WO2021100829A1 - 神経系細胞の凍結方法 - Google Patents

神経系細胞の凍結方法 Download PDF

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WO2021100829A1
WO2021100829A1 PCT/JP2020/043275 JP2020043275W WO2021100829A1 WO 2021100829 A1 WO2021100829 A1 WO 2021100829A1 JP 2020043275 W JP2020043275 W JP 2020043275W WO 2021100829 A1 WO2021100829 A1 WO 2021100829A1
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Prior art keywords
cells
cell
positive
dopamine
producing
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PCT/JP2020/043275
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English (en)
French (fr)
Japanese (ja)
Inventor
里恵 平松
隆 中川
吉田　賢司
高橋　淳
大輔 土井
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Kyoto University NUC
Sumitomo Pharma Co Ltd
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Sumitomo Dainippon Pharma Co Ltd
Kyoto University NUC
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Priority to AU2020387259A priority Critical patent/AU2020387259A1/en
Priority to EP20890883.0A priority patent/EP4063496A4/en
Priority to CN202080093441.4A priority patent/CN115175989A/zh
Priority to IL293137A priority patent/IL293137A/en
Priority to JP2021558457A priority patent/JP7491533B2/ja
Priority to KR1020227020620A priority patent/KR20220104764A/ko
Priority to US17/778,250 priority patent/US20230000071A1/en
Priority to MYPI2022002608A priority patent/MY209020A/en
Application filed by Sumitomo Dainippon Pharma Co Ltd, Kyoto University NUC filed Critical Sumitomo Dainippon Pharma Co Ltd
Priority to CA3162268A priority patent/CA3162268A1/en
Publication of WO2021100829A1 publication Critical patent/WO2021100829A1/ja
Anticipated expiration legal-status Critical
Priority to JP2024075610A priority patent/JP7787527B2/ja
Priority to JP2025205398A priority patent/JP2026026215A/ja
Ceased legal-status Critical Current

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Definitions

  • the present application relates to a method for freezing cell aggregates containing neural cells.
  • Dopamine-producing (DA) nerve transplantation is considered to be a promising therapeutic method for Parkinson's disease (PD) based on reports of clinical trials using fetal midbrain cells (Non-Patent Document 1).
  • PD Parkinson's disease
  • PSC pluripotent stem cells
  • ES cells or ESC embryonic stem cells
  • iPS cells or iPSC induced pluripotent stem cells
  • the group of the present inventors has already reported a method for producing dopamine-producing neurons, dopamine-producing neural progenitor cells, and the like from human induced pluripotent stem cells (Non-Patent Document 2).
  • Other groups have also reported the production of PSC-derived dopamine-producing nerves (Non-Patent Documents 3 and 4).
  • cryopreserved cells In pharmaceutical products containing cells as an active ingredient, achievement of cryopreservation of the final product is an indispensable element for spreading cell therapy (Patent Documents 1 to 4). Unlike cell biology studies, for clinical use, cryopreserved cells should be immediately transplanted after thawing without recovery culture. Therefore, it is important for frozen cells to maintain their engraftment ability, function / activity, and cell viability after thawing.
  • Non-Patent Document 5 It has been suggested that the presence of donor blood vessels and antigen-presenting cells as they are in solid tissue transplantation induces a stronger immune response than cell suspension transplantation.
  • VM ventral midbrain
  • FIG. 6 Shows behavioral recovery.
  • mechanical and enzymatic dissociation processes for obtaining cell suspensions can alter cell properties and cause cell damage. Therefore, in clinical use, it is desirable to administer the transplanted cells as a cell mass rather than as a cell suspension.
  • cell clusters are more difficult to cryopreserve than single cells.
  • Non-Patent Document 7 When a single cell suspension of cryopreserved PSC-derived dopamine-producing nerves is transplanted into the rat striatum, the proportion of viable TH + cells is reduced to about 60% as compared with non-frozen cells (Non-Patent Document 7). .. On the other hand, in most studies in which human or rat ventral midbrain (VM) tissue was cryopreserved, the survival rate of dopaminergic nerves in vivo was reduced to less than 20% compared to non-frozen tissue (in vivo). Non-Patent Documents 8 to 10). Therefore, there is a need to develop a freezing method capable of maintaining the survival of PSC-derived dopamine-producing neuronal cell clusters.
  • VM ventral midbrain
  • Non-Patent Documents 11 to 13 there are two cell cryopreservation methods (Non-Patent Documents 11 to 13).
  • the slow method is a method in which cells are frozen at about 1 ° C./min together with a low concentration of cryoprotectant (CPA) (10% dimethyl sulfoxide (DMSO), etc.) (Patent Document 5, Non-Patent Documents 14, 15).
  • CPA cryoprotectant
  • the vitrification method is a high-speed cooling method in which cells are immediately transferred into liquid nitrogen after adding a high-concentration cryoprotectant substance (Patent Document 6 and Non-Patent Document 16). Since the vitrification method requires strict time control, its application to clinical cell production is technically difficult (Non-Patent Document 17).
  • An object of the present application is to provide a method for freezing cell aggregates containing neural cells.
  • a method for freezing cell aggregates containing neural cells having a three-dimensional structure which comprises the following steps (1) and (2): (1) A step of bringing cell aggregates containing neural cells having a three-dimensional structure into contact with a preservation solution at 0 ° C. or higher and 30 ° C. or lower before freezing to prepare cell aggregates immersed in the preservation solution, and; (2) The cell aggregates immersed in the preservation solution obtained in step (1) are averaged from 2 to at least from a temperature about 5 ° C. higher than the freezing point of the preservation solution to a temperature about 5 ° C.
  • the freezing point of the storage liquid is -1 ° C to ⁇ 10 ° C.
  • the storage liquid is an aqueous liquid containing 7% to 12% of dimethyl sulfoxide and / or propylene glycol, and the step (2) is from 0 ⁇ 5 ° C. to ⁇ 30 ⁇ 5 ° C., with an average of 2 to 5 ° C.
  • step (3) A step of cooling the frozen cell aggregate obtained in step (2) to ⁇ 50 ° C. or lower.
  • step (3) A step of cooling the frozen cell aggregate obtained in step (2) to ⁇ 50 ° C. or lower.
  • the cell aggregate containing neural cells is a cell aggregate containing neural cells derived from pluripotent stem cells.
  • the cell aggregate containing nervous system cells contains cells in which at least one of FOXA2, TH and NURR1 is positive.
  • the cell aggregate containing the nervous system cells contains FOXA2-positive and LMX1A-positive cells.
  • [18] Cell coagulation containing neural cells having a three-dimensional structure, which comprises holding the frozen cell aggregate obtained by the method according to any one of [1] to [17] above at ⁇ 80 ° C. or lower. Long-term storage method for aggregates. [19] The method according to any one of [1] to [18] above, which comprises obtaining frozen cell aggregates which do not require culturing for recovery after thawing. [20] A composition for transplantation containing, as an active ingredient, cell aggregates frozen or stored for a long period of time by the method according to any one of the above [1] to [19].
  • Cell aggregates having a circle-equivalent diameter of 150 ⁇ m to 1000 ⁇ m and containing 500 to 150,000 cells, containing 60% or more of dopamine-producing neural progenitor cells and dopamine-producing neurons derived from pluripotent stem cells, and 7 It contains a cryopreservation solution having a freezing point of -1 ° C to -10 ° C containing% to 12% dimethylsulfoxide or propylene glycol, and has the following properties: (1) About 60% or more of the total number of cells is alive after thawing. (2) It has a neurite outgrowth activity of 50% or more as compared with that before freezing.
  • a frozen transplant composition showing that the change in the positive rate of FOXA2, LMX1A, NURR1 and TH in living cells after thawing is within ⁇ 10%.
  • the number of cells is 80,000 to 5,000,000 cells / mL, FOXA2-positive and LMX1A-positive cells are contained in an amount of 40% or more of the total cell number, and TH-positive and NURR1-positive cells are contained in an amount of 40% or less of the total cell number.
  • the number of cells is 80,000 to 5,000,000 cells / mL, and FOXA2-positive and LMX1A-positive cells are contained in an amount of 40% or more of the total number of cells.
  • a transplant composition containing dopamine-producing neural progenitor cells as an active ingredient which comprises freezing cell aggregates having a circle-equivalent diameter of 150 ⁇ m to 1000 ⁇ m, which contains 40% or less of the total number of TH-positive and NURR1-positive cells. Production method.
  • a method for treating a disease requiring regeneration of dopaminergic nerves which comprises the following steps: (1) A step of thawing the transplantation composition according to any one of the above [20] to [27] at 30 ° C. to 40 ° C., preferably 37 ° C.
  • the present application provides a method for cryopreserving cell aggregates containing neural cells.
  • Nervous system cells cryopreserved by the method of the present application show high cell viability and maintain functional properties.
  • Time-temperature curves for the sample (straight line), refrigeration chamber (dashed line) and program (dotted line).
  • Bambanker hRM was used as a sample.
  • the figure below is an enlargement of the temperature change due to the release of latent heat in the figure above.
  • Characteristics of cryopreserved cell clusters in vitro Live cell recovery rate after thawing. Characteristics of cryopreserved cell clusters in vitro. Neurite elongation of the cell mass. Characteristics of cryopreserved cell clusters in vitro. Immunostaining of cell clusters on day 35 and 7 days after thawing. The left figure shows the FOXA2 / DAPI, the center figure shows the NURR1 / TH, and the right figure shows the immunostaining image of SOX1 / KI67 / PAX6 / DAPI. The scale bar indicates 100 ⁇ m. Characteristics of cryopreserved cell clusters in vitro.
  • the expression level of undifferentiated cells was set to 1. Characteristics of cryopreserved cell clusters in vitro. Principal component analysis of microarray data. The time variation of gene expression in non-frozen cells (circular) and frozen cells (triangle) is shown. Characteristics of cryopreserved cell clusters in vitro. A scatter plot of microarray data of non-frozen cells and frozen cells of the same lot on day 35 (X-axis) or 7 days after thawing (Y-axis) is shown. Black circles indicate genes with a signal intensity of 50 or more in either sample, and white circles indicate genes with a signal intensity of 50 or less in both samples. Characteristics of cryopreserved cell clusters in vitro.
  • a scatter plot of non-frozen cell microarray data between different lots at day 35 is shown. Black circles indicate genes with a signal intensity of 50 or more in either sample, and white circles indicate genes with a signal intensity of 50 or less in both samples.
  • Characteristics of cryopreserved cell clusters in vitro Immunostaining image of TUBB3, TH and DAPI in iPSC-derived dopamine-producing neurons after thawing on day 28 + 21. The scale bar indicates 50 ⁇ m. Characteristics of cryopreserved cell clusters in vitro. Representative action potentials of iPSC-derived dopamine-producing neurons after thawing on days 28 + 21. Characteristics of cryopreserved cell clusters in vitro. Results of high-concentration potassium stimulation-induced dopamine release on day 56 or 28 + 28 days after thawing.
  • Time-dependent changes in dopamine-producing neural progenitor cell marker expression after thawing Percentage of FOXA2 + cells to total cells on days 28, 29, 31, 35 and 0, 1, 3, 7 after thawing.
  • Time-dependent changes in dopamine-producing neural progenitor cell marker expression after thawing Percentage of NURR1 + cells to total cells on days 28, 29, 31, 35 and 0, 1, 3, 7 after thawing.
  • Time-dependent changes in dopamine-producing neural progenitor cell marker expression after thawing Expression of TH gene in cell mass against GAPDH as measured by quantitative RT-PCR. The expression level of undifferentiated cells (day 0) was set to 1.
  • Marker expression of cell clusters derived from cells not sorted with anti-CORIN antibody Ratio of SOX1 + cells, PAX6 + cells, and KI67 + cells to total cells. Marker expression of the cell mass immediately before freezing (day 28) in the same lot as the cell mass shown in FIG. 6C. Immunostained images of LMX1A, FOXA2, DAPI (upper), NURR1, FOXA2, TH, DAPI (middle), SOX1, KI67, PAX6, DAPI (lower) in the cell mass. The scale bar indicates 100 ⁇ m.
  • the present application provides a method for freezing cell aggregates containing neural cells having a three-dimensional structure.
  • Neural cells include a nerve cell (Neuronal cell or Neuron) and a precursor cell of the nerve cell, that is, a neural progenitor cell or a neural precursor cell and the like.
  • Nervous system cells are derived from any site, such as the nervous system cells of the central nervous system, or the somatic nervous system cells of the motor nerve or sensory system, or the nervous system cells of the peripheral nervous system of the nervous system cells of the autonomic nerve. These include nerves (neurons), nerve ridge-derived cells, glial cells such as oligodendrocytes or astrocytes, and stem cells or precursor cells thereof. Nervous system cells include cells that express a nervous system cell marker.
  • Nervous system cell markers include, for example, NCAM, ⁇ III-Tubulin (TUJ1), tyrosine hydroxylase (TH), serotonin, nestin, MAP2, MAP2AB, NEUN, GABA, glutamate, CHAT, SOX1, BF1, EMX1, VGLUT1, PAX, NKX, GSH, Nestinphalin, GLUR1, CAMKII, CTIP2, TBR1, Reelin, TBR1, BRN2, OTX2, LMX1A, LMX1B, EN1, NURR1, PITX3, DAT, GIRK2 and TH. It can be confirmed that the cells are nervous system cells by the expression of one or more of the neural system cell markers.
  • examples of neural cells include cells expressing 1 or more, 2 or more, or 3 or more of the above nervous system cell markers.
  • Nervous system cells of the central nervous system can be classified according to the difference in the site where the nervous system cells are present. That is, nerve cells derived from the forebrain, the telencephalon, the diencephalon, the cerebrum, the hypothalamus, the midbrain, the retencephalon, the mesencephalon-posterior border region, the cerebellum, the retina, the pituitary gland, or the spinal cord, and their precursor cells. ..
  • Forebrain-derived neurons are neurons that exist in forebrain tissues (that is, the telencephalon, cerebrum, hippocampus or choroid, diencephalon, hypothalamus, etc.). Forebrain neurons can be identified by the expression of forebrain neurons markers.
  • the forebrain nerve cell marker include OTX1 (forebrain), BF1 (also referred to as FOXG1), and SIX3 (also a marker for the telencephalon or cerebrum).
  • examples of nervous system cells include cells expressing 1 or more, 2 or more, or 3 or more of the above-mentioned forearn nerve cell markers, telencephalon or cerebral markers.
  • cerebral-derived neurons examples include dorsal cells (eg, cerebral cortex cells, Kahar-retius cells, hippocampal neurons, etc.) or ventral cells (eg, basal ganglia cells, etc.).
  • ventral cerebral nerve cell marker examples include basal ganglia nerve cell markers (eg, GSH2, MASH1, NKX2.1, NOZ1).
  • dorsal cerebral nerve cell marker examples include cerebral cortical nerve cell markers (for example, PAX6, EMX1, TBR1).
  • neural cells include cells expressing one or more, two or more, or three or more of the above-mentioned cerebral nerve cell markers, basal ganglia nerve cell markers, or cerebral cortex nerve cell markers.
  • Examples of the nervous system cells derived from the midbrain include ventral midbrain-derived neurons, dopamine-producing neurons (also referred to as dopamine neurons or Dopaminergic neurons) or dopamine-producing neurons (dopamine neurons or Dopaminergic projectors). ), Etc. can be mentioned.
  • Examples of markers of nervous system cells derived from the midbrain include FOXA2, EN2, TUJ1 and the like.
  • Examples of FOXA2-positive and TUJ1-positive neural cells include dopamine-producing neural progenitor cells and dopamine-producing neurons.
  • dopamine-producing neurons can be identified using FOXA2-positive, NURR1-positive, and TH-positive as indicators.
  • dopamine-producing neural progenitor cells can be identified using FOXA2-positive and LMX1A-positive as indicators. More preferably, one or more of OTX2, LMX1A, LMX1B, CORIN, SHH, AADC, ⁇ III-Tubulin, EN1, NURR1, PITX3, DAT, GIRK2 and TH contain positive cells.
  • the cell aggregate containing dopamine-producing neural progenitor cells may include dopamine-producing neurons, dopaminergic neurons, and the like, unless otherwise specified.
  • examples of neural cells include cells expressing 1 or more, 2 or more, or 3 or more of markers of midbrain-derived nervous system cells, dopamine-producing neural progenitor cells, or dopamine-producing nerve cells. Be done.
  • dopamine-producing neural progenitor cells cells expressing FOXA2 and / or LMX1A (FOXA2-positive and / or LMX1A-positive), preferably in addition to FOXA2 and LMXA1, OTX2, LMX1B, CORIN, SH, Examples thereof include cells expressing 1 or more, 2 or more, or 3 or more selected from the group consisting of AADC and ⁇ III-Tubulin.
  • dopamine-producing neurons dopamine neurons
  • cells expressing TH and / or NURR1 TH-positive and / or NURR1-positive
  • FOXA2 AADC
  • DAT DAT in addition to TH and NURR1.
  • Examples of nerve cells derived from the midbrain-metencephalon boundary region include nerve cells existing in the cerebellum, cerebellar plate tissue, ventricular zone, hindbrain lip and the like.
  • Examples of the midbrain-hindbrain border region marker include EN2 (midbrain), GBX2 (hindbrain), and N-Cadherin (nerve progenitor cells in the midbrain-hindbrain border region).
  • Examples of the cerebellar nerve progenitor cell marker include GABAergic nerve progenitor cell markers KIRREL2, PTF1A or SOX2, and cerebellar granule cell progenitor cell markers ATOH1 or BARHL1.
  • the nervous system cells one or more, two or more, or three or more of the above-mentioned mesencephalic brain border region marker, cerebral nerve progenitor cell marker, GABAergic nerve progenitor cell marker or cerebral granule cell progenitor cell marker.
  • Examples include cells that express.
  • Examples of retinal-derived neural cells include photoreceptor cells, photoreceptor progenitor cells, retinal pigment epithelial cells, and corneal cells.
  • Neural cells can also be classified according to the difference in neurotransmitters produced (secreted). For example, dopamine-producing neurons, dopamine-producing neural progenitor cells, GABA neurons, GABA neural progenitor cells, cholinergic neurons, etc. Examples thereof include cholinergic neurons, serotonin neurons, serotonin neurons, glutamate neurons, glutamate neurons, noradrenaline neurons, noradrenaline neurons, adrenaline neurons, adrenaline neurons and the like.
  • Examples of the neural cells of the motor nerve and the sensory organ system include cholinergic neurons or progenitor cells thereof.
  • Examples of the nervous system cells of the autonomic nerve include cholinergic neurons, adrenergic neurons, and progenitor cells thereof.
  • Preferred examples of the nervous system cells in the present specification include dopamine-producing neurons (dopamine neurons) and dopamine-producing neural progenitor cells (dopamine neural progenitor cells).
  • Living-derived nervous system cells are cells isolated from mammals such as humans.
  • cells isolated from human brain tissue include Nature Neuroscience, 2,1137 (1999) or N. Engl. J. Med. .; 344: 710-9 (2001) exemplifies cells contained in fetal mesencephalic tissue.
  • Nervous system cells may also be cells obtained by inducing differentiation from pluripotent stem cells such as embryonic stem cells (ES cells) and iPS cells.
  • pluripotent stem cells such as embryonic stem cells (ES cells) and iPS cells.
  • Methods for inducing differentiation of nervous system cells from pluripotent stem cells include, for example, the methods described in Non-Patent Documents 3 and 4 and WO2015 / 034012 (dopamine-producing neural progenitor cells), WO2009 / 148170 (nervous system such as cerebral brain).
  • WO2013 / 065763 WO2016 / 013669 or WO2017 / 126551 (pituitary or hypothalamic nervous system cells), WO2016 / 039317 (cerebral nervous system cells), WO2015 / 076388 (terminal brain nervous system cells), Numasawa -Kuroiwa, Y et al., Stem Cell Reports, 2: 648-661 (2014) (neural precursor cells), Qiu, L et al., Stem Cells Transl Med. 6 (9): 1803-1814 (2017) (dopamine-producing neural precursors) Cells) are exemplified.
  • the nervous system cell may be a cell obtained by inducing differentiation from a multipotent stem cell such as a mesenchymal stem cell (MSC).
  • a multipotent stem cell such as a mesenchymal stem cell (MSC).
  • MSC mesenchymal stem cell
  • Examples of the method for inducing differentiation of neural cells from mesenchymal stem cells include the method described in J Chem Neuroanat. 96: 126-133 (2019).
  • the pluripotent stem cell means a stem cell having pluripotency capable of differentiating into almost all cells existing in a living body and also having proliferative ability.
  • Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, reproductive stem cells, tissue stem cells, somatic cells and the like.
  • the pluripotent stem cells are not particularly limited, but are, for example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem cells (GS cells), and embryonic germ cells.
  • Pluripotent stem cells may be ES cells, ntES cells, or iPS cells. From an ethical point of view, the pluripotent stem cell may be an iPS cell. Embryonic stem cells were established from embryos within 14 days of fertilization.
  • Embryonic stem cells were first established in 1981 and have been applied to the production of knockout mice since 1989. Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Embryonic stem cells can be produced by culturing the inner cell mass on feeder cells or in a medium containing LIF (leukemia inhibitory factor). Methods for producing embryonic stem cells are described in, for example, WO96 / 22362, WO02 / 10157, US5,843,780, US6,200,806, US6,280,718 and the like. Embryonic stem cells can be obtained from a given institution or can be purchased commercially. 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 KhES-1 strain) is available from RIKEN. The mouse embryonic stem cells, EB5 cell line and D3 cell line, are available from RIKEN and ATCC, respectively.
  • LIF
  • ntES cells Nuclear transplanted embryonic stem cells
  • ntES cells which are one of the embryonic stem cells, can be established from cloned embryos produced by transplanting somatic nuclei into eggs from which the nuclei have been removed.
  • EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF and bFGF (Cell, 70: 841-847, 1992).
  • the “artificial pluripotent stem cell” in the present specification is a cell in which pluripotency is induced by reprogramming a somatic cell by a known method or the like.
  • differentiated somatic cells such as fibroblasts or peripheral blood mononuclear cells are used in OCT3 / 4, SOX2, KLF4, MYC (c-MYC, N-MYC, L-MYC), GLIS1, NANOG, etc.
  • Examples thereof include cells in which pluripotency is induced by reprogramming by expression of any combination of a plurality of genes selected from a group of reprogramming genes including SALL4, LIN28, ESRRB and the like.
  • Preferred combinations of reprogramming factors include (1) OCT3 / 4, SOX2, KLF4, and MYC (c-MYC or L-MYC), (2) OCT3 / 4, SOX2, KLF4, LIN28 and L-MYC (Stem). Cells, 2013; 31: 458-466), (3) OCT3 / 4, SOX2, NANOG, LIN28 (Science 2007; 318: 1917-1920) and the like can be mentioned.
  • Induced pluripotent stem cells were established in mouse cells by Yamanaka et al. In 2006 (Cell, 2006, 126 (4), pp.663-676). Induced pluripotent stem cells were also established in human fibroblasts in 2007 and have pluripotency and self-renewal ability similar to embryonic stem cells (Cell, 2007, 131 (5), pp.861-872; Science. , 2007, 318 (5858), pp. 1917-1920; Nat. Biotechnol., 2008, 26 (1), pp. 101-106).
  • Induced pluripotent stem cells can be produced not only by a method of direct reprogramming by gene expression, but also by a method of inducing induced pluripotent stem cells from somatic cells by adding a compound or the like (Science, 2013, 341). , Pp. 651-654).
  • induced pluripotent stem cells for example, 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells established at Kyoto University.
  • 1231A3 cells and other human induced pluripotent stem cell cell lines are available from Kyoto University.
  • the induced pluripotent stem cells for example, Ff-I01 cells, Ff-I01s04 cells, QHJ-I01 and Ff-I14 cells established at Kyoto University are available from Kyoto University.
  • the somatic cells used in producing artificial pluripotent stem cells are not particularly limited, but are tissue-derived fibroblasts, blood cell lineage cells (for example, peripheral blood mononuclear cells (PBMC), T cells), and liver.
  • tissue-derived fibroblasts for example, peripheral blood mononuclear cells (PBMC), T cells
  • PBMC peripheral blood mononuclear cells
  • liver examples include cells, pancreatic cells, intestinal epithelial cells, smooth muscle cells and the like.
  • the means for expressing the genes is not particularly limited.
  • an infection method using a virus vector for example, a retrovirus vector, a lentivirus vector, a Sendai virus vector, an adenovirus vector, or an adeno-associated virus vector
  • a plasmid vector for example, a plasmid vector, or an episomal vector
  • RNA vector for example, calcium phosphate method, lipofection method, or electroporation method
  • examples include a method of directly injecting a protein (for example, a method using a needle, a lipofection method, or an electroporation method).
  • Induced pluripotent stem cells can be produced in the presence of feeder cells or in the absence of feeder cells (feeder-free).
  • the induced pluripotent stem cells can be produced in the presence of undifferentiated maintenance factors by a known method.
  • the medium used for producing induced pluripotent stem cells in the absence of feeder cells is not particularly limited, but is a known embryonic stem cell and / or a maintenance medium for induced pluripotent stem cells, or a feeder-free artificial pluripotent stem cell.
  • a medium for establishing pluripotent stem cells can be used.
  • Examples of the medium for establishing a feeder-free induced pluripotent stem cell include Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit medium. Feeder-free medium can be mentioned.
  • feeder-free somatic cells are genetically subjected to four factors, OCT3 / 4, SOX2, KLF4, and MYC (L-MYC or C-MYC), using a Sendai viral vector. By introducing it, artificial pluripotent stem cells can be produced.
  • the pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (eg, mouse or rat) or primate (eg, human or monkey) pluripotent stem cells. More preferably, it is a human or mouse pluripotent stem cell, and even more preferably, it is a human induced pluripotent stem cell (iPS cell) or a human embryonic stem cell (ES cell).
  • rodent eg, mouse or rat
  • primate eg, human or monkey
  • iPS cell human induced pluripotent stem cell
  • ES cell human embryonic stem cell
  • cell aggregate has a three-dimensional structure
  • cells are a three-dimensional cell population formed by adhering cells to each other, for example, by suspension culture or three-dimensional culture. It means that it forms an aggregate (Cell aggregate or sphere).
  • Cell aggregates of nervous system cells are also called neurospheres.
  • the shape of the cell aggregate is not particularly limited, and may be spherical or non-spherical.
  • the cell aggregate in the present specification is preferably a cell aggregate having a three-dimensional shape close to a spherical shape.
  • a three-dimensional shape that is close to a sphere is a shape that has a three-dimensional structure and, when projected onto a two-dimensional surface, shows, for example, a circular or elliptical shape.
  • the size of the cell aggregate containing the neural cells having a three-dimensional structure is not particularly limited, but is usually 150 ⁇ m to 1000 ⁇ m in equivalent circle diameter, for example, 200 ⁇ m to 800 ⁇ m, or 300 ⁇ m to 500 ⁇ m in one embodiment.
  • cell aggregates containing neural cells having a three-dimensional structure usually contain 500 to 150,000 cells, and in one embodiment, for example, 1,000 to 100,000 cells, 1,000 to 70,000 cells, or 3,000 to 30,000 cells.
  • the cell aggregate containing the nervous system cells may contain other cells together with the nervous system cells. Examples thereof include cell aggregates containing 60% or more, 70% or more, 80% or more, and more preferably 90% or more of nervous system cells.
  • the cell aggregate containing the nervous system cells may contain 60% or more, 70% or more, or 80% or more of dopamine-producing neural progenitor cells and / or dopamine-producing neurons. That is, the cell aggregate containing neural cells contains 60% or more, 70% or more, or 80% or more of neural cells expressing one or more markers selected from FOXA2, LMX1A, LMX1B, NURR1 and TH. You may be.
  • the cell aggregate containing nervous system cells contains 40% or more, 60% or more, 70% or more, 80% or more, 85% or more or 90% or more of dopamine-producing neural progenitor cells.
  • the cell aggregate containing neural cells contains 40% or more, 60% or more, 70% or more, 80% or more of cells expressing 1 or more, 2 or more or 3 or more of the markers of dopamine-producing neural progenitor cells. , 85% or more or 90% or more.
  • the cell aggregate containing nervous system cells contains 40% or more, 60% or more, 70% or more, 80% or more, 85% or more or 90% or more of FOXA2-positive and LMX1A-positive cells. In one aspect, the cell aggregate further comprises 40% or less of TH-positive and NURR1-positive cells.
  • the cell aggregate containing nervous system cells may contain 0% or more, 10% or more, or 20% or more of FOXA2-positive, TH-positive, and NURR1-positive cells.
  • cell aggregates containing dopamine-producing neural progenitor cells contain 60% or less, 50% or less, 40% or less, 5-50%, 5-40% or 5-20% of NURR1-positive cells. May be good.
  • cell aggregates containing dopaminergic neural progenitor cells and / or dopaminergic neurons are 30% or less, 20% or less, 1-30%, 5-30%, 1-20% TH-positive cells. It may contain 5 to 20%, or 5 to 15%.
  • cell aggregates containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons contain 30% or less, 1-25%, 1-20%, or 5-20% of KI67-positive cells. May be good.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons may contain SOX1-positive cells in an amount of 20% or less, 10% or less, 5% or less, or 1% or less.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons may contain PAX6 positive cells in an amount of 5% or less, 2% or less, 1% or less, or 0.5% or less. Good.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons further comprises 20% or less of TH-positive and NURR1-positive cells, specifically 1% to 20%, more specifically. Contains 5% to 15%.
  • the 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.
  • TH-positive and NURR1-positive cells are contained in an amount of 20% or less, 1% to 20%, and more specifically, 5% to 15%.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons further contains 10% or less, preferably 7% or less, more preferably 3% or less of SOX1-positive cells, and PAX6-positive cells. Is 5% or less, preferably 4% or less, and more preferably 2% or less.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons contains 60% or more of FOXA2-positive and LMX1A-positive cells, and 1% to 20% of TH-positive and NURR1-positive cells.
  • SOX1-positive cells are 10% or less, preferably 7% or less, more preferably 3% or less
  • PAX6-positive cells are 5% or less, preferably 4% or less, still more preferably 2% or less.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons contains 60% or more of the total number of FOXA2-positive and LMX1A-positive cells, and TH-positive and NURR1-positive cells are all cells. It may contain 20% or less of the number, 1 to 20%, or 5 to 15%.
  • the above-mentioned cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons is a cell aggregate having a circle-equivalent diameter of 150 ⁇ m to 1000 ⁇ m.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons contains 60% or more of FOXA2-positive and LMX1A-positive cells, and 1% to 20% of NURR1-positive and TH-positive cells. Moreover, it is a cell aggregate having a circle-equivalent diameter of 150 ⁇ m to 1000 ⁇ m.
  • the cryopreservation liquid means an aqueous liquid containing a cryoprotectant.
  • the cryoprotectant has a high affinity with water molecules and means a substance having a high effect of suppressing the growth of ice crystals in a cryopreservation solution.
  • DMSO dimethylsulfoxide
  • EG ethylene glycol
  • PG propylene glycol
  • 1,2-propanediol (1,2-PD) 1,3-propanediol (1,3-PD)
  • BG butylene glycol
  • IPG isopylene glycol
  • DPG dipropylene glycol
  • Or glycerin and the like are included.
  • the cryoprotectant is preferably dimethyl sulfoxide and / or propylene glycol.
  • the concentration of the cryoprotectant in the cryopreservation solution is usually 7-12%, preferably about 10% when dimethyl sulfoxide and / or propylene glycol is used as the cryoprotectant.
  • aqueous liquid examples include physiological saline, buffer solutions such as PBS, EBSS, and HBSS, culture solutions for culturing cells and tissues such as DMEM, GMEM, and RPMI, serum, serum substitutes, or mixtures thereof. Can be used.
  • cryopreservation solution a commercially available cryopreservation solution containing dimethyl sulfoxide (DMSO) and / or propylene glycol as a substantial component can be used.
  • DMSO dimethyl sulfoxide
  • SCB ZENOAQ
  • SCB DMSO free SCB DMSO free; ZENOAQ
  • Bambanker hRM Bambanker hRM
  • CryoStor CS5 CS5; BioLife Solutions
  • CryoStor CS10 CryoStor CS10; BioLife Solutions
  • commercially available cryopreservation solutions such as Synth-a- Freeze (SaF; Thermo Fisher Scientific).
  • cryopreservation solutions containing 7-12%, preferably about 10% dimethyl sulfoxide and / or propylene glycol (eg, STEM-CELL BANKER, Bambanker hRM, CryoStor CS10 and Synth-a-Freeze) are used. Is desirable. More preferably, Bambanker hRM can be used.
  • the number of cells (cell packing density) with respect to the cryopreservation solution is 80,000 to 5,000,000 cells / mL, 100,000 to 4000,000 cells / mL, or 200,000 to 2000,000 cells / mL, 300,000 to 1,000,000 cells / mL. is there.
  • the equivalent circle diameter of the cell aggregate is 150 to 1000 ⁇ m, 150 ⁇ m to 600 ⁇ m, or 300 ⁇ m to 500 ⁇ m.
  • 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.
  • the cell aggregate 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.
  • 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 the cryopreservation liquid in the present specification include an aqueous liquid containing 7 to 12%, preferably about 10% dimethyl sulfoxide and having a freezing point of -1 ° C to -10 ° C as a substantial component.
  • cryopreservation liquid in the present specification an aqueous liquid containing 7 to 12%, preferably about 10% of dimethyl sulfoxide and having a freezing point of -3 ° C to -6 ° C can be mentioned as a substantial component.
  • the temperature at which cell aggregates containing nervous system cells are brought into contact with a cryopreservation solution is usually 0 ° C. or higher and 30 ° C. or lower, preferably 0 ° C. or higher and 20 ° C. or lower, more preferably 0 ° C. or higher and 10 ° C. or lower, and even more preferably. Is 0 ° C or higher and 4 ° C or lower.
  • the time for contacting the cell aggregate containing the nervous system cells with the cryopreservation solution is usually 5 minutes to 240 minutes, 5 minutes to 120 minutes, preferably 5 minutes to 60 minutes, 15 minutes to 240 minutes, and 15 minutes to. It is 180 minutes, 15 minutes to 150 minutes, preferably 15 minutes to 120 minutes, 15 minutes to 90 minutes, and more preferably 15 minutes to 60 minutes.
  • the method of the present application also causes the cell aggregates immersed in the preservation solution obtained in step (2) step (1) to be at least about 5 ° C. higher than the freezing point of the preservation solution and about 5 ° C. lower than the freezing point. It includes a step of cooling and freezing to a temperature at an average temperature decrease rate of 2 to 7 ° C., 2.5 to 7 ° C., or 3 to 7 ° C./min.
  • the cell aggregates immersed in the preservation solution are averaged at a temperature of about 5 ° C. higher than the freezing point of the preservation solution to a temperature of about 5 ° C. lower than the freezing point, averaging 2 to 7 ° C./min. It is cooled at a temperature decrease rate of 5 to 7 ° C./min, or an average of 3 to 7 ° C./min, preferably an average of 2 to 5.5 ° C./min, 2.5 to 5.5 ° C./min, or 3 to 5.5 ° C./min.
  • the cell aggregates soaked in the preservation solution are from 0 ⁇ 5 ° C to ⁇ 30 ° C. ⁇ 5 ° C, averaging 2-5 ° C / min, 2.5-5 ° C / min, or 3-5. It is cooled at a temperature decrease rate of ° C / min.
  • the cooling means is not particularly limited as long as the above steps are achieved, and a commercially available freezer can be used, and a program freezer (also referred to as a controlled rate freezer) capable of controlling the temperature may be used.
  • a program freezer also referred to as a controlled rate freezer
  • the cell aggregates soaked 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 is preferably a fixed frequency. Further, for example, it can be frozen under an electrostatic field of 10 to 2000 gauss, preferably 50 to 1000 gauss, and more preferably 100 to 150 gauss.
  • the method for achieving the above conditions is not particularly limited, and for example, a program freezer equipped with an electromagnetic field and a device for generating a magnetic field, which is sold as a proton freezer (Ryoho Freeze Systems Co., Ltd.), can be used. That is, a device having a magnetic field (Static Magnetic Field (SMF)), an electromagnetic wave (Alternating electrostatic field (AEF)), and an ultra-cooling air flow (Ultra-cold air) can be used. Specifically, by using an electromagnetic wave of 300 kHz to 2 MHz, damage to cell aggregates due to ice formation can be suppressed.
  • SMF Static Magnetic Field
  • AEF Alternating electrostatic field
  • Ultra-cold air ultra-cooling air flow
  • the method of the present application may further include a step of cooling the frozen cell aggregate obtained in step (3) step (2) to ⁇ 50 ° C. or lower, preferably ⁇ 80 ° C. or lower, more preferably ⁇ 150 ° C. or lower. ..
  • the cooling means is not particularly limited, and examples thereof include use in a deep freezer, a program freezer, a proton freezer, and a low-temperature medium (for example, liquid nitrogen).
  • the frozen cell aggregate obtained in step (2) or step (3) may be held at ⁇ 80 ° C. or lower, preferably ⁇ 150 ° C. or lower, and stored for a long period of time.
  • Means for long-term storage include, for example, deep freezer, program freezer, proton freezer, and low temperature medium (for example, liquid nitrogen).
  • Frozen cell aggregates can be appropriately thawed and used.
  • the thawing method is not particularly limited, but from the viewpoint of function / activity and cell viability, it is desirable to thaw at a temperature of about body temperature in a short time. Specifically, it is desirable to thaw at 30 ° C. to 40 ° C., preferably 35 ° C. to 38 ° C., more preferably a temperature near human body temperature, for example, about 37 ° C.
  • 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 after thawing, or may be transplanted into a living body without performing recovery culture.
  • the cell aggregates frozen by the method of the present invention can maintain the same properties as the cell aggregates that have not been frozen.
  • the cell aggregates frozen by the method of the present invention have the same marker expression rate as the cell aggregates before freezing when they are subjected to recovery culture for 7 days after thawing.
  • the markers may include FOXA2, LMX1A, NURR1 or TH.
  • the same marker expression rate means that the difference in the ratio of marker-expressing cells to the total number of cells is about 10% or less between before freezing and after thawing or culturing for 7 days after thawing. means.
  • the cell aggregates frozen by the method of the present invention are useful in that they can be transplanted into a living body without performing recovery culture.
  • composition containing a cell aggregate frozen or stored for a long period of time by the above method as an active ingredient, that is, a composition (formulation) for transplantation.
  • composition for transplantation is a concept including both a pharmaceutical composition frozen by the method of the present invention and a pharmaceutical composition obtained by thawing the pharmaceutical composition. That is, as the pharmaceutical composition (composition for transplantation) of the present invention, a frozen or non-frozen composition containing cell aggregates containing nervous system cells and a cryopreservation solution, and a cryopreservation solution after thawing are used as a medium for administration. Examples thereof include cell aggregates containing neural cells replaced with and a composition containing a medium for administration.
  • Examples of the pharmaceutical composition (transplantation composition) of the present invention include the transplantation compositions described in the above [20] to [27].
  • the present application contains 500 to 150,000 cells having a circle equivalent diameter of 150 ⁇ m to 1000 ⁇ m, which contains 60% or more of dopamine-producing neural progenitor cells and dopamine-producing neurons derived from pluripotent stem cells. It contains cell aggregates and a cryopreservation solution containing 7% to 12% dimethylsulfoxide or propylene glycol at a freezing point of -1 ° C to -10 ° C, preferably Bambanker hRM, and has the following properties: (1) About 60% or more of the total number of cells is alive after thawing. (2) It has a neurite outgrowth activity of 50% or more as compared with that before freezing.
  • the present invention comprises FOXA2-positive and LMX1A-positive cells having a total cell count of 80,000 to 5,000,000 cells / mL, 100,000 to 4,000,000 cells / mL or 200,000 to 2000,000 cells / mL, 300,000 to 1,000,000 cells / mL.
  • Compositions for transplantation comprising 5% to 15% are included.
  • the equivalent circle diameter of the cell aggregate is 150 to 1000 ⁇ m, 150 ⁇ m to 600 ⁇ m, or 300 ⁇ m to 500 ⁇ m.
  • 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.
  • the cell aggregate and the preservation solution may be filled in a container of 0.5 ml to 15 ml, 0.5 ml to 5 ml, or 1 ml to 2 ml.
  • the present invention includes a composition for transplantation, which is characterized in that it does not require culturing for recovery after thawing.
  • the cell aggregate contains 8 to 192 cells / ml, the average particle size of the cell aggregate is 150 ⁇ m to 1000 ⁇ m, and the number of cells per container is 80,000 to 2400000.
  • the transplant composition according to any one of [24] is included.
  • the cell aggregate is useful as a pharmaceutical composition for transplantation for a patient suffering from a disease requiring transplantation of nervous system cells, and treats a disease associated with degeneration, damage or dysfunction of nervous system cells. It can be used as a medicine such as a medicine. That is, a pharmaceutical composition containing the cell aggregate of the present invention and a pharmaceutically acceptable carrier is also within the scope of the present invention.
  • Diseases that require transplantation of neurological cells, or diseases associated with neurological cell damage or dysfunction include, for example, spinal cord injury, motor neurological disease, multiple sclerosis, muscle atrophic lateral atrophic sclerosis, atrophic side. Sclerosis, Huntington butoh disease, multilineage atrophy, spinocerebellar degeneration, Alzheimer's disease, retinal pigment degeneration, age-related yellow spot degeneration, Parkinson's syndrome (including Parkinson's disease).
  • One aspect of the present invention is a pharmaceutical composition for treating Parkinson's disease, which comprises the dopamine-producing neural progenitor cells and / or cell aggregates containing dopamine-producing neurons of the present invention as an active ingredient.
  • the number of dopamine-producing neuroprogenitor cells and / or dopamine-producing neurons contained in the Parkinson's disease therapeutic agent is not particularly limited as long as the graft can be engrafted after administration, and is, for example, 1.0 ⁇ 10 per transplant. 4 or more can be included. In addition, it may be adjusted by increasing or decreasing as appropriate according to the symptom and the size of the body. Transplantation of dopamine-producing neural progenitor cells into diseased sites is described, for example, in Nature Neuroscience, 2,1137 (1999) or N Engl J Med. It can be done by the method described in 344: 710-9 (2001).
  • the pharmaceutical composition of the present invention (also referred to as a composition for transplantation) contains 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 a frozen solid form and a liquid form before or after thawing.
  • the pharmaceutical composition may appropriately contain additives used for maintaining cell survival within a range that does not affect the freezing rate and freezing temperature. Examples of the cryopreservation solution include those described above.
  • the pharmaceutical composition or the composition for transplantation of the present invention is used for transplantation after thawing, removing the cryopreservation solution, replacing with a administration medium that can be administered to a living body. That is, a composition containing thawed cell aggregates and a medium for administration is also in the category of the pharmaceutical composition of the present invention (also referred to as a composition for transplantation).
  • the pharmaceutical composition (transplantation composition) according to the above-mentioned [20] to [27] can be produced by the freezing method according to any one of the above-mentioned [1] to [17]. That is, the present invention includes the method for producing the above-mentioned pharmaceutical composition (composition for transplantation).
  • ⁇ Method of treatment As one aspect of the present invention, there is a method for treating a disease requiring supplementation of nervous system cells, which comprises a step of transplanting the cell aggregate of the present invention into a patient suffering from a disease requiring transplantation of nervous system cells. Can be mentioned.
  • the cell aggregate containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons obtained in the present invention is administered to a Parkinson's disease patient as a pharmaceutical composition, specifically as a material for transplantation. Can be done.
  • a frozen pharmaceutical composition containing a cell aggregate containing dopamine-producing nerve progenitor cells and / or dopamine-producing nerve cells of the present invention and a cryopreservation solution is thawed, and appropriately transplanted with physiological saline or the like. It is done by suspending in a medium and implanting it in a patient's dopaminergic nerve-deficient area, such as the striatum.
  • the pharmaceutical composition may be thawed, washed with a medium containing a suitable carrier, and replaced with a transplant medium for suspending cell aggregates when the cryopreservation solution is transplanted into humans.
  • the thawing temperature is not particularly limited, but as described above, 30 ° C.
  • the cell aggregate contained in the pharmaceutical composition (composition for transplantation) of the present invention can be transplanted into a living body by substituting a cryopreservation solution with a medium for administration after thawing without culturing for recovery. is there.
  • the carrier used for the transplantation medium (administration medium) used for the dopamine-producing neural progenitor cells and / or the cell aggregate containing the dopamine-producing neurons may be a substance used for maintaining the survival of the cells.
  • a physiological aqueous solvent physiological saline solution, buffer solution, serum-free medium, etc.
  • a pharmaceutical composition containing a tissue or cell to be transplanted may be blended with a preservative, a stabilizer, a reducing agent, an isotonic agent, etc., which are usually used.
  • the thawed cell aggregates may be stored in a medium necessary for maintaining the viability of each cell aggregate.
  • the "medium necessary for maintaining viability" include a medium, a physiological buffer solution, etc., but are particularly limited as long as a cell population containing dopamine-producing neural progenitor cells and / or dopamine-producing neurons survives.
  • a person skilled in the art can appropriately select it.
  • a medium prepared by using a medium usually used for culturing animal cells as a basal medium can be mentioned.
  • basal medium for example, BME medium, BGJb medium, CMRL 1066 medium, GMEM medium, Improved MEM Zinc Option medium, Neurobasal medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, F-12 medium. , DMEM / F12 medium, IMDM / F12 medium, ham medium, RPMI 1640 medium, Fisher's medium, or a mixed medium thereof, and the like, which can be used for culturing animal cells.
  • the transplanted dopamine-producing neural progenitor cells and / or dopamine-producing neurons were administered to the treated patients. Engraft functionally.
  • transplantation means that the transplanted cells survive for a long period of time (eg, 30 days or more, 60 days or more, 90 days or more) and adhere to the organs. Means.
  • the "functional engraftment rate" in the present specification means the proportion of transplanted cells that have achieved functional engraftment.
  • the functional engraftment rate of transplanted dopamine-producing neural progenitor cells can be determined, for example, by measuring the number of TH-positive cells in the graft.
  • the functional engraftment rate of the transplanted cells and the dopamine-producing neural progenitor cells and / or dopamine-producing neurons induced after transplantation is 0.1% or more, preferably 0.2. % Or more, more preferably 0.4% or more, still more preferably 0.5% or more, still more preferably 0.6% or more.
  • One aspect of the present invention includes a method for treating a disease requiring regeneration of dopamine-producing nerves, which comprises the following steps.
  • One aspect of the present invention includes a therapeutic method characterized in that, after thawing, the cryopreservation solution is replaced with an administration medium and the step (2) is performed without culturing.
  • mammals to be transplanted in the present specification include humans, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys and the like, and are preferable.
  • Rodents eg, mice, rats
  • primates eg, humans, monkeys
  • Example 1 The outline of Example 1 is shown in FIG.
  • the differentiation medium is a medium containing 8% KSR, 0.1 mM MEM non-essential amino acids (all Invitrogen), sodium pyruvate (Sigma-Aldrich) and GMEM supplemented with 0.1 mM 2-mercaptoethanol.
  • the differentiation medium was changed daily from the day after sowing to the 12th day. 10 ⁇ M Y-27632 (Wako) was added on day 1 to increase cell viability after seeding. LDN193189 (STEMGENT) and A83-01 (Wako) were added to efficiently induce neural differentiation.
  • CORIN bottom plate marker in developing brain
  • dopamine-producing neural progenitor cells were concentrated, and sorting (FACS method) was performed.
  • FACS method sorting
  • cultured cells were stained with PE-labeled anti-CORIN antibody (100 ng / mL; Catalent / BD) for 20 minutes. Dead cells and debris were eliminated by 7-AAD staining. Analysis was performed using the FACSAria II or III cell sorter and the FACSDiva software program (BD Biosciences).
  • the sorted cells were placed on cell low-adhesion U-bottom 96-well plates (Sumitomo Bakelite) with B27 supplements, 2 ⁇ M Glutamax-I (all Invitrogen), 10 ng ml- 1 GDNF, 200 mM. Reseeded at a density of 2-3 ⁇ 10 4 cells / well in neurodifferential medium containing neurobasal medium supplemented with ascorbic acid, 20 ng ml- 1 BDNF (all Wako) and 400 ⁇ M dbcAMP (Sigma-Aldrich), floating cells It was cultured as a mass until the 28th day.
  • Unsorted cells were also reseeded at a density of 1.5 ⁇ 10 4 cells / well. Half of the medium was changed every 3 days and 30 ⁇ M Y-27632 (Wako) was added only to the first medium. For long-term culture, the floating cell mass was cultured in a neural differentiation medium (see FIG. 1).
  • Cryopreservation method The cell mass collected on the 28th day was cryopreserved and used for subsequent experiments. That is, they were placed in cryovials containing 1 mL of ice-cold cryopreservation solution listed in Table 1 and held on ice until frozen. For cryopreservation, the vials were transferred into a freezing container: BICELL (NIHON FREEZER), programmed freezer: PDF-150, 250 (STREX), cryomed (ThermoFisher Scientific) or proton freezer (RYOHO FREEZER SYSTEMS). In this experiment, 6 types of cooling profiles (shown in FIG. 3) were used.
  • BICELL was transferred to a deep freezer (-80 ° C) and held for 4 hours or more (upper left in FIG. 3).
  • the vial is frozen at a rate of 0.5 ° C / min (top center of Figure 3) or 1 ° C / min (top right of Figure 3) to -40 ° C, then faster 3-5 ° C / min. It was cooled to ⁇ 80 ° C. in minutes.
  • the shock cooling method a step of freezing to ⁇ 35 ° C. at a rate of ⁇ 25 ° C./min and then heating to ⁇ 12 ° C. at a rate of + 10 ° C./min was inserted at a temperature of ⁇ 4 ° C. during freezing.
  • FIG. 3 Vials frozen in a proton freezer were held in the chamber for 30-60 minutes (bottom right of FIG. 3).
  • the proton freezer has a static magnetic field, electromagnetic waves, and cold air. It is believed that static and electromagnetic fields affect the orientation of water molecules, causing the formation of small ice crystals and thus preventing cell destruction. However, these mechanisms are not yet fully understood.
  • cryovials were stored in the gas phase of a liquid nitrogen tank. Frozen cells were thawed at 37 ° C. for about 2 minutes and transferred to 15 mL tubes containing 10 mL neurobasal medium. After removing the supernatant, cells were rinsed with PBS and used for each assay or transplant.
  • the floating cell mass picked up for the neurite outgrowth assay was cultured on a 24-well plate coated with iMatrix 511 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 without cell bodies was measured using Photoshop (Adobe systems) and WinRoof (Mitani Corporation).
  • This patch pipette 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). Recording by the voltage-fixed method and the current-fixed method was performed using a patch clamp amplifier (EPC-8; HEKA). The resistance at the time of giga seal was in the range of 10 to 20 GW.
  • the current signal from the patch clamp amplifier is passed through a 4-pole low-pass filter (UF-BL2; NF) with Bessel characteristics, filtered at 5 kHz, sampled using a 12-bit analog-to-digital converter, and sampled using a 32-bit computer (PC-). Saved in 9821Ra333; NEC). All experiments were performed at room temperature.
  • the floating cell mass picked up for the dopamine release assay was cultured on an O / F / L coated 12-well plate for an additional 28 days and washed twice with a low concentration KCl solution (4.7 mM). And incubated in low concentration KCl solution for 15 minutes. The medium was then replaced with a high concentration KCl solution (60 mM) for 15 minutes. The solution was recovered and the dopamine concentration was determined by LC / MS / MS. The cells remaining on the plate were collected in PBS and sonicated. The DNA concentration of the cell lysate was measured using the Quant-iT TM dsDNA Assay Kit (Thermo Fisher) and used to correct the dopamine concentration.
  • Microarray analysis cDNA Microarray analysis was performed at the Bio-Medical Department of Kurabo Industries Ltd. Undifferentiated cells, cultured cells on day 12, iPSC-derived dopamine-producing neurons (day 28, 29, 31, 35) and after thawing on days 28 + 0,1,3,7 the iPSC-derived dopamine-producing nerve cells total RNA were treated with Genechip (TM) 3'IVT pico Reagent Kit and Human Genome U133 Plus 2.0 array (Affymetrix ). Data analysis was performed using Genechip operating software ver1.4 (Affymetrix). Signal detection and quantification was performed using the MAS5 algorithm. Global normalization was performed so that the average signal intensity of the entire probe set was equal to 100. Analysis was performed using probe set data showing signal intensities greater than 50 at p ⁇ 0.05 and more than 2-fold variation between samples.
  • 6-OHDA was injected into the medial forebrain bundle in the right hemisphere of SD rats in the following arrangement (A, -4.0; L, -1.3; V, -7.0). A total of 19.2 mg of 6-OHDA (in 3 ⁇ L saline containing 0.02% ascorbic acid) was injected per rat. From 1 day before transplantation, the immunity of SD rats was suppressed daily with cyclosporine (10 mg / kg, intraperitoneal, LC Laboratories). 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 ⁇ l; 200,000 cells / ⁇ l).
  • Parkinson's disease model nude rats were used for long-term transplantation studies.
  • Methamphetamine-induced rotational movement assessments were performed prior to transplantation and every 4 weeks post-transplantation using a video-monitored rotating bowl. Methamphetamine (Dainippon Sumitomo Pharma) at a dose of 2.5 mg / kg was injected intraperitoneally and rotations were recorded for 90 minutes.
  • the conditions of 0.5 ° C./min and 1 ° C./min without the proton freezer and shock cooling steps resulted in relatively higher cell viability than the other conditions (Fig. 4A).
  • the conditions of the proton freezer and 0.5 ° C./min showed a cell viability of about 80% as compared with the case of non-frozen cells.
  • the proton freezer showed significantly greater neurite outgrowth than other freezing conditions, except for the 1 ° C / min condition with a shock cooling step, which is about 60% compared to non-frozen cells. It was the size of (Fig. 4B).
  • the size of the cryopreserved cell mass was 200 ⁇ m to 500 ⁇ m, and the number of cells was 5500 to 12000. Estimated to be 2000 to 15000).
  • the resulting cell mass exhibited 52 ⁇ 8% cell viability and 51 ⁇ 19% neurite outgrowth (FIGS. 6A, 6B). Protein markers and gene expression were examined 7 days after thawing to confirm that they were dopamine-producing neural progenitor cells.
  • cryopreserved cell clusters have almost the same levels of dopamine-producing neural progenitor cell markers (FOXA2), dopamine-producing neural markers (NURR1 and TH), and proliferative cell markers (KI67) compared to non-frozen cell clusters. ) was expressed (FIGS. 6C to E). Due to the high enrichment of dopamine-producing neural progenitor cells by the process of cell sorting, very few cells expressed neural stem cell markers (SOX1 and PAX6) in both non-frozen and frozen cell clusters. This indicates that the cryopreservation process did not cause overgrowth.
  • FOXA2 dopamine-producing neural progenitor cell markers
  • NURR1 and TH dopamine-producing neural markers
  • KI67 proliferative cell markers
  • qPCR analysis revealed that the expression levels of dopamine-producing neural progenitor cell markers (FOXA2, LMX1A and EN1) and dopamine-producing neural markers (NURR1, PITX3 and TH) did not change with or without cryopreservation (Fig. 6G). ). Expression levels of pluripotency markers (POU5F1 and NANOG) were maintained below 1% on day 0 in both cell clusters (Fig. 6F).
  • Electrophysiological analysis and dopamine release measurements were performed to confirm the functional maturation of iPSC-derived dopamine-producing neural progenitor cell clusters.
  • cryopreserved cell clusters were dissociated and cultured on plates for further maturation, most cells developed TH + / TUBB3 + dopaminergic nerves on days 28 + 21 (Fig. 6K).
  • continuous action potentials from mature dopaminergic nerves were detected by the current-immobilized whole-cell patch clamp technique (FIG. 6L).
  • dopamine secretion in the non-frozen cell mass on day 56 and the cryopreserved cell mass on day 28 + 28 was detected by LC / MS / MS.
  • the amount of dopamine released was similar to that of non-frozen cell mass (Fig. 6M). From the above, it was found that the cell number and function of the cryopreserved iPSC-derived dopamine-producing neural progenitor cell mass were maintained.
  • non-frozen cell clusters (4 ⁇ 10 5 cells) and cryopreserved cell clusters (8 ⁇ 10 5 cells) were transplanted into 6-OHDA lesion PD disease model rats. , Methanphetamine-induced rotational movement was measured. Twenty-four weeks after transplantation, abnormal rotation decreased in both groups (Fig. 8A).
  • qPCR analysis revealed that the expression level of TH in non-frozen cells increased with the culture period until the 35th day. It was confirmed that the expression of TH remained constant in the cells 0 to 3 days after thawing, but the expression of TH increased on the 7th day (Fig. 7C). From the above, it was found that the cryopreserved iPSC-derived dopamine-producing neural progenitor cell mass retains the ability to mature into dopamine-producing neurons. Table 8 below shows the results of comparing the case where the non-frozen cells were cultured up to the 35th day and the case where the non-frozen cells were frozen on the 28th day and cultured for 7 days after thawing in a plurality of lots.

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