0056] In one embodiment, the cell aggregates containing neural cells contain 40% or more, 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more cells expressing one or more, two or more, or three or more markers of dopamine-producing neural progenitor cells.

[0057] In one embodiment, the cell aggregate containing neural cells comprises 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 embodiment, the cell aggregate further comprises 40% or less of TH-positive and NURR1-positive cells.

[0058] In one embodiment, a cell aggregate containing nervous system cells may contain FOXA2-positive, TH-positive, and NURR1-positive cells at 0% or more, 10% or more, or 20% or more.

[0059] In one embodiment, a cell aggregate containing dopamine-producing neural progenitor cells may contain NURR1-positive cells at 60% or less, 50% or less, 40% or less, 5 to 50%, 5 to 40%, or 5 to 20%.

[0060] In one embodiment, a cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells may contain TH-positive cells at 30% or less, 20% or less, 1 to 30%, 5 to 30%, 1 to 20%, 5 to 20%, or 5 to 15%.

[0061] In one embodiment, a cell aggregate containing dopaminergic neural progenitor cells and / or dopaminergic neural cells may contain KI67-positive cells at 30% or less, 1 to 25%, 1 to 20%, or 5 to 20%.

[0062] In one embodiment, a cell aggregate containing dopaminergic neural progenitor cells and / or dopaminergic neural cells may contain SOX1-positive cells at 20% or less, 10% or less, 5% or less, or 1% or less.

[0063] In one embodiment, a cell aggregate containing dopaminergic neural progenitor cells and / or dopaminergic neural cells may contain PAX6-positive cells at 5% or less, 2% or less, 1% or less, or 0.5% or less.

[0064] In one embodiment, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells further contains 20% or less, specifically 1% to 20%, more specifically 5% to 15% TH-positive and NURR1-positive cells.

[0065] In one embodiment, a cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons contains 50% or more, preferably 60% or more, 70% or more, or 80% or more, of FOXA2-positive and LMX1A-positive cells, and 20% or less, 1% to 20%, more specifically 5% to 15% of TH-positive and NURR1-positive cells.

[0066] In one embodiment, a cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neural cells further comprises SOX1-positive cells at 10% or less, preferably 7% or less, and more preferably 3% or less, and PAX6-positive cells at 5% or less, preferably 4% or less, and more preferably 2% or less.

[0067] In one embodiment, a cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons contains 60% or more FOXA2-positive and LMX1A-positive cells, 1% to 20% TH-positive and NURR1-positive cells, 10% or less, preferably 7% or less, and more preferably 3% or less SOX1-positive cells, and 5% or less, preferably 4% or less, and more preferably 2% or less PAX6-positive cells.

[0068] In one embodiment, a cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons may contain FOXA2-positive and LMX1A-positive cells at 60% or more of the total number of cells, and TH-positive and NURR1-positive cells at 20% or less, 1 to 20%, or 5 to 15% of the total number of cells.

[0069] In one embodiment, the cell aggregate containing dopaminergic neural progenitor cells and / or dopaminergic neural cells is a cell aggregate having a circle-equivalent diameter of 150 μm to 1000 μm.

[0070] In one embodiment, the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons is a cell aggregate containing 60% or more FOXA2-positive and LMX1A-positive cells, 1% to 20% NURR1-positive and TH-positive cells, and having a circle-equivalent diameter of 150 μm to 1000 μm.

[0071] [Freezing method] The method of the present application includes the step of (1) contacting a cell aggregate containing neural cells having a three-dimensional structure with a preservation solution at a temperature of 0°C or higher and 30°C or lower before freezing, thereby preparing a cell aggregate immersed in the preservation solution.

[0072] In this application, the cryopreservation solution (preservation solution) refers to an aqueous liquid containing a cryoprotectant. A cryoprotectant is a substance that has a high affinity for water molecules and is highly effective in suppressing ice crystal growth in the cryopreservation solution. Examples include 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 / or glycerin. In this application, the cryoprotectant is preferably dimethyl sulfoxide and / or propylene glycol. When dimethyl sulfoxide and / or propylene glycol are used as the cryoprotectant, the concentration of the cryoprotectant in the cryopreservation solution is typically 7-12%, preferably about 10%.

[0073] Examples of aqueous liquids that can be used include buffer solutions such as physiological saline, PBS, EBSS, and HBSS; culture media for culturing cells and tissues such as DMEM, GMEM, and RPMI; serum; serum substitutes; and mixtures thereof.

[0074] Commercially available cryopreservation solutions containing dimethyl sulfoxide (DMSO) and / or propylene glycol as essential components can be used. Specific examples of cryopreservation solutions include STEM-CELL BANKER (SCB; ZENOAQ), STEM-CELL BANKER DMSO-free (SCB DMSO-free; ZENOAQ), Bambanker hRM (BBK; NIPPON Genetics), CryoStor CS5 (CS5; BioLife Solutions), CryoStor CS10 (CS10; BioLife Solutions), and Synth-a-Freeze (SaF; ThermoFisher Scientific). For example, cryopreservation solutions containing 7-12%, preferably about 10%, of dimethyl sulfoxide and / or propylene glycol (e.g., STEM-CELL BANKER, Bambanker hRM, CryoStor CS10, and Synth-a-Freeze) are recommended. More preferably, Bambanker hRM can be used.

[0075] In this specification, when cell aggregates are frozen, the number of cells in the cryopreservation solution (cell packing density) is 80,000 to 5,000,000 cells / mL, 100,000 to 4,000,000 cells / mL, 200,000 to 2,000,000 cells / mL, or 300,000 to 1,000,000 cells / mL.

[0076] In the present specification, when the cell aggregate is frozen, the equivalent circle diameter of the cell aggregate is 150 to 1000 μm, 150 μm to 600 μm, or 300 μm to 500 μm.

[0077] In this specification, 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.

[0078] In the present 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.

[0079] The freezing point of the cryopreservation solution in the present application is not particularly limited, but is usually −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. Examples of cryopreservation solutions in this specification include aqueous liquids that contain 7 to 12%, preferably about 10%, of dimethyl sulfoxide as a substantial component and have a freezing point of −1° C. to −10° C. Examples of cryopreservation solutions in this specification also include aqueous liquids that contain 7 to 12%, preferably about 10%, of dimethyl sulfoxide as a substantial component and have a freezing point of −3° C. to −6° C.

[0080] The temperature at which cell aggregates containing nervous system cells are brought into contact with a cryopreservation solution is typically between 0°C and 30°C, preferably between 0°C and 20°C, more preferably between 0°C and 10°C, and even more preferably between 0°C and 4°C.

[0081] Furthermore, the time for contacting cell aggregates containing nervous system cells with the cryopreservation solution is typically 5 to 240 minutes, 5 to 120 minutes, preferably 5 to 60 minutes, 15 to 240 minutes, 15 to 180 minutes, 15 to 150 minutes, preferably 15 to 120 minutes, 15 to 90 minutes, and more preferably 15 to 60 minutes.

[0082] The method of the present application also includes (2) a step of cooling and freezing the cell aggregates immersed in the preservation solution obtained in step (1) from a temperature at least about 5°C higher than the freezing point of the preservation solution to a temperature about 5°C lower than the freezing point at an average rate of 2 to 7°C, 2.5 to 7°C, or 3 to 7°C / min.

[0083] In the method of the present application, the cell aggregate immersed in the preservation solution is cooled from a temperature about 5°C higher than the freezing point of the preservation solution to a temperature about 5°C lower than the freezing point at an average rate of 2 to 7°C / min, 2.5 to 7°C / min, or 3 to 7°C / min, preferably an average rate of 2 to 5.5°C / min, 2.5 to 5.5°C / min, or 3 to 5.5°C / min. In a preferred embodiment, the cell aggregate immersed in the preservation solution is cooled from 0±5°C to -30°C±5°C at an average rate of 2 to 5°C / min, 2.5 to 5°C / min, or 3 to 5°C / min.

[0084] The cooling means is not particularly limited as long as the above steps are achieved, and a commercially available freezer can be used, or a programmable freezer (also called a controlled rate freezer) that can control the temperature may also be used.

[0085] In step (2), the cell aggregates immersed in the preservation solution may be exposed to an electromagnetic field and / or a magnetic field. The frequency of the electromagnetic field is, for example, about 300 kHz to about 2 MHz, preferably about 500 kHz to about 1 MHz, and more preferably about 600 kHz to about 1 MHz. The frequency of the magnetic field is not particularly limited, but a fixed frequency is preferred. Alternatively, the cell aggregates may be frozen under an electrostatic field of, for example, 10 to 2000 gauss, preferably 50 to 1000 gauss, and more preferably 100 to 150 gauss. The method for achieving these conditions is not particularly limited. For example, a programmable freezer equipped with a device that generates electromagnetic and magnetic fields, such as a proton freezer (Ryoho Freeze Systems Co., Ltd.), can be used. That is, a device that combines a static magnetic field (SMF), an alternating electric field (AEF), and ultra-cold air can be used. Specifically, by using electromagnetic waves of 300 kHz to 2 MHz, damage to cell aggregates due to ice formation can be suppressed.

[0086] The method of the present application may further comprise the step of (3) cooling the frozen cell aggregates obtained in step (2) to -50°C or below, preferably -80°C or below, more preferably -150°C or below.

[0087] The cooling means is not particularly limited, but examples thereof include a deep freezer, a program freezer, a proton freezer, and exposure to a low-temperature medium (such as liquid nitrogen).

[0088] The frozen cell aggregates obtained in step (2) or (3) may be stored for a long period of time by keeping them at -80°C or below, preferably at -150°C or below.

[0089] Examples of means for long-term storage include deep freezers, program freezers, proton freezers, and subjecting to low temperature media (eg, liquid nitrogen).

[0090] Frozen cell aggregates can be thawed as needed for use. The thawing method is not particularly limited, but from the viewpoint of function, activity, and cell viability, it is desirable to thaw the aggregates in a short time at a temperature around body temperature. Specifically, it is desirable to thaw the aggregates at a temperature between 30°C and 40°C, preferably between 35°C and 38°C, and more preferably at a temperature around human body temperature, for example, about 37°C.

[0091] After thawing, the cell aggregates frozen by the method of the present invention may be subjected to recovery culture by replacing the cryopreservation solution with a medium, or may be transplanted into a living body without recovery culture.

[0092] That is, cell aggregates frozen by the method of the present invention can maintain properties equivalent to those of cell aggregates that have not been frozen. For example, when cell aggregates frozen by the method of the present invention are thawed and cultured for 7 days to recover, 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, the markers can include FOXA2, LMX1A, NURR1, or TH. Here, "equivalent marker expression rate" means that the difference in the numerical value of the proportion of marker-expressing cells to the total number of cells before freezing and after thawing or after 7 days of culture after thawing is approximately 10% or less.

[0093] The cell aggregates frozen by the method of the present invention are useful in that they can be transplanted into a living body without recovery culture.

[0094] [Pharmaceutical composition] Furthermore, the present application provides a pharmaceutical composition, i.e., a transplant composition (preparation), containing as an active ingredient the cell aggregate frozen or long-term preserved by the above-mentioned method.

[0095] The pharmaceutical composition (composition for transplantation) of the present invention conceptually includes both a pharmaceutical composition frozen by the method of the present invention and a pharmaceutical composition obtained by thawing the same. That is, the pharmaceutical composition (composition for transplantation) of the present invention includes a frozen or unfrozen composition comprising a cell aggregate containing nervous system cells and a cryopreservation solution, as well as a composition comprising a cell aggregate containing nervous system cells in which the cryopreservation solution has been replaced with an administration vehicle after thawing and an administration vehicle.

[0096] The pharmaceutical composition (composition for transplantation) of the present invention includes the compositions for transplantation described in

[20] to

[27] above.

[0097] In one embodiment, the present application provides a cell aggregate having a circle equivalent diameter of 150 μm to 1,000 μm and containing 500 to 150,000 cells, which contains 60% or more dopamine-producing neural progenitor cells and dopamine-producing neurons derived from pluripotent stem cells, and a cryopreservation solution having a freezing point of −1° C. to −10° C., which contains 7% to 12% dimethyl sulfoxide or propylene glycol, preferably Bambanker hRM, and which has the following properties: (1) Approximately 60% or more of the total number of cells survives after thawing. (2) have neurite outgrowth activity of 50% or more compared to before freezing; (3) The change in the positive rates of FOXA2, LMX1A, NURR1, and TH in surviving cells after thawing is within ±10%, preferably the change in the positive rates of FOXA2, LMX1A, NURR1, TH, EN1, and PITX3 is within ±10%; The present invention provides a frozen transplant composition comprising:

[0098] In one embodiment, the present invention encompasses a transplant composition having a cell count of 80,000 to 5,000,000 cells / mL, 100,000 to 4,000,000 cells / mL, 200,000 to 2,000,000 cells / mL, or 300,000 to 1,000,000 cells / mL, wherein FOXA2-positive and LMX1A-positive cells account for 40% or more of the total cell count, preferably 60% or more, 60% or more, 80% or more, 85% or more, or 90% or more, and wherein TH-positive and NURR1-positive cells account for 40% or less, 1% to 20%, or 5% to 15% of the total cell count.

[0099] In one embodiment, 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 embodiment, 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 embodiment, the cell aggregates 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 embodiment, the present invention includes a transplant composition that does not require culture for recovery after thawing.

[0103] In one embodiment, the transplant composition is described in any one of

[20] to

[24] above, which contains 8 to 192 cell aggregates / ml, has an average particle size of 150 μm to 1000 μm, and has a cell count of 80,000 to 2,400,000 cells per container.

[0104] The cell aggregate is useful as a pharmaceutical composition for transplantation for patients suffering from diseases requiring the transplantation of nervous system cells, and can be used as a pharmaceutical, such as a therapeutic agent for diseases accompanied by degeneration, damage, or dysfunction of nervous system cells. That is, a pharmaceutical composition comprising the cell aggregate of the present invention and a pharmaceutically acceptable carrier also falls within the scope of the present invention.

[0105] Diseases requiring transplantation of nervous system cells or diseases involving 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, and Parkinson's syndromes (including Parkinson's disease).

[0106] One aspect of the present invention is a pharmaceutical composition for treating Parkinson's disease, which contains, as an active ingredient, a cell aggregate containing dopaminergic neural progenitor cells and / or dopaminergic neurons of the present invention. The number of dopaminergic neural progenitor cells and / or dopaminergic neurons contained in the therapeutic agent for Parkinson's disease is not particularly limited as long as the graft can survive after administration. For example, 1.0 × 10 cells per transplant can be used. 4The amount of dopamine-producing neural progenitor cells may be increased or decreased depending on the symptoms and body size. Transplantation of dopamine-producing neural progenitor cells into the diseased area can be performed, for example, by the method described in Nature Neuroscience, 2, 1137 (1999) or N Engl J Med.; 344: 710-9 (2001).

[0107] In one embodiment, the pharmaceutical composition of the present invention (also referred to as a transplant composition) comprises a cell aggregate containing neural cells to be transplanted into a human and a cryopreservation solution. The pharmaceutical composition of the present invention includes both frozen solid compositions and liquid compositions before freezing or after thawing. The pharmaceutical composition may optionally contain additives used to maintain cell viability, provided that they do not affect the freezing rate or temperature. Examples of cryopreservation solutions include those described above.

[0108] As described below, the pharmaceutical composition or transplant composition of the present invention is thawed, the cryopreservation solution is removed, and the cryopreservation solution is replaced with an administration vehicle that can be administered to a living body before being used for transplantation. That is, a composition containing a thawed cell aggregate and an administration vehicle also falls within the scope of the pharmaceutical composition (also referred to as a transplant composition) of the present invention.

[0109] [Method for producing a transplant composition] The pharmaceutical compositions (compositions for transplantation) described in

[20] to

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

[17] above. That is, the present invention encompasses a method for producing the pharmaceutical compositions (compositions for transplantation) described above.

[0110] [Treatment method] One aspect of the present invention is a method for treating a disease requiring replenishment of nervous system cells, comprising the step of transplanting the cell aggregate of the present invention into a patient suffering from a disease requiring transplantation of nervous system cells.

[0111] In one embodiment of the present invention, the cell aggregates containing dopaminergic neural progenitor cells and / or dopaminergic neural cells obtained by the present invention can be administered to Parkinson's disease patients as a pharmaceutical composition, specifically as a transplant material.

[0112] Specifically, a frozen pharmaceutical composition containing a cell aggregate containing the dopaminergic neural progenitor cells and / or dopaminergic neural cells of the present invention and a cryopreservation solution is thawed, suspended in an appropriate transplantation medium such as physiological saline, and transplanted into a region of the patient lacking dopaminergic neurons, such as the striatum. For example, after thawing, the pharmaceutical composition may be washed with a medium containing an appropriate carrier, and the cryopreservation solution may be replaced with a transplantation medium for suspending the cell aggregate when transplanted into a human. The thawing temperature is not particularly limited, but may be, as mentioned above, 30°C to 40°C, preferably 35°C to 38°C, and more preferably a temperature around human body temperature, such as approximately 37°C. The cell aggregates contained in the pharmaceutical composition (transplant composition) of the present invention can be transplanted into a living body by replacing the cryopreservation solution with an administration medium without culturing for recovery after thawing.

[0113] The carrier used in the transplantation medium (administration medium) for cell aggregates containing dopaminergic neural progenitor cells and / or dopaminergic neurons is not particularly limited, and any substance known to those skilled in the art can be used as long as it is used to maintain cell viability. Specifically, physiological aqueous solvents (e.g., physiological saline, buffer solution, serum-free medium, etc.) can be used. If necessary, commonly used preservatives, stabilizers, reducing agents, isotonicity agents, etc. in transplantation medicine may be incorporated into pharmaceutical compositions containing tissues or cells to be transplanted.

[0114] Furthermore, prior to transplantation, thawed cell aggregates may be stored in a medium necessary to maintain the viability of each cell aggregate. Examples of "medium necessary to maintain viability" include culture media, physiological buffer solutions, etc., but 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 select an appropriate medium. Examples of basal media include media prepared using media commonly used for culturing animal cells as basal media. Examples of basal media 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's medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof, which can be used to culture animal cells.

[0115] By transplanting the above-mentioned cell aggregates, the transplanted dopamine-producing neural progenitor cells and / or dopamine-producing neurons, and the dopamine-producing neural progenitor cells and / or dopamine-producing neurons induced after transplantation, are functionally engrafted in the patient to whom they are administered.

[0116] Here, "engraftment" in this specification means that transplanted cells survive in the body for a long period of time (for example, 30 days or more, 60 days or more, 90 days or more) and adhere to and remain within the organ.

[0117] As used herein, the term "functional engraftment" refers to a state in which transplanted cells have engrafted and are performing their inherent functions in vivo.

[0118] As used herein, the term "functional engraftment rate" refers to the percentage of cells that have achieved functional engraftment among transplanted cells. The functional engraftment rate of transplanted dopamine-producing neural progenitor cells can be determined, for example, by counting the number of TH-positive cells in the graft.

[0119] By transplanting the above-mentioned cell aggregates, the functional engraftment rate of the transplanted cells and the dopamine-producing neural progenitor cells and / or dopamine-producing neural cells 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] One aspect of the present invention includes a method for treating a disease requiring regeneration of dopamine-producing neurons, comprising the following steps: (1) thawing the transplant composition according to any one of

[20] to

[27] at 30°C to 40°C, preferably at 37°C ± 3°C; (2) A step of transplanting the transplant composition obtained in (1) into the striatum region of a patient. One embodiment of the present invention includes a method of treatment characterized by replacing the cryopreservation solution with an administration medium after thawing without culturing, and then carrying out step (2).

[0121] In this specification, examples of mammals to be transplanted include humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, and monkeys, preferably rodents (e.g., mice and rats) or primates (e.g., humans and monkeys), and more preferably humans. [Example]

[0122] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples. Example 1 An outline of Example 1 is shown in FIG.

[0123] Materials and Methods Maintenance of human iPS cells and differentiation into neural cells iPSC-derived dopamine-producing neural progenitors were induced according to the dual SMAD inhibition and bottom plate induction protocol described by Doi et al., 2014. Human iPSCs (1231A3) (Kyoto University) were maintained in StemFit medium (Ajinomoto) on six-well plates coated with iMatrix511 (Nippi). To initiate neural differentiation, iPSCs were incubated with TrypLE select (Invitrogen) for 10 minutes, then dissociated into single cells and plated on six-well plates coated with iMatrix511 (Nippi) at 5 × 10 cells. 6 Cells were seeded at a density of 1000 cells / well in differentiation medium. The differentiation medium was 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 increase cell viability after seeding, 10 μM Y-27632 (Wako) was added on day 1. To efficiently induce neural differentiation, LDN193189 (STEMGENT) and A83-01 (Wako) were added. Additionally, to induce floor plate cells, 2 μM palmorfamine and 100 ng ml -1 FGF8 (Wako) was added, and 3 μM CHIR99021 (Wako) was added on days 3 to 12 (see Figure 1).

[0124] Cell sorting and culture On day 12, cells expressing CORIN (a floor plate marker in the developing brain) were isolated to enrich for dopaminergic neural progenitors and sorted (FACS method). First, cultured cells were stained with PE-labeled anti-CORIN antibody (100 ng / mL; Catalent / BD) for 20 minutes. Dead cells and debris were excluded by 7-AAD staining. Analysis was performed using a FACSAria II or III cell sorter and the FACSDiva software program (BD Biosciences). After cell sorting on day 12, sorted cells were plated onto low-attachment U-bottom 96-well plates (Sumitomo Bakelite) in a medium containing B27 supplement, 2 μM Glutamax-I (all Invitrogen), and 10 ng / mL ATP. -1 GDNF, 200 mM ascorbic acid, 20 ng ml -1 2-3 × 10 cells in neural differentiation medium containing neurobasal medium supplemented with BDNF (all from Wako) and 400 μM dbcAMP (Sigma-Aldrich). 4 The cells were replated at a density of 1.5 × 10 cells / well and cultured as floating cell masses until day 28. Unsorted cells were also cultured at a density of 1.5 × 10 cells / well. 4 The cells were replated at a density of 1000 cells / well. Half of the medium was replaced every 3 days, and 30 μM Y-27632 (Wako) was added to the initial medium only. For long-term culture, floating cell clusters were cultured in neural differentiation medium (see Figure 1).

[0125] Cryopreservation method Cell masses collected on day 28 were cryopreserved for further experiments. Specifically, they were placed in cryovials containing 1 mL of the cryopreservation solution listed in Table 1 and kept on ice until frozen. For cryopreservation, vials were transferred into a freezing container: BICELL (NIHON FREEZER), a programmed freezer: PDF-150, 250 (STREX), cryomed (ThermoFisher Scientific), or a proton freezer (RYOHO FREEZER SYSTEMS). Six cooling profiles (shown in Figure 3) were used in this experiment. Specifically, BICELL was transferred to a deep freezer (-80 °C) and held there for at least 4 hours (Figure 3, top left). For the controlled-rate freezing method, vials were frozen to -40 °C at a rate of 0.5 °C / min (Figure 3, top center) or 1 °C / min (Figure 3, top right) and then cooled to -80 °C at a faster rate of 3–5 °C / min. The shock cooling method involved freezing to -35°C at a rate of -25°C / min, followed by a subsequent warming step to -12°C at a rate of +10°C / min, with the temperature at -4°C during freezing (Figure 3, bottom left and center). Vials frozen in a proton freezer were kept in a chamber for 30–60 minutes (Figure 3, bottom right). A proton freezer combines a static magnetic field, electromagnetic waves, and cold air. It is believed that the static and electromagnetic fields affect the orientation of water molecules, causing the formation of small ice crystals, thereby preventing cell destruction. However, these mechanisms are not yet fully understood. After freezing, the cryovials were stored in the vapor phase of a liquid nitrogen tank. The frozen cells were thawed at 37°C for approximately 2 minutes and transferred to a 15-mL tube containing 10 mL of neurobasal medium. After removing the supernatant, the cells were rinsed with PBS and used for each assay or transplantation. To estimate cell number after cryopreservation, approximately 50 aggregates were dissociated and counted with a hemocytometer to calculate cell concentration 1 day after thawing and before freezing. Table 1. Commercially available xeno-free cryopreservation solutions used in this example [Table 1]

[0126] Quantitative RT-PCR Total RNA was extracted from undifferentiated cells, iPSC-derived dopaminergic neurons (days 28, 29, 31, and 35), and thawed iPSC-derived dopaminergic neurons on days 28+0, 29, 31, and 35 using the RNeasy Mini Kit or RNeasy Micro Kit (Qiagen), and cDNA was synthesized using the Super Script III First-Strand Synthesis System (Invitrogen). Quantitative PCR reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems) in StepOne. Data were evaluated using the delta Ct method and normalized by GAPDH expression. The primer sequences used are listed in Table 2. [Table 2]

[0127] Immunofluorescence studies For in vitro studies, cultured cells were fixed with 4% paraformaldehyde on day 35 or 7 after thawing. For in vivo studies, fixed and frozen brains were sliced ​​at 40 μm thickness. The slices were immunostained using the free-floating method. The primary antibodies used are listed in Table 3. Cells were visualized using a fluorescence microscope (BZ-9000; Keyence) and a confocal laser microscope (Fluoview FV1000D; Olympus). The number of immunoreactive cells was quantified in every six sections throughout the graft and corrected using the Abercrombie method. [Table 3] *TUBB3 stands for βIII-Tubulin.

[0128] Neurite outgrowth assay On day 28, floating cell clusters were picked for neurite outgrowth assays and cultured on iMatrix511-coated 24-well plates for 5 days and fixed with 4% paraformaldehyde. Cell clusters were stained with PE-labeled anti-PSA-NCAM antibody (1:100; Milteny) and visualized using a fluorescence microscope (BZ-9000; Keyence). The area of ​​PSA-NCAM-positive neurites, excluding cell bodies, was measured using Photoshop (Adobe Systems) and WinRoof (Mitani Corporation).

[0129] Electrophysiological analysis On day 28, floating cell clusters were dissociated with papain and cultured on plates coated with poly-l-ornithine, fibronectin, and laminin (O / F / L) for up to day 49. Nerves with large cell bodies and neurite-like structures were selected for whole-cell patch clamp recording. Cells were maintained in a saline solution containing the following components: 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, and 17 mM glucose. Patch pipettes were fabricated from borosilicate glass capillaries (GC150TF-10; Clark). These patch pipettes had a resistance of 3–4 MW when filled with an internal solution consisting of 140 mM KCl, 10 mM HEPES, and 0.2 mM EGTA (pH 7.3). Voltage-clamp and current-clamp recordings were performed using a patch-clamp amplifier (EPC-8; HEKA). The resistance during gigaseal contact was in the range of 10–20 GW. The current signal from the patch-clamp amplifier was filtered at 5 kHz through a four-pole low-pass filter (UF-BL2; NF) with Bessel characteristics, sampled using a 12-bit A / D converter, and stored on a 32-bit computer (PC-9821Ra333; NEC). All experiments were performed at room temperature.

[0130] Dopamine release assay On day 28, the floating cell clusters picked for the dopamine release assay were cultured on O / F / L-coated 12-well plates for an additional 28 days. They were washed twice with low-concentration KCl solution (4.7 mM) and incubated in low-concentration KCl solution for 15 minutes. The medium was then replaced with high-concentration KCl solution (60 mM) for 15 minutes. The solution was collected, and the dopamine concentration was determined by LC / MS / MS. Cells remaining on the plate were collected in PBS and sonicated. The DNA concentration of the cell lysate was measured using Quant-iT (商標) The concentration was measured using a dsDNA Assay Kit (ThermoFisher) and used to correct for dopamine levels.

[0131] Microarray analysis cDNA microarray analysis was performed at the Bio-Medical Department of Kurabo Industries Ltd. Total RNA from undifferentiated cells, day 12 cultured cells, iPSC-derived dopaminergic neurons (days 28, 29, 31, and 35), and thawed iPSC-derived dopaminergic neurons on days 28+0, 28+1, 28+2, 28+3, and 28+7 was analyzed using Genechip. (登録商標) Data were processed using the 3'IVT pico Reagent Kit and Human Genome U133 Plus 2.0 array (Affymetrix). Data analysis was performed using Genechip operating software version 1.4 (Affymetrix). Signal detection and quantification were performed using the MAS5 algorithm. Global normalization was performed so that the average signal intensity of all probe sets was equal to 100. Analysis was performed using data from probe sets that showed a signal intensity higher than 50 and a 2-fold or greater variation between samples at p<0.05.

[0132] cell transplantation Experimental animals were treated and handled in accordance with the Sumitomo Dainippon Pharma Animal Experiment Ethics Guidelines, the Kyoto University Animal Experiment Conduct Guidelines, and the Institute for Laboratory Animal Resources (ILAR) Guide for the Care and Use of Laboratory Animals. Male F344 / NJcl-rnu / rnu (nude) rats, Parkinson's disease model F344 NJcl-rnu / rnu rats (CLEA Japan), and Sprague-Dawley (SD) rats (SHIMIZU LABORATORY SUPPLIES) were used for short-term transplantation studies. 6-OHDA was injected into the medial forebrain bundle in the right hemisphere of SD rats at the following location: A, -4.0; L, -1.3; V, -7.0. A total of 19.2 mg of 6-OHDA (in 3 μL of saline containing 0.02% ascorbic acid) was injected per rat. SD rats were immunosuppressed daily with cyclosporine (10 mg / kg, intraperitoneally, LC Laboratories) starting 1 day before transplantation. Cell transplantation was performed using stereotactic injection of cell mass (A, +1.0; L, -3.0; V, -5.0 and -4.0; and TB, 0 (2 μl; 200,000 cells / μl)).

[0133] Nude rats modeling Parkinson's disease were used for long-term transplantation studies. Cell transplantation was performed by stereotactic injection of fresh cell mass (A, +1.0; L, -3.0; V, -5.0 and -4.0; and TB, 0) (2 μl; 200,000 cells / μl) or cryopreserved cell mass (A, +1.0; L, -3.5 and 2.5; V, -5.5 and -4.5; and TB, 0) (4 μl; 200,000 cells / μl) into the right hemisphere of the striatum. Experimental animals were anesthetized and perfused transcardially with PBS followed by 4% paraformaldehyde.

[0134] behavior analysis Methamphetamine-induced rotational activity was assessed using a video-monitored rotation bowl before implantation and every 4 weeks after implantation. Methamphetamine (Dainippon Sumitomo Pharma) was injected intraperitoneally at a dose of 2.5 mg / kg, and rotation was recorded for 90 min.

[0135] statistical analysis Statistical analysis was performed using a commercially available software package (GraphPad Prism 6; GraphPad Software). In vitro data were analyzed by one-way ANOVA and Tukey's post hoc analysis (Figures 2A, 2B, 4A, 4B, 5A, and 5B). Behavioral data were analyzed by two-way ANOVA with Tukey's multiple comparison test (Figure 8A). Differences with p<0.05 were considered statistically significant. Data are presented as mean ± SD, except for behavioral data (mean ± SEM).

[0136] [result] (1) Results of cryopreservation solution study (reference example) As described above, in order to establish cryopreservation conditions suitable for iPSC-derived dopamine-producing neural progenitor cell clusters, the clinically applicable cryopreservation solutions listed in Table 1 above were compared.

[0137] First, we screened cryopreservation conditions using unsorted CORIN-sorted neural progenitor cell clumps, as described in Doi et al., 2014. It has been reported that cell numbers decrease after thawing due to necrosis and subsequent delayed apoptosis and necrosis (Milosevic et al., 2005). Proliferative capacity begins to recover during this process, with cell numbers reaching their lowest point approximately 24–48 hours after thawing (Malpique et al., 2010; Mitchell et al., 2014; Baust et al., 2017). Furthermore, it has been reported that neurotoxic compounds or diseases associated with genetic abnormalities can cause neurite abnormalities (Radio et al., 2008; Reinhardt et al., 2013; Ryan et al., 2016). These phenomena suggest that neurite morphology is a good indicator of neuronal characteristics. Therefore, we evaluated the recovery rate of viable cells one day after thawing as the cell viability, and further investigated the area of ​​neurite outgrowth as a new functional evaluation. The results are shown in Figure 2A, Figure 2B, and Figure 2C.

[0138] Cell viability and neurite outgrowth significantly decreased after freezing in all cryopreservation media (Figure 2). All media containing 10% DMSO demonstrated significantly higher viability than STEM-CELL BANKER DMSO-free (SCB DMSO-free) (21 ± 7%) and CryoStor CS5 (CS5) (16 ± 6%) (Figure 2A). Bambanker hRM (BBK) and Synth-a-Freeze (SaF) tended to result in relatively greater neurite outgrowth than the other freezing conditions (Figures 2B and 2C). In the SCB DMSO-free, CS5, and CryoStor CS10 conditions, 8 ± 17%, 50 ± 43%, and 13 ± 25% of the cell aggregates failed to adhere to the plate, respectively. Because BBK was already registered in the Japanese Drug Master File, we used it in subsequent experiments.

[0139] (2) Effects of intermediate cooling rates and suppression of supercooling Next, unsorted cell aggregates in BBK were cryopreserved using six different protocols. The temperature profiles for each protocol are shown in Figure 3. To verify the effectiveness of ice nucleation and latent heat control at the freezing point, we also investigated a shock cooling method (Morris and Acton, 2013), which involves a temporary temperature drop. In addition to magnetic fields and electromagnetic waves, proton freezers have a unique cooling profile. Proton freezers cool samples from -4°C to -30°C at approximately 5°C / min without supercooling. This cooling rate is intermediate between conventional slow freezers and vitrification methods. Specifically, when cooling samples in a proton freezer, the actual measured values ​​are approximately 2–7°C / min.

[0140] The proton freezer and the 0.5°C / min and 1°C / min conditions without the shock cooling step resulted in relatively higher cell viability than the other conditions (Figure 4A). In particular, the proton freezer and 0.5°C / min conditions showed approximately 80% cell viability compared to unfrozen cells. Furthermore, the proton freezer showed significantly greater neurite outgrowth than the other freezing conditions, except for the 1°C / min condition with the shock cooling step, which was approximately 60% greater than unfrozen cells (Figure 4B). These results demonstrate that the proton freezer condition maintains cell function. Even when the pre-freezing soaking time in the cryoprotectant was extended to 1 hour, the proton freezer condition still produced similarly favorable cell viability and neurite outgrowth results as a 15-minute soaking time (Figures 5A and B). Although cell viability decreased under the 0.5°C / min condition, no significant differences in cell viability or neurite outgrowth were observed under the other conditions (Figures 5A and B). These results demonstrate that intermediate cooling rates and suppression of supercooling are effective for cryopreserving iPSC-derived dopamine-producing neural progenitor cell clusters. The cell aggregates used above were induced to differentiate in the same manner as in Figure 1, except that sorting with an anti-CORIN antibody was not performed. The results of measuring marker expression in the cell aggregates before freezing are shown in Figure 10 and Table 4. [Table 4]

[0141] (3) Cell number and function of cryopreserved iPSC-derived dopaminergic neural progenitor cell clusters Based on the results of the above screening, we investigated the post-thaw properties of iPSC-derived dopaminergic neural progenitor cell clusters cryopreserved using BBK and a proton-activated freezer. iPSC-derived dopaminergic neural progenitor cells were sorted using a CORIN antibody to enrich them to over 80%. They were then cultured in suspension under the conditions described above to form cell aggregates, which were then cryopreserved using BBK and a proton-activated freezer (see Figure 1). The BBK permeation time was 1 hour.

[0142] The cryopreserved cell clusters measured 200 μm–500 μm in size and contained 5,500–12,000 cells (cell counts were estimated to be 2,000–15,000 based on the size of the clusters, as the entire cluster was not counted). The resulting cell clusters exhibited a cell viability of 52 ± 8% and neurite outgrowth of 51 ± 19% (Figure 6A, B). To confirm their identity as dopamine-producing neural progenitor cells, protein markers and gene expression were examined 7 days after thawing. Immunocytochemistry revealed that the cryopreserved cell clusters expressed almost the same levels of the dopamine-producing neural progenitor cell marker (FOXA2), dopamine-producing neural markers (NURR1 and TH), and proliferative cell marker (KI67) as non-frozen cell clusters (Figure 6C–E). Because the cell sorting process highly enriched dopaminergic neural progenitor cells, cells expressing neural stem cell markers (SOX1 and PAX6) were extremely rare in both unfrozen and frozen cell clusters, indicating that the cryopreservation process did not result in abnormal proliferation. Furthermore, qPCR analysis revealed that the expression levels of dopaminergic neural progenitor markers (FOXA2, LMX1A, and EN1) and dopaminergic neural markers (NURR1, PITX3, and TH) were unchanged by cryopreservation (Figure 6G). The expression levels of pluripotency markers (POU5F1 and NANOG) were maintained at less than 1% of day 0 in both cell clusters (Figure 6F).

[0143] Furthermore, marker expression in cell clusters of the same lot as the cell clusters shown in FIG. 6C immediately before freezing is shown in FIG. 11, and the results of measuring marker expression levels in multiple lots are shown in Table 5. [Table 5] The expression rates of FOXA2 and LMX1A were also compared (Table 6). [Table 6]

[0144] To obtain an overview of the temporal changes in maturation after the cryopreservation / thawing process, we also performed a global PCA analysis using the microarray data. All 21,882 probe sets containing significant signals were used for the analysis. The contributions of PC1 and PC2 were 48.0% and 15.7%, respectively. The PC score plots clustered into three major groups: day 0 (iPSC), day 12, and day 28 or later (Figure 6H). The PCA plots for both non-frozen and cryopreserved samples shifted in the positive direction of the PC1 and PC2 axes, regardless of whether they were cryopreserved on day 28 or later. Only 206 of 23,349 probe sets (0.88%) showed a more than two-fold increase or decrease between non-frozen and cryopreserved samples from the same differentiation batch. On the other hand, 222 of 23,275 probe sets (0.95%) showed a more than two-fold increase or decrease between non-frozen samples from different batches (Figure 6I, J). This result strongly indicates that the cryopreservation process does not affect the cellular properties of dopamine-producing neural progenitor cells.

[0145] To confirm the functional maturation of iPSC-derived dopamine-producing neural progenitor cell clusters, we performed electrophysiological analysis and dopamine release assays. When cryopreserved cell clusters were dissociated and cultured on plates for further maturation, most cells were TH-positive by day 28 + 21. + / TUBB3 + Dopaminergic neurons were generated (Figure 6K). At this time point, continuous action potentials from mature dopaminergic neurons were detected using the current-clamp whole-cell patch clamp method (Figure 6L). Furthermore, dopamine secretion from non-frozen cell clusters on day 56 and cryopreserved cell clusters on day 28+28 was detected by LC / MS / MS. The amount of released dopamine was comparable to that of non-frozen cell clusters (Figure 6M). These results demonstrate that the cell number and function of cryopreserved iPSC-derived dopaminergic neural progenitor cell clusters are maintained.

[0146] (4) Engraftment of cryopreserved iPSC-derived dopamine-producing neural progenitor cell clusters and behavior of 6-OHDA-lesioned rats To confirm that cryopreserved iPSC-derived dopamine-producing neural progenitor cells survived after transplantation, unfrozen and cryopreserved cell masses were transplanted into the striatum of several types of rats, and after short-term observation, the graft survival rate and number of surviving cells were counted. The results are shown in Table 7. There was no difference in the number of rats with graft survival between the two groups. When cell masses cryopreserved using a proton freezer and BBK were transplanted, the total number of viable cells (HNA) was 1.2. + ) was 42±20% of that in unfrozen cells. Table 7. Results of a short-term validation experiment evaluating graft survival of cryopreserved cell masses. [Table 7]

[0147] As a result of short-term validation experiments using transplantation, it was found that in order to achieve cell viability similar to that of unfrozen cells in vivo, more than twice as many cryopreserved cells as unfrozen cells should be transplanted. Therefore, to evaluate the viability and pharmacological effects of cryopreserved cells, unfrozen cell masses (4 × 10 5 cells) and cryopreserved cell mass (8 × 10 5 The cells were transplanted into 6-OHDA-lesioned PD model rats, and methamphetamine-induced rotational activity was measured. 24 weeks after transplantation, abnormal rotation was reduced in both groups (Fig. 8A). Immunofluorescence staining at 24 weeks confirmed cell engraftment in all rats that showed behavioral recovery. The number of HNAs in the non-frozen cell mass-derived grafts was 55,996 ± 3,603. + Cells survived, and 36,486 ± 3,578 HNAs were detected in the cryopreserved cell mass-derived grafts. + The cells were viable (Fig. 8B, C). + There was no significant difference in cell viability between the two groups. + The cells survived and showed normal neurite outgrowth (Fig. 8D-G). +There was no difference in cell numbers (3603 ± 1576 cells / graft vs. 3578 ± 1490 cells / graft) (Figure 8H). + The percentage of TH cells was 1.1±0.4% and 0.5±0.2% of the injected cells, respectively. This result shows that the TH cells were approximately 50% lower than those in the case of non-frozen cell masses. + This means that nerves survived in the cryopreserved cell mass-derived grafts. Furthermore, the majority of surviving cells in both groups were FOXA2 + Dopaminergic neural progenitor cells accounted for 71±6% of the unfrozen cell clusters and 76±4% of the cryopreserved cell clusters (Figure 8I, J). On the other hand, KI67 cells, which may pose a risk of abnormal proliferation, were also involved. + Few proliferating cells were observed in either group (Fig. 8I, K). These results demonstrate that cryopreserved iPSC-derived dopaminergic neural progenitor cell clusters can engraft and improve the behavior of 6-OHDA-lesioned rats.

[0148] (5) Expression of dopaminergic neural progenitor cell markers To confirm that thawed cells retained the same potential to mature into dopaminergic neurons as non-frozen cells, we examined protein marker and gene expression over time up to 7 days after thawing. Immunocytochemistry revealed that cryopreserved cell clusters continued to express FOXA2 at almost the same levels as non-frozen cell clusters (Figure 7A). NURR1+ cells increased in non-frozen cells from days 28 to 35. Although the increase in NURR1+ cells was slightly delayed compared to non-frozen cells, it was observed by day 7 after thawing (Figure 7B). Furthermore, qPCR analysis revealed that TH expression levels increased with the culture period in non-frozen cells up to day 35. TH expression remained constant from days 0 to 3 after thawing, but increased by day 7 (Figure 7C). These results demonstrate that cryopreserved iPSC-derived dopaminergic neural progenitor cell clusters retain the potential to mature into dopaminergic neurons. In addition, the results of comparing non-frozen cells cultured for multiple lots up to day 35 with cells frozen on day 28 and cultured for 7 days after thawing are shown in Table 8 below. [Table 8]

[0149] (6) Comparison of thawing conditions (reference) We investigated the thawing conditions for cryopreserved cell aggregates frozen in a proton freezer using BBK as a cryopreservation medium. Tubes stored in a liquid nitrogen vapor layer were removed and thawed under three conditions: (i) room temperature, (ii) a 37°C water bath, and (iii) a programmable freezer at a thawing rate of approximately 3°C / min (actual measurement). The thawing times were (i) 17 minutes, (ii) 2 minutes, and (iii) approximately 25 minutes, respectively. The viability (viable cell recovery rate) and neurite outgrowth activity of the thawed cell aggregates were measured using the methods described above. As shown in Figure 9, thawing in a 37°C water bath provided the best results.

Claims

[Claim 1] A method for freezing a cell aggregate containing neural cells having a three-dimensional structure, comprising the following steps (1) and (2): (1) contacting a cell aggregate containing neural cells having a three-dimensional structure with a preservation solution at 0°C or higher and 30°C or lower before freezing to prepare a cell aggregate immersed in the preservation solution; and (2) A step of freezing the cell aggregates immersed in the preservation solution obtained in step (1) by cooling them from a temperature at least about 5°C higher than the freezing point of the preservation solution to a temperature at least about 5°C lower than the freezing point at an average rate of 2 to 7°C / min.

Images