Cellular pharmaceutical composition, disease treatment kit, and cell suspension solution

Abstract

An objective of the present invention is to provide a cell pharmaceutical composition that can maintain the cell condition in good condition and maintain a high survival rate for a long period of time. The present invention relates to a method for suspending a cell suspension comprising: (A) cells; and (B) a cell suspension solution, wherein the cell suspension solution contains Na as an electrolyte. + , Cl - , K. + and Ca 2+ (B) The cell suspension solution is an isotonic electrolyte solution containing CH3COO - , HCO3 - The aqueous solution may further contain at least one electrolyte selected from the group consisting of citrate ions and lactate ions.

Description

[Technical field]

[0001] The present invention relates to a cell pharmaceutical composition, a disease treatment kit, and a cell suspension solution. [Background technology]

[0002] The technology for using pharmaceuticals containing cells to treat diseases is improving year by year. In particular, stem cells such as iPS cells, hematopoietic stem cells, and mesenchymal stem cells, skin cells, and cardiomyocytes have moved from the basic research stage to the development stage, and some of them are now actually being used in clinical settings. In treating diseases with cells, it is expected that the functions of the cells themselves will be used directly or indirectly to treat diseases, and that the functions of damaged cells and tissues of patients will be compensated for by newly differentiated cells and organs from stem cells.

[0003] For example, mesenchymal stem cells are precursor cells with multipotency that were first isolated from bone marrow by Friedenstein (1982) (Non-Patent Document 1). It has been revealed that these mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected to be a new treatment method for various intractable diseases (Patent Documents 1 to 4). Recently, it has been known that cells with equivalent functions exist in the interstitial cells of fetal appendages such as adipose tissue, placenta, umbilical cord, and fetal membrane. Therefore, mesenchymal stem cells are sometimes called mesenchymal stromal cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2002-506831 [Patent Document 2] Special Publication No. 2000-508911 [Patent Document 3] JP 2012-157263 A [Patent Document 4] Special Publication No. 2012-508733 [Non-patent literature]

[0005] [Non-Patent Document 1] Pittenger FMet al.,Science,1999,284,pp.143-147 Summary of the Invention [Problem to be solved by the invention]

[0006] In pharmaceuticals containing cells such as mesenchymal stem cells, the cells are sometimes suspended in a solution for the purpose of ensuring safety, facilitating administration of the cells, etc. When cells such as mesenchymal stem cells are transferred or injected into a living body as a suspension by drip infusion, injection, etc., there are concerns that the viability of the cells in the suspension will gradually decrease, resulting in insufficient pharmacological action, that the cells will aggregate and clog the cannula, and that embolism will form in the patient's pulmonary vein, etc. Therefore, an objective of the present invention is to provide a cell pharmaceutical composition that can maintain a high cell viability for a long period of time. [Means for solving the problem]

[0007] As a result of intensive research to solve the above problems, the present inventors have discovered that cells for disease treatment are in a solution containing sodium chloride as an electrolyte, such as Ringer's solution, acetate Ringer's solution, or bicarbonate Ringer's solution. + , Cl - , K + and Ca 2+ The inventors have found that when cells are suspended in an isotonic electrolyte solution containing 1,000,000,000,000, and the cells are administered by drip infusion or the like, the decrease in cell viability during administration is significantly suppressed, and the present invention has been arrived at. According to the present invention, a pharmaceutical composition containing cells can maintain the cell condition in good condition and maintain the cell viability at a high level for a long period of time, and therefore can provide excellent therapeutic effects against various diseases. That is, the gist of the present invention is as follows.

[0008] [1] (A) a cell, and (B) Cell suspension solution Contains (B) The cell suspension solution contains Na as an electrolyte. + , Cl - , K + and Ca 2+ The cell medicine composition is an isotonic electrolyte solution containing the above and not containing any carbohydrate. [2] (B) The cell suspension solution is CH3COO - , HCO3 - The cellular pharmaceutical composition described in [1], further comprising at least one electrolyte selected from the group consisting of citrate ions and lactate ions. [3] (B) The cell pharmaceutical composition according to [1] or [2], wherein the cell suspension solution is at least one selected from the group consisting of Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution and lactate Ringer's solution. [4] (A) The cell pharmaceutical composition according to any one of [1] to [3], wherein the cell is a mesenchymal stem cell or a peripheral blood mononuclear cell. [5] The cell pharmaceutical composition described in [4], wherein the mesenchymal stem cells are derived from adipose tissue, umbilical cord tissue, or bone marrow. [6] (A) a cell, and (B) Cell suspension solution Contains (B) The cell suspension solution contains Na as an electrolyte. + , Cl - , K + and Ca 2+ and a carbohydrate-free, isotonic electrolyte solution. [7] Sodium as an electrolyte + , Cl - , K + and Ca 2+ The cell suspension solution for a cell pharmaceutical composition is an isotonic electrolyte solution containing the above and containing no carbohydrates. Effect of the Invention

[0009] The cell pharmaceutical composition of the present invention can maintain the cell condition in a good condition and maintain the cell survival rate at a high level for a long period of time, and is therefore expected to have an excellent therapeutic effect against various diseases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The cell pharmaceutical composition, the disease treatment kit, and the cell suspension solution for injection of the present invention will be described in detail below.

[0011] <Cell medicine composition> The cell medicine composition of the present invention contains (A) cells and (B) a cell suspension solution, and (B) the cell suspension solution contains Na as an electrolyte. + , Cl - , K + and Ca 2+ The present invention is characterized in that the cell pharmaceutical composition is an isotonic electrolyte solution containing the above and containing no carbohydrates. In the present invention, the term "cell pharmaceutical composition" refers to a pharmaceutical composition containing cells, which exerts a therapeutic effect against a disease by the function of the cells. The cell pharmaceutical composition of the present invention can maintain the cell viability at a high level for a long period of time by suspending the cells in the above specific solution, and therefore can exert an excellent therapeutic effect against various diseases. The cell pharmaceutical composition of the present invention may contain other drugs having a therapeutic effect against a disease in addition to the above essential components (A) cells and (B) cell suspension solution. Furthermore, other components may be contained as long as they do not impair the effect of the present invention. The following describes in detail the (A) cells, (B) cell suspension solution, other drugs, and other components contained in the cell pharmaceutical composition of the present invention.

[0012] [(A) Cell] In the present invention, the (A) cells are not particularly limited as long as they are effective in treating a disease, and examples thereof include mesenchymal stem cells, peripheral blood mononuclear cells (including neutrophils, eosinophils, basophils, lymphocytes, monocytes, etc.), erythrocytes, T cells, NK cells, NKT cells, NKM cells, LAK cells, dendritic cells, fibroblasts, hematopoietic stem cells, iPS cells, ES cells, bone marrow cells, cardiac myocytes, hepatic cells, nerve cells, skin cells, adipocytes, and other cells that constitute various tissues. Among these, mesenchymal stem cells, peripheral blood mononuclear cells, and bone marrow cells are preferred from the viewpoint of excellent viability maintenance effect by the (B) cell suspension solution described below.

[0013] (Mesenchymal stem cells) In the present invention, mesenchymal stem cells refer to cells that have the ability to differentiate into one or more types of cells belonging to the mesenchymal system, preferably two or more types, more preferably three or more types of cells (such as bone cells, cardiac muscle cells, chondrocytes, tendon cells, and adipocytes), and can proliferate while maintaining this ability. The term mesenchymal stem cells used in the present invention refers to the same cells as interstitial cells, and does not particularly distinguish between the two. They may also be simply referred to as mesenchymal cells. Examples of tissues that contain mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, and tooth germ. For example, adipose tissue-derived mesenchymal stem cells refer to mesenchymal stem cells contained in adipose tissue, and may also be called adipose-derived mesenchymal stem cells. Among these, from the standpoint of effectiveness in treating various diseases and ease of availability, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells are preferred, and adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are more preferred.

[0014] The mesenchymal stem cells of the present invention may be derived from the same species as the subject (specimen) to be treated, or may be derived from a different species. The species of mesenchymal stem cells of the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, and are preferably derived from the same species as the subject (specimen) to be treated. The mesenchymal stem cells of the present invention may be derived from the subject (specimen) to be treated, i.e., autologous cells (allogeneic), or may be derived from another subject of the same species, i.e., allogeneic cells (allogeneic). Allogeneic cells (allogeneic) are preferred.

[0015] Since mesenchymal stem cells are unlikely to cause rejection even in allogeneic subjects, donor cells prepared in advance can be expanded and cryopreserved and used as the mesenchymal stem cells (A) in the cell pharmaceutical composition of the present invention. Therefore, compared to the case of preparing and using autologous mesenchymal stem cells, from the viewpoints of ease of commercialization and ease of obtaining a stable and consistent effect, it is more preferable that the mesenchymal stem cells in the present invention are allogeneic.

[0016] In the present invention, mesenchymal stem cells refer to any cell population containing mesenchymal stem cells, in which at least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% are mesenchymal stem cells.

[0017] In the present invention, adipose tissue means tissue containing adipocytes and stromal cells including microvascular cells, and is, for example, tissue obtained by surgically excising or aspirating subcutaneous fat of a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferable to obtain it from the same animal as the subject of administration of adipose-derived mesenchymal stem cells described later, and more preferably human subcutaneous fat considering administration to humans. The individual that supplies the subcutaneous fat may be alive or dead, but the adipose tissue used in the present invention is preferably tissue collected from a living individual. When collecting from an individual, liposuction can be exemplified by, for example, PAL (power-assisted) liposuction, Erchonia laser liposuction, or body jet liposuction, and it is preferable not to use ultrasound from the viewpoint of maintaining the state of the cells.

[0018] In the present invention, the umbilical cord refers to a white tubular tissue that connects the fetus and the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's jelly), the umbilical cord matrix itself, etc., and contains a large amount of mesenchymal stem cells. The umbilical cord is preferably obtained from an animal of the same species as the subject (administration subject) in which the cellular pharmaceutical composition of the present invention will be used, and when considering administration of the cellular pharmaceutical composition of the present invention to humans, a human umbilical cord is more preferable.

[0019] In the present invention, bone marrow refers to the soft tissue that fills the cavity of bone and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and cells present therein are called bone marrow cells. Bone marrow cells include red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, vascular endothelial precursor cells, etc. Bone marrow cells can be collected, for example, from human iliac bone, long bone, or other bones.

[0020] In the present invention, the term "tissue-derived mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells" refers to any cell population containing tissue-derived mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. At least 20%, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% of the cell population are tissue-derived mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

[0021] The mesenchymal stem cells of the present invention may be characterized by growth characteristics (e.g., population doubling capacity from passaging to senescence, doubling time), karyotype analysis (e.g., normal karyotype, maternal lineage or neonatal lineage), surface marker expression by flow cytometry (e.g., FACS analysis), immunohistochemistry and / or immunocytochemistry (e.g., epitope detection), gene expression profiling (e.g., gene chip arrays; polymerase chain reaction such as reverse transcription PCR, real-time PCR, conventional PCR), miRNA expression profiling, protein arrays, protein secretion such as cytokines (e.g., plasma clotting analysis, ELISA, cytokine arrays), metabolites (metabolomic analysis), and other methods known in the art.

[0022] (Method for preparing mesenchymal stem cells) Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. As an example, a method for preparing adipose-derived mesenchymal stem cells will be described below. Adipose-derived mesenchymal stem cells may be obtained, for example, by the production method described in U.S. Patent No. 6,777,231, and can be produced, for example, by a method including the following steps (i) to (iii): (i) obtaining a cell suspension by enzymatic digestion of adipose tissue; (ii) sedimenting the cells and resuspending the cells in an appropriate medium; and (iii) culturing the cells on a solid surface and removing cells that do not exhibit binding to the solid surface.

[0023] The adipose tissue used in step (i) is preferably washed. Washing can be performed by vigorous agitation and sedimentation using a physiologically compatible saline solution (e.g., phosphate buffered saline (PBS)). This is to remove contaminants contained in the adipose tissue (also called debris, e.g., damaged tissue, blood, red blood cells, etc.) from the tissue. Therefore, washing and sedimentation are generally repeated until the debris is totally removed from the supernatant. Since the remaining cells exist as clumps of various sizes, it is preferable to treat the washed cell clumps with an enzyme (e.g., collagenase, dispase, trypsin, etc.) that weakens or destroys intercellular bonds in order to dissociate the cells while minimizing damage to the cells themselves. The amount and duration of such enzymes vary depending on the conditions used and are known in the art. Instead of or in combination with such enzyme treatment, the cell clumps can be decomposed by other treatment methods such as mechanical agitation, ultrasonic energy, and heat energy, but it is preferable to perform only enzyme treatment in order to minimize damage to the cells. When an enzyme is used, it is desirable to inactivate the enzyme using a medium or the like after an appropriate period of time in order to minimize harmful effects on cells.

[0024] The cell suspension obtained by step (i) contains a slurry or suspension of aggregated cells as well as various contaminating cells, such as red blood cells, smooth muscle cells, endothelial cells, and fibroblasts. The aggregated cells and the contaminating cells may then be separated and removed, but this step may be omitted since they can be removed by adhesion and washing in step (iii) described below. The separation and removal of the contaminating cells may be achieved by centrifugation, which forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing the contaminating cells is suspended in a physiologically compatible solvent. The suspended cells may contain red blood cells, but the lysis step is not necessarily required since the red blood cells are excluded by selection by adhesion to a solid surface, as described below. Methods known in the art can be used to selectively lyse red blood cells, such as lysis with ammonium chloride followed by incubation in a hypertonic or hypotonic medium. After lysis, the lysate may be separated from the desired cells, for example by filtration, centrifugation, or density fractionation.

[0025] In step (ii), the cells in suspension may be washed once or multiple times, centrifuged, and resuspended in culture medium to increase the purity of the mesenchymal stem cells. Alternatively, the cells may be separated based on cell surface marker profiles or based on cell size and granularity.

[0026] The medium used for resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, but such a medium may be prepared by adding serum to a basal medium and / or adding one or more serum substitutes such as albumin, transferrin, fatty acids, insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc. These media may further contain substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc., as necessary.

[0027] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, MCDB201 medium, and mixed media thereof.

[0028] Examples of the serum include, but are not limited to, human serum, fetal bovine serum (FBS), bovine serum, calf serum, goat serum, horse serum, porcine serum, sheep serum, rabbit serum, rat serum, etc. When serum is used, it may be added to the basal medium at 5 v / v% to 15 v / v%, preferably 10 v / v%.

[0029] Examples of the fatty acid include, but are not limited to, linoleic acid, oleic acid, linoleic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of the lipid include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of the amino acid include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, and L-glycine. Examples of the protein include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharide include, but are not limited to, glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid, heparan sulfate, and the like. Examples of growth factors include, but are not limited to, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), etc. From the viewpoint of using the adipose-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a xeno-free medium that does not contain xenogeneic components such as serum. Such a medium is provided as a medium prepared in advance for mesenchymal stem cells (stromal cells) by, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, Rohto, etc.

[0030] Next, in step (iii), the cells in the cell suspension obtained in step (ii) are cultured on a solid surface without differentiation, using the appropriate cell culture medium described above, at an appropriate cell density and culture conditions. In the present invention, the term "solid surface" refers to any material that allows the adipose-derived mesenchymal stem cells of the present invention to bind and adhere to the solid surface. In a specific embodiment, such a material is a plastic material that has been treated to promote the binding and adhesion of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but a petri dish or a flask is preferably used. To remove unbound cells and cell debris, the cells are washed after incubation.

[0031] In the present invention, cells that ultimately remain bound and adhered to the solid surface can be selected as a cell population of adipose-derived mesenchymal stem cells.

[0032] The selected cells may be analyzed for surface antigens by conventional methods such as flow cytometry to confirm that they are adipose-derived mesenchymal stem cells of the present invention. Furthermore, they may be examined for their ability to differentiate into each cell lineage, and such differentiation may be performed by conventional methods.

[0033] The mesenchymal stem cells of the present invention can be prepared as described above, but may also be defined as cells having the following characteristics: (1) When cultured in standard medium, it exhibits adhesion to plastic. (2) surface antigens CD44, CD73, and CD90 are positive, and CD31 and CD45 are negative; and (3) They can be differentiated into bone cells, adipocytes, and chondrocytes under appropriate culture conditions.

[0034] (peripheral blood mononuclear cells) In the present invention, peripheral blood mononuclear cells refer to a fraction including lymphocytes, neutrophils, eosinophils, basophils, and monocytes obtained from human or animal peripheral blood. Peripheral blood mononuclear cells can be separated from peripheral blood by density gradient centrifugation using Ficoll-hypaque (registered trademark) or the like. Peripheral blood mononuclear cells as (A) cells in the present invention may be cells separated from peripheral blood, or may be cells proliferated and activated by culturing, etc., with various factors, low molecular weight compounds, antibodies, etc., as necessary.

[0035] (A) Cryopreservation of cells The (A) cells in the present invention may be cells that have been repeatedly cryopreserved and thawed as appropriate, so long as they have a therapeutic effect against various diseases. In the present invention, cryopreservation can be performed by suspending the (A) cells in a cryopreservation solution known to those skilled in the art and cooling them. Suspension can be performed by detaching the cells with a detaching agent such as trypsin as necessary, transferring them to a cryopreservation container, treating them appropriately, and then adding a cryopreservation solution.

[0036] The cryopreservation solution may contain DMSO (Dimethyl sulfoxide) as a cryoprotectant, but since DMSO has cytotoxicity, it is preferable to reduce the DMSO content. It is also known that DMSO has the property of inducing differentiation of mesenchymal stem cells. Examples of substitutes for DMSO include glycerol, propylene glycol, and polysaccharides. When DMSO is used, it contains 5% to 20%, preferably 5% to 10%, and more preferably 10%. In addition, it may contain additives described in WO2007 / 058308. As such a cryopreservation solution, for example, cryopreservation solutions provided by Bio Verde, Japan Genetics, ReproCell, Zenoac, Cosmo Bio, Kohjin Bio, Thermo Fisher Scientific, etc. may be used.

[0037] When the above-mentioned suspended cells are cryopreserved, they may be stored at a temperature between -80°C and -100°C (for example, -80°C), and any freezer capable of achieving said temperature may be used. Although not particularly limited, in order to avoid abrupt temperature changes, the cooling rate may be appropriately controlled using a programmable freezer. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the manufacturer's instructions for the cryopreservation solution.

[0038] The storage period is not particularly limited as long as the cells cryopreserved under the above conditions retain the same properties as before freezing after thawing, and may be, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Since cell damage can be suppressed by storing at a lower temperature, the cells may be transferred to the gas phase above liquid nitrogen (about -150°C or less to -180°C or less) for storage. When storing in the gas phase above liquid nitrogen, storage containers well known to those skilled in the art can be used. Although not particularly limited, for example, when storing for 2 weeks or more, it is preferable to store in the gas phase above liquid nitrogen.

[0039] The thawed (A) cells may be appropriately cultured before the next cryopreservation. For example, the mesenchymal stem cells may be cultured using a medium capable of culturing the above-mentioned mesenchymal stem cells, and may be cultured at a culture temperature of, but not limited to, about 30 to 40°C, preferably about 37°C, in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. In the culture, after the cells reach an appropriate confluency for the culture vessel (for example, the cells occupy 50% to 80% of the culture vessel), the cells may be detached with a detaching agent such as trypsin, and seeded at an appropriate cell density in a separately prepared culture vessel to continue the culture. When seeding the cells, a typical cell density is 100 cells / cm. 2 ~100,000 cells / cm 2 , 500 cells / cm 2 ~50,000 cells / cm 2 , 1,000~10,000 cells / cm 2, 2,000~10,000 cells / cm 2 In a specific embodiment, the cell density is 2,000 to 10,000 cells / cm. 2 It is preferable to adjust the period until the appropriate confluency is reached to 3 to 7 days. During the culture, the medium may be appropriately replaced as necessary.

[0040] Cryopreserved cells can be thawed by methods well known to those skilled in the art, such as by leaving the cells to stand or shake them in a 37°C incubator or water bath.

[0041] (A) Cell morphology The (A) cells contained in the cell pharmaceutical composition of the present invention may be in any state, for example, cells recovered by detaching cells during culture, or cells frozen in a cryopreservation solution. The use of cells obtained by expansion culture and aliquotting and cryopreserving the same lot is preferable in terms of stably obtaining the same action and effect, excellent handling, etc.

[0042] The frozen preserved (A) cells may be thawed immediately before use and directly mixed with the (B) cell suspension solution described below while still suspended in the cryopreservation solution. Alternatively, the cryopreservation solution may be removed by a method such as centrifugation before the cells are suspended in the (B) cell suspension solution.

[0043] The dose (administration amount) of the (A) cells of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the dosage form, etc., but from the viewpoint of achieving a sufficient therapeutic effect, the amount tends to be more preferable, while from the viewpoint of suppressing the occurrence of side effects, the amount tends to be less preferable. Generally, when administered to an adult, the number of cells is about 1 x 10 3 ~1x10 12 Pieces / time, preferably 1x10 4 ~1x10 11 pcs / time, more preferably 1x10 5 ~1x10 10 Pieces / time, preferably 5x10 6 ~1x109 This dose may be administered multiple times as a single dose, or this dose may be divided and administered multiple times.

[0044] The dose (administration amount) of the (A) cells of the present invention may vary depending on the condition of the patient (body weight, age, symptoms, physical condition, etc.) and the dosage form of the composition of the present invention. In general, when administered to an adult, the number of cells is 1x10 to 5x10 10 Pieces / kg, preferably 1x10 2 ~5x10 9 / kg, more preferably 1x10 3 ~5x10 8 pieces / kg, particularly preferably 1x10 4 ~5x10 7 This dosage may be administered multiple times as a single dose, or may be administered in multiple divided doses.

[0045] [(B) Cell suspension solution] The (B) cell suspension solution of the present invention contains Na as an electrolyte. + , Cl - , K + and Ca 2+ (B) The cell suspension solution is an isotonic electrolyte solution containing CH3COO - , HCO3 - , and may further contain at least one electrolyte selected from the group consisting of citrate ion and lactate ion. (B) Cell suspension solution can be said to be an isotonic electrolyte infusion preparation having an effect of replenishing extracellular fluid in a living body, and specifically, at least one selected from the group consisting of Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution, and lactate Ringer's solution is preferred. By employing (B) cell suspension solution having such a composition, the cell pharmaceutical composition of the present invention can maintain the state of (A) cells in a good condition and maintain their survival rate at a high level for a long period of time.

[0046] (B) The cell suspension solution does not contain carbohydrates and contains sodium as an electrolyte. + , K + , Ca 2+ , Cl- Contains Na + 140mEq / L~160mEq / L, K + 1mEq / L~10mEq / L, Ca 2+ 1mEq / L~10mEq / L, Cl - A Ringer's solution having a concentration of 140 mEq / L to 170 mEq / L is preferred. The Ringer's solution can be prepared with each electrolyte in the above-mentioned concentration range, but commercially available products such as Ringer's solution "Otsuka" (Japanese Pharmacopoeia Ringer's solution, Otsuka Pharmaceutical Factory, Inc.) and Ringer's solution "Fuso" (Japanese Pharmacopoeia Ringer's solution, Fuso Pharmaceutical Industries, Ltd.) can also be used.

[0047] (B) The cell suspension solution does not contain carbohydrates and contains sodium as an electrolyte. + , K + , Ca 2+ , Cl - , CH3COO - Contains Na + 120mEq / L~140mEq / L, K + 1mEq / L~10mEq / L, Ca 2+ 1mEq / L~10mEq / L, Cl - 90mEq / L~130mEq / L, CH3COO - An example of a preferred acetate Ringer's solution is one having an electrolyte concentration of 10 mEq / L to 40 mEq / L. The acetate Ringer's solution can be prepared so that each electrolyte falls within the above-mentioned concentration range, but commercially available products such as Veen (registered trademark) F Infusion (Kowa Co., Ltd.), Solugen (registered trademark) F Injection (Kyowa Critical Care Co., Ltd.), and Solaceto (registered trademark) F Infusion (Terumo Corporation) can also be used.

[0048] (B) The cell suspension solution does not contain carbohydrates and contains sodium as an electrolyte. + , K + , Ca 2+ , Mg 2+ , Cl - , HCO3 - , citrate ion 3+ ) and the electrolyte concentration of each is Na +120mEq / L~145mEq / L, K + 1mEq / L~10mEq / L, Ca 2+ 1mEq / L~10mEq / L, Mg 2+ 0.1mEq / L~10mEq / L, Cl - 95mEq / L~135mEq / L, HCO3 - 10mEq / L~40mEq / L, citrate ion (Citrate 3+ A preferred example is bicarbonate Ringer's solution having an electrolyte concentration of 1 mEq / L to 10 mEq / L. The bicarbonate Ringer's solution can be prepared so that each electrolyte falls within the above-mentioned concentration range, but commercially available products such as Bicanate (registered trademark) infusion (Otsuka Pharmaceutical Factory, Inc.) and Bicarbon (registered trademark) infusion (AY Pharmaceuticals, Inc.) can also be used.

[0049] (B) The cell suspension solution does not contain carbohydrates and contains sodium as an electrolyte. + , K + , Ca 2+ , Cl - , lactate ion -) The electrolyte concentration is Na + 120mEq / L~140mEq / L, K + 1mEq / L~10mEq / L, Ca 2+ 1mEq / L~10mEq / L, Cl - 90mEq / L~130mEq / L, Lactate - A preferred example is lactated Ringer's solution with an electrolyte concentration of 15 mEq / L to 45 mEq / L. The lactated Ringer's solution can be prepared with each electrolyte in the above-mentioned concentration range, but commercial products such as Sollact (registered trademark) infusion (Terumo Corporation), Nisori (registered trademark) infusion (Mylan Pharmaceutical Co., Ltd.), Hartmann's solution "Kobayashi" (Kyowa Critical Care Co., Ltd.), Hartmann's infusion "NP" (Nipro Corporation), Hartmann's infusion pH8 "NP" (Nipro Corporation), Lactec (registered trademark) injection (Otsuka Pharmaceutical Factory, Inc.), and Lactated Ringer's solution "Fuso" (Fuso Pharmaceutical Industries, Ltd.) can also be used.

[0050] [Other drugs] The cellular pharmaceutical composition of the present invention may contain one or more other drugs having a therapeutic effect against a disease. Examples of other drugs include any drug that can be used as a drug for treating liver disease, heart disease, inflammatory bowel disease, respiratory system, nervous system, circulatory system, cerebral circulation improving drug, or immunosuppressant.

[0051] Examples of therapeutic agents for liver disease include therapeutic agents for hepatitis B (lamivudine, adefovir, entecavir, tenofovir, etc.), interferon preparations (interferon α, interferon α-2b, interferon β, peginterferon α-2a, peginterferon α-2b, etc.), therapeutic agents for hepatitis C (ribavirin, telapirevir, simeprevir, vaniprevir, daclatasvir, asunaprevir, sofosbuvir, etc.), adrenal corticosteroids (prednisolone, methylprednisolone sodium succinate, etc.), anticoagulants (dried human antithrombin III concentrate, gabexate mesilate, thrombomodulin α, etc.), antidotes (edetate calcium disodium hydrate, glutathione, dimethycaprol, sodium thiosulfate hydrate, sugamadesc sodium, etc.), human serum albumin, liver extract, ursodeoxycholic acid, glycyrrhizic acid, azathioprine, bezafibrate, amino acids (glycine, L-cysteine, L-isoleucine, L-leucine, L-valine, L-threonine, L-serine, L-alanine, L-methionine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine, and salts thereof, etc.), vitamins (tocopherol, flavin adenine dinucleotide, thiamine disulfide phosphate, pyridoxine, cyanocobalamin, and salts thereof, etc.), antibiotics (sulbactam sodium, cefoperazone sodium, meropenem hydrate, vancomycin hydrochloride, etc.).

[0052] Examples of therapeutic agents for cardiac diseases include ACE inhibitors, angiotensin II receptor antagonists, β-blockers, antiplatelet agents, warfarin, calcium antagonists, nitrates, diuretics, HMG-CoA reductase inhibitors, and ancalon.

[0053] Examples of the therapeutic agents for inflammatory bowel disease include salazosulfapyridine, mesalazine, and the like.

[0054] Respiratory medications include, for example, dimorpholamine, doxapram hydrochloride hydrate, sivelestat sodium hydrate, pirfenidone, pulmonary surfactant, dornase alfa, and the like.

[0055] Examples of nervous system drugs include edaravone, interferon beta-1a, interferon beta-1b, fingolimod hydrochloride, riluzole, taltirelin hydrate, and the like.

[0056] Examples of circulatory system drugs include hepronicate, midodrine hydrochloride, amezinium methyl methyl sulfate, etilefrine hydrochloride, phenylephrine hydrochloride, etc.

[0057] Examples of cerebral circulation improvers include ifenprodil tartrate, nicergoline, ipdilast, dihydroergotoxine mesylate, nizofenone fumarate, fasudil hydrochloride hydrate, and the like.

[0058] Examples of immunosuppressants include cyclosporine, azathioprine, mizoribine, basiliximab, tacrolimus hydrate, gusperimus hydrochloride, mycophenolate mofetil, everolimus, and the like.

[0059] When the cell pharmaceutical composition of the present invention contains the other drug, the other drug may be stored in a container separate from the (A) cells and the (B) cell suspension solution during storage, or may be contained in a form mixed with either of them. Depending on the type of disease, treatment method, patient condition, etc., the other drug may be administered simultaneously with the (A) cells and the (B) cell suspension solution, or may be administered at a certain interval.

[0060] In addition to the above (A) cells and (B) cell suspension solution, the cell pharmaceutical composition of the present invention may contain other components such as pharma- ceutically acceptable carriers and additives according to the usual method depending on the application and form of the composition, so long as the effects of the present invention are not impaired. Such carriers and additives may be contained in the (B) cell suspension solution or may be contained separately from the (B) cell suspension solution. Examples of such carriers and additives include isotonicity agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH regulators, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, sweeteners, sour agents, colorants, flavoring agents, fragrances, or refreshing agents, but are not limited thereto.

[0061] The cellular pharmaceutical composition of the present invention can be used in various forms depending on the purpose, for example, in the form of an injection (including an infusion solution, an implantable injection, a sustained injection, and an injection prepared just before use), a dialysis agent, a patch, a cataplasm, etc. The cellular pharmaceutical composition of the present invention can also be applied to the affected area by spraying, and the cellular pharmaceutical composition of the present invention can also be used in the form of a gel or sheet at the affected area after spraying. The cellular pharmaceutical composition of the present invention can also be applied to the affected area after forming the above (A) cells into a sheet or three-dimensional structure.

[0062] In the cell medicine composition of the present invention, (A) cells, (B) cell suspension solution, other drugs, and other components may be stored in separate containers and mixed at the time of use. During storage, the (A) cells, (B) cell suspension solution, other drugs, and other components may be stored under conditions appropriate for each, such as frozen conditions, refrigerated conditions, room temperature conditions, etc.

[0063] The pH of the cellular pharmaceutical composition of the present invention is not particularly limited as long as it is within a medicamentously, pharmacologically (pharmaceutical) or physiologically acceptable range, but an example is a range of 2.0 to 9.0, preferably 2.5 to 8.5, and more preferably 4.0 to 8.0.

[0064] The osmotic pressure of the cellular pharmaceutical composition of the present invention is not particularly limited as long as it is within a range acceptable to the living body. An example of the osmotic pressure ratio of the cellular pharmaceutical composition of the present invention is preferably in the range of 0.6 to 1.5, more preferably 0.7 to 1.2, and even more preferably 0.8 to 1.0. The osmotic pressure can be adjusted by a method known in the art using the above-mentioned electrolytes and the like. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 15th revised Japanese Pharmacopoeia, and the osmotic pressure is measured with reference to the osmotic pressure measurement method (freezing point depression method) described in the Japanese Pharmacopoeia. The standard solution for osmolality ratio measurement (0.9 w / v% sodium chloride aqueous solution) is prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500-650°C for 40-50 minutes, allowing it to cool in a desiccator (silica gel), and then accurately weighing out 0.900 g of it and dissolving it in purified water to make exactly 100 mL, or use a commercially available standard solution for osmolality ratio measurement (0.9 w / v% sodium chloride aqueous solution).

[0065] The concentration of the (A) cells in the cell pharmaceutical composition of the present invention, i.e., the concentration when the (A) cells are suspended in the (B) cell suspension solution and used for administration, may vary depending on the type of cells and the cell suspension solution. Generally, the concentration is 1 x 10 2 ~2.5x10 8 / mL, preferably 1x10 3 ~2.5x10 7 / mL, more preferably 1x10 4 ~2.5x10 6 pieces / mL.

[0066] In a preferred embodiment of the cell pharmaceutical composition of the present invention, human mesenchymal stem cells are suspended in a cell suspension solution at 1×10 4 ~2.5×10 6 The cell suspension solution contains 125 to 150 mEq / L sodium ions, 100 to 160 mEq / L chloride ions, 3 to 5 mEq / L potassium ions, and 2 to 6 mEq / L calcium ions, has an osmotic pressure ratio of 0.8 to 1.0 relative to physiological saline, and has a pH of 4.0 to 8.0.

[0067] The routes of administration of the cellular pharmaceutical composition of the present invention to a subject include subcutaneous administration, intramuscular administration, intravenous administration, intra-arterial administration, intrathecal administration, intraperitoneal administration, rectal administration, vaginal administration, transdermal administration, implant, and direct administration to an organ. From the viewpoint of the efficacy of the cellular pharmaceutical composition of the present invention, preferred are implant, intra-arterial administration, intravenous administration, and direct administration to an organ, and more preferred are intravenous administration and direct administration to an organ.

[0068] The administration rate of the cellular pharmaceutical composition of the present invention to a subject may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the administration route of the non-alcoholic steatohepatitis therapeutic agent of the present invention, but when administered to an adult, it is usually 50 mL / h to 1,000 mL / h, preferably 75 mL / h to 500 mL / h, and more preferably 100 mL / h to 250 mL / h.

[0069] The temperature at which the cellular pharmaceutical composition of the present invention is administered to a subject may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the administration route of the cellular pharmaceutical composition of the present invention, but is generally 4°C to 45°C, preferably 15°C to 37°C, and more preferably room temperature to 37°C.

[0070] The cell pharmaceutical composition of the present invention can be administered to a subject using an infusion set. Specifically, commercially available infusion sets such as Wand Disposable Infusion Tube Set (manufactured by Yoshida Seisakusho Co., Ltd.), infusion set (manufactured by Forte Grow Medical Co., Ltd.), Terufusion (registered trademark) infusion set (manufactured by Terumo Corporation), JMS infusion set (manufactured by JMS Co., Ltd.), SurePlug infusion set (manufactured by Terumo Corporation), infusion set (manufactured by Nipro Corporation), Top infusion set NP (manufactured by Top Co., Ltd.), infusion set with filter (EX type) (manufactured by Toray Medical Co., Ltd.) can also be used.

[0071] The cell pharmaceutical composition of the present invention can be administered to a subject using an infusion tube. Specifically, commercially available infusion tubes such as LectroCath (manufactured by Samic International Co., Ltd.), JMS extension tube (manufactured by JMS Co., Ltd.), Safeed extension tube (manufactured by Terumo Corporation), extension tube (manufactured by Top Co., Ltd.), connecting tube (Chuatsu) (Medicos Hirata Co., Ltd.), Safty AP tube (manufactured by Kawasumi Chemical Industries Co., Ltd.), BioNectar 2 with extension tube (Toray Medical Co., Ltd.), Medicut extension tube set B (manufactured by Japan Sherwood Co., Ltd.), Wand disposable infusion tube set (manufactured by Yoshida Seisakusho Co., Ltd.), and infusion tube (manufactured by Forte Grow Medical Co., Ltd.) can also be used.

[0072] The materials that can be used for the infusion tube used when administering the cellular pharmaceutical composition of the present invention to a subject include polyvinyl chloride, thermoplastic elastomer, TPE thermoplastic elastomer, silicone, silicone rubber, polyethylene, polybutadiene, Teflon (registered trademark), polyurethane, polypropylene, natural rubber, polyolefin, PVC (plasticizer: TOTM, DOA), plasticizer-free PVC, and mixtures thereof.

[0073] The cell pharmaceutical composition of the present invention can be suitably used for the treatment of various diseases, for example, visceral diseases, specifically, cardiac diseases, gastric and duodenal diseases, small and large intestinal diseases, liver diseases, biliary tract diseases, pancreatic diseases, kidney diseases, lung diseases, mediastinal diseases, diaphragmatic diseases, pleural diseases, peritoneal diseases, neurological diseases, central nervous system (CNS) disorders, peripheral arterial diseases, and peripheral venous diseases.

[0074] Specific diseases include, for example, liver diseases such as autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver (NAFL), hepatic fibrosis, liver cirrhosis, liver cancer, fatty liver, drug allergic liver disease, hemochromatosis, hemosiderosis, Wilson's disease, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver abscess, chronic active hepatitis, and chronic persistent hepatitis; myocardial infarction, heart failure, arrhythmia, palpitations, cardiomyopathy, ischemic cardiomyopathy, angina pectoris, congenital heart disease, valvular heart disease, myocarditis, and family heart diseases such as chronic hypertrophic cardiomyopathy, dilated cardiomyopathy, acute coronary syndrome, atherothrombosis, and restenosis; gastric and duodenal diseases such as acute gastritis, chronic gastritis, gastric and duodenal ulcers, gastric cancer, and duodenal cancer; small and large intestinal diseases such as ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, simple ulcer, intestinal Behcet's disease, small intestinal cancer, and large intestinal cancer; biliary tract diseases such as acute cholecystitis, acute cholangitis, chronic cholecystitis, bile duct cancer, and gallbladder cancer; acute pancreatic pancreatic diseases such as acute nephritis, chronic nephritis, and pancreatic cancer; kidney diseases such as acute nephritis, chronic nephritis, acute renal failure, and chronic renal failure; pneumonia, emphysema, pulmonary fibrosis, interstitial pneumonia, idiopathic interstitial pneumonia, desquamative interstitial pneumonia, acute interstitial pneumonia, nonspecific interstitial pneumonia, drug-induced pulmonary disease, eosinophilic lung disease, pulmonary hypertension, pulmonary tuberculosis, sequelae of pulmonary tuberculosis, acute respiratory distress syndrome, cystic fibrosis, chronic obstructive pulmonary disease, pulmonary embolism, pneumoconiosis, and aspiration lung disease Pulmonary diseases such as pulmonary fibrosis, acute upper respiratory tract infection, chronic lower respiratory tract infection, pneumothorax, diseases with damage to the alveolar epithelium, lymphangioleiomyoma, lymphocytic interstitial pneumonia, pulmonary alveolar proteinosis, and pulmonary Langerhans cell granulomatosis; mediastinal diseases such as mediastinal tumors, mediastinal cystic diseases, and mediastinitis; diaphragmatic diseases such as diaphragmatic hernia; pleural diseases such as pleurisy, empyema, pleural tumors, carcinomatous pleurisy, and pleural mesothelioma; peritonitis, peritoneal tumors, and other peritoneal diseases;Neurological disorders including cerebral palsy syndromes including childhood cerebral palsy, aseptic meningitis, Guillain-Barré syndrome, amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, polyneuropathy, spinal muscular atrophy, spinal disorders, acute transverse myelitis, spinal cord infarction (ischemic spinal cord injury), intracranial tumors, spinal tumors, etc.; CNS disorders including Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, and Parkinson's disease; fibromuscular dysplasia, peripheral arterial disease (PAD), and occlusive thromboembolism Peripheral arterial diseases such as vascular endothelial ulcer (Buerger's disease) and Kawasaki disease (KD); peripheral venous diseases such as deep vein thrombosis, chronic venous insufficiency, postphlebitic syndrome, and superficial venous thrombosis; immunodeficiency diseases such as graft-versus-host disease (GVHD), secondary immunodeficiency, primary immunodeficiency, B cell deficiency, T cell deficiency, combined B and T cell deficiency, phagocyte deficiency, classical pathway complement deficiency, MBL pathway complement deficiency, alternative pathway complement deficiency, complement regulatory protein deficiency, and complement receptor deficiency;

[0075] Among these, liver disease, heart disease, lung disease, neurological disease, peripheral arterial disease, and immunodeficiency disease, which have been confirmed to have a sufficient therapeutic effect by mesenchymal stem cells, are preferred, and among them, they can be more preferably used to treat liver fibrosis, liver cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, interstitial pneumonia, pediatric cerebral palsy, amyotrophic lateral sclerosis (ALS), peripheral arterial disease (PAD), and graft-versus-host disease (GVHD), and can be more preferably used to treat liver fibrosis, liver cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, and interstitial pneumonia. They can also be preferably used to treat cancers of various tissues, which have been confirmed to have a sufficient therapeutic effect by peripheral blood mononuclear cells.

[0076] <Disease treatment kit> The present invention relates to a method for suspending cells in a cell suspension comprising the steps of: (A) cells; and (B) a cell suspension solution, the cell suspension solution containing Na as an electrolyte. + , Cl - , K + and Ca 2+and (B) a cell suspension solution, and is an isotonic electrolyte solution containing no carbohydrates. The disease treatment kit of the present invention is a kit containing the above-mentioned cellular pharmaceutical composition of the present invention, and the explanation in the section on the cellular pharmaceutical composition can be applied to (A) the cells, (B) the cell suspension solution, and other components that may be contained in the cellular pharmaceutical composition of the present invention. The disease treatment kit of the present invention can maintain the condition of cells in a good condition and maintain a high survival rate for a long period of time, thereby providing excellent therapeutic effects against various diseases.

[0077] The disease treatment kit of the present invention can also be expressed as including the cellular pharmaceutical composition of the present invention, a container, and a label. Suitable containers included in the disease treatment kit of the present invention include, but are not limited to, cryotubes for freezing cells, bottles for cell suspension solutions, vials, test tubes, dialysis bags, and the like. These containers may be made of various materials such as glass, metal, plastic, or a combination thereof. The labels on these containers contain information explaining the contents, such as cells and cell suspension solutions.

[0078] The present disease treatment kits can include other materials desirable from a commercial and user standpoint, including other additives, other agents, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0079] <Cell suspension solution> Sodium as an electrolyte + , Cl - , K + and Ca 2+ The present invention also includes a cell suspension solution for a cell pharmaceutical composition, which is an isotonic electrolyte solution containing Na and does not contain carbohydrates. + , Cl - , K + and Ca 2+The present inventors have newly discovered that the use of an isotonic electrolyte solution containing and not containing carbohydrates can maintain high cell viability for a long period of time. For details of the cell suspension solution, the explanation of (B) Cell suspension solution in the section on cell pharmaceutical composition can be applied.

[0080] <Disease treatment methods> The present invention relates to a method for treating a disease, which comprises suspending (A) cells in (B) a cell suspension solution and administering the resulting solution to a patient, the cell suspension solution containing Na + , Cl - , K + and Ca 2+ The present invention also includes a treatment method characterized in that the electrolyte solution is an isotonic electrolyte solution containing (A) and not containing carbohydrates. According to the treatment method of the present invention, the cell viability can be maintained high for a long period of time, and therefore excellent therapeutic effects can be achieved against various diseases. The disease treatment method of the present invention is a treatment method using the above-mentioned cellular pharmaceutical composition of the present invention, and the explanation in the section on the cellular pharmaceutical composition can be applied to (A) the cells, (B) the cell suspension solution, and other components that may be contained in the cellular pharmaceutical composition of the present invention. EXAMPLES

[0081] The present invention will be described in detail below with reference to examples and test examples, but the present invention is not limited to these examples.

[0082] [Example 1] (Preparation of adipose-derived mesenchymal stem cells) After obtaining consent from a human donor, subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To achieve the destruction of the extracellular matrix and the isolation of cells, collagenase (Roche diagnostics) (solvent: physiological saline) was added, and the cells were shaken and dispersed at 37°C for 90 minutes. The above suspension was then centrifuged at 800g for 5 minutes to obtain a precipitate of stromal vascular cells. Serum-free medium for mesenchymal stem cells (Rohto) was added to the above cell precipitate, the cell suspension was centrifuged at 400g for 5 minutes, and after removing the supernatant, the cells were resuspended in serum-free medium for mesenchymal stem cells (Rohto), and the cells were seeded in a flask. The cells were cultured at 37°C for several days in 5% CO2. After several days, the culture was washed with PBS to remove blood cells and residual adipose tissue contained in the culture solution, and mesenchymal stem cells attached to a plastic container were obtained.

[0083] The obtained adipose-derived mesenchymal stem cells were dispensed into centrifuge tubes and centrifuged at 400g for 5 minutes to obtain cell precipitates. After removing the supernatant, an appropriate amount of cell freezing storage solution (STEM-CELLBANKER (Zenoac)) was added and suspended. The cell suspension was dispensed into cryotubes, stored in a freezer at -80 degrees, and then transferred to the gas phase above liquid nitrogen for continued storage.

[0084] In a 15 mL centrifuge tube (Sumitomo Bakelite Co., Ltd., product number: MS-56150), add Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory, Inc., Lot: K4K73), KN No. 2 infusion solution (No. 2 solution (dehydration replacement solution)) (Otsuka Pharmaceutical Factory, Inc., Lot: K6E92), KN No. 3 infusion solution (No. 3 solution (maintenance solution)) (Otsuka Pharmaceutical Factory, Inc., Lot: K6D96), KN No. 4 infusion solution (No. 4 solution ( After dispensing 5 mL of each of Physio (registered trademark) 70 (2.5% glucose acetate Ringer's solution) (Otsuka Pharmaceutical Factory, Inc., Lot: M6D91), the frozen adipose tissue-derived mesenchymal stem cells were rapidly thawed in a water bath (37 ± 1 ° C.), and 62.5 μL of the cell suspension of adipose tissue-derived mesenchymal stem cells was added. After suspending by inversion and mixing, the cells were stored at room temperature, and 1 mL was taken from the middle layer with a 5 mL syringe with a 21G needle immediately after preparation, 2 hours, 4 hours, and 7 hours later, and transferred to a 1.5 mL tube. 10 μL of trypan blue (Trypan Blue Stain (0.4%); Life technologies, 15250-061) was added to 10 μL of the cell suspension, and live and dead cells were distinguished and measured using a phase contrast microscope (OLYMPUS, product number: CKX41SF). A disposable cell counting chamber (WAKEN, product number: WC2-100) was used to count the cells, and 18 sections were counted five times. The cell viability was calculated using the average of the three measurements excluding the maximum and minimum values, according to the formula below. If the cell viability was below 70% after four hours, no further measurements were performed after seven hours. The composition of each infusion is shown in Table 1 below, and the cell viability results are shown in Table 2 below. Cell viability (%) = number of live cells / total number of cells x 100

[0085] [Table 1]

[0086] [Table 2]

[0087] When adipose-derived mesenchymal stem cells were suspended in KN2 infusion solution and Physio (registered trademark) 70, the cell viability after 4 hours was below 70%. When adipose-derived mesenchymal stem cells were suspended in KN3 infusion solution and KN4 infusion solution, the cell viability after 7 hours was below 70%. In contrast, when adipose-derived mesenchymal stem cells were suspended in Ringer's solution, the cell viability was 80% or more even after 7 hours. From the above results, it was found that by suspending adipose-derived mesenchymal stem cells in Ringer's solution, the cell viability can be maintained at a significantly high level for a long period of time.

[0088] [Example 2] In a 15 mL centrifuge tube (Sumitomo Bakelite Co., Ltd., product number: MS-56150), 5 mL of Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory, Inc., Lot: K4K73) and bicarbonate Ringer's solution (Bicanate (registered trademark) infusion, Otsuka Pharmaceutical Factory, Inc.) were dispensed, and then, as in Example 1, the cryopreserved adipose-derived mesenchymal stem cells were rapidly thawed in a water bath (37 ± 1 ° C.), and 62.5 μL of the cell suspension of adipose-derived mesenchymal stem cells was added. After suspending by inversion and mixing, the cells were stored at room temperature, and the number of live cells and the number of dead cells immediately after preparation, 1 hour, 2 hours, and 4 hours after preparation were measured in the same manner as in Example 1 to calculate the cell viability, and the cell viability was calculated relative to that immediately after preparation. The composition of each Ringer's solution is shown in Table 3 below, and the results of the cell viability are shown in Table 4 below.

[0089] [Table 3]

[0090] [Table 4]

[0091] When adipose-derived mesenchymal stem cells were suspended not only in Ringer's solution but also in bicarbonate Ringer's solution, the cell viability was high at about 90% for all storage times, including after 4 hours. These results show that by suspending adipose-derived mesenchymal stem cells in Ringer's solution and bicarbonate Ringer's solution, the cell viability can be maintained at a significantly high level for a long period of time.

[0092] [Example 3] The adipose-derived mesenchymal stem cells of Example 1 were replaced with bone marrow-derived mesenchymal stem cells (manufactured by Lonza), and a similar test was performed using Ringer's solution (Otsuka Ringer's solution, Otsuka Pharmaceutical Factory, Lot: K4K73). The number of live cells and the number of dead cells were measured immediately after preparation, and after 2 hours, 3 hours, and 7 hours to calculate the cell survival rate. The results are shown in Table 5 below.

[0093] [Table 5]

[0094] When bone marrow-derived mesenchymal stem cells were suspended in Ringer's solution, the cell viability was over 95% after 7 hours. These results show that by suspending bone marrow-derived stromal cells in Ringer's solution, the cell viability can be maintained at a significantly high level for a long period of time.

[0095] [Example 4] A similar test was performed using Ringer's solution (Otsuka Ringer's solution, Otsuka Pharmaceutical Factory, Lot: K4K73) instead of the adipose-derived mesenchymal stem cells of Example 1, and the number of live cells and dead cells was measured immediately after preparation, and after 2 hours, 4 hours, and 6 hours, and the cell survival rate was calculated as in Example 1. The results are shown in Table 6 below.

[0096] [Table 6]

[0097] When umbilical cord-derived stromal cells were suspended in Ringer's solution, the cell viability was over 90% after 6 hours. Therefore, it was found that Ringer's solution can maintain the cell viability of umbilical cord-derived mesenchymal stem cells at a significantly high level for a long period of time, similar to that of adipose-derived mesenchymal stem cells.

[0098] [Example 5] A similar test was performed using Ringer's solution (Otsuka Ringer's solution, Otsuka Pharmaceutical Factory, Lot: K4K73) instead of peripheral blood mononuclear cells (ACCUCELL (registered trademark) PBMCs derived from normal donors, manufactured by Precision Bioservices (PRECISION FOR MEDICINE), Lot. 13134-10) instead of the adipose-derived mesenchymal stem cells of Example 1, and the number of live and dead cells was measured immediately after preparation, and after 2 hours, 4 hours, and 6 hours to calculate the cell viability. The results are shown in Table 7 below.

[0099] [Table 7]

[0100] When peripheral blood mononuclear cells were suspended in Ringer's solution, the cell viability was about 80% after 6 hours. Therefore, it was found that Ringer's solution can maintain the cell viability of peripheral blood mononuclear cells at a remarkably high level for a long period of time, just like adipose-derived mesenchymal stem cells. [Industrial Applicability]

[0101] The cell pharmaceutical composition of the present invention can maintain the cell condition in a good condition and maintain the cell survival rate at a high level for a long period of time, and is therefore expected to have an excellent therapeutic effect against various diseases.

Claims

1. (A) a cell, and (B) Cell suspension solution Contains (A) the cell is a human mesenchymal stem cell or a human peripheral blood mononuclear cell; (B) A cell pharmaceutical composition in which the cell suspension solution is Ringer's solution, acetate Ringer's solution, or bicarbonate Ringer's solution (excluding those containing carbohydrates, and excluding those containing at least one selected from the group consisting of dimethyl sulfoxide, serum albumin, and hyaluronic acid).

2. The cell pharmaceutical composition according to claim 1 , wherein the mesenchymal stem cells are derived from adipose tissue, umbilical cord tissue, or bone marrow.

3. (A) a cell, and (B) Cell suspension solution Contains (A) the cell is a human mesenchymal stem cell or a human peripheral blood mononuclear cell; (B) A disease treatment kit in which the cell suspension solution is Ringer's solution, acetate Ringer's solution, or bicarbonate Ringer's solution (excluding those containing carbohydrates, and excluding those containing at least one selected from the group consisting of dimethyl sulfoxide, serum albumin, and hyaluronic acid).

Images