WO2023054317A1

WO2023054317A1 - Cell preparation used for treatment of neuropathy due to radiation

Info

Publication number: WO2023054317A1
Application number: PCT/JP2022/035846
Authority: WO
Inventors: 登紀子長村; 雅充原田; トランタイビンパム
Original assignee: 国立大学法人東京大学; ヒューマンライフコード株式会社
Priority date: 2021-09-30
Filing date: 2022-09-27
Publication date: 2023-04-06

Abstract

Provided is a cell preparation that can treat neuropathy due to radiation.　The present invention is a cell preparation used for treatment of neuropathy due to radiation, the cell preparation comprising umbilical cord-derived cells.

Description

Cell preparation for treatment of radiation-induced neuropathy

The present disclosure relates to cell preparations for use in the treatment of radiation-induced neuropathy.

Radioactive materials are used in various fields such as medicine, agriculture, industry, and energy generation. On the other hand, in handling the radioactive materials, the operator may be exposed to radiation, resulting in health hazards. Therefore, establishment of an effective treatment method for radiation exposure is desired.

In the medical field, radiation exposure occurs during radiotherapy. The radiotherapy is one of cancer treatment methods along with surgery and drug therapy, and is a treatment method applied to cancer treatment such as brain tumors. The radiation therapy is used to completely cure cancer or alleviate symptoms by irradiating a lesion site with radiation (Non-Patent Documents 1 and 2).

However, there is a problem that the radiation therapy causes radiation injury to normal tissues. Neuropathy is known as radiation injury. As a treatment method for neuropathy caused by irradiation, administration of steroids is performed. However, although the symptoms can be alleviated by administering steroids, there is no therapeutic method that can be expected to have a sufficient therapeutic effect on the neuropathy.

Therefore, the present disclosure aims to provide a cell preparation that can treat radiation-induced neuropathy.

In order to achieve the above object, the present disclosure is a cell preparation (hereinafter also referred to as "cell preparation") used for treatment of radiation-induced neuropathy, wherein the cell preparation contains umbilical cord-derived cells.

According to the present disclosure, radiation-induced neuropathy can be treated.

1 is a graph showing the ratio of GAP43-positive cells in Example 1. FIG. 2 is a photograph showing an immunostained image of cortical neuron cells in Example 1. FIG. 3 is a graph showing the length of neurites in Example 1. FIG. 4 is a photograph showing an immunostained image of cortical neuron cells in Example 1. FIG. 5 is a photograph showing a fluorescent image and a bright field image of cortical neuron cells in Example 1. FIG. 6 is a graph showing the ratio of fluorescence-positive cells due to active oxygen in Example 1. FIG. 7 is a photograph showing a fluorescence image and a bright field image of necrosis in Example 1. FIG. 8 is a graph showing the ratio of viable cells, apoptotic cells and necrotic cells in Example 1. FIG. 9 is a graph showing the expression level of each gene in Example 1. FIG. FIG. 10 is a photograph showing a histologically stained image of the mouse brain tissue in Example 1, showing the thickness of the myelin sheath (myelin sheath).

<Definition>
As used herein, “radiation neuropathy” means a state in which normal nerve cells (neuron cells) and/or nerve tissue are damaged by radiation exposure such as radiation therapy. The neuropathy includes, for example, neuronal and/or neurological tissue disorders associated with demyelination, axonal degeneration, coagulative necrosis, and the like. The neuropathy caused by radiation includes, for example, radiation encephalopathy, radiation neuritis, radiation myelopathy, radiation brain necrosis, radiation neurosis, leukoencephalopathy, hypoglossal nerve palsy, facial nerve palsy, trigeminal neuropathy, and radiation-induced brachial plexopathy. etc.

As used herein, "radiation" means particle beams or electromagnetic waves emitted when an unstable atomic nucleus structure changes to a stable atomic nucleus structure. The radiation includes, for example, ionizing radiation or non-ionizing radiation. Examples of the ionizing radiation include particle beams and electromagnetic waves. Examples of the particle beam include α-rays, β-rays, proton beams, deuteron beams, triple proton beams, heavy ion beams, charged meson beams, uncharged meson beams, neutrinos, and neutron beams. Examples of the electromagnetic waves include X-rays and γ-rays. Examples of the non-ionizing radiation include radio waves, microwaves, infrared rays, visible rays, and ultraviolet rays.

As used herein, "nerve" means a tissue that transmits information. Examples of the nerves include central nerves and peripheral nerves. The central nervous system includes the cerebrum; brain stems such as the diencephalon, midbrain, pons, and medulla oblongata; the cerebellum; the brain; and the spinal cord. Examples of the peripheral nerves include motor nerves, sensory nerves, and autonomic nerves.

As used herein, "treatment" means therapeutic treatment and/or prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, arresting, ameliorating, ameliorating a disease, condition, or disorder, or halting, arresting, reducing, or delaying the progression of a disease, condition, or disorder. do. As used herein, "prevention" means reducing the likelihood of developing a disease or condition or delaying the onset of a disease or condition. The “treatment” may be, for example, treatment of a subject (patient) who develops the target disease, or treatment of a model animal of the target disease.

As used herein, "cell preparation" means a cell population containing desired cells or a composition containing desired cells. As such, a cell preparation can also be referred to herein, for example, as a cell population or composition. In the cell preparation, the ratio of desired cells to all cells (also referred to as “purity”) can be quantified, for example, as the ratio of cells expressing one or more markers that desired cells express. The purity is, for example, the percentage in viable cells. The purity can be measured, for example, by flow cytometry, immunohistochemistry, in situ hybridization, and the like.

As used herein, the purity of the cell preparation is, for example, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher , 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

As used herein, the term “mesenchymal cell” means a cell that constitutes connective tissue derived from mesoderm or neural crest and/or a cell that has the potential to differentiate into said cell. When the mesenchymal cells have self-renewal ability, the mesenchymal cells can also be called mesenchymal stem cells (MSCs). Said mesenchymal cells are usually present in blood vessels; in and around organs such as liver or pancreas; fat; bone marrow or umbilical cord; The mesenchymal stem cells are one of multipotent stem cells, and mean cells that have the potential to differentiate into adipocytes, osteocytes, chondrocytes, muscle cells, hepatocytes, tendon cells, and/or nerve cells. do. For each tissue to be collected, the mesenchymal stem cells are, for example, adipose tissue-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, and umbilical cord-derived mesenchymal stem cells. They are called stem cells.

As used herein, "positive" is defined as a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen by an analysis method such as flow cytometry that is detected using an antigen-antibody reaction. By comparison, it means that a higher signal or the like is detected. As used herein, "negative" means that a signal or the like that is equal to or lower than that in a negative control reaction using a negative control cell that does not express the antigen or an antibody that does not react with the antigen is detected. means.

As used herein, "under inflammatory conditions" refers to conditions in which inflammatory cytokines such as interferon γ are contacted or added.

As used herein, "nerve cell" or "neuron cell" means a cell composed of dendrites that receive information and axons (neurites) that transmit information. Examples of the neuron cells include mature neuron cells and immature neuron cells. The mature neuron cells are morphologically cells with well-developed dendrites. The mature neuron cells can be identified by expression of mature neuron marker genes such as MAP2. The immature neuronal cells are morphologically simple cells with dendrites compared to the mature neuronal cells. The immature neuron cells can be identified by expression of immature neuron marker genes such as GAP43.

As used herein, "reactive oxygen species" (hereinafter referred to as "active oxygen") means atoms, molecules, radicals, compounds, etc. containing oxygen atoms in a chemically activated state. The radical means an atom, molecule or ion having an unpaired electron. Said active oxygen is, for example, non-radical species such as singlet oxygen ( ¹ O ₂ ), ozone (O ₃ ), and hydrogen peroxide (H ₂ O ₂ ); ), peroxy radicals (LOO.), hydroperoxy radicals (HOO.), nitric oxide (NO.), nitrogen dioxide ( _NO.sub.2 .), superoxide anions ( ^O.sub.2- ), radical species such as lipid radicals; is given.

As used herein, "cell death" means the death of cells that are constituents of living organisms. The cell death includes, for example, apoptosis and necrosis. Said apoptosis refers to programmed cell death controlled by molecular mechanisms. Said necrosis refers to passive cell death caused when cells are physically and/or chemically damaged.

As used herein, "isolated" or "isolated" means the state of being identified and separated, and/or the state of being recovered from components in their natural state. Said "isolation" or "isolated" can be carried out, for example, by going through at least one purification step. When the "isolated" or "isolated" is used in combination with cells, the "isolated" or "isolated" refers to a state in which the cells of interest are separated and/or purified from tissue. may mean. Examples of the tissue include organs such as blood vessels, liver, and pancreas; fat; bone marrow; umbilical cord; Examples of the cells include mesenchymal cells.

<Preparation for treating neuropathy caused by radiation>
In one aspect, the description provides a cell preparation for use in treating radiation-induced neuropathy. The cell preparation of the present disclosure, as described above, is a cell preparation for use in the treatment of radiation-induced neuropathy, said cell preparation comprising umbilical cord-derived cells. The cell preparation of the present disclosure is characterized by containing umbilical cord-derived cells, and other configurations and conditions are not particularly limited. Further, the therapeutic agent for radiation neuropathy of the present invention is characterized by containing the cell preparation of the present disclosure.

As a result of extensive research, the present inventors have found that the umbilical cord-derived cells suppress neurite retraction of mature neuron cells caused by irradiation, suppress cell death of immature neuron cells caused by irradiation, We have found that it suppresses the generation of reactive oxygen in neuronal cells caused by irradiation, suppresses necrosis of neuronal cells caused by irradiation, and suppresses inflammation in the nervous system caused by irradiation. The present inventors have found that demyelination in radiation-induced neuropathy patients can be suppressed by using umbilical cord-derived cells exhibiting one or more of these activities, and have established the present disclosure. . The umbilical cord-derived cells are presumed to suppress symptoms of radiation-induced neuropathy by suppressing damage to neuronal cells caused by radiation and/or promoting recovery from damage caused by radiation. Therefore, it can be said that the cell preparation of the present disclosure can suitably treat radiation-induced neuropathy by containing the umbilical cord-derived cells.

As used herein, "umbilical cord" is a white tubular tissue that connects the fetus and placenta, and means tissue that does not contain placenta and cord blood. In the present specification, the origin of the "umbilical cord" is not particularly limited. is the umbilical cord of a primate mammal, more preferably a human umbilical cord.

As used herein, the umbilical cord may be an umbilical cord collected from a subject for administration, treatment, or treatment (hereinafter collectively referred to as "administration subject"), or an umbilical cord collected from a subject other than the administration subject. There may be. It is preferable to use an umbilical cord collected from a person other than the subject of administration from the viewpoint of not being subject to restrictions during preparation. The umbilical cord-derived cells of the present disclosure may be umbilical cord-derived cells (autologous cells) collected from an administration subject, or umbilical cord-derived cells collected from a non-administration subject, as shown in Examples described later. (allogeneic cells). It has been confirmed that the umbilical cord-derived cells have therapeutic effects without being eliminated by immune rejection or the like, for example.

In the present specification, the umbilical cord can be collected by appropriately removing the placenta from, for example, postpartum tissue containing the placenta and/or umbilical cord delivered by vaginal delivery or cesarean section. In the present disclosure, the umbilical cord may be one obtained by removing cord blood from the collected umbilical cord, and may be one subjected to aseptic or bacteriostatic treatment. Removal of the cord blood can be performed, for example, by rinsing or perfusion with a solution containing an anticoagulant such as heparin. Said aseptic or bactericidal treatment is not particularly limited, for example, application of disinfectants such as povidone-iodine; addition of antibiotics such as penicillin, streptomycin, amphotericin B, gentamicin, and/or nystatin, and/or antimycotics immersion in a medium or buffer; The umbilical cord may also selectively lyse red blood cells, for example, if desired. As a method for selectively lysing the red blood cells, methods well known in the art can be used, such as incubation in hypertonic or hypotonic medium by lysis with ammonium chloride.

As used herein, the term "umbilical cord-derived cells" means a cell population prepared using umbilical cord as a raw material. The "umbilical cord-derived cells" may be composed of a single type of cell, or may be composed of a plurality of types of cells. The umbilical cord-derived cells preferably contain umbilical cord-derived mesenchymal cells. The umbilical cord-derived cells may be a cell population composed of multiple types of cells including the umbilical cord-derived mesenchymal cells.

The umbilical cord-derived cells of the present disclosure may be, for example, a cell population having any one or more characteristics and/or markers of (a) to (c) below, preferably a cell population having all characteristics is.
(a) shows adhesion (adherence) to plastic in culture in the presence of a medium;
(b) CD105, CD73, CD90, CD44, HLA-classI, HLA-G5 and PD-L (Programmed cell death 1 ligand) 2 positive, CD45, CD34, CD11b, CD19 and HLA-ClassII negative ;
(c) expression of IDO (indoleamine 2,3-dioxygenase), PGE ₂ (prostaglandin E2) and PD-L1 genes and/or proteins is induced under inflammatory conditions;

As used herein, the "HLA-class I" means HLA-A, B, or C. As used herein, the "HLA-Class II" means HLA-DR, DQ, or DP.

In the present specification, the umbilical cord-derived cells may be extracts and/or secretions of the umbilical cord-derived cells. The umbilical cord-derived cell extract can be obtained by, for example, subjecting the umbilical cord-derived cells to concentration treatment, centrifugation treatment, drying treatment, freeze-drying treatment, solvent treatment, surfactant treatment, protease, glycolytic enzyme, or the like. processed products obtained by enzymatic treatment, protein extraction treatment, ultrasonic treatment and/or grinding treatment, or processed products obtained by a combination of these treatments, and the like. Examples of the secretions of the umbilical cord-derived cells include exosomes and cell culture supernatants of umbilical cord-derived cells.

In the present specification, the cell preparation preferably exhibits an inhibitory effect on neurite retraction of mature neuron cells caused by irradiation. The mature neuron cells are, for example, central nerve neuron cells, preferably cerebral mature neuron cells.

The neurite retraction inhibitory action of the mature neuron cells is, for example, when the irradiated mature neuron cells and the subject coexist, compared to the absence of the subject, the mature neuron It can be evaluated by whether it can suppress retraction of neurites of cells. Specifically, the neurite retraction inhibitory action can be evaluated based on the rate of inhibition of neurite length retraction. The inhibition rate was calculated by comparing the neurite length (L _nc ) of non-irradiated mature neuronal cells with that of irradiated mature neurons treated identically but cultured in the absence of the specimen. Length of neurites of mature neuron cells irradiated and cultured in the presence of the subject, with reference to the difference (L _nc −L _c ) from the neurite length (L _c ) of the cells The ratio of the difference (L _e −L _c ) between the length (L _e ) and the length (L _c ) of the neurite ((L _e −L _c )/(L _nc −L _c )×100(%) ) can be calculated as The type and irradiation dose of radiation used to calculate the inhibition rate, mature neuron cells, and the length of each neurite can be measured according to Example 1 (7) described below, and more specifically, the radiation As the mature neuron cells, mouse fetal-derived mature neuron cells can be used for measurement. In the evaluation, for example, the suppression rate is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more % or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% In the case of the above, the test object can be evaluated as having an inhibitory effect on neurite retraction.

In the present specification, the cell preparation preferably exhibits an inhibitory effect on cell death of immature neuronal cells caused by irradiation. The immature neuron cell is, for example, a central nervous immature neuron cell, preferably a brain immature neuron cell.

The effect of suppressing cell death of the immature neuron cells is, for example, when the irradiated immature neuron cells and the test object coexist, compared to the absence of the test object, the immature neuron cell It can be evaluated by whether it can suppress cell death of mature neuron cells. Specifically, the cell death inhibitory action can be evaluated based on the cell death inhibition rate of the immature neuron cells. The inhibition rate was calculated by comparing the number of surviving immature neuronal cells (C _nc ) that were not irradiated and the immature neurons that were irradiated and treated in the same manner except that they were cultured in the absence of the test substance. Surviving cell number (C _e ) of immature neuron cells that have been irradiated and cultured in the presence of the subject, and the surviving cells, based on the difference from the surviving cell number (C _c ) of the cells It can be calculated as the ratio of the difference (C _e −C _c ) from the number (C _c ) ((C _e −C _c )/(C _nc −C _c )×100(%)). As the viable cell count, the ratio of the viable cell count to the total cell count may be used. The type and irradiation dose of radiation, the number of immature neuron cells, and the number of viable cells used for calculating the inhibition rate can be measured according to Example 1 (9) described later. It can be measured using γ-rays and immature neuron cells derived from mouse embryos as the immature neuron cells. In the evaluation, for example, the suppression rate is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more % or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% In the above case, the test substance can be evaluated as having an effect of suppressing cell death.

In the present specification, it is preferable that the cell preparation exhibits an action of suppressing the generation of reactive oxygen in neuronal cells caused by irradiation. The neuron cells are, for example, central nerve neuron cells, preferably brain neuron cells.

The effect of suppressing the generation of reactive oxygen in the neuron cells is, for example, when the irradiated neuron cells and the test object are allowed to coexist, compared to the absence of the test object, in the neuron cells It can be evaluated by whether or not generation of active oxygen can be suppressed. Specifically, the inhibitory effect on the generation of reactive oxygen can be evaluated based on the rate of inhibition of the generation of reactive oxygen in the neuron cells. The inhibition rate is calculated by comparing the number of reactive oxygen marker-positive cells (R _nc ) of non-irradiated neuronal cells and neurons that have been irradiated and treated in the same manner except that they were cultured in the absence of the test substance. The number of reactive oxygen marker-positive cells ₍ _Re ) and the ratio of the difference (R _e -R _c ) between the number of reactive oxygen marker-positive cells (R _c ) ((R _e -R _c )/(R _nc -R _c ) × 100 (%)) , can be calculated. As the number of reactive oxygen marker-positive cells, the ratio of the number of reactive oxygen marker-positive cells to the total number of cells may be used. The type and irradiation dose of radiation used to calculate the inhibition rate, neuron cells, the reactive oxygen marker to be used, and the number of cells positive for each reactive oxygen marker can be measured according to Example 1 (8) described below. Specifically, gamma rays can be used as the radiation, and mouse fetal neuron cells can be used as the neuron cells. In the evaluation, for example, the suppression rate is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more % or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% In the above case, the test object can be evaluated as having an effect of suppressing the generation of active oxygen.

In the present specification, the cell preparation preferably exhibits an inhibitory effect on necrosis of neuronal cells caused by irradiation. The neuron cells are, for example, central nerve neuron cells, preferably brain neuron cells.

The effect of suppressing necrosis in neuronal cells is, for example, when the irradiated neuronal cells and the test substance are allowed to coexist, compared to the absence of the test substance, suppresses necrosis in the neuronal cells. It can be evaluated according to whether it can be done. Specifically, the necrosis-suppressing action can be evaluated based on the necrosis-suppressing rate of the neuronal cell. The inhibition rate was calculated by comparing the number of necrotic cells (N _nc ) in non-irradiated neuronal cells and the number of irradiated neuronal cells treated in the same manner but cultured in the absence of the test substance. The number of necrotic cells (N _e ) of irradiated neuronal cells cultured in the presence of the subject, and the necrotic cells, based on the difference from the number of necrotic cells (N _c ) can be calculated as a ratio ((N _e -N _c )/(N _nc -N _c )×100(%)) of the difference (N _e −N c ) from the number of cells (N _c ) of the cell number (N _c ). As the number of necrotic cells, the ratio of the number of necrotic cells to the total number of cells may be used. The type and irradiation dose of radiation used to calculate the inhibition rate, the detection of neuronal cells, and each necrotic cell can be measured according to Example 1 (9) described below, and more specifically, the radiation It can be measured using γ-rays and mouse embryo-derived neuron cells as the neuron cells. In the evaluation, for example, the suppression rate is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more % or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% In the case of the above, the test object can be evaluated as having an inhibitory effect on necrosis.

As used herein, the method for producing (preparing) umbilical cord-derived cells includes, for example, a step of isolating cells from the umbilical cord, and optionally a step of subculturing the isolated cells. As a specific example, the preparation method includes, for example, (1) cutting the umbilical cord, (2) culturing the umbilical cord segment, and (3) subculturing. As another example, the preparation method includes, for example, (A) a step of cutting the umbilical cord, a step of enzymatic treatment, or a step of dissociating the tissue by both, (B) a step of culturing the umbilical cord tissue, and ( C) including the step of passaging. The umbilical cord-derived cells may be a uniform cell population or a heterogeneous cell population.

When the umbilical cord-derived cells are prepared by the method including the steps (1) to (3) or the method including the steps (A) to (C), an example can be carried out as follows. The method for preparing the umbilical cord-derived cells is not limited to the following example.

First, the method including the steps (1) to (3) will be described. In the (1) step of cutting the umbilical cord, for example, the umbilical cord obtained by the above method is subjected to mechanical force (shredding force or shear force) in a state containing amniotic membrane, blood vessels, perivascular tissue and/or Walton's jelly. It can be implemented by cutting. The size of the umbilical cord segment obtained by cutting is not particularly limited, and examples thereof include 1 to 10 mm ³ , 1 to 5 mm ³ , 1 to 4 mm ³ , 1 to 3 mm ³ and 1 to 2 mm ³ .

Next, in the step (2) of culturing the umbilical cord segment, for example, the cut umbilical cord segment is seeded in an incubator such as a Petri dish, dish, or flask, and cultured in a culture medium suitable for umbilical cord-derived cells. . In the step (2), it is preferred that the umbilical cord segment is not treated with a digestive enzyme.

In this specification, the "incubator" may be, for example, an incubator having a solid surface. As the incubator, for example, an incubator used for culturing cells, tissues, and/or organs can be used. Said "solid surface" means, for example, any material capable of binding with said umbilical cord-derived cells. Specifically, the material includes, for example, a plastic material that has been treated (eg, hydrophilicity-enhancing treatment) to promote binding of mammalian cells to its surface. The type of culture vessel having the solid surface is not particularly limited, and examples thereof include petri dishes, dishes, flasks, and the like.

As used herein, the "culture medium suitable for umbilical cord-derived cells" can be prepared, for example, by adding additives such as serum to the basal medium. Said additives are, for example, serum and/or one or more of albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, etc. of serum replacement. For example, if necessary, the culture solution further contains lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antifungal agents, antioxidants, pyruvic acid, buffers, and inorganic salts. You may add substances, such as. The basal medium is not particularly limited. Ham's F10 Medium (F10), Ham's F-12 Medium (F12), Iscove's Modified Dulbecco's (IMDM) Medium, Fischer's Medium, Mesenchymal Stem Cell Growth Medium (MSCGM), DMEM/F12, RPMI 1640, CELL-GRO-FREE, and mixed media of these. Examples of the serum include animal-derived serum such as human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, pig serum, sheep serum, rabbit serum and rat serum. The amount of serum added to the basal medium is, for example, 5 v/v % to 15 v/v %, preferably about 10 v/v %. The fatty acid is not particularly limited, and examples thereof include linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoic acid, palmitic acid, and stearic acid. The lipid is not particularly limited, and examples thereof include phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and the like. The amino acid is not particularly limited, and examples thereof include amino acids such as L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. L-isomers, D-isomers thereof, or mixtures thereof (DL-isomers). The protein is not particularly limited, and examples thereof include ecotin, reduced glutathione, fibronectin, β2-microglobulin and the like. The polysaccharide is not particularly limited, and examples thereof include glycosaminoglycans such as hyaluronic acid and heparan sulfate. The growth factor is not particularly limited, and examples include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1), leukocyte inhibitory factor (LIF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) , erythropoietin (EPO) and the like. The antibiotic and/or antifungal agent is not particularly limited, and examples thereof include penicillin G, streptomycin sulfate, amphotericin B, gentamicin, nystatin, and mixtures thereof.

In the above step (2), in order to prevent the seeded umbilical cord segments from floating in the culture medium, it is preferable to hold down the umbilical cord segments using a plate or the like during the culture period. Examples of the plate include plates described in JP-A-2015-70824.

In the step (2), culture conditions are not particularly limited, and for example, general culture conditions for cells, tissues, organs, etc. can be referred to. As a specific example, the CO ₂ concentration in the step (2) is, for example, 0 to 5%. The O ₂ concentration in the step (2) is, for example, 2 to 25%, preferably 5 to 20%. The culture temperature in the step (2) is, for example, 25 to 40°C, preferably about 37°C (35 to 39°C).

In the step (2), the culture period is not particularly limited. It is preferable to culture until the cells become confluent.

In the step (2), for example, in order to remove unbound cells and cell debris after the culture, the cells are washed and treated with a solution containing a chelating agent such as EDTA, trypsin, collagenase, dispase, or the like. protease, sugar chain degrading enzyme such as hyaluronidase, or a stripping agent containing a mixture thereof. Then, in the step (2), for example, by filtering the detachment solution containing the cells and the umbilical cord segment using a cell strainer or the like, only cells can be obtained as umbilical cord-derived cells. The obtained umbilical cord-derived cells can be seeded, for example, in the culture vessel described above and cultured using the culture medium described above.

In the step (3), the umbilical cord-derived cells can be appropriately grown to the required number by subculturing. In the step (3), in the subculture, the cells may be detached with the detachment agent, seeded at an appropriate cell density in a separately prepared culture vessel, and culture continued. The cell density (seeding density) when seeding the cells is, for example, 1×10 ² to 1×10 ⁵ cells/cm ² , 5×10 ² to 5×10 ⁴ cells/cm ² , 1×10 ³ 1×10 ⁴ cells/cm ² , 2×10 ³ to 1×10 ⁴ cells/cm ² and the like, preferably 2×10 ³ to 1×10 ⁴ cells/cm ² . The seeding density is preferably adjusted, for example, so that the period to reach a suitable confluency is 3 to 7 days. In the subculture of the step (3), the medium may be changed as appropriate, if necessary.

The number of passages in the step (3) is not particularly limited, and may be performed, for example, until senescence, when cell division stops. Regarding the number of passages in the step (3), for example, from the viewpoint of use for treatment, it is preferably subcultured 3 to 25 times, more preferably subcultured 4 to 12 times.

Next, a method including the steps (A) to (C) will be described. In the step of (A) enzymatic treatment, the umbilical cord obtained by the method described above is enzymatically treated in a state containing amniotic membrane, blood vessels, perivascular tissue and/or Walton's Jelly to dissociate the tissues. The enzyme used for the enzymatic treatment is not particularly limited, and examples thereof include proteases such as collagenase and dispase; glycolytic enzymes such as hyaluronidase; and the like.

Next, the step of (B) culturing the umbilical cord tissue and the step of (C) subculturing are, for example, the same as the step of (2) culturing the umbilical cord tissue and the step of (3) subculturing, respectively. can be implemented as

As a result, the umbilical cord-derived cells can be obtained after the step (3) or (C).

In order to confirm that the cells obtained by the method for preparing umbilical cord-derived cells are umbilical cord-derived cells, surface antigens and the like may be analyzed by conventional methods using flow cytometry and the like. In addition, whether or not the cells obtained by the method for preparing umbilical cord-derived cells are umbilical cord-derived cells may be evaluated by measuring the amounts of various proteins produced from the cells.

The cells obtained by the method for preparing umbilical cord-derived cells may be directly prepared for treatment or may be cryopreserved. The cryopreservation is performed, for example, by suspending the cells in a cryopreservation solution capable of preserving the umbilical cord-derived cells and storing the cells at -80°C to -180°C. The cryopreservation solution is not particularly limited, and examples thereof include an aqueous solution containing a cryoprotectant and glucose. Examples of the antifreeze agent include dimethyl sulfoxide (hereinafter also referred to as "DMSO"), dextran, glycerol, propylene glycol, and 1-methyl-2-pyrrolidone, preferably DMSO and/or propylene glycol. Yes, more preferably DMSO. The cryoprotectant is contained, for example, in the cryopreservation solution at 1 to 15 v/v%, preferably 5 to 15 v/v%, more preferably 5 to 12v/v%, More preferably, it is contained in an amount of 8 to 11 v/v%.

Glucose contained in the cryopreservation solution is, for example, 0.5 to 10 w/v%, preferably 1 to 10 w/v%, more preferably 2 to 10 w/v% in the cryopreservation solution. 8 w/v %, more preferably 2 to 5 w/v %.

The cryopreservation solution may further contain other components. Other components include, for example, pH adjusters and thickeners. Examples of the pH adjuster include sodium hydrogen carbonate, HEPES, and phosphate buffer. In addition, when no phosphate buffer is added to the basic stock solution (BSS), as the pH adjuster, for example, sodium chloride having a function of imparting a buffering capacity around a pH suitable for the umbilical cord-derived cells was added. can also be used. A phosphate buffer is preferably used as the pH adjuster. The pH adjuster is preferably used to adjust the pH in the cryopreservation solution to, for example, about 6.5-9, preferably 7-8.5. In the present specification, the "phosphate buffer" includes, for example, sodium chloride, monosodium phosphate (anhydrous), monopotassium phosphate (anhydrous), disodium phosphate (anhydrous), trisodium phosphate ( Anhydrous), potassium chloride, and potassium dihydrogen phosphate (anhydrous), etc., especially sodium chloride, monosodium phosphate (anhydrous), potassium chloride, or potassium dihydrogen phosphate (anhydrous). A buffer containing The pH adjuster is contained, for example, in the cryopreservation solution at 0.01 to 1 w/v%, preferably 0.05 to 0.5 w/v%.

The cryopreservation solution may or may not contain natural animal-derived components. Examples of the natural animal-derived components include the aforementioned serum and basal medium. Preferably, the cryopreservation solution does not contain natural animal-derived components. The cryopreservation solution that does not contain the natural animal-derived components does not cause quality differences between lots of natural animal-derived components, and components such as various cytokines, growth factors, and hormones contained in serum. It is possible to suppress the possibility of changes in the properties of the cells in the umbilical cord tissue caused by , and also to suppress the influence of components of unknown origin contained in the basal medium. Therefore, cryopreservation solutions free of said natural animal-derived components are very useful, especially in clinical use.

The cryopreservation solution may further contain a thickening agent. The thickening agent is not particularly limited, and examples thereof include those capable of constituting a cryopreservation solution capable of sufficiently preserving the umbilical cord tissue. Examples of the thickener include carboxymethyl cellulose (hereinafter also referred to as "CMC"), carboxymethyl cellulose sodium (hereinafter also referred to as "CMC-Na"), organic acid polymers, propylene glycol alginate, sodium alginate and the like. As the thickening agent, CMC and CMC-Na are preferred, and CMC-Na is particularly preferred. The organic acid polymer is preferably sodium polyacrylate. The thickening agent is contained, for example, in the cryopreservation solution at 0.1 to 1 w/v%, preferably 0.1 to 0.5 w/v%, more preferably 0.2 Contains ~0.4 w/v%.

The cryopreservation solution is preferably an aqueous solution. The osmotic pressure of the cryopreservation solution is, for example, preferably 1000 mOsm or more, more preferably 1000 to 2700 mOsm, in order to maintain performance as a preservation solution.

The cryopreservation solution is preferably an aqueous solution that contains a thickener, a cryoprotectant, and glucose, and does not contain natural animal-derived components. The cryopreservation solution is more preferably an aqueous solution containing CMC-Na, DMSO, and glucose and free of natural animal-derived components. The cryopreservation solution more preferably contains 0.1 to 1 w/v% CMC-Na, 1 to 15 v/v% DMSO, 0.5 to 10 w/v% glucose, and natural It is an aqueous solution containing no animal-derived components.

The cells obtained by the method for preparing umbilical cord-derived cells may be used as cell preparations for various applications, for example, by mixing with infusion preparations. In addition, when the umbilical cord-derived cells are cryopreserved, the cryopreserved umbilical cord-derived cells may be suspended in the cryopreservation solution, and after thawing, may be used directly as a cell preparation for various purposes, or may be used after thawing. It may be mixed with an infusion formulation and the resulting mixture used as a cell preparation for various uses. When mixed with the infusion preparation, the culture medium or cryopreservation solution in which the umbilical cord-derived cells are suspended may be mixed with the infusion preparation or the like, and the culture medium or cryopreservation solution may be separated by centrifugation or the like. After separating the cells from the solvent, the cells alone may be mixed with the infusion formulation. In the preparation method, for example, in order to avoid the complexity of the procedure, the step of culturing the frozen cells after thawing is not included, or the cryopreservation solution in which the thawed cells are suspended is directly used as an infusion preparation. Mixing is preferred.

In the present specification, the above-mentioned "infusion preparation" includes, for example, solutions such as infusion solutions used for human treatment, and specific examples include physiological saline, 5% glucose solution, Ringer's solution, lactated Ringer's solution, and acetated Ringer's solution. , No. 1 liquid, No. 2 liquid, No. 3 liquid, No. 4 liquid, and the like.

The cell preparation of the present disclosure may be a kit containing the infusion preparation in addition to the umbilical cord-derived cells.

The cell preparation of the present disclosure may contain a pharmaceutically acceptable carrier in addition to or instead of the infusion preparation. The carrier includes suspending agents, solubilizers, stabilizers, tonicity agents, preservatives, antiadsorption agents, surfactants, diluents, vehicles, pH adjusters, Examples include soothing agents, buffering agents, sulfur-containing reducing agents, antioxidants, and the like, and can be added appropriately within a range that does not interfere with the effects of the present disclosure.

The suspending agent is not particularly limited, and examples thereof include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic (gum arabic), tragacanth powder, carboxymethylcellulose sodium, polyoxyethylene sorbitan monolaurate, and the like.

The solution adjuvant is not particularly limited, and examples thereof include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

The stabilizer is not particularly limited, and examples include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, and the like.

The tonicity agent is not particularly limited, and examples thereof include D-mannitol and sorbitol.

The preservative is not particularly limited, and examples thereof include methyl paraoxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

The antiadsorption agent is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, and polyethylene glycol.

The sulfur-containing reducing agent is not particularly limited. Those having a sulfhydryl group such as sodium sulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms are included.

The antioxidant is not particularly limited, and examples thereof include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stear. sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate and the like.

Cell preparations of the present disclosure may further include inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate; organic salts such as sodium citrate, potassium citrate, sodium acetate; Saccharides such as glucose; and other commonly added components may be added as appropriate. Further, as an anticoagulant and/or pH adjuster, for example, ACD-A solution (composition composed of sodium citrate hydrate, citric acid hydrate, glucose, etc.) may be added.

Cellular preparations of the present disclosure may be mixed with, for example, for topical administration, organics such as biopolymers; inorganics such as hydroxyapatite; specific examples include collagen matrices, polylactic polymers or copolymers, polyethylene It may be mixed with glycol polymers or copolymers and chemical derivatives thereof.

A cell preparation of the present disclosure may be used, for example, in vitro or in vivo. The cell preparations of the present disclosure can be used, for example, as research reagents and can be used as pharmaceuticals.

The subject of administration of the cell preparation of the present disclosure is not particularly limited. When the cell preparation of the present disclosure is used in vivo, the administration subject includes, for example, humans or non-human animals other than humans. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions; birds; fish; When the cell preparation of the present disclosure is used in vitro, the subject of administration includes, for example, cells, tissues, organs, and the like, and the cells include, for example, cells collected from living organisms, cultured cells, and the like. The tissue or organ includes, for example, a tissue (living tissue) or organ collected from a living body.

When the cell preparation of the present disclosure is used in vivo, the administration subject is, for example, a subject diagnosed as having neuropathy due to radiation, a subject suspected of having neuropathy due to radiation, or a subject having neuropathy due to radiation. There are targets that may cause The neuropathy includes, for example, radiation encephalopathy, radiation neuritis, radiation myelopathy, radiation brain necrosis, radiation neurosis, leukoencephalopathy, hypoglossal nerve palsy, facial nerve palsy, trigeminal neuropathy, and radiation-induced brachial plexopathy. is given.

The administration subject may be a subject whose administration has been determined based on the radiation exposure dose. In this case, the exposure dose of the radiation to be administered may be the exposure dose that causes neuropathy or the exposure dose that is predicted to cause neuropathy. Specifically, the lower limit of the exposure dose is, for example, 0.1 Gy or more, 1 Gy or more, 2 Gy or more, 3 Gy or more, 4 Gy or more, 5 Gy or more, 6 Gy or more, 7 Gy or more, 8 Gy or more, 9 Gy or more, or 10 Gy or more. is. The upper limit of the exposure dose is, for example, 100 Gy or less, 50 Gy or less, 40 Gy or less, 30 Gy or less, 20 Gy or less, or 15 Gy or less. The numerical range of the exposure dose can be, for example, any combination of the upper limit and the lower limit, and specific examples are 0.1 to 100 Gy, 1 to 100 Gy, 2 to 100 Gy, 3 to 50 Gy, 4 to 50 Gy, 6-40 Gy, 7-40 Gy, 8-30 Gy, 9-20 Gy, or 10-15 Gy. The exposure dose may be an actually measured value or an estimated value. The radiation exposure dose may be the total amount of radiation exposure dose in a predetermined period. In this case, the total radiation exposure dose may be a single radiation exposure dose or a total radiation exposure dose for a plurality of times. The predetermined period is preferably a period during which acute radiation-induced symptoms occur, for example, up to 6 weeks before the determination of administration (for example, Non-Patent Document 1). The radiation exposure may be, for example, radiation therapy exposure, occupational accident exposure, accident exposure, terrorism exposure, or the like. The site of radiation exposure in the administration subject may be a part of the body or the whole body. The part of the body is, for example, a part containing nerves, preferably the head containing the brain, the back containing the spinal cord, and the like.

The usage conditions (administration conditions) of the cell preparation of the present disclosure are not particularly limited, and for example, the dosage form, administration period, dosage, etc. can be appropriately set according to the type of administration subject.

Examples of administration methods of the cell preparation of the present disclosure include intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, and intravenous administration. Intravenous administration is preferred because it can be administered rapidly and stably.

The dosage of the cell preparation of the present disclosure is such that when the cell preparation is administered to the administration subject, compared to when the cell preparation is not administered to the administration subject, the therapeutic effect of neuropathy, the nerve of mature neuron cells It is the amount of cells (therapeutically effective amount) capable of obtaining the effect of suppressing process regression, the effect of suppressing cell death of immature neuronal cells, the effect of suppressing the generation of reactive oxygen in neuronal cells, or the effect of suppressing necrosis of neuronal cells. The dosage can be appropriately determined according to, for example, the age, body weight, symptoms, etc. of the subject. As a specific example, the dose is, for example, 10 ⁴ to 10 ⁹ cells/kg body weight, 10 ⁴ to 10 ⁸ cells/kg body weight, 10 ⁴ to 10 ⁷ cells/kg as the number of umbilical cord-derived cells per administration. body weight, preferably 10 ⁴ to 10 ⁸ cells/kg body weight, 10 ⁴ to 10 ⁷ cells/kg body weight.

The number of administrations of the cell preparation of the present disclosure is one or more. The plurality of times is, for example, 2 times, 3 times, 4 times, 5 times or more. The frequency of administration may be determined as appropriate while confirming the effect of treatment on the subject. When administering multiple times, the administration interval can be determined appropriately while confirming the therapeutic effect of the subject, for example, once a day, once a week, once every two weeks, once a month, once every three months. , once every six months, etc.

The cell preparations of the present disclosure may be used in combination with agents and/or methods used for other neurological disorders, for example. Examples of drugs used for the neuropathy include steroids such as dexamethasone; osmotic diuretics such as glyceol; anticonvulsants; Methods used for the neuropathy include, for example, hyperbaric oxygen therapy.

The cell preparations of the present disclosure can treat radiation-induced neuropathy, as described above. As such, the present disclosure may include methods of treating a subject with radiation-induced neuropathy. In this case, the present disclosure is a method of treating a subject with radiation-induced neuropathy using the cell preparation of the present disclosure in the subject. A method of treating a subject with radiation neuropathy of the present disclosure comprises, for example, administering to the subject the cell preparation of the present disclosure. The above description can be used for the administration conditions in the administration step.

<Application of cell preparation>
In another aspect, the present disclosure provides a cell preparation for use in inhibiting radiation-induced neurite retraction of mature neuronal cells or a method for inhibiting radiation-induced neurite retraction of mature neuronal cells. In this case, the present disclosure is a cell preparation for use in inhibiting radiation-induced neurite retraction of mature neuronal cells, wherein the cell preparation comprises umbilical cord-derived cells. The present disclosure also provides a method for suppressing neurite retraction of mature neuron cells caused by radiation, wherein a cell preparation used in the method for suppressing neurite retraction of mature neuron cells caused by radiation of the present disclosure is used as a subject. . The present disclosure can incorporate the description of the cell preparation of the present disclosure above.

In another aspect, the present disclosure provides a cell preparation for use in inhibiting radiation-induced immature neuronal cell death or a method for inhibiting radiation-induced immature neuronal cell death. In this case, the disclosure is a cell preparation for use in inhibiting radiation-induced cell death of immature neuronal cells, said cell preparation comprising umbilical cord-derived cells. The present disclosure also provides a method for suppressing radiation-induced immature neuronal cell death, wherein a cell preparation used in the method for suppressing radiation-induced immature neuronal cell death of the present disclosure is used as a subject. . The present disclosure can incorporate the description of the cell preparation of the present disclosure above.

In another aspect, the present disclosure provides a cell preparation for use in suppressing generation of reactive oxygen species in neuronal cells caused by radiation or a method for suppressing generation of reactive oxygen species in neuronal cells caused by radiation. In this case, the present disclosure is a cell preparation used for suppressing generation of reactive oxygen in neuronal cells caused by radiation, wherein the cell preparation contains umbilical cord-derived cells. The present disclosure also provides a method for suppressing the generation of reactive oxygen in neuronal cells caused by radiation, and uses a cell preparation used in the method for suppressing the generation of reactive oxygen in neuronal cells caused by radiation of the present disclosure as an object. The present disclosure can incorporate the description of the cell preparation of the present disclosure above.

In another aspect, the present disclosure provides a cell preparation for use in inhibiting radiation-induced necrosis of neuronal cells or a method of inhibiting radiation-induced neuronal cell necrosis. In this case, the present disclosure is a cell preparation for use in inhibiting radiation-induced necrosis of neuronal cells, said cell preparation comprising umbilical cord-derived cells. The present disclosure also provides a method of inhibiting radiation-induced necrosis of neuronal cells, wherein the cell preparation used in the method of inhibiting radiation-induced necrosis of neuronal cells of the present disclosure is used as a subject. The present disclosure can incorporate the description of the cell preparation of the present disclosure above.

In another aspect, the present disclosure provides cell preparations for use in suppressing radiation-induced nervous system inflammation or methods of suppressing radiation-induced nervous system inflammation. In this case, the disclosure is a cell preparation for use in suppressing radiation-induced nervous system inflammation, wherein the cell preparation comprises umbilical cord-derived cells. The present disclosure also provides a method of suppressing inflammation of the nervous system caused by radiation, wherein the cell preparation used in the method of suppressing inflammation of the nervous system caused by radiation of the present disclosure is used as a subject. The present disclosure can incorporate the description of the cell preparation of the present disclosure above.

Each of the above-described aspects can be preferably applied to, for example, human nervous system or human central nervous system neuronal cells, immature neuronal cells, or mature neuronal cells.

<Treatment method>
In another aspect, the present disclosure provides a treatable method of radiation neuropathy. The method of treating neuropathy by radiation according to the present disclosure (hereinafter also referred to as "treatment method") includes an administration step of administering the cell preparation of the present disclosure to a subject (administration subject). The treatment method of the present disclosure is characterized by administering the cell preparation of the present disclosure, and other steps and conditions are not particularly limited. Since the treatment method of the present disclosure uses the cell preparation of the present disclosure, suppression of neurite retraction of mature neuron cells caused by radiation, suppression of cell death of immature neuron cells caused by radiation, and suppression of cell death of immature neuron cells caused by radiation and/or suppress necrosis of neuronal cells caused by radiation. Therefore, the treatment method of the present disclosure can be suitably used for treatment of radiation-induced neuropathy. Further, the method for treating radiation neuropathy of the present invention is a treatment method for prophylactic or therapeutic treatment of radiation neuropathy, comprising the step of administering the therapeutic agent for radiation neuropathy of the present invention to an administration subject. include. The administration subject may be an administration subject including humans or an administration subject other than humans.

<Use of cell preparation>
The present disclosure provides treatment of neuropathy by radiation, suppression of neurite retraction of mature neuron cells caused by radiation, suppression of cell death of immature neuron cells caused by radiation, suppression of generation of active oxygen in neuronal cells caused by radiation, and A cell preparation for use in inhibiting radiation-induced necrosis of neuronal cells, said cell preparation comprising umbilical cord-derived cells. The present disclosure provides treatment of neuropathy by radiation, suppression of neurite retraction of mature neuron cells caused by radiation, suppression of cell death of immature neuron cells caused by radiation, suppression of generation of active oxygen in neuronal cells caused by radiation, and and/or the use of umbilical cord-derived cells to manufacture cell preparations for use in inhibiting radiation-induced necrosis of neuronal cells. The present disclosure can incorporate the description of the cell preparation of the present disclosure above. A use of the present invention is also the use of a cell preparation of the present disclosure for use in treating radiation-induced neuropathy.

The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples. Unless otherwise indicated, commercially available reagents, kits, etc. were used according to their protocols.

[Example 1]
It was confirmed that the umbilical cord-derived cells of the present invention can reduce radiation damage to the brain.

(1) Preparation of umbilical cord-derived mesenchymal stem cells Umbilical cord mesenchymal stem cells (UC-MSCs) were collected by the method described in Cytotherapy, 18, 229-241, 2016. Specifically, with the approval of the ethics committee of the Institute of Medical Science, the University of Tokyo, all tissue elements of the umbilical cord (amniotic membrane, blood vessels, perivascular tissue, and Walton's jelly) collected with the consent of the donor. ) were chopped into 1-2 mm ³ pieces and seeded onto culture dishes. Then, it was covered with cell amigo (manufactured by Tsubakimoto Chain Co., Ltd.) and placed in α-minimal essential medium (αMEM) (manufactured by Fuji Film Co., Ltd.) to which 10% fetal bovine serum (FBS) (manufactured by Gibco) and an antibiotic were added. UC-MSCs were obtained by a modified explant method of culturing. The property of the obtained UC-MSCs cells is that they adhere to plastic.

(2) Confirmation of surface antigen Next, the presence or absence of surface antigen expression was confirmed for the obtained UC-MSCs. The presence or absence of expression of the surface antigen was confirmed by FACS analysis using a specific antibody against each antigen. bottom. As surface antigens of UC-MSCs, CD73, CD105, CD90, CD44 and HLA-class I were positive, and HLA-Class II, CD34, CD45, CD19, CD80, CD86, CD40 and CD11b were negative. In addition, HLA-G5 and PD-L2 were positive. In addition, HLA-G was weakly positive, CD49d (ITGA4) and CD184 (CXCR4) were negative to weakly positive, and CD29 (ITGB1) was positive.

In addition, UC-MSCs highly express the HGF (Hepatic Growth Factor) gene under normal conditions, and the IDO (Indoleamine 2,3-dioxygenase) gene under inflammatory conditions (IFN-γ 100 ng/ml). Induction of expression was confirmed by Realtime PCR. In particular, HGF expression was found to be higher in UC-MSCs than in bone marrow-derived mesenchymal stem cells. In addition, it was confirmed by ELISA that PGE2 secretion was induced by co-culturing UC-MSCs with MLR (mixed allogeneic lymphocyte reaction).

(3) Preparation of Cell Preparation The obtained UC-MSCs were seeded on a Corning (registered trademark) CellBIND (registered trademark) surface 100 mm dish (manufactured by Corning, Product Number: #3292) and subjected to Passage 1 to Passage 4. Passed and propagated. In addition, the culture medium during the subculture is CiMS (trademark)-BM (Nipro Co., Ltd., product code: 87-070), which is a basic culture medium for human mesenchymal stem cells. CiMS (trademark)-sAF (Nipro Co., Ltd., product code: 87-072), which is an animal-free additive to the basal culture medium, was added at a ratio according to the package insert. Thereafter, TrypLE (trademark) Select Enzyme (1X), no phenol red (ThermoFisher, product number: ^12563011 ) was used to detach the cells, and the cell preparation and

(4) Primary Culture of Cortical Neuron Cells Cortical neuron cells were prepared from an albino strain of B6 strain background mice (B6N-Tyr ^c-Brd /BrdCrCrl, manufactured by Charles River Japan). Specifically, a 17-day-embryonic fetal mouse was obtained from a pregnant mouse, and the brain and cerebral cortex of the fetal mouse were removed under a microscope (manufactured by Olympus). The removed brain and cortex were used for cortical neuron cell primary culture using a nerve cell dispersion (manufactured by Wako Pure Chemical Industries, Ltd.). After isolation of cortical neuron cells, the cortical neuron cells were resuspended in Neurobasal medium supplemented with 2% B27 and seeded in Poly-D-Lysine Culture Dishes (BioCoat™, Corning). The cortical neuron cells were cultured at 37° C. and 5% CO ₂ , and half of the medium was replaced with fresh medium on day 3-5 of culture.

(5) Preparation of Cortical Neuron Cells for Radiation Injury Model cells for brain radiation injury were prepared using the cortical neuron cells obtained in Example 1(4). Specifically, the cortical neuron cells in the dish were irradiated with radiation. Using a γ-ray irradiation apparatus (IBL 437C III, manufactured by Cis Bio International) using cesium 137 as a radiation source, the cortical neuron cells were irradiated with a set dose of 12 Gy.

(6) Co-Culture of Irradiated Cortical Neuron Cells and UC-MSCs It was examined whether the above-mentioned cell preparation (UC-MSCs) reduces radiation damage to cortical neuron cells. Specifically, immediately after irradiation of the cortical neuron cells of Example 1 (5), the cortical neuron cells and the cell preparation (UC-MSCs) obtained in Example 1 (3) are co-cultured. bottom. A 24-well chamber (manufactured by Corning) with a 3 μm filter membrane attached to the top was used for the co-culture. The cortical neuron cells were seeded in the bottom dish of the 24-well chamber. The UC-MSCs were seeded at 1.5×10 ⁴ cells/well in the upper chamber. After the seeding, the 24-well chambers were incubated at 37°C, 5% _CO2 for 72 hours. Neurobasal medium was used as the medium for the co-culture.

(7) Evaluation of Cortical Neuron Cells by Immune Cell Staining It was examined by observing the morphology of cortical neuron cells whether co-culturing with UC-MSCs alleviates radiation damage to the cortical neuron cells. For the observation of the cortical neuron morphology obtained in Example 1(6), immune cell staining was used using nerve markers. Specifically, the cortical neuron cells were fixed with 4% paraformaldehyde at room temperature (approximately 25° C., hereinafter the same) for 20 minutes. After the fixation, the cortical neuron cells were permeabilized with 0.3% Triton-X100 and then blocked with 5% goat serum at room temperature for 60 minutes. After the blocking, the cortical neuron cells were allowed to react with the primary antibody at 4° C. overnight, and stained with the secondary antibody at room temperature for 1 hour. For nuclear staining, 4',6-diamidino-2-phenylindole (DAPI, manufactured by Sigma-Aldrich) was used and added to the secondary antibody solution. As the primary antibody, mouse anti-human MAP2 antibody (1000-fold dilution, Cat.No: MA5-12826, Thermo Fisher Scientific), rabbit anti-GAP43 antibody (200-fold dilution, Cat.No: 8945S , Cell Signaling Technology) was used. The secondary antibodies included Alexa Fluor R 488-labeled donkey anti-mouse IgG (H + L) (1000-fold dilution, Abcam) and Alexa Fluor R 594-labeled donkey anti-rabbit IgG (H + L) antibody (1000-fold dilution). Dilution, Abcam) and were used.

After the staining, cortical neuron cells were observed using a fluorescence microscope (Nikon Eclipse Ti, manufactured by Nikon Solutions) and microscope image integrated software (NIS-Elements software version 4.10). Under observation with a fluorescence microscope set at a magnification of 200, the number of immature neuron cell marker-positive cells and all cells present in 10 randomly selected fields (view fields) were counted. Then, the ratio of the immature neuron cell marker-positive cells to the total number of cells was calculated. Neurite length was measured using ImageJ. MAP2-positive neurites were measured for length in at least 10 randomly selected fields. The negative control group (NC) was performed in the same manner, except that cortical neuron cells that were not irradiated were used. Immunocyte staining and cell count were performed in the same manner except that neuron cells were used, the percentage of immature neuron cell marker-positive cells was calculated, and the neurite length of the MAP2-positive cells was calculated. It was measured. The length of the neurite was measured from the root to the tip of the neurite. If there were multiple such neurites, the longest neurite was used to measure neurite length. These results are shown in FIGS. 1-4.

Figure 1 is a graph showing the percentage of GAP43-positive cells. In FIG. 1, the vertical axis indicates the percentage of GAP43-positive cells, and the horizontal axis indicates the type of sample. As shown in FIG. 1, the percentage of immature neuronal cells in the radiation group (radiation) was significantly decreased compared to the negative control group (NC). In contrast, in the co-culture group with UC-MSCs (co-culture), the percentage of immature neuron cells was significantly higher than in the radiation group. From these results, it was found that immature neuronal cells are damaged by irradiation, but co-culture with UC-MSCs can reduce radiation damage to immature neuronal cells.

Figure 2 is a photograph showing an immunostained image of cortical neuron cells. In FIG. 2, the scale bar indicates 100 μm. As shown in Figure 2, the radiation group had reduced expression of immature neuronal cell markers. On the other hand, in the co-culture group with UC-MSCs, expression of immature neuron cell markers was observed as in the control group.

Fig. 3 is a graph showing the length of neurites. In FIG. 3, the vertical axis indicates the length of neurites, and the horizontal axis indicates the type of sample. As shown in Figure 3, the neurite length of mature neuronal cells in the radiation group was significantly reduced compared to the negative control group. In contrast, the neurite length of mature neuron cells was significantly longer in the co-culture group with UC-MSCs than in the radiation group. From these results, it was found that radiation injury to mature neuron cells is caused by irradiation, and that radiation injury to mature neuron cells can be alleviated by co-culturing with UC-MSCs.

Fig. 4 is a photograph showing an immunostained image of cortical neuron cells. In the photographs in each row in FIG. 4, the upper photograph shows a stained image of MAP2 (MAP-2), the middle photograph shows a stained image of GAP43 (GAP43), and the lower photograph shows a stained image of GAP43. and a stained image of MAP2 are superimposed (Merged). In addition, in the photographs of each column in FIG. 4, the photographs in the left column show the results of the negative control group (NC), the photographs in the middle row show the results of the radiation group (radiation), and the photographs in the right column show The results of the co-culture group (co-culture) are shown. In FIG. 4, the scale bar indicates 100 μm. As shown in FIG. 4, the radiation group had reduced expression of immature neuronal cell markers and mature neuronal cell markers. On the other hand, in the co-culture group with UC-MSCs, expression of immature neuron cell markers and mature neuron cell markers was observed as in the negative control group.

From the above, when cortical neuron cells are exposed to radiation, radiation damage such as reduction of immature neuron cells and retraction of neurites of mature neuron cells is induced. In contrast, co-culture of irradiated cortical neuron cells with UC-MSCs was found to alleviate the radiation injury.

(8) Evaluation of Cortical Neuron Cells by Measurement of Active Oxygen Content Radiation damage to cortical neuron cells is reduced by co-culturing with UC-MSCs. In order to examine whether the reduction in the radiation injury is due to the reduction in the amount of active oxygen, the amount of active oxygen was measured. Specifically, the active oxygen in cortical neuron cells obtained in Example 1(6) was measured using the DCFDA-Cellular ROS Detection Assay kit (Cat No. ab113851, manufactured by Abcam). When the cell-permeable fluorescent probe DCFDA (2',7'-Dichlorodihydrofluorescin diacetate, also referred to as "H2DCFDA", "DCFH-DA" or "DCFH") reacts with active oxygen, fluorescence observation becomes possible. 20 mmol/l DCFDA was diluted to a final concentration of 20 μmol/l with 1×buffer from the kit. The cortical neuron cells obtained in Example 1(6) were cultured in the presence of the diluted DCFDA solution at 37° C. for 45 minutes in the dark. After the culture, the DCFDA solution was removed, and the 1×buffer was added at 20 μl/well. After the addition, the cortical neuron cells were washed two more times with the 1×buffer. After the washing, cortical neuron cells were observed using the fluorescence microscope and the integrated software for microscopic images. The number of fluorescent probe-positive cells and the total number of cells were counted in 10 randomly selected fields (fields of view) under observation with a fluorescence microscope set at a magnification of 200 times. Then, the ratio of the fluorescent probe-positive cells to the total number of cells was calculated. For the negative control group (NC), cortical neuron cells that were not irradiated were used in the same manner, and for the comparative example, the radiation group (radiation) was co-cultured with UC-MSCs after irradiation. The percentage of fluorescent probe-positive cells was calculated in the same manner, except that cortical neuron cells without cytotoxicity were used. These results are shown in FIGS. 5-6.

FIG. 5 is a photograph showing a fluorescent image and a bright field image of cortical neuron cells. In FIG. 5, the upper photograph shows the fluorescence image, and the lower photograph shows the bright field image. In FIG. 5, the scale bar indicates 100 μm. As shown in FIG. 5, in the radiation group, the number of fluorescent probe-positive cells, which is an indicator of active oxygen, was greater than in the negative control group. In contrast, there was no difference in the number of fluorescent probe-positive cells, which is an indicator of active oxygen, between the co-culture group with UC-MSCs and the negative control group. Also, in the co-culture group with UC-MSCs, there were fewer fluorescent probe-positive cells than in the radiation group.

FIG. 6 is a graph showing the ratio of fluorescent probe-positive cells, which is an index of active oxygen. In FIG. 6, the vertical axis indicates the percentage of fluorescent probe-positive cells, and the horizontal axis indicates the type of sample. As shown in FIG. 6, the proportion of fluorescent probe-positive cells in the radiation group was significantly higher than that in the negative control group. In contrast, in the co-culture group with UC-MSCs, the percentage of fluorescent probe-positive cells was significantly lower than in the radiation group. From these results, it was found that the generation of reactive oxygen increased in irradiated cortical neuron cells, whereas the generation of reactive oxygen could be suppressed by co-culturing with UC-MSCs.

(9) Evaluation of cell death of cortical neuron cells Radiation injury of cortical neuron cells is reduced by co-culturing with UC-MSCs. Cell death was measured to determine whether the reduction in radiation injury was due to reduced cell death of cortical neuronal cells. Specifically, viable cortical neuron cells, apoptotic cells, and necrotic cells obtained in Example 1 (6) were separated using an Apoptotic/Necrotic/Healthy Cells Detection Kit (manufactured by Promokine), The percentage of the above cells was calculated. Apoptotic cells of the cortical neuron cells were identified by Annexin V staining. Necrotic and late apoptotic (secondary necrotic) cells of the cortical neuronal cells were distinguished by ethidium homodimer III staining. Hoechst33342 was used for nuclear staining. The 5x binding buffer of the kit was diluted with distilled water to 1x binding buffer, and the cortical neuron cells obtained in Example 1(6) were washed with the 1x binding buffer. After the washing, 5 μl of FITC-annexin V, 5 μl of ethidium homodimer III, and 100 μl of the 1×Binding buffer were added to the cortical neuron cells. The cortical neuron cells were then incubated at room temperature for 15 minutes in the dark. After the culture, cortical neuron cells were observed using the fluorescence microscope and the integrated software for microscopic images. The number of viable cells, the number of apoptotic cells, and the number of necrotic cells were counted in 10 randomly selected fields (fields of view) under observation with a fluorescence microscope set at 200x magnification. The percentages of viable, apoptotic, and necrotic cells were then calculated. In the control group (NC), cortical neuron cells that were not irradiated were used in the same manner, and in the comparative radiation group (radiation), co-culture with UC-MSCs was not performed after irradiation. Percentages of viable, apoptotic, and necrotic cells were calculated in the same manner, except cortical neuronal cells were used. These results are shown in FIGS. 7-8.

FIG. 7 is a photograph showing a fluorescent image and a bright field image of cortical neuron cells. The upper photographs show fluorescence images, and the lower photographs show bright field images. In FIG. 7, the scale bar indicates 100 μm. Arrows in the figure indicate necrotic cells. As shown in FIG. 7, in the radiation group, there were more fluorescent probe-positive cells, which are indicators of necrosis, compared to the negative control group (NC). On the other hand, in the co-culture group with UC-MSCs, the number of fluorescent probe-positive cells, which is an indicator of necrosis, was smaller than in the radiation group. Therefore, it was found that UC-MSCs can suppress cell death of cortical neuron cells.

FIG. 8 is a graph showing the percentage of viable cells, apoptotic cells, and necrotic cells. In FIG. 8, the vertical axis indicates the type of sample, and the horizontal axis indicates the ratio of viable cells, apoptotic cells, and necrotic cells. As shown in FIG. 8, the ratio of surviving cells was significantly lower in the radiation group and co-culture group than in the negative control group (NC). However, the percentage of viable cells was significantly higher in the co-culture group compared to the radiation group. Furthermore, the proportion of necrotic cells was significantly higher in the radiation group and co-culture group than in the negative control group. However, the percentage of viable cells was significantly lower in the co-culture group than in the radiation group. From these results, it was found that irradiated cortical neuron cells decreased viable cells due to necrosis, whereas co-culture with UC-MSCs could suppress necrosis and suppress the decrease in viable cells. .

(10) Generation of Radiation Injury Model Mice For generation of radiation injury model mice, 9-week-old albino strains of B6 strain background mice (B6N-Tyr ^c-Brd /BrdCrCrl, manufactured by Charles River Japan) were used. Specifically, a radiation injury model mouse was prepared by irradiating the mouse. Using an X-ray irradiator (MBR-1520R-3, manufactured by Hitachi, Ltd.), the mice were irradiated three times at a set dose of 6 Gy to give a total radiation dose of 18 Gy.

(11) Preparation of mouse model for treatment of radiation injury by UC-MSCs It was confirmed whether US-MSCs could suppress radiation injury in a mouse model of radiation injury. Specifically, UC-MSCs were intravenously injected into the mice at 1×10 ⁶ cells/mouse 24 hours after irradiation in Example 1 (10). The UC-MSCs contain dibenzyl azodicarboxylate as a cryoprotectant. For this reason, the negative control group (NC) was not irradiated and was injected with sterile dibenzyl azodicarboxylate. The comparative radiation group (Radiation) received an injection of sterile dibenzyl azodicarboxylate 24 hours after the irradiation of Example 1 (10). Upon injection of the mice, anesthesia used medetomidine in the amount of 0.75 mg/kg, midazolam in the amount of 4.0 mg/kg, and butorphanol in the amount of 5.0 mg/kg. The mice were used 3 weeks after injection of the UC-MSCs.

(12) Evaluation of Cortical Neuron Cells by Gene Expression Whether administration of UC-MSCs to radiation-damaged mice alleviates radiation injury in brain tissue was examined by expression of inflammatory cytokine genes. Specifically, the brain tissue of each group of mice obtained in Example 1 (11) was collected, and TRI Reagent (manufactured by Invitrogen) and chloroform (manufactured by Wako Pure Chemical Industries, Ltd.) were used to treat the brain tissue. Total RNA was extracted. Using the obtained total RNA and SYBR (registered trademark) Green PCR Mix (manufactured by Takara Bio Inc.), cDNA was synthesized. Then, a reaction solution prepared from the cDNA, the following primer set, and an RT-PCR kit (manufactured by Takara Bio Inc.) was measured using a real-time PCR measuring device (PIKOREAL96, manufactured by Thermo Fisher Scientific) to measure IFN-γ, Gene expression levels of IL-12, IL-1β, IL-6, and TNFα were measured. GAPDH was used as an internal standard gene, and the expression level of each gene was calculated as a relative expression level with respect to the expression level of the internal standard gene. These results are shown in FIG.

・ IFN-γ primer set Forward primer (SEQ ID NO: 1)
5'-AGGAACTGGCAAAAGGATGGT-3'
Reverse primer (SEQ ID NO: 2)
5'-ACGCTTATGTTGTTGCTGATGG-3'
・ Primer set for IL-12 forward primer (SEQ ID NO: 3)
5'-GGAAGCACGGCAGCAGAATA-3'
Reverse primer (SEQ ID NO: 4)
5'-AACTTGAGGGAGAAGTAGGAATGG-3'
・ Primer set for IL-1β Forward primer (SEQ ID NO: 5)
5'-GCCCATCCTCTGTGACTCAT-3'
Reverse primer (SEQ ID NO: 6)
5'-AGGCCACAGGTATTTTGTCG-3'
・ Primer set for IL-6 forward primer (SEQ ID NO: 7)
5'-AGTTGCCTTCTTGGGACTGA-3'
Reverse primer (SEQ ID NO: 8)
5'-TCCACGATTTCCCAGAGAAC-3'
・Primer set for TNFα Forward primer (SEQ ID NO: 9)
5'-CGTCAGCCGATTTGCTATCT-3'
Reverse primer (SEQ ID NO: 10)
5'-CGGACTCCGCAAAGTCTAAG-3'
・ GAPDH primer set Forward primer (SEQ ID NO: 11)
5'-CACTGAGCATCTCCCTCACA-3'
Reverse primer (SEQ ID NO: 12)
5'-GTGGGTGCAGCGAACTTTAT-3'

FIG. 9 is a graph showing the expression level of each gene. In FIG. 9, (A) shows the results of IFN-γ, (B) shows the results of IL-12, (C) shows the results of IL-1β, and (D) shows the results of IL- 6, and (E) shows the results for TNFα. In FIGS. 9A to 9E, the vertical axis indicates the expression level of each gene, and the horizontal axis indicates the type of sample. As shown in FIG. 9, gene expression levels of inflammatory cytokines in the radiation group (radiation) were significantly increased compared to the negative control group (NC). In contrast, the treated mouse group (radiation + MSCs) injected with UC-MSCs had significantly lower gene expression levels of inflammatory cytokines compared to the radiation group. These results show that the gene expression of inflammatory cytokines is enhanced in irradiated mouse brain tissue, whereas the administration of UC-MSCs can suppress the gene expression of inflammatory cytokines. .

(13) Evaluation of cortical neuron cells by tissue staining Examination of whether radiation injury of brain tissue is reduced by administering UC-MSCs to radiation-damaged mice, and the presence or absence of demyelination of myelin marrow by tissue staining with KB staining bottom. Specifically, brain tissue was collected from each group of mice obtained in Example 1 (11) and fixed with 10% or 20% formalin. After the fixation, the brain tissue was embedded in paraffin to prepare 6 μm-thick paraffin-embedded sections. The sections were deparaffinized and hydrated with 95% alcohol. After the hydration, the sections were stained in Luxol fast blue solution (manufactured by CHROMA) at 75° C. for 5 minutes. After the staining, excess staining was washed off using 95% alcohol and then washed with sterilized water. After the washing, the sections were fractionated with an aqueous lithium carbonate solution (Cat. No: 122-01132, Wako Pure Chemical Industries, Ltd.), further fractionated with 70% alcohol, and placed in sterile water. After that, the sections were counterstained with cresyl violet acetate (manufactured by CHROMA). After the counterstaining, the cells were washed three times with 95% alcohol and quickly washed with 100% ethanol. Thereafter, mouse brain tissue was observed using the fluorescence microscope and the integrated software for microscopic images. These results are shown in FIG.

FIG. 10 is a photograph showing a histological staining image of mouse brain tissue, showing the thickness of the myelin sheath (myelin sheath). In FIG. 10, in each row of photographs, the upper photograph shows a stained image at a magnification of 40 times, and the lower photograph shows a stained image at a magnification of 200 times. As shown in FIG. 10, the radiation group (radiation) had demyelinating myelin sheaths compared to the negative control group (NC). In contrast, in the group of treated mice injected with UC-MSCs (radiation + MSCs), demyelination of the myelin sheath as in the radiation group was not observed, and the thickness of the myelin sheath was similar to that of the negative control group. From these results, it was found that the demyelination of the myelin sheath is suppressed in the irradiated mouse brain tissue, whereas the administration of UC-MSCs can suppress the demyelination of the myelin sheath.

From the above, it was found that the umbilical cord-derived cells can reduce brain radiation damage such as demyelination. In addition, the umbilical cord-derived cells suppress neurite retraction of the neuronal cells, suppress generation of reactive oxygen in the neuronal cells, suppress cell death of the immature neuronal cells, and suppress the neuron cells after the irradiation. It was presumed that this was due to suppression of necrosis, etc. However, the present disclosure is in no way limited by the presumption.

Although the present invention has been described with reference to the embodiments, the present invention is not limited to the above embodiments. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

<Appendix>
Some or all of the above-described embodiments and examples can be described as in the following appendices, but are not limited to the following.
<Cell preparation used for treatment of radiation-induced neuropathy>
(Appendix 1)
A cell preparation for the treatment of radiation neuropathy, comprising:
A cell preparation, wherein the cell preparation comprises umbilical cord-derived cells.
(Appendix 2)
The cell preparation of paragraph 1, wherein the umbilical cord-derived cells are umbilical cord-derived mesenchymal cells.
(Appendix 3)
The umbilical cord-derived cells are
(i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2; 2. Cell preparation according to 2.
(Appendix 4)
The umbilical cord-derived cells are
(iii) A cell preparation according to any one of Appendices 1 to 3, wherein the expression of the gene and/or protein of any one of IDO, PGE2, PD-L1 is induced under conditions of inflammation.
(Appendix 5)
5. A cell preparation according to any one of Appendices 1 to 4, wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue comprising amnion, blood vessels, perivascular tissue and/or Walton's Jelly.
(Appendix 6)
6. The cell preparation of Appendix 5, wherein the umbilical cord-derived cells are cells prepared from the umbilical cord tissue substantially free of proteases (e.g., collagenase, dispase, etc.) (without degradation by proteases).
(Appendix 7)
7. The cell preparation according to

appendix

5 or 6, wherein the umbilical cord-derived cells are adherent cells obtained by cutting the umbilical cord tissue into pieces and culturing the pieces.
(Appendix 8)
8. A cell preparation according to any one of Supplements 1 to 7, wherein said cell preparation comprises 1×10 ⁶ to 1×10 ⁹ umbilical cord-derived cells.
(Appendix 9)
9. A cell preparation according to any one of Supplements 1 to 8, wherein said cell preparation comprises an extract and/or secretion of said umbilical cord-derived cells.
(Appendix 10)
10. A cell preparation according to any one of Appendices 1 to 9, which is for intravenous administration.
(Appendix 11)
11. A cell preparation according to any one of Appendices 1 to 10, which inhibits neurite retraction of mature neuronal cells caused by irradiation.
(Appendix 12)
12. The cell preparation according to any one of Appendices 1 to 11, which suppresses cell death of immature neuronal cells caused by irradiation.
(Appendix 13)
13. The cell preparation according to any one of Appendices 1 to 12, which suppresses generation of reactive oxygen in neuronal cells caused by irradiation.
(Appendix 14)
14. A cell preparation according to any one of Appendices 1 to 13, which inhibits necrosis of neuronal cells caused by irradiation.
(Appendix 15)
15. A cell preparation according to any of Supplements 1 to 14, wherein said nerve is a central nerve.
(Appendix 16)
16. A cell preparation according to any of Supplements 1 to 15, wherein said nerve is the brain or the spinal cord.
(Appendix 17)
The radiation-induced neuropathy consists of radiation encephalopathy, radiation neuritis, radiation myelopathy, radiation brain necrosis, radiation optic nerve, leukoencephalopathy, hypoglossal nerve palsy, facial nerve palsy, trigeminal neuropathy, and radiation-induced brachial plexopathy. 17. A cell preparation according to any one of appendices 1 to 16, selected from the group.
<Application of cell preparation>
(Appendix 18)
A cell preparation for suppressing neurite retraction of mature neuronal cells caused by radiation,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 19)
A method for suppressing neurite retraction of mature neuron cells caused by radiation, comprising:
19. A method of using the cell preparation of Supplementary Note 18 in a subject.
(Appendix 20)
20. The method of paragraph 19, comprising administering said cell preparation to said subject.
(Appendix 21)
21. The method of paragraphs 19 or 20, wherein said cell preparation is used in vitro or in vivo.
(Appendix 22)
A cell preparation for use in suppressing radiation-induced cell death of immature neuronal cells, comprising:
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 23)
A method for suppressing cell death of immature neuronal cells caused by radiation, comprising:
A method of using the cell preparation of Supplementary Note 22 in a subject.
(Appendix 24)
24. The method of Clause 23, comprising administering said cell preparation to said subject.
(Appendix 25)
25. The method of paragraphs 23 or 24, wherein said cell preparation is used in vitro or in vivo.
(Appendix 26)
A cell preparation used to suppress the generation of reactive oxygen in neuronal cells caused by radiation,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 27)
A method for suppressing the generation of reactive oxygen in neuronal cells caused by radiation, comprising:
A method of using the cell preparation of Supplementary Note 26 in a subject.
(Appendix 28)
28. The method of Clause 27, comprising administering said cell preparation to said subject.
(Appendix 29)
29. The method of paragraphs 27 or 28, wherein said cell preparation is used in vitro or in vivo.
(Appendix 30)
A cell preparation for use in inhibiting radiation-induced necrosis of neuronal cells, comprising:
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 31)
A method for suppressing the occurrence of neuronal necrosis caused by radiation, comprising:
A method of using the cell preparation of Supplementary Note 30 in a subject.
(Appendix 32)
32. The method of paragraph 31, comprising administering said cell preparation to said subject.
(Appendix 33)
33. The method of paragraphs 31 or 32, wherein said cell preparation is used in vitro or in vivo.
(Appendix 34)
A cell preparation for use in suppressing radiation-induced nervous system inflammation, comprising:
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 35)
A method of inhibiting inflammation of the nervous system caused by radiation, comprising:
A method of using the cell preparation of Supplementary Note 34 in a subject.
(Appendix 36)
36. The method of paragraph 35, comprising administering said cell preparation to said subject.
(Appendix 37)
37. The method of paragraphs 35 or 36, wherein said cell preparation is used in vitro or in vivo.
<Treatment method>
(Appendix 38)
A method of treating neuropathy with radiation, comprising:
18. A method, wherein the subject uses a cell preparation according to any one of Appendices 1-17 and/or an umbilical cord-derived cell according to any one of Appendices 1-17.
(Appendix 39)
385. The method of Clause 384, comprising administering said cell preparation and/or umbilical cord-derived cells to said subject.
(Appendix 40)
40. A method according to Appendix 38 or 39, wherein said cell preparation is used in vitro or in vivo.
<Use of cell preparation>
(Appendix 41)
A cell preparation for use in the treatment of radiation neuropathy,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 42)
A cell preparation for use in inhibiting neurite retraction of mature neuronal cells caused by radiation,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 43)
A cell preparation for use in suppressing radiation-induced cell death of immature neuronal cells,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 44)
A cell preparation for use in suppressing generation of reactive oxygen species in neuronal cells caused by radiation,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 45)
A cell preparation for use in inhibiting necrosis of neuronal cells caused by radiation,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.
(Appendix 46)
A cell preparation for use in suppressing radiation-induced nervous system inflammation,
18. A cell preparation comprising a cell preparation according to any one of Appendices 1 to 17 and/or an umbilical cord-derived cell according to any one of Appendices 1 to 17.

As described above, according to the present invention, it is possible to suppress neurite retraction of mature neuron cells caused by irradiation, suppress cell death of immature neuronal cells caused by irradiation, and reduce active oxygen in neuronal cells caused by irradiation. It can suppress development, suppress necrosis of neuronal cells caused by irradiation, and suppress inflammation of the nervous system caused by irradiation. Therefore, according to the present invention, it can be said that radiation-induced neuropathy can be treated. Therefore, the present invention is extremely useful in, for example, the medical field.

This application claims priority based on Japanese Patent Application No. 2021-162249 filed on September 30, 2021, and incorporates all of its disclosure herein.

Claims

A cell preparation for the treatment of radiation neuropathy, comprising:
A cell preparation, wherein the cell preparation comprises umbilical cord-derived cells.
2. The cell preparation of claim 1, wherein said umbilical cord-derived cells are umbilical cord-derived mesenchymal cells.
The umbilical cord-derived cells are
(i) positive for CD105, CD73, CD90, CD44, HLA-class I, HLA-G5 and PD-L2; and (ii) negative for CD45, CD34, CD11b, CD19 and HLA-Class II. or the cell preparation according to 2.
The umbilical cord-derived cells are
(iii) The cell preparation of any one of claims 1 to 3, wherein expression of any one of IDO, PGE2, PD-L1 gene and/or protein is induced under conditions of inflammation.
5. The cell preparation of any one of claims 1-4, wherein the umbilical cord-derived cells are cells prepared from umbilical cord tissue, including amniotic membrane, blood vessels, perivascular tissue and/or Walton's Jelly.
6. The cell preparation of any one of claims 1-5, wherein the cell preparation comprises 1 x 106 to 1 x 109 umbilical cord-derived cells.
7. The cell preparation of any one of claims 1-6, wherein said cell preparation comprises an extract and/or secretion of said umbilical cord-derived cells.
8. The cell preparation according to any one of claims 1 to 7, which is for intravenous administration.
9. The cell preparation according to any one of claims 1 to 8, which inhibits neurite retraction of mature neuronal cells caused by irradiation.
10. The cell preparation according to any one of claims 1 to 9, which suppresses cell death of immature neuronal cells caused by irradiation.
11. The cell preparation according to any one of claims 1 to 10, which suppresses generation of active oxygen in neuronal cells caused by irradiation.
12. The cell preparation according to any one of claims 1 to 11, which inhibits necrosis of neuronal cells caused by irradiation.
13. The cell preparation of any one of claims 1-12, wherein the nerve is the central nerve.
14. The cell preparation of any one of claims 1-13, wherein the nerve is the brain or spinal cord.
The radiation-induced neuropathy consists of radiation encephalopathy, radiation neuritis, radiation myelopathy, radiation brain necrosis, radiation optic nerve, leukoencephalopathy, hypoglossal nerve palsy, facial nerve palsy, trigeminal neuropathy, and radiation-induced brachial plexopathy. 15. A cell preparation according to any one of claims 1 to 14, selected from the group.
16. A therapeutic agent for radiation neuropathy for therapeutic or prophylactic treatment of radiation neuropathy, comprising the cell preparation according to any one of claims 1 to 15.
17. A treatment method for therapeutic or prophylactic treatment of radiation neuropathy, comprising the administration step of administering the therapeutic agent for radiation neuropathy according to claim 16 to an administration subject.
16. Use of the cell preparation according to claims 1-15 for treating radiation-induced neuropathy.