WO2019059741A1 - Stem cell dosage formulation containing cerebrospinal fluid and method for producing same - Google Patents

Abstract

The present invention relates to a stem cell dosage formulation and a method for producing the same. The stem cell dosage formulation and the method for producing the same according to one aspect of the present invention are safe in terms of the immune system and also have the effect in which gene or protein expression of a stem cell is changed in a patient-customized manner while retaining stem cell properties, or gene or protein expression is further activated.

Description

Stem cell dosage formulations containing cerebrospinal fluid and method for producing the same

A stem cell dosage form and a method for producing the same.

Stem cells are cells capable of self-replication and capable of differentiating into two or more cells. They are totipotent stem cells, pluripotent stem cells, multipotential stem cells multipotent stem cells).

A totipotent stem cell is an all-purpose cell that can develop into a complete individual. Cells from oocyte to spermatozoa until 8th gestation have these properties. Can be generated as a complete entity. Pluripotent stem cells are cells that can be formed in various cells and tissues derived from ectoderm, mesoderm, and endodermal layer. The inner cell mass located inside the blastocyst after 4-5 days of fertilization ), Which is called an embryonic stem cell and differentiates into a variety of different tissue cells but does not form new life forms. Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs containing the cells.

The stem cells, which have been able to be obtained in large quantities, are increasingly used as a cell therapy agent for injecting stem cells themselves for medical purposes, including treatment of incurable diseases, and beauty and molding.

As a conventional technique for preserving stem cells, there is a technique of suspending human adipose tissue-derived mesenchymal stem cells in physiological saline under refrigeration conditions. However, in this case, it is difficult to achieve survival rate of 70% or more when stored for 48 hours or more. In addition, in the case of a conventional stem cell treatment agent, a culture solution or the like was used for administration to a patient. However, it is difficult to precisely analyze the constituents, the proportion of the additives, and the effect on the cells, thus posing a problem in verification of safety.

As a result of efforts to develop a dosage form of stem cells, the present inventors have found that the patient-derived body fluid is the same as the constituent components in the body, and in particular, the body fluid of the patient is not only safe from immunity, The present inventors completed the present invention by confirming that gene or protein expression of a stem cell is changed or expression of a gene or protein is further activated in a patient-customized manner.

One aspect is to provide a stem cell dosage formulation comprising CSF.

Another aspect is the use of CSF for the manufacture of a stem cell dosage form.

Another aspect is to provide a method for treating a disease comprising administering to a subject a formulation comprising cerebrospinal fluid and stem cells.

Another aspect is the use of CSF and stem cells for use in the manufacture of a medicament for the treatment of disease.

Another aspect of the present invention is to provide a method for producing a patient-tailored stem cell, which comprises the step of retaining stem cells in cerebrospinal fluid, or a method for producing a stem cell dosage form.

One aspect is to provide a stem cell dosage formulation comprising CSF.

Another aspect is the use of CSF for the manufacture of a stem cell dosage form.

Specifically, the formulation is a cerebrospinal fluid; And a stem cell dosage form containing stem cells suspended in the cerebrospinal fluid.

As used herein, the term " cerebrospinal fluid " may refer to body fluids present in the brain or spinal cord. Specifically, it may be a liquid that circulates in the brain and spinal cord along the ventricle and sub arachnoid space produced in the brain. It is produced in the cerebral cortex and is decomposed and absorbed by the spider membrane granules in the subterranean space, so that a constant amount is always maintained. Cerebrospinal fluid circulates around the brain and spinal cord and acts as a buffer for external shocks and acts as a transport medium for hormones and waste products. In one embodiment, the cerebrospinal fluid may be a patient-derived cerebrospinal fluid. Specifically, it may be a cerebrospinal fluid derived from an individual to which a stem cell is to be administered.

In the present specification, stem cells may be suspended in cerebrospinal fluid. As used herein, " suspended " or " floating " may mean that a cell (e.g., a stem cell) is not adhered to a container for incubation and is stored in a container for final administration to a patient. More specifically, it may not be a stem cell which is suspended in a culture container for three-dimensional culture. Thus, the stem cells may be cells that have undergone QC in the GMP process. That is, the stem cells are cultured in a culture medium in a GMP facility, and then they are manufactured into a formulation for administration to a patient through a QC (Quality Control) process. At this time, in the existing technique, the final stem cells to be administered to the patient are stored in the same medium as the culture medium.

In one embodiment, the formulation may be patient-tailored. The final stem cells to be administered to the patient are stored in cerebrospinal fluid (e. G., Patient-derived). By doing so, the stem cell dosage form can change the expression of some genes or proteins in a patient-tailored manner, while retaining stem cell properties and not changing activity. Examples of some of the above-mentioned patient-customized genes may include TNFα and the like. In the case of the TNFa gene, expression may be decreased as compared with the case where it is stored in an existing culture medium (for example, MEM? Medium). Accordingly, the stem cells in the formulation according to one embodiment are kept in the cerebrospinal fluid, so that the expression of genes or proteins of the stem cells can be changed in a patient-customized manner or can be further activated (for example, , Cell adhesion, neovascularization, activation of genes or proteins associated with neuronal regeneration).

As used herein, the term " stem cells " may refer to undifferentiated cells that have the ability to differentiate into other cells. The stem cells may be embryonic stem cells, adult stem cells, induced pluripotent stem cells, or mesenchymal stem cells. The mesenchymal stem cells may be isolated from various tissues, from various races, or from humans of various ages. For example, the mesenchymal stem cells may be mesenchymal stem cells derived from adipose tissue, placenta-derived, umbilical cord blood-derived, muscle tissue-derived, corneal tissue-derived, or bone marrow-derived tissue. In addition, for example, the mesenchymal stem cell may be an adipocyte stem cell, a bone marrow stem cell, a cord blood stem cell, a neural stem cell, a placental stem cell, or a cord blood stem cell.

In one embodiment, the formulation can contain a therapeutically effective amount of stem cells. As used herein, the term " treatment-effective amount " refers to an amount of a compound that, in a patient or an individual in need of stem cell treatment, is ameliorated, the condition of the patient (e.g., one or more symptoms) Etc. < / RTI >&Quot; Treat " as used herein refers to the treatment of a disease or condition at an individual who is at risk of developing the disease, an improvement in the condition of the individual (e.g., one or more symptoms) Slowing the onset of symptoms or slowing the progression of symptoms, and the like. Thus, the term " treatment " also includes prophylactic treatment of the individual to prevent the occurrence of symptoms. The therapeutically effective amount may be, for example, 1.0 x 10 ⁵ to 1.0 x 10 ⁸ cells / kg body weight, or 1.0 x 10 ⁷ to 1.0 x 10 ⁸ cells / kg body weight. However, the dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, These factors can be taken into account to appropriately adjust the dosage. The number of administrations may be one or two or more times within the range of clinically acceptable side effects, and the administration site may be administered at one site or two or more sites. For an animal other than a human, the dosage may be the same as that of a human per kg, or the dosage may be converted into a dose in terms of a volume ratio (for example, an average value) One dose may be administered. Examples of animals to be treated in accordance with one embodiment include humans and other mammals for the purpose of administration. Specifically, humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, .

The formulation according to one embodiment may comprise stem cells as the active ingredient and a pharmaceutically acceptable carrier and / or additive. Examples thereof include sterilized water, physiological saline, buffering agents (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonic agents, can do. For topical administration, it is also possible to combine with organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically with collagen matrix, polylactic acid polymer or copolymer, polyethylene glycol polymer or copolymer and its chemical derivatives have. When formulated into a formulation suitable for injection, the formulation according to one embodiment may be one wherein the stem cells are frozen in a solution state in which the stem cells are dissolved or dissolved in a pharmaceutically acceptable carrier.

In one embodiment, the formulation may comprise 1 x 10 ⁵ cells to 1 x 10 ⁸ cells or 1 x 10 ⁵ cells to 1 x 10 ⁷ cells of stem cells per 0.5 to 10 ml of CSF.

In one embodiment, the formulation may be for the treatment of disease.

In one embodiment, the present disclosure provides a method of treating a disease comprising administering to a subject a formulation comprising cerebrospinal fluid and a therapeutically effective amount of stem cells.

The stem cells according to one embodiment, for example, mesenchymal stem cells may be those having anti-inflammatory, vascular regeneration, and nerve regeneration effects. Thus, the formulations may be useful in cell therapy for the prevention or treatment of a variety of diseases, including inflammatory diseases, ischemic diseases, and / or neurological diseases. Examples of such ischemic diseases include ischemic stroke, myocardial infarction, ischemic heart disease, ischemic brain disease, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, . As used herein, the term "ischemic stroke" or "stroke" may refer to a disease caused by brain tissue or necrosis of the brain due to a decrease in cerebral blood flow for a certain period of time or more. "Cerebral infarction ) &Quot;. < / RTI > Examples of such neurological diseases may include neurodegenerative diseases. Examples of such neurodegenerative diseases include spinal cord injury, multiple sclerosis, Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Pick's disease ), Or boxer dementia (Dementia pugilistica, DP).

In one embodiment, the formulation can be administered to a lesion of an individual to achieve the desired effect. For example, the formulation may be a parenteral dosage form.

Another aspect provides a method of preparing a dosage form of stem cells.

The method may comprise the step of keeping the stem cells suspended in the cerebrospinal fluid. The holding step may be performed in vitro.

In addition, the stem cell may further include a step of changing the gene or protein expression of the stem cell to a patient-customized state or activating gene or protein expression while maintaining stem cell characteristics. Changes in the gene or protein are as described above. Thus, the method of preparing the dosage form of the stem cell may be a method of preparing a dosage form of a patient-customized stem cell, or a method of producing a stem cell according to the present invention, Or a gene or protein associated with function of the stem cell) expression or activity of the stem cell.

In the method, the holding step may be at least 6 hours or more. The retention time may be a time at which the stem cells are stabilized or changed patiently, for example, 6 hours to 1 week, or 6 hours to 3 days.

In addition, the fat can be stored at room temperature. The stem cells may be lyophilized.

According to one aspect of the present invention, there is provided a stem cell dosage form and a method for producing the stem cell according to one aspect of the present invention, which is not only safe from an immunological point of view, but also capable of changing stem cell gene or protein expression, There is an effect that is activated.

FIG. 1A is a graph showing cell viability by flow cytometry when umbilical cord-derived mesenchymal stem cells according to one embodiment are maintained in MEM medium and cerebrospinal fluid derived from Alzheimer's disease patients.

FIG. 1B is a graph quantifying the results of flow cytometry analysis of the viability of the umbilical cord-derived mesenchymal stem cells according to one embodiment when they are maintained in MEM medium and CSF derived from Alzheimer's disease patients.

FIG. 2 is a graph showing the viability of the umbilical cord-derived mesenchymal stem cells according to one embodiment with the CCK-8 assay.

FIG. 3A is a flow cytometric analysis result of positive markers for confirming stem cell characteristics of umbilical cord-derived mesenchymal stem cells according to one embodiment.

FIG. 3B is a flow cytometric analysis result of negative markers (CD14, CD11b, HLA-DR) for confirming the stem cell characteristics of umbilical cord mesenchymal stem cells according to one embodiment.

FIG. 3c is a flow cytometry result of negative markers (CD19, CD34, CD45) for confirming the stem cell characteristics of the umbilical cord-derived mesenchymal stem cells according to one embodiment.

FIG. 4A is a microphotograph showing the ability of the umbilical cord-derived mesenchymal stem cells to differentiate according to one embodiment. FIG.

FIG. 4B is a graph showing the ability of the umbilical cord-derived mesenchymal stem cells to differentiate according to one embodiment.

FIG. 5 is a graph showing changes in RNA expression levels of umbilical cord-derived mesenchymal stem cells according to one embodiment.

6 is a graph showing changes in RNA expression levels of umbilical cord-derived mesenchymal stem cells according to one embodiment; Green showed decreased expression, black showed no change in expression, and red showed increased expression.

7 is a graph showing changes in RNA expression levels of umbilical cord-derived mesenchymal stem cells according to one embodiment.

8A is a graph showing a result of comparing the gene expression level of umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid with umbilical cord-derived mesenchymal stem cells stored in cerebrospinal fluid derived from normal subjects; Red: Increased gene expression; Green: Reduced gene expression.

FIG. 8B is a Venn diagram showing a gene increased in umbilical cord-derived mesenchymal stem cells stored in a cerebrospinal fluid derived from a patient as compared with umbilical cord-derived mesenchymal stem cells stored in a normal human-derived CSF according to an embodiment.

FIG. 8c is a table that comprehensively analyzes functions of genes increased in umbilical cord-derived mesenchymal stem cells stored in a patient-derived cerebrospinal fluid according to an embodiment.

FIG. 9 is a graph showing the results of analyzing the functions of genes increased in FIG. 8C; FIG. a: cell component; b: Molecular function.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 Isolation of Mesenchymal Stem Cells

Umbilical mesenchymal stem cells were used for mesenchymal stem cells.

Specifically, they received informed consent from healthy pregnant women who had been delivered normally and separated umbilical cord from about 15 cm to about 20 cm at the time of the first cesarean section. The separated umbilical cord was washed 2 to 5 times with PBS to remove blood. Thereafter, the umbilical cord was cut to a size of about 1.5 cm. In order to separate the cells from the whiten jelly, each piece was forcepsed to remove blood in the blood vessel. Each piece was cut with sterile scissors, and cord blood was removed. The gelatin pieces surrounding the blood were cut to a size of 1 to 3 mm, and then collagenase I was added thereto and reacted in a shaking incubator. The disrupted tissues were filtered using a strainer and washed with 10% FBS (Biowest, Nuaille ', France), 0.5% gentamicin (10 mg / ml) (Gibco, NY, USA) modified Minimum Essential Media (Gibco, NY, USA). The culture conditions were maintained at 37 ° C and 5% CO ₂ . Subsequently, the culture medium was changed every 3 to 4 days to remove unattached cells from the bottom of the flask. For the first subculture, trypLE, an animal component free (ACF) recombinant enzyme, and cells attached to the bottom of the flask were reacted in a 37 ° C incubator for about 3 minutes to recover mesenchymal stem cells.

EXPERIMENTAL EXAMPLE 1. Analysis of viability of mesenchymal stem cells

The umbilical cord-derived mesenchymal stem cells isolated from Example 1 were stored at 5.5 × 10 ⁶ cells in a vial (1.5 ml) containing MEM medium. The vials (1.5 ml) containing the cerebrospinal fluid derived from Alzheimer's disease patients were stored at 5.5 x 10 ⁶ cells and analyzed by flow cytometry. Flow cytometry analysis results are shown in Fig. In all figures, CSF1-CSF4 are the identification numbers of cerebrospinal fluid from Alzheimer's disease patients.

In addition, the viability of the cells was further analyzed via a CCK-8 assay. Specifically, mesenchymal stem cells, which had been suspended by each formulation (MEM medium and cerebrospinal fluid), were divided into 3 × 10 ³ cells per well in a 96-well plate and viability was analyzed for 24 hours in a 24-hour unit. After cell attachment was confirmed, CCK-8 solution (10 ul) was added to each well and incubated for 1 hour at 37 ° C. The absorbance of CCK-8 was analyzed at 450 nm with a microplate reader (x-Mark ^™ , Bio-Rad Laboratories, Inc.), and the results are shown in FIG.

FIG. 1A is a graph showing cell viability by flow cytometry when umbilical cord-derived mesenchymal stem cells according to one embodiment are maintained in MEM medium and cerebrospinal fluid derived from Alzheimer's disease patients.

FIG. 1B is a graph quantifying the results of flow cytometry analysis of the viability of the umbilical cord-derived mesenchymal stem cells according to one embodiment when they are maintained in MEM medium and CSF derived from Alzheimer's disease patients.

FIG. 2 is a graph showing the viability of the umbilical cord-derived mesenchymal stem cells according to one embodiment with the CCK-8 assay.

As shown in Figs. 1 and 2, even if the umbilical cord-derived mesenchymal stem cells are maintained in the cerebrospinal fluid derived from the patient, the viability of the cells is not significantly different from that of the control group.

Experimental Example 2. Analysis of Stem Cellularity of Mesenchymal Stem Cells

In the same manner as in Experimental Example 1, mesenchymal stem cells were maintained in MEM medium and patient-derived cerebrospinal fluid, and the cell viability was analyzed.

CD90, CD73, CD105 and CD166 were used as positive surface markers for stem cell function and CD14, CD11b, CD19, CD34, CD45 and HLA-DR were used as negative surface markers. For analysis of flow cytometry, cells were washed with DPBS and loaded in DPBS containing 2% FBS to remove CD90, CD73, CD105, CD166, CD14, CD11b, CD19, CD34, CD45 and HLA- The reaction was carried out for 20 minutes. Thereafter, surface antigens were analyzed using a flow cytometer (FACS Calibur, Becton Bickinson), and the results are shown in FIG.

In addition, the differentiation potential of stem cells was analyzed as follows.

For analysis of adipocyte differentiation ability, the umbilical cord-derived mesenchymal stem cells were cultured in an adipogenesis differentiation medium (StemPro® Adipogenesis Differentiation Kit, Life Technology) for 3 days at the intervals of 2 weeks. The culture was then removed, washed with Ca / Mg-free DPBS, and incubated at room temperature for 15 min with 4% paraformaldehyde. After washing with 60% isopropanol, Oil Red O was added thereto and reacted for 10 minutes. After washing with purified water, adipocytes were observed under a microscope. The results are shown in FIG.

For analysis of bone cell differentiation ability, umbilical cord mesenchymal stem cells were cultured in an osteogenesis differentiation medium (StemPro® Osteogenesis Differentiation Kit, Life Technology) for 3 days at 2-week intervals. After incubation, the medium was washed with Ca / Mg-free DPBS, and 4% paraformaldehyde was added. The reaction was carried out at room temperature for 15 minutes. After the reaction, add 1% silver nitrate solution, incubate at room temperature for 5 minutes, wash with purified water, add 5% sodium thiosulfate solution and incubate at room temperature And reacted for 5 minutes. Next, after washing with purified water, 0.1% Nuclear Fast Red Solution was added, and the reaction was allowed to proceed at room temperature for 5 minutes. Thereafter, the sample was washed with purified water and the calcium-accumulated sample was analyzed under a microscope. The results are shown in FIG.

For analysis of chondrocyte differentiation ability, umbilical cord mesenchymal stem cells were inoculated with chondrogenesis differentiation media (StemPro® Chondrogenesis Differentiation Kit, Life Technology) and the lid was loosely closed for 3 weeks at 3 days intervals Exchange culture. Cell clumps were cut into paraffin blocks and then cut and Alcian blue staining was performed. Thereafter, blue-stained chondrocytes were analyzed with an optical microscope. The results are shown in FIG. 4A and quantified as shown in FIG. 4B.

FIG. 3A is a flow cytometric analysis result of positive markers for confirming stem cell characteristics of umbilical cord-derived mesenchymal stem cells according to one embodiment.

FIG. 3B is a flow cytometric analysis result of a negative marker for confirming stem cell characteristics of a umbilical cord-derived mesenchymal stem cell according to an embodiment.

FIG. 4A is a microphotograph showing the ability of the umbilical cord-derived mesenchymal stem cells to differentiate according to one embodiment. FIG.

FIG. 4B is a graph showing the ability of the umbilical cord-derived mesenchymal stem cells to differentiate according to one embodiment.

As shown in Fig. 3, the marker expression of umbilical mesenchymal stem cell stored in AD CSF derived from Alzheimer's disease patients was significantly different from that of umbilical mesenchymal stem cells stored in MEM-a, .

In addition, as shown in FIG. 4, it was confirmed that the umbilical cord-derived mesenchymal stem cells stored in the cerebrospinal fluid (AD CSF) maintained their ability to differentiate into adipocytes, bone cells, and chondrocytes.

Experimental Example 3. Analysis of gene expression level of mesenchymal stem cells

In order to analyze the genes expressed in umbilical cord mesenchymal stem cells, RNA was extracted from stem cells, and labeling and purification were performed. The labeled cDNA was hybridized to an Illumina Expression BeadChip and the results were obtained. The data were subjected to statistical processing and the results are shown in FIGS. 5 to 7. FIG.

The list of analyzed RNA expression levels is shown below.

As shown in FIG. 5, Stemness Markers: FGF2, INS, LIF, POU5F1, SOX2, TERT, WNT3A, ZFP42; MSC-Specific Markers: ALCAM, ANPEP, BMP2, CASP3, CD44, ENG, ERBB2, FUT4, FZD9, ITGA6, ITGAV, KDR, MCAM, NGFR, NT5E, PDGFRB, PROM1, THY1, VCAM1; Other Genes Associated with MSC: ANXA5, BDNF, BGLAP, BMP7, COL1A1, CSF2, CSF3, CTNNB1, EGF, FUT1, GTF3A, HGF, ICAM1, IFNG, IGF1, IL10, IL1B, IL6, ITGB1, KITLG, MITF, NES, NUDT6, PIGS, PTPRC, SLC17A5, TGFB3, TNF, VEGFA, VIM, VWF; MSC Differentiation Markers; (i) Genes Involved in Osteogenesis: BMP2, BMP6, FGF10, HDAC1, HNF1A, KDR, PTK2, RUNX2, SMURF1, SMURF2, TBX5; (ii) Gene Involved in Adipogenesis: PPARG, RHOA, RUNX2; (iii) Gene Involved in Chondrogenesis: ABCB1, BMP2, BMP4, BMP6, GDF5, GDF6, GDF7, HAT1, ITGAX, KAT2B, SOX9, TGFB1; (iv) Gene Involved in Myogenesis: JAG1, NOTCH1; (v) Gene Involved in Tenogenesis (Tendon Development): BMP2, GDF15, SMAD4, and TGFB1 are related to stem cell characteristics.

However, as shown in Fig. 6 and Fig. 7, there was a difference in the expression pattern of some genes, for example, TNFa in each patient. In particular, a decrease in inflammation-related factors was commonly observed.

As a result, it was confirmed that the gene expression of the umbilical cord mesenchymal stem cells changed to the patient-customized state when stored in the patient-derived CSF.

In addition, we also compared changes in cellular RNA expression levels when umbilical mesenchymal stem cells were exposed to cerebrospinal fluid from patients with Alzheimer's disease and when exposed to normal CSF.

Specifically, the mesenchymal stem cells were stored in the same manner as in Example 1, except that the normal human derived CSF was used. The MeV program was used to analyze gene expression of mesenchymal stem cells in the same manner as in the above experiment, and the results are shown in FIG.

Also, in order to analyze how the genes expressing the respective genes function as a result of the gene expression analysis, Database for Annotation, Visualization and Integrated Discovery (DAVID) was used. The results are shown in FIG. The fold enrichment and count in FIG. 9 indicate how many genes are involved in the function shown in the result. Count indicates how many genes are involved in the function. Fold enrichment is calculated as the ratio of the gene involved in the function among the total genes analyzed / the ratio of the gene involved in the function in the known human genome.

8A is a graph showing a result of comparing the gene expression level of umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid with umbilical cord-derived mesenchymal stem cells stored in cerebrospinal fluid derived from normal subjects; Red: Increased gene expression; Green: Reduced gene expression.

FIG. 8B is a Venn diagram showing a gene increased in umbilical cord-derived mesenchymal stem cells stored in a cerebrospinal fluid derived from a patient as compared with umbilical cord-derived mesenchymal stem cells stored in a normal human-derived CSF according to an embodiment.

FIG. 8c is a table that comprehensively analyzes functions of genes increased in umbilical cord-derived mesenchymal stem cells stored in a patient-derived cerebrospinal fluid according to an embodiment.

FIG. 9 is a graph showing the results of analyzing the functions of the genes increased in FIG. 8C.

As shown in FIG. 8, both the umbilical mesenchymal stem cell exposed to normal CSF and the umbilical cord mesenchymal stem cell exposed to CSF from patient are activated, but the gene of umbilical mesenchymal stem cell exposed to CSF from patient is normal It was confirmed that the umbilical cord exposed to cerebrospinal fluid was slightly activated more than mesenchymal stem cells. In addition, there are many genes that are commonly activated in CSF from patient, but there are genes that are activated in each, indicating that there is also a patient - specific gene activation reaction. Next, the function of the genes increased in the mesenchymal stem cells exposed to the patient-derived cerebrospinal fluid was analyzed. As a result, it was confirmed that the functions due to the secreted factors such as the signal peptide, the growth factor activity, or the cytokine activity were greatly increased. In addition, it was confirmed that the functions of activating cell migration and regeneration such as cell proliferation, cell migration, cell adhesion, and neuron formation were greatly increased in the new blood.

As shown in FIG. 9, it was confirmed where the cellular components were located in the cells, and it was confirmed that most of the expression-increasing genes were expressed or secreted in the cell membrane to go outside the cell. It was also found that the molecular function is related to various growth factor activity, cell adhesion, and cytokine activity. The above results indicate that mesenchymal stem cells stored in cerebrospinal fluid according to one embodiment can secrete various active proteins and can be used as a cell therapy agent.

Claims (13)

1. Cerebrospinal fluid; And a stem cell containing a therapeutically effective amount of stem cells suspended in said cerebrospinal fluid.
2. The dosage form according to claim 1, wherein the stem cell is a mesenchymal stem cell.
3. The dosage form according to claim 1, wherein the cerebrospinal fluid is derived from a patient.
4. The dosage form according to claim 1, wherein said administration is intracerebral administration.
5. The dosage form of claim 1, wherein the dosage form is a patient-customized dosage form.
6. The dosage form according to claim 1, wherein the stem cell comprises 1 x 10 ⁵ cells to 1 x 10 ⁸ cells per 0.5 to 10 ml of cerebrospinal fluid.
7. [Claim 2] The dosage form according to claim 1, wherein the stem cell is stored in cerebrospinal fluid, and the expression of the gene or protein of the stem cell is changed to be patient-customized or activated while maintaining stem cell characteristics.
8. The dosage form according to claim 1, which is for the treatment of neurological diseases.
9. And maintaining the stem cells in suspension in the cerebrospinal fluid in vitro.
10. [Claim 11] The method according to claim 9, wherein the stem cell further comprises a step of changing the gene or protein expression of the stem cell to a patient-customized state or activating gene or protein expression while maintaining stem cell characteristics.
11. The method according to claim 9, wherein said holding step is at least 6 hours or more.
12. The method according to claim 9, wherein the cerebrospinal fluid is a cerebrospinal fluid derived from a patient.
13.  Use of cerebrospinal fluid for the manufacture of a stem cell dosage form.

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