Method for producing frozen cells

Abstract

To provide a cell freezing technique which allows cellular functions (e.g., spheroid-forming ability, neurite-extending ability) to be preserved in an elevated state.SOLUTION: A method for producing frozen cells involves freezing cells under irradiation with an electromagnetic wave having an electric field intensity of 100 V / m or more.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing frozen cells and the like.

Background Art

[0002] In recent years, research and development of cell pharmaceuticals using iPS cells have been underway. For example, it has been reported that spheroids of dopaminergic neurons are used for the treatment of Parkinson's disease (Non-Patent Documents 1 and 2). For this technology, a physician-led clinical trial has already been started. However, currently, the cells used have not undergone a freezing process from differentiation induction to spheroid formation and neurite outgrowth. When using cells that have not undergone such a freezing process, it is difficult to store them for a long period after the completion of culture. Therefore, consistent schedule management from cell production to administration to patients is essential.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a cell freezing technique capable of maintaining higher cell functions (for example, spheroid formation ability and neurite outgrowth ability after freezing and thawing).

Means for Solving the Problems

[0005] As a result of intensive research in view of the above problems, the present inventors have found that the above problems can be solved by a method for producing frozen cells, which includes freezing cells under irradiation with radio waves having an electric field strength of 100 V / m or more. That is, the present invention includes the following aspects.

[0006] Item 1. A method for producing frozen cells, which includes freezing cells under irradiation with radio waves having an electric field strength of 100 V / m or more.

[0007] Item 2. The production method according to Item 1, wherein the cells are nervous system cells.

[0008] Item 3. The production method according to Item 2, wherein the nervous system cells are at least one selected from the group consisting of neural stem cells, neural progenitor cells, and nerve cells.

[0009] Item 4. The production method according to Item 2 or 3, wherein the nervous system cells are derived from stem cells.

[0010] Item 5. The production method according to Item 4, wherein the stem cells are induced pluripotent stem cells.

[0011] Item 6. The production method according to any one of Items 2 to 5, wherein the nervous system cells include single cells or cell aggregates.

[0012] Item 7. The production method according to any one of Items 1 to 6, wherein the electric field strength of the radio waves is 150 V / m or more and 300 V / m or less.

[0013] Item 8. The production method according to any one of Items 1 to 7, wherein the cells are frozen at -20°C or lower.

[0014] Item 9. The production method according to any one of Items 1 to 8, wherein the cells are frozen at -100°C or higher and -20°C or lower.

[0015] Item 10. The production method according to any one of Items 1 to 9, wherein the cells are frozen in the presence of a magnetic field.

[0016] Item 11. The manufacturing method according to Item 10, wherein the magnetic field strength of the magnetic field is 5 μT or more.

[0017] Item 12. The manufacturing method according to Item 10 or 11, wherein the magnetic field strength of the magnetic field is 5 μT or more and 50 μT or less.

[0018] Item 13. Frozen cells obtained by the manufacturing method according to any one of Items 1 to 12, or thawed cells of the frozen cells.

[0019] Item 14. A medicine containing thawed cells of frozen cells obtained by the manufacturing method according to any one of Items 1 to 12, or cells induced to differentiate from the thawed cells.

Advantages of the Invention

[0020] According to the present invention, it is possible to provide a cell freezing technique capable of maintaining cell functions (for example, spheroid formation ability, neurite outgrowth ability) in a higher state, cells obtained by the technique, a medicine containing the cells, and the like.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0022] In this specification, the expressions "containing" and "comprising" include the concepts of "containing", "comprising", "substantially consisting of", and "consisting only of".

[0023] In this specification, when upper and lower limits are respectively indicated, not only the ranges defined by the upper limit and the ranges defined by the lower limit, but also ranges formed by arbitrarily combining the upper limit and the lower limit can be adopted.

[0024] 1. Method for producing frozen cells In one aspect, the present invention relates to a method for producing frozen cells (which may also be referred to as "the production method of the present invention" in this specification), including freezing cells under irradiation with radio waves having an electric field strength of 100 V / m or more. This will be described below.

[0025] The cells to be frozen by the production method of the present invention are not particularly limited. According to the production method of the present invention, cells can be frozen while maintaining cell functions (for example, the spheroid formation ability and neurite outgrowth ability of cells after thawing) in a higher state. Examples of cells include blood cells, hematopoietic stem cells / hematopoietic progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratin-producing cells, muscle cells, epidermal cells, endocrine cells, pluripotent stem cells (ES cells, iPS cells, etc.), tissue stem cells, cancer cells, and the like.

[0026] The cells to be frozen by the production method of the present invention are particularly preferably nervous system cells. Since nervous system cells do not regenerate in the living body, they are particularly cells for which cell transplantation is expected. Therefore, there is a particular need for a method of cryopreserving cells prepared in vitro for transplantation while maintaining their functions. According to the production method of the present invention, cells can be frozen while maintaining the cell functions (for example, spheroid formation ability, neurite outgrowth ability) of nervous system cells in a higher state.

[0027] Nervous system cells include neuronal cells (Neuronal cell or Neuron), progenitor cells of such neuronal cells, namely neural progenitor cells (neural progenitor cell or neural precursor cell), neural stem cells, and the like.

[0028] Nervous system cells can be nervous system cells derived from any site, such as nervous system cells of the central nervous system, or somatic nervous system cells of motor nerves and sensory organs or nervous system cells of the peripheral nervous system of the nervous system cells of the autonomic nervous system, and include nerves (neurons), cells derived from the neural crest, glial cells such as oligodendrocytes or astrocytes, and their stem cells or progenitor cells, etc. Examples of nervous system cells include cells that express a nervous system cell marker. Examples of nervous system 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, and GIRK2, etc. The fact that a cell is a nervous system cell can be confirmed by the expression of one or more of the nervous system cell markers. In this specification, examples of nervous system cells include cells that express one or more, two or more, or three or more of the above-mentioned nervous system cell markers.

[0029] 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, it includes nerve cells and their progenitor cells derived from the forebrain, telencephalon, diencephalon, cerebrum, hypothalamus, midbrain, hindbrain, midbrain-hindbrain boundary region, cerebellum, retina, pituitary gland, or spinal cord.

[0030] Neurons derived from the forebrain are neurons present in forebrain tissues (i.e., telencephalon, cerebrum, hippocampus or choroid, diencephalon, hypothalamus, etc.). Neurons of the forebrain can be confirmed by the expression of forebrain neuron markers. Examples of forebrain neuron markers include OTX1 (forebrain), BF1 (also called FOXG1), or SIX3 (which is also a marker for the telencephalon or cerebrum). In this specification, examples of nervous system cells include cells that express one or more, two or more, or three or more of the above-mentioned forebrain neuron markers, telencephalon or cerebrum markers.

[0031] Examples of neurons derived from the cerebrum include dorsal cells (e.g., cerebral cortex cells, Cajal-Retzius cells, hippocampal neurons, etc.) or ventral cells (e.g., basal ganglia cells of the cerebrum, etc.). Examples of ventral cerebral neuron markers include, for example, basal ganglia neuron markers (e.g., GSH2, MASH1, NKX2.1, NOZ1). Examples of dorsal cerebral neuron markers include, for example, cerebral cortex neuron markers (e.g., PAX6, EMX1, TBR1). In this specification, examples of nervous system cells include cells that express one or more, two or more, or three or more of the above-mentioned cerebral neuron markers, basal ganglia neuron markers, or cerebral cortex neuron markers.

[0032] Examples of nervous system cells derived from the midbrain include neural progenitor cells derived from the ventral part of the midbrain, dopamine-producing neurons (also called dopaminergic neurons or Dopaminergic neuron), or dopamine-producing neural progenitor cells (also called dopaminergic progenitor cells or Dopaminergic progenitor). Examples of markers for nervous system cells derived from the midbrain include FOXA2, EN2, TUJ1, etc. Examples of FOXA2-positive and TUJ1-positive nervous system cells include dopamine-producing neural progenitor cells and dopamine-producing neurons. In addition, dopamine-producing neurons can be identified using as an indicator the fact that they are FOXA2-positive, NURR1-positive, and TH-positive.

[0033] In addition, dopamine-producing neural progenitor cells can be identified using the criteria of being positive for both FOXA2 and LMX1A. More preferably, they contain cells that are positive for one or more of OTX2, LMX1A, LMX1B, CORIN, SHH, AADC, βIII-Tubulin, EN1, NURR1, PITX3, DAT, GIRK2, and TH. In this specification, unless otherwise specified, dopamine-producing neural progenitor cells may include, for example, dopamine-producing neurons or dopaminergic neurons.

[0034] Examples of neurons derived from the midbrain-hindbrain boundary region include neurons present in the cerebellum, cerebellar plate tissue, ventricular zone, rhombic lip, etc. Examples of midbrain-hindbrain boundary region markers include EN2 (midbrain), GBX2 (hindbrain), and N-Cadherin (neural progenitor cells in the midbrain-hindbrain boundary region). Examples of cerebellar neural progenitor cell markers include KIRREL2, PTF1A, or SOX2, which are GABAergic neural progenitor cell markers, and ATOH1 or BARHL1, which are cerebellar granule cell progenitor cell markers. In this specification, examples of nervous system cells include cells that express one or more, two or more, or three or more of the above midbrain-hindbrain boundary region markers, cerebellar neural progenitor cell markers, GABAergic neural progenitor cell markers, or cerebellar granule cell progenitor cell markers.

[0035] Examples of neural system cells derived from the retina include photoreceptor cells, photoreceptor cell progenitors, etc.

[0036] In addition, nervous system cells can also be classified according to the differences in neurotransmitters they produce (secrete). For example, dopamine-producing neurons, dopamine-producing neural progenitor cells, GABA neurons, GABA neural progenitor cells, cholinergic neurons, cholinergic neural progenitor cells, serotonin neurons, serotonin neural progenitor cells, glutamic acid neurons, glutamic acid neural progenitor cells, noradrenergic neurons, noradrenergic neural progenitor cells, adrenergic neurons, adrenergic neural progenitor cells, etc.

[0037] Examples of nervous system cells of motor nerves and sensory organs include cholinergic neurons or their progenitor cells, etc.

[0038] Examples of nervous system cells of the autonomic nervous system include cholinergic neurons, adrenergic neurons, or progenitor cells thereof, etc.

[0039] Examples of the nervous system cells in this specification preferably include dopamine-producing neurons (dopaminergic neurons) and dopamine-producing neural progenitor cells (dopamine neural progenitor cells).

[0040] Biologically-derived nervous system cells 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).

[0041] The nervous system cells are also preferably cells obtained by inducing differentiation from stem cells, particularly preferably pluripotent stem cells such as embryonic stem cells (ES cells) and iPS cells, and the differentiation induction can be carried out by a method known to those skilled in the art as appropriate. Methods for inducing differentiation of nervous system cells from stem cells include, for example, the methods described in WO2015 / 034012 (dopamine-producing neural progenitor cells), WO2009 / 148170 (nervous system cells such as the cerebrum), WO2013 / 065763, WO2016 / 013669 or WO2017 / 126551 (nervous system cells of the pituitary gland or hypothalamus), WO2016 / 039317 (nervous system cells of the cerebellum), WO2015 / 076388 (nervous system cells of the telencephalon), Numasawa-Kuroiwa, Y et al., Stem Cell Reports, 2: 648-661 (2014) (neural progenitor cells), Qiu, L et al., Stem Cells Transl Med. 6(9): 1803-1814 (2017) (dopamine-producing neural progenitor cells), WO2013 / 067362 (dopamine-producing neural progenitor cells), Nolblant, S et al., Nat Protoc. 12(9):1962-1979 (2017) (dopamine-producing neural progenitor cells), Cederquist, Y et al., Nat Biotechnol. 37(4):436-44 (2019) (nervous system cells such as the cerebrum), Sundberg, M et al., Stem Cells. 31(8): 1548-1562 (2013) (dopamine-producing neural progenitor cells), Kuwahara, A et al., Sci Rep. 12;9(1):18936 (2019) (photoreceptor cells, photoreceptor cell progenitors), WO2018 / 074567 (peripheral nerve cells).

[0042] A pluripotent stem cell is a stem cell that has the pluripotency to differentiate into many cells existing in a living body and also has a proliferative ability, and includes any cell induced into an intermediate mesoderm cell used in the present invention. The pluripotent stem cells are not particularly limited, and examples thereof include, but are not limited to, embryonic stem (ES) cells (note that human embryonic stem cells are preferably established from a human embryo within 14 days after fertilization), embryonic stem (ntES) cells derived from a cloned embryo obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells, and the like. A preferred pluripotent stem cell is an iPS cell, and more preferably a human iPS cell, from the viewpoints that the effect of maintaining cell functions by the production method of the present invention is particularly remarkable and that it can be obtained without destroying embryos, eggs, etc. in the production process.

[0043] Methods for producing iPS cells are known in the art and can be produced by introducing reprogramming factors into any somatic cell. Here, reprogramming factors include, for example, genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, etc. These reprogramming factors can be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells.26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol.26:1269-1275, Shi Y, et al.(2008), Cell Stem Cell, 3, 568 - 574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475 - 479, Marson A, (2008), Cell Stem Cell, 3, 132 - 135, Feng B, et al. (2009), Nat. Cell Biol. 11:197 - 203, R.L. Judson et al., (2009), Nat. Biotechnol., 27:459 - 461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912 - 8917, Kim JB, et al. (2009), Nature. 461:649 - 643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491 - 503, Heng JC, et al. (2010), Cell Stem Cell. 6:167 - 74, Han J, et al. (2010), Nature. 463:1096 - 100, Mali P, et al. (2010), Stem Cells. 28:713 - 720, Maekawa M, et al. (2011), Nature. 474:225 - 9. The combinations described therein are exemplified.

[0044] Cells (particularly, nervous system cells) may be single cells or two or more cells that adhere or aggregate to form cell masses (including two - dimensional sheet - like structures). When single cells are included, after freezing and thawing, the cells of the single cells are suitable for being used for spheroid formation. When cell masses (particularly, spheroids) are included, after freezing and thawing, the cell masses are suitable for differentiating into a more highly differentiated state such as neurite outgrowth in vitro or in vivo (for example, in a living body after cell transplantation).

[0045] A single cell, as the name implies, is a cell that exists in a state where it is not adhered to other cells. As one aspect of this specification, the cells to be frozen include a cell population of single cells, and in this cell population, there may be a part of cell clusters in a state where a small number (for example, within 5, specifically 2, 3, 4, or 5) of cells are aggregated. A single cell can be obtained by treating the cells cultured in a culture vessel with a drug such as trypsin as needed and stirring by pipetting or the like.

[0046] The number of cells constituting the cell cluster is not particularly limited, but is usually 500 to 150,000, and as one aspect, for example, 1,000 to 100,000, 1,000 to 70,000, or 3,000 to 30,000.

[0047] A spheroid means a cell aggregate (Cell aggregate or sphere) which is a three-dimensional cell population formed by cultured cells adhering to each other. A spheroid includes a self-organized so-called organoid. A cell aggregate of nervous system cells is also called a neurosphere. The shape of the spheroid is not particularly limited and may be spherical or non-spherical. The spheroid in this specification preferably has a three-dimensional shape close to spherical. A three-dimensional shape close to spherical is a shape having a three-dimensional structure, and when projected onto a two-dimensional plane, it shows, for example, a circular or elliptical shape.

[0048] The size of the spheroid is not particularly limited, but is usually 150 μm to 1,000 μm in equivalent circle diameter, and as one aspect, for example, 200 μm to 800 μm, or 300 μm to 500 μm. Also, a spheroid usually contains 500 to 150,000, and as one aspect, for example, 1,000 to 100,000, 1,000 to 70,000, or 3,000 to 30,000 cells.

[0049] The production method of the present invention includes freezing cells under irradiation with an electric wave having an electric field strength of 100 V / m or more.

[0050] Typically, when freezing, it is performed by placing a container (cryopreservation vial) containing a cryopreservation solution containing cells under cell freezing conditions.

[0051] In the present application, the cryopreservation solution (preservation solution) means an aqueous liquid containing a cryoprotective substance. The cryoprotective substance means a substance having a high affinity for water molecules and having a high effect of suppressing the growth of ice crystals in the cryopreservation solution. For example, dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), 1,2-propanediol (1,2-PD), 1,3-propanediol (1,3-PD), butylene glycol (BG), isoprene glycol (IPG), dipropylene glycol (DPG) and / or glycerin are included. In the present application, the cryoprotective substance is preferably dimethyl sulfoxide and / or propylene glycol. When dimethyl sulfoxide and / or propylene glycol is used as the cryoprotective substance, the concentration of the cryoprotective substance in the cryopreservation solution is usually 7 to 12%, preferably about 10%.

[0052] As the aqueous liquid, for example, buffers such as physiological saline, PBS, EBSS, HBSS, culture solutions for culturing cells and tissues such as DMEM, GMEM, RPMI, serum, serum substitutes, or mixtures thereof can be used.

[0053] As the cryopreservation solution, commercially available cryopreservation solutions containing dimethyl sulfoxide (DMSO) and / or propylene glycol as substantial components can be used. Specifically, as the cryopreservation solution, there are commercially available cryopreservation solutions such as STEM-CELL BANKER (SCB; ZENOAQ), STEM-CELL BANKER DMSO free (SCB DMSO free; ZENOAQ), Bambanker hRM (BBK; NIPPON Genetics), CryoStor CS5 (CS5; BioLife Solutions), CryoStor CS10 (CS10; BioLife Solutions), and Synth-a-Freeze (SaF; Thermo Fisher Scientific). For example, it is desirable to use a cryopreservation solution containing 7 to 12%, preferably about 10% dimethyl sulfoxide and / or propylene glycol (such as STEM-CELL BANKER, Bambanker hRM, CryoStor CS10, and Synth-a-Freeze). More preferably, Bambanker hRM can be used.

[0054] In this specification, when freezing cells, the number of cells (cell filling density) relative to the cryopreservation solution is 80000 - 5000000 cells / mL, 100000 - 4000000 cells / mL, or 200000 - 2000000 cells / mL, 300000 - 1000000 cells / mL.

[0055] In this specification, the volumes of the cells and the preservation solution are 0.2 mL - 2 mL, 0.5 mL - 1.5 mL, or 0.5 mL - 1 mL.

[0056] In this specification, the cells and the preservation solution may be filled in a container of 0.5 mL - 15 mL, 1 mL - 5 mL, or 1 mL - 2 mL.

[0057] The material of the container is not particularly limited as long as it can completely shield radio waves, but preferably it has excellent radio wave permeability. Examples of the material of the container include plastics such as polypropylene.

[0058] 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.

[0059] As the cryopreservation solution in this specification, those known to those skilled in the art can be appropriately used, and commercially available cryopreservation solutions can also be appropriately used. Specifically, for example, an aqueous liquid containing 7 to 12%, preferably about 10% dimethyl sulfoxide as a substantial component and having a freezing point of -1°C to -10°C can be mentioned. Also, as the cryopreservation solution in this specification, an aqueous liquid containing 7 to 12%, preferably about 10% dimethyl sulfoxide as a substantial component and having a freezing point of -3°C to -6°C can be mentioned.

[0060] The cell freezing conditions in the production method of the present invention include that it is under the irradiation of radio waves with an electric field strength of 100 V / m or more (Condition 1).

[0061] The electric field strength is the electric field strength measured near the location where the vial for cryopreservation is placed. From the perspective of being able to maintain cell functions (e.g., spheroid formation ability, neurite outgrowth ability) in a higher state, the electric field strength is preferably 120 V / m or more, more preferably 150 V / m or more, still more preferably 170 V / m or more, even more preferably 180 V / m or more, particularly preferably 190 V / m or more, especially more preferably 195 V / m or more, and most preferably 200 V / m or more. The upper limit of the electric field strength is not particularly limited, but for example, it is 500 V / m, 400 V / m, 300 V / m, or 250 V / m. The electric field strength is, for example, 120 V / m or more and 500 V / m or less, preferably 150 V / m or more and 500 V / m or less, more preferably 170 V / m or more and 500 V / m or less, still more preferably 180 V / m or more and 500 V / m or less, even more preferably 190 V / m or more and 400 V / m or less, particularly preferably 195 V / m or more and 300 V / m or less, and most preferably 200 V / m or more and 250 V / m or less.

[0062] The method for measuring the electric field strength is as follows, for example. A plurality (3 to 5) of electromagnetic wave measurement sensors are arranged at the location where the vial is placed, and measurement is performed at room temperature (25°C) using a measuring instrument (Gauss meter, RIGOL DSA815 or its equivalent), and the average value of the measured values at a plurality of locations is calculated.

[0063] As the radio wave, for example, a radio wave in the region having a frequency of 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 is preferred.

[0064] The method of irradiating the radio waves is not particularly limited, and a method according to or analogous to a known method can be adopted. The radio waves are usually irradiated using a radio wave transmitting antenna. As the radio wave transmitting antenna, various conventionally known ones can be used. However, from the viewpoint that even at low power, the radio waves can be transmitted more reliably and the dimensions do not become too large in consideration of the frequencies described later, it is preferable to use a coil antenna. Further, the coil antenna can be composed of an electric wire or a coaxial cable, etc., and various electric wires and coaxial cables can be used from the viewpoints of insulation, dielectric constant, cold-resistant temperature, and dimensions. As the electric wire, for example, an electric wire composed of a copper wire and an external insulating coating (for example, made of a fluororesin) covering the copper wire can be mentioned. Further, as the coaxial cable, for example, a coaxial cable composed of a copper internal conductor, an insulator made of polyethylene covering the internal conductor, an external conductor composed of a copper wire mesh conductor covering the insulator, and an external insulating coating (for example, made of a fluororesin) covering the external conductor can be mentioned. Regarding the number of turns, thickness, etc. of the coil antenna, those skilled in the art can appropriately select them in consideration of the frequency.

[0065] The production method of the present invention freezes cells at a temperature below the freezing point of the cryopreservation solution. From this viewpoint, it is preferable that the cell freezing conditions in the production method of the present invention include freezing the cells at -20°C or lower (Condition 2).

[0066] The temperature at the time of freezing is preferably -25°C or lower, more preferably -30°C or lower. The lower limit of the temperature is preferably -100°C, more preferably -85°C, still more preferably -60°C, even more preferably -45°C, and particularly preferably -40°C.

[0067] It is preferable that the cell freezing conditions in the production method of the present invention include freezing the cells in the presence of a magnetic field (Condition 3).

[0068] The magnetic field strength is the magnetic field strength measured near the location where the vial for cryopreservation is placed. The magnetic field strength of the magnetic field is not particularly limited. For example, it is 5 μT or more, preferably 10 μT or more, more preferably 15 μT or more. The upper limit of the magnetic field strength is not particularly limited and is, for example, 50 μT, 40 μT, 30 μT, or 25 μT. The combination of the upper and lower limits of the magnetic field strength is arbitrary. However, the magnetic field strength is, for example, 5 μT or more and 50 μT or less, preferably 5 μT or more and 40 μT or less, more preferably 10 μT or more and 40 μT or less, still more preferably 10 μT or more and 30 μT or less, even more preferably 15 μT or more and 30 μT or less, and particularly preferably 15 μT or more and 25 μT or less.

[0069] The method for measuring the magnetic field strength is as follows, for example. A plurality (3 to 5) of magnetic flux density measurement sensors are arranged at the location where the vial is placed, and measurement is performed at room temperature (25 °C) using a measuring instrument (magnetic flux density meter), and the average value of the measured values at a plurality of locations is calculated.

[0070] The method of applying the magnetic field is not particularly limited, and a method according to or analogous to a known method can be adopted. The magnetic field is preferably a unidirectional and substantially uniform static magnetic field. The "unidirectional and substantially uniform static magnetic field" means that the magnetic field lines run substantially linearly in one direction and the magnetic flux density is substantially constant. The magnetic field can be applied by arranging a magnet body around the vial. The magnet body is not particularly limited as long as it can generate a static magnetic field with the above-described magnetic field strength, and various magnets can be used. From the viewpoint of maintaining the properties of the magnet for a relatively long period without receiving supply of a magnetic field or current from the outside, it is preferable to use a permanent magnet such as an alnico magnet, a ferrite magnet, or a neodymium magnet as the magnet that can be used for the magnet body. Among them, a ferrite magnet, which is inexpensive because it uses iron oxide as the main raw material, has a high magnetic permeability, and is used in various applications, is preferable. An alnico magnet is a magnet (cast magnet) cast using aluminum (Al), nickel (Ni), cobalt (Co), etc. as raw materials, and iron, copper, etc. may be added as additives. A ferrite magnet is obtained by adding a small amount of barium, strontium, etc. to iron oxide as the main raw material, sintering it after molding and pulverizing the sintered product, and magnetizing it with an electromagnet. A neodymium magnet is a rare earth magnet (rare earth magnet) mainly composed of neodymium, iron, and boron. Among these, a ferrite magnet, which is inexpensive because it uses iron oxide as the main raw material, has a high magnetic permeability, and is used in various applications, is preferable.

[0071] The time for freezing the cells under the above conditions is not particularly limited as long as it is a time sufficient for the cells to freeze. For example, it is 10 minutes to 5 hours, preferably 15 minutes to 3 hours, and more preferably 20 minutes to 2 hours.

[0072] After freezing the cells under the above conditions, they can be stored as they are under those conditions. Preferably, however, they are transferred and stored under conditions where the cells are not thawed (for example, liquid nitrogen, a freezer, etc., for example, conditions of -80°C or lower, preferably -150°C or lower).

[0073] The frozen cells can be thawed as appropriate and then used. The thawing method is not particularly limited, but from the viewpoints of function, activity, and cell viability, it is desirable to thaw the cells at a temperature around body temperature in a short time. Specifically, it is desirable to thaw at a temperature of 30°C to 40°C, preferably 35°C to 38°C, and more preferably at a temperature near human body temperature, for example, about 37°C.

[0074] In one aspect, the present invention also includes the frozen cells obtained by the production method of the present invention, or the thawed cells of the frozen cells.

[0075] 2. Medicine In one aspect, the present invention relates to a medicament (which may also be referred to as "the medicament of the present invention" in this specification) containing the thawed cells of the frozen cells obtained by the production method of the present invention, or the cells induced to differentiate from the thawed cells. This will be described below.

[0076] The induction of differentiation of the thawed cells can be carried out according to the method described in the above "1. Method for producing frozen cells".

[0077] When the cells are nervous system cells, examples of the target diseases include spinal cord injury, motor neuron diseases, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, Alzheimer's disease, retinitis pigmentosa, age-related macular degeneration, Parkinson's syndrome (including Parkinson's disease).

[0078] The medicament of the present invention contains a therapeutically effective amount of the above cells. For example, for a single administration, it contains 1×10 4 to 1×10 8 cells. Components such as dimethyl sulfoxide (DMSO) and serum albumin for the purpose of protecting the cells, and antibiotics for the purpose of preventing the contamination of bacteria may be contained in the medicament of the present invention.

[0079] The administration route of the medicine of the present invention is not particularly limited. For example, it is administered by intravenous injection, intra-arterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. Instead of systemic administration, local administration may be performed. As local administration, direct injection into the target tissue, organ, or organ can be exemplified. The administration schedule may be prepared in consideration of the gender, age, weight, pathological condition, etc. of the subject (patient). In addition to single administration, multiple administrations may be performed continuously or periodically.

Examples

[0080] Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited by these examples.

[0081] Test Example 1. Evaluation 1 of spheroid formation ability after freezing treatment Dopamine neuron differentiation-inducing cells differentiated from stem cells were cryopreserved by various methods, and the spheroid formation ability after cryopreservation was examined. Specifically, it was carried out as follows.

[0082] Stem cells (Ff-I01s04 cells (human iPS cells, established at the Institute for Integrated Cell-Material Sciences, Kyoto University, available from the Kyoto University iPS Cell Research and Development Initiative Foundation: https: / / www.cira-foundation.or.jp / j / research-institution / ips-stock-project / homozygous.html), 1231A3 cells (human iPS cells: https: / / cellbank.brc.riken.jp / cell_bank / CellInfo / ?cellNo=HPS0381&lang=En), KhES-1 cells (human ES cells: https: / / cellbank.brc.riken.jp / cell_bank / CellInfo / ?cellNo=HES0001&lang=Ja)) were seeded in a single cell state in a 24-well plate (product name: Nunc 24-well plate, round / flattened bottom, manufactured by ThermoFisher, coated with iMatrix-511 silk 0.5 μg / cm 2 at). 6cells / well or 5.3x10 5 cells / cm 2 ) and cultured in an induction medium (medium composition: Glasgow MEM, NEAA, Pyruvate, 2-ME, 8% Knockout Serum Replacement) for 13 days. On day 0 from the start of culture, Y27632 was added to the medium (final concentration: 20 μM). From day 0 to day 6 from the start of culture, A-83-01 was added to the medium (final concentration: 500 nM). From day 0 to day 12 from the start of culture, LDN193189 was added to the medium (final concentration: 100 nM). From day 1 to day 6 from the start of culture, Purmorphamine and FGF8 were added to the medium (final concentration: 2 μM, 100 ng / mL). From day 3 to day 12 from the start of culture, CHIR99021 was added to the medium (final concentration: 3 μM). Also, during the culture, the medium was changed daily. After 13 days of culture, the cells were dissociated into single cells using trypsin, then the medium was removed, and the cells were suspended in a cryopreservation solution (Bambanker (registered trademark) hRM, manufactured by Nippon Genetics Co., Ltd.), and the suspension was dispensed into cryopreservation vials (product name: Nalgene Cryoware Cryogenic Vial, manufactured by Thermo scientific) (3 - 8 x10 5 cells / 200 μL / vial). The cryopreservation vials were set in a proton freezer (manufactured by Hisho Freeze Systems Co., Ltd.) improved so that radio waves with a high electric field strength could be irradiated, and the cells in the vials were frozen. The specifications of the proton freezer are as follows. · Power supply: 100V 15A · Size: approximately 90 cm (front width) x approximately 60 cm (depth) x 90 cm (height) · Weight: approximately 80 kg (low-temperature storage + proton system) · Magnetic field: fixed (magnetic field strength near the vial: 18 μT) · Electric field strength of radio waves: variable (0 - 10 scales) · Air volume: three levels (strong, medium, weak) · Temperature: -20°C to -45°C.

[0083] The method for measuring the electric field strength is as follows. A plurality (3 to 5) of electromagnetic wave measurement sensors were placed at the location where the vial was placed, and measurements were taken at room temperature (25 °C) using a measuring instrument (Gauss meter·RIGOL DSA815), and the average value of the measured values at multiple locations was calculated.

[0084] The method for measuring the magnetic field strength is as follows. A plurality (3 to 5) of magnetic flux density measurement sensors were placed at the location where the vial was placed, and measurements were taken at room temperature (25 °C) using a measuring instrument (magnetic flux density meter), and the average value of the measured values at multiple locations was calculated.

[0085] The freezing conditions were set to 3 patterns shown in Table 1.

[0086]

Table 1

[0087] After freezing the vial in a freezer for 1 hour, it was put into liquid nitrogen and completely frozen (for 1 day). Then, the vial was put into a 37 °C water bath to thaw the cells. The obtained cells were seeded in a 96-well U-bottom low-adhesion plate (product name: PrimeSurface 96 U plate, manufactured by Sumitomo Bakelite Co., Ltd.) (the cells contained in 1 vial were suspended in 1300 μL and dispensed at 150 μL / well each), and cultured in a spheroid formation medium (medium composition: Neurobasal, B27, 200 μM L-glutamine, 200 μM Ascorbic acid, 400 μM dbc AMP, 10 ng / mL GDNF, 20 ng / mL BDNF, 20 μM Y27632) for 4 days. After the culture was completed, 7 or 8 wells of samples of the same cell type and the same freezing conditions were observed, and spheres were recognized using Incucyte (registered trademark) (manufactured by Sartorius Japan Co., Ltd.). The area of the sphere with the largest area in each well was taken as the sphere size of that well.

[0088] The results are shown in Fig. 1. As shown in Fig. 1, it was found that increasing the electric field strength of the radio wave irradiated during freezing improved the sphere size of the spheroids formed after thawing. The sphere size in the case of Proton 10 was comparable to that when spheroid formation was similarly performed using the cells before freezing. From this, it was found that by increasing the electric field strength of the radio wave irradiated during freezing, cells can be frozen while maintaining cell functions at a higher level.

[0089] Test Example 2. Evaluation 2 of spheroid formation ability after freezing treatment As stem cells, 201B7-Ff cells (human iPS cells (feeder-free): https: / / cellbank.brc.riken.jp / cell_bank / CellInfo / ?cellNo=HPS0063&lang=Ja) and 1231A3 cells (human iPS cells) were used to perform dopamine neuron differentiation induction culture in the same manner as in Test Example 1, and the cells were dispensed into vials for cryopreservation. The vials for cryopreservation were set in the proton freezer used in Test Example 1 (the freezing conditions were Proton 10 in Table 1), a small programmable freezer (PF-NP-200, manufactured by Nepagene Co., Ltd.), or a cell freezing container (CoolCell (registered trademark), manufactured by Corning Inc.) to freeze the cells in the vials. The freezing temperature of both the small programmable freezer and the cell freezing container was -80°C, and the cooling rate of the small programmable freezer was -1°C / min.

[0090] After the freezing was completed, the samples were immersed in liquid nitrogen for complete freezing (for 8 days). Then, the vials were placed in a 37°C water bath to thaw the cells. The obtained cells were seeded in a 96-well U-bottom low-attachment plate (product name: PrimeSurface 96 U plate, manufactured by Sumitomo Bakelite Co., Ltd.) (the cells contained in 1 vial were suspended in 1300 μL and dispensed at 150 μL / well), and cultured in a spheroid formation medium (medium composition: Neurobasal, B27, 200 μM L-glutamine, 200 μM Ascorbic acid, 400 μM dbc AMP, 10 ng / mL GDNF, 20 ng / mL BDNF, 20 μM Y27632) for 4 days. After the culture was completed, multiple wells were observed for samples of the same cell type and under the same freezing conditions.

[0091] Observation images of wells representing typical characteristics of each sample are shown in Figure 2. Spheroid formation was confirmed only in the case of the proton freezer (freezing conditions: Proton 10 in Table 1), but not when other devices and instruments were used.

[0092] Test Example 3. Evaluation of neurite outgrowth ability after freezing treatment As stem cells, 1231A3 cells (human iPS cells) were used, and dopamine neuron differentiation induction culture was performed in the same manner as in Test Example 1. After 13 days of culture, the cells were dissociated into single cells using trypsin, and then seeded in a 96-well U-bottom low-adhesion plate (product name: PrimeSurface 96 U plate, manufactured by Sumitomo Bakelite Co., Ltd.) (the cells contained in 1 vial were suspended in 1300 μL and dispensed at 150 μL / well each), and cultured in a spheroid formation medium (medium composition: Neurobasal, B27, 200 μM L-glutamine, 200 μM Ascorbic acid, 400 μM dbc AMP, 10 ng / mL GDNF, 20 ng / mL BDNF) for 14 days. In addition, for the first 3 days of the culture, Y27632 was added to the medium to a final concentration of 20 μM. The formed spheroids were dispensed into a cryopreservation vial (product name: Cryotube Inner Cap 1.8 mL non-self-standing type, round bottom type, manufactured by Nunc) in a cryopreservation solution (Bambanker (registered trademark) hRM, manufactured by Japan Genetics Co., Ltd.). The cryopreservation vial was set in the proton freezer used in Test Example 1 (the freezing conditions were Proton 10 in Table 1), a small programmable freezer (PF-NP-200, manufactured by Nepagene Co., Ltd.), or a cell freezing container (CoolCell (registered trademark), manufactured by Corning Inc.) to freeze the cells in the vial. The freezing temperature of both the small programmable freezer and the cell freezing container was -80°C, and the cooling rate of the small programmable freezer was -1°C / min.

[0093] After the freezing was completed, the samples were immersed in liquid nitrogen and completely frozen for 8 days. Then, the vials were thawed by mixing with PBS warmed to 37°C. The spheroids were seeded directly into a 24-well plate (product name: Tissue Culture Plate VTC-P24, manufactured by Viola Bio) and subjected to adherent culture for 3 days in a medium (medium composition: Neurobasal, B27, 200 μM L-glutamine, 200 μM ascorbic acid, 400 μM dbcAMP, 10 ng / mL GDNF, 20 ng / mL BDNF, 20 μM Y27632). After the culture was completed, the spheroids were immunostained using a primary antibody (product name: Purified anti-Tubulin β-3 (TUBB3) Antibody, manufactured by BioLegend) and a secondary antibody (product name: Donkey anti-Rabbit IgG(H+L) Highly Cross-Absorbed Secondary Antibody, Alexa Fluor® 488, manufactured by Invitrogen), and neurite outgrowth was evaluated. The area of the neurite portion was measured by image analysis.

[0094] The results are shown in Figures 3 and 4. The neurite outgrowth was the greatest in the case of the proton freezer (freezing conditions: Proton 10 in Table 1).

Claims

1. A method for producing frozen cells, comprising freezing cells under irradiation with radio waves having an electric field strength of 100 V / m or more.

2. The production method according to Claim 1, wherein the cells are nervous system cells.

3. The production method according to Claim 2, wherein the nervous system cells are at least one selected from the group consisting of neural stem cells, neural progenitor cells, and nerve cells.

4. The production method according to Claim 2, wherein the nervous system cells are derived from stem cells.

5. The production method according to Claim 4, wherein the stem cells are induced pluripotent stem cells.

6. The production method according to Claim 2, wherein the nervous system cells contain single cells or cell aggregates.

7. The production method according to Claim 1, wherein the electric field strength of the radio waves is 150 V / m or more and 300 V / m or less.

8. The production method according to Claim 1, wherein the cells are frozen at -20°C or lower.

9. The production method according to Claim 1, wherein the cells are frozen at -100°C or higher and -20°C or lower.

10. The production method according to Claim 1, wherein the cells are frozen in the presence of a magnetic field.

11. The production method according to Claim 10, wherein the magnetic field strength of the magnetic field is 5 μT or more.

12. The production method according to Claim 10, wherein the magnetic field strength of the magnetic field is 5 μT or more and 50 μT or less.

13. Frozen cells obtained by the production method according to any one of Claims 1 to 12, or thawed cells of the frozen cells.

14. A medicament containing thawed cells of frozen cells obtained by the production method according to any one of Claims 1 to 12, or cells induced to differentiate from the thawed cells.

Images