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

Abstract

To provide a cell pharmaceutical composition capable of maintaining a cell state well and a high survival rate over the long term.SOLUTION: This cell pharmaceutical composition contains cells (A) and a cell suspension solution (B). The cell suspension solution (B) is an isotonic electrolytic solution that includes Na+, Cl-, K+ and Ca2+ as electrolytes and does not include carbohydrates. The cell suspension solution (B) may further include at least one electrolyte selected from the group consisting of CH3COO-, HCO3-, citrate ions, and lactate ions.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cell pharmaceutical composition, a kit for treating a disease, and a cell suspension solution.

Background Art

[0002] Techniques for using pharmaceuticals containing cells for treating diseases have been advancing year by year. In particular, for stem cells such as iPS cells, hematopoietic stem cells, and mesenchymal stem cells, as well as skin cells and cardiomyocytes, they have shifted from the basic research stage to the development stage, and currently, there are also those actually used in clinical settings. In the treatment of diseases with cells, it is expected to directly or indirectly use the functions possessed by the cells themselves for disease treatment, and to supplement the functions of cells and tissues of patients who have been damaged with newly differentiated cells and organs from stem cells.

[0003] For example, mesenchymal stem cells are pluripotent progenitor cells first isolated from bone marrow by Friedenstein (1982) (Non-Patent Document 1). It has been clarified that these mesenchymal stem cells exist in various tissues such as bone marrow, umbilical cord, and adipose tissue, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases (Patent Documents 1 to 4). Recently, it has been known that there are cells having the same functions as the stromal cells of fetal appendages such as adipose tissue, placenta, umbilical cord, and egg membrane. Therefore, mesenchymal stem cells are sometimes referred to as mesenchymal stromal cells.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] In pharmaceuticals containing cells such as mesenchymal stem cells, for the purpose of ensuring safety and facilitating cell administration, cells may be suspended in a solution and used. When cells such as mesenchymal stem cells are transferred or injected into the body as a suspension by methods such as intravenous drip or injection, there are concerns that the viability of the cells in the suspension gradually decreases and sufficient pharmacological effects cannot be obtained, that the cells aggregate and clog the cannula, and that embolisms are formed in the patient's pulmonary vein or the like. Therefore, an object of the present invention is to provide a cell pharmaceutical composition capable of maintaining a high cell viability over a long period of time.

Means for Solving the Problems

[0007] As a result of intensive research to solve the above problems, the present inventors have found that when cells for treating diseases are suspended in an isotonic electrolyte solution containing Na + , Cl - , K + and Ca 2+ as electrolytes and not containing carbohydrates and are subjected to intravenous drip injection or the like, the decrease in the viability of the cells during administration is significantly suppressed, and the present invention has been achieved. According to the present invention, since the pharmaceutical composition containing cells can keep the cell state good and maintain a high viability over a long period of time, it can exhibit excellent therapeutic effects against various diseases. That is, the gist of the present invention is as follows.

[0008] [1](A) Cells, and (B) Solution for cell suspension containing (B) The solution for cell suspension contains Na as an electrolyte + , Cl - , K + and Ca 2+ and is an isotonic electrolyte solution that does not contain carbohydrates, a cell pharmaceutical composition. [2] The solution for cell suspension according to [1], further containing at least one electrolyte selected from the group consisting of CH 3 COO - , HCO 3 - , citrate ions and lactate ions. [3] The cell pharmaceutical composition according to [1] or [2], wherein (B) the solution for cell suspension 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] The cell pharmaceutical composition according to any one of [1] to [3], wherein (A) the cells are mesenchymal stem cells or peripheral blood mononuclear cells. [5] The cell pharmaceutical composition according to [4], wherein the mesenchymal stem cells are derived from adipose tissue, umbilical cord or bone marrow. [6] A kit for treating diseases, containing (A) cells, and (B) a solution for cell suspension (B) The solution for cell suspension contains Na as an electrolyte + , Cl - , K + and Ca 2+ and is an isotonic electrolyte solution that does not contain carbohydrates. [7] A solution for cell suspension for a cell pharmaceutical composition, which contains Na + , Cl - , K + and Ca 2+ as electrolytes and is an isotonic electrolyte solution that does not contain carbohydrates. [Advantages of the Invention]

[0009] The cell pharmaceutical composition of the present invention can maintain the state of cells well and keep their survival rate at a high level for a long time, so excellent therapeutic effects can be expected for various diseases.

Mode for Carrying Out the Invention

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

[0011] <Cell Pharmaceutical Composition> The cell pharmaceutical composition of the present invention contains (A) cells and (B) a cell suspension solution, and (B) the cell suspension solution is an isotonic electrolyte containing Na as an electrolyte + , Cl - , K + and Ca 2+ and does not contain carbohydrates. In the present invention, the "cell pharmaceutical composition" refers to a pharmaceutical composition containing cells, and those having a therapeutic effect on diseases due to the functions of the cells. The cell pharmaceutical composition of the present invention can maintain the survival rate of cells at a high level for a long time by suspending the cells in the above specific solution, so it is possible to exhibit excellent therapeutic effects on various diseases. The cell pharmaceutical composition of the present invention may contain other drugs having a therapeutic effect on diseases in addition to the essential components (A) cells and (B) the cell suspension solution. Furthermore, other components may be contained as long as the effects of the present invention are not impaired. Hereinafter, (A) cells, (B) the cell suspension solution, other drugs, and other components contained in the cell pharmaceutical composition of the present invention will be described in detail.

[0012] [(A) Cells] In the present invention, the cell (A) is not particularly limited as long as it has an effect on the treatment of diseases. For example, mesenchymal stem cells, peripheral blood mononuclear cells (including neutrophils, eosinophils, basophils, lymphocytes, monocytes, etc.), red blood cells, T cells, NK cells, NKT cells, NKM cells, LAK cells, dendritic cells, fibroblasts, hematopoietic stem cells, iPS cells, ES cells, bone marrow cells, cardiomyocytes, hepatocytes, nerve cells, skin cells, adipocytes, and cells constituting other tissues can be mentioned. Among these, from the viewpoint of excellent survival rate maintenance effect by the cell suspension solution (B) described later, mesenchymal stem cells, peripheral blood mononuclear cells, and bone marrow cells are preferable.

[0013] (Mesenchymal stem cells) In the present invention, mesenchymal stem cells mean cells having the ability to differentiate into one or more, preferably two or more, more preferably three or more types of cells (such as osteocytes, cardiomyocytes, chondrocytes, tendon cells, adipocytes, etc.) belonging to the mesenchymal system and capable of proliferating while maintaining this ability. The term mesenchymal stem cells used in the present invention means the same cells as stromal cells and does not particularly distinguish between the two. Also, it may be simply referred to as mesenchymal cells. Examples of tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amnion, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, dental pulp, tooth germ, etc. For example, adipose tissue-derived mesenchymal stem cells mean mesenchymal stem cells contained in adipose tissue and may be referred to as adipose-derived mesenchymal stem cells. Among these, from the viewpoints of effectiveness for the treatment of various diseases and ease of acquisition, 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 preferable, and adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are more preferable.

[0014] The mesenchymal stem cells in the present invention may be derived from the same species as the subject (test subject) to be treated or from a different species. Examples of the species of mesenchymal stem cells in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, and preferably they are cells derived from the same species as the subject (test subject) to be treated. The mesenchymal stem cells in the present invention may be derived from the subject (test subject) to be treated, that is, autologous cells (syngeneic), or from another subject of the same species, that is, allogeneic cells (allogeneic). Preferably, they are allogeneic cells (allogeneic).

[0015] Since mesenchymal stem cells are less likely to cause rejection reactions in allogeneic subjects, the mesenchymal stem cells prepared in advance from a donor and cryopreserved after expansion culture can be used as the (A) cells, i.e., mesenchymal stem cells, in the cell pharmaceutical composition of the present invention. Therefore, from the viewpoints of being easier to commercialize and being more likely to stably obtain a certain effect as compared with the case of preparing and using autologous mesenchymal stem cells, the mesenchymal stem cells in the present invention are more preferably allogeneic.

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

[0017] In the present invention, adipose tissue means a tissue containing adipocytes and stromal cells including microvascular cells, etc., for example, a tissue obtained by surgically excising or aspirating subcutaneous fat of a mammal. Adipose tissue can be obtained from subcutaneous fat. It is preferably obtained from the same species of animal as the administration target of the adipose-derived mesenchymal stem cells described later, and more preferably human subcutaneous fat in consideration of administration to humans. The donor of subcutaneous fat may be alive or dead, but the adipose tissue used in the present invention is preferably a tissue collected from a living individual. When collecting from an individual, examples of fat aspiration include PAL (power-assisted) fat aspiration, Elconia laser fat aspiration, or Body Jet fat aspiration, etc. From the viewpoint of maintaining the cell state, it is preferable not to use ultrasonic waves.

[0018] In the present invention, the umbilical cord is a white tubular tissue connecting the fetus and the placenta, and is composed of the umbilical vein, umbilical artery, gelatinous tissue (Wharton's Jelly), the umbilical cord stroma itself, etc., and contains a large number of mesenchymal stem cells. The umbilical cord is preferably obtained from the same species of animal as the subject (administration target) using the cell pharmaceutical composition of the present invention, and more preferably a human umbilical cord in consideration of administration of the cell pharmaceutical composition of the present invention to humans.

[0019] In the present invention, bone marrow refers to the soft tissue filling the lumen of bone and is a hematopoietic organ. Bone marrow fluid exists in the bone marrow, and the cells present therein are called bone marrow cells. Bone marrow cells include, in addition to red blood cells, granulocytes, megakaryocytes, lymphocytes, adipocytes, etc., mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells, etc. Bone marrow cells can be collected, for example, from the human ilium, long bones, or other bones.

[0020] In the present invention, mesenchymal stem cells derived from various tissues, such as adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, mean any cell population containing mesenchymal stem cells derived from various tissues, such as adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. At least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98% or 99% of the cell population is mesenchymal stem cells derived from various tissues, 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 in the present invention can be characterized by growth characteristics (e.g., population doubling ability, doubling time from passage to senescence), karyotype analysis (e.g., normal karyotype, maternal 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 array; polymerase chain reaction such as reverse transcription PCR, real-time PCR, conventional PCR), miRNA expression profiling, protein array, protein secretion such as cytokines (e.g., plasma coagulation assay, ELISA, cytokine array), metabolite (metabolome 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. Hereinafter, as an example, a method for preparing adipose-derived mesenchymal stem cells will be described. Adipose-derived mesenchymal stem cells can be obtained, for example, by the production method described in US Patent No. 6,777,231, and can be produced, for example, by a method including the following steps (i) to (iii): (i) A step of obtaining a cell suspension by enzymatic digestion of adipose tissue; (ii) A step of sedimenting the cells and resuspending the cells in an appropriate medium; and (iii) A step of culturing the cells on a solid surface and removing the cells that do not show binding to the solid surface.

[0023] For the adipose tissue used in step (i), it is preferable to use washed adipose tissue. Washing can be performed by vigorously stirring and sedimenting with a physiologically compatible aqueous saline solution (e.g., phosphate buffered saline (PBS)). This is to remove contaminants (also referred to as debris, e.g., damaged tissue, blood, red blood cells, etc.) contained in the adipose tissue from the tissue. Therefore, washing and sedimentation are generally repeated until debris is completely removed from the supernatant. Since the remaining cells exist as clumps of various sizes, in order to dissociate them while minimizing damage to the cells themselves, it is preferable to treat the washed cell clumps with an enzyme (e.g., collagenase, dispase, or trypsin, etc.) that weakens or breaks cell-cell junctions. The amount of such an enzyme and the treatment period vary depending on the conditions used, but 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 stirring, ultrasonic energy, thermal energy, etc., but in order to minimize cell damage, it is preferable to perform only enzyme treatment. When using an enzyme, in order to minimize the harmful effects on the cells, it is desirable to inactivate the enzyme using a medium or the like after an appropriate period of time.

[0024] The cell suspension obtained by step (i) contains an agglomerated cell slurry or suspension, as well as various contaminating cells, such as red blood cells, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, although the agglomerated cells and these contaminating cells may subsequently be separated and removed, since they can be removed by adhesion and washing in step (iii) described below, this separation and removal may be omitted. When separating and removing contaminating cells, it can be achieved by centrifugation that forcibly separates the cells into supernatant and precipitate. The obtained precipitate containing the contaminating cells is suspended in a physiologically compatible solvent. Although the suspended cells may contain red blood cells, since red blood cells are excluded by the selection by adhesion to the individual surface described below, the step of lysing is not necessarily required. As a method for selectively lysing red blood cells, for example, methods well known in the art, such as incubation in a hypertonic medium or a hypotonic medium by lysis with ammonium chloride, can be used. After lysis, the lysate may be separated from the desired cells, for example, by filtration, centrifugation, or density fractionation.

[0025] In step (ii), in the suspended cells, in order to increase the purity of the mesenchymal stem cells, they may be washed one or multiple times continuously, centrifuged, and resuspended in a medium. In addition to this, the cells may be separated based on the cell surface marker profile or based on the cell size and granularity.

[0026] The medium used for resuspension is not particularly limited as long as it can culture mesenchymal stem cells. 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. To these media, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. may be added as necessary.

[0027] Examples of the above-mentioned 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, etc.

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

[0029] Examples of the fatty acids include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid, and stearic acid. Examples of the lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine. Examples of the amino acids 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 proteins include, but are not limited to, echotins, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharides include glycosaminoglycans, and particularly, hyaluronic acid, heparan sulfate, and the like, but are not limited thereto. Examples of the 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), and vascular endothelial growth factor (VEGF). 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 medium that does not contain xenogeneic components such as serum (xeno-free). Such media are provided, for example, as media prepared in advance for mesenchymal stem cells (stromal cells) from PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning, and Rohto.

[0030] Subsequently, in step (iii), the cells in the cell suspension obtained in step (ii) are cultured on a solid surface without differentiating the cells, using the above-mentioned appropriate cell medium, at an appropriate cell density and culture conditions. In the present invention, the "solid surface" means any material that enables the binding and adhesion of adipose-derived mesenchymal stem cells in the present invention. In a specific embodiment, such a material is a plastic material 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, a flask, etc. are preferably used. In order to remove non-bound cells and cell fragments, the cells are washed after incubation.

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

[0032] For the selected cells, in order to confirm that they are adipose-derived mesenchymal stem cells in the present invention, they may be analyzed by a conventional method using flow cytometry or the like for surface antigens. Furthermore, the ability to differentiate into each cell lineage may be examined, and such differentiation can be carried out by a conventional method.

[0033] The mesenchymal stem cells in the present invention can be prepared as described above, but may also be defined as cells having the following characteristics; (1) It shows adhesiveness to plastic under culture conditions in a standard medium, (2) It is positive for surface antigens CD44, CD73, CD90 and negative for CD31, CD45, and (3) It can differentiate into osteocytes, adipocytes, and chondrocytes under culture conditions.

[0034] (Peripheral blood mononuclear cells) In the present invention, peripheral blood mononuclear cells refer to a fraction containing lymphocytes, neutrophils, eosinophils, basophils, and monocytes, which are obtained from the peripheral blood of humans or animals. Peripheral blood mononuclear cells can be separated from peripheral blood by density gradient centrifugation using Ficoll-hypaque (registered trademark) or the like. The peripheral blood mononuclear cells as the cells (A) in the present invention may be cells in a state separated from peripheral blood, or may be cells that have been proliferated and activated by culturing them together with various factors, low molecular weight compounds, antibodies, etc. as necessary.

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

[0036] The cryopreservation solution may contain DMSO (Dimethyl sulfoxide) as a cryoprotectant. However, since DMSO has cytotoxicity, it is preferable to reduce the DMSO content. It is also known that DMSO has the property of inducing differentiation in mesenchymal stem cells. Examples of alternatives to DMSO include glycerol, propylene glycol, or polysaccharides. When using DMSO, it contains a concentration of 5% to 20%, preferably 5% to 10%, more preferably 10%. In addition to this, it may also contain the additives described in WO2007 / 058308. As such a cryopreservation solution, for example, a cryopreservation solution provided by Bioverde, Nippon Genetics Co., Ltd., Reprocell, Zenoaq, Cosmo Bio Co., Ltd., Kojin Bio Inc., Thermo Fisher Scientific, etc. may be used.

[0037] When cryopreserving the above-mentioned suspended cells, it is sufficient to store them at a temperature between -80°C and -100°C (for example, -80°C), and this can be done using any freezer capable of reaching this temperature. Although not particularly limited, to avoid rapid temperature changes, a programmable freezer can be used to appropriately control the cooling rate. The cooling rate may be appropriately selected according to the components of the cryopreservation solution and can be carried out in accordance with the instructions of the cryopreservation solution manufacturer.

[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. For example, it can be 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 longer. Since cell damage can be suppressed by storing at a lower temperature, it may be transferred to the gas phase above liquid nitrogen (from about -150°C to -180°C or lower) for storage. When storing in the gas phase above liquid nitrogen, it can be carried out using a storage container well-known to those skilled in the art. 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 until the next cryopreservation. For example, the culture of mesenchymal stem cells is carried out using a medium capable of culturing the above-mentioned mesenchymal stem cells. Although not particularly limited, at a culture temperature of about 30 - 40°C, preferably about 37°C, it may be carried out in an atmosphere containing CO 2 The concentration of CO 2 is about 2 - 5%, preferably about 5%. In the culture, after reaching an appropriate confluence for the culture vessel (for example, the cells occupy 50% to 80% of the culture vessel), the cells are detached with a detachment 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, typical cell densities include 100 cells / cm 2 ~100,000 cells / cm 2 、500 cells / cm 2 ~50,000 cells / cm 2 、1,000 - 10,000 cells / cm2 、2,000 to 10,000 cells / cm 2 and the like are exemplified. In a specific embodiment, the cell density is 2,000 to 10,000 cells / cm 2 . It is preferable to adjust the period until an appropriate confluence is reached to be 3 to 7 days. During the culture, the medium may be appropriately replaced as necessary.

[0040] The thawing of the cryopreserved cells can be carried out by methods well known to those skilled in the art. For example, a method of performing static or shaking in a constant temperature bath at 37°C or in a hot water bath is exemplified.

[0041] ((A) Cell morphology) The (A) cells contained in the cell pharmaceutical composition of the present invention can be cells in any state. For example, they may be cells recovered by detaching cultured cells, or cells in a frozen state in a cryopreservation solution. Using cells obtained by subculturing and cryopreserving the same lot of cells in small aliquots is preferable in terms of stably obtaining similar effects and excellent handleability.

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

[0043] The dose (administered 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 dosage form, etc. From the viewpoint of achieving a sufficient therapeutic effect, the amount preferably tends to be larger. On the other hand, from the viewpoint of suppressing the occurrence of side effects, the amount preferably tends to be smaller. Usually, when administered to adults, as the number of cells, 1x10 3 ~1x10 12 cells / time, preferably 1x10 4 ~1x10 11 cells / time, more preferably 1x10 5 ~1x10 10 cells / time, particularly preferably 5x10 6~1x10 9 per administration. Note that this dosage may be administered multiple times as a single dose, or may be divided and administered multiple times.

[0044] The dosage (administered amount) of the cells (A) of the present invention may vary depending on the patient's condition (weight, age, symptoms, physical condition, etc.) and the dosage form of the composition of the present invention. Usually, when administered to adults, as the number of cells, it is 1x10 to 5x10 10 cells / kg, preferably 1x10 2 ~5x10 9 cells / kg, more preferably 1x10 3 ~5x10 8 cells / kg, particularly preferably 1x10 4 ~5x10 7 cells / kg. Note that this dosage may be administered multiple times as a single dose, or may be divided and administered multiple times.

[0045] [Solution for suspending cells (B)] The solution for suspending cells (B) of the present invention is an isotonic electrolyte solution containing Na + , Cl - , K + and Ca 2+ and not containing carbohydrates. The solution for suspending cells (B) may further contain at least one electrolyte selected from the group consisting of CH 3 COO - , HCO 3 - , citrate ions and lactate ions. The solution for suspending cells (B) can also be said to be an isotonic electrolyte infusion preparation having an extracellular fluid replenishing effect in the living body. 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 preferably mentioned. By adopting the solution for suspending cells (B) having such a composition, in the cell pharmaceutical composition of the present invention, the state of the cells (A) can be kept good and its survival rate can be maintained at a high level for a long time.

[0046] (As the solution for suspending cells (B), it does not contain carbohydrates and, as electrolytes, Na + , K+ 、 Ca 2+ 、 Cl - and contains Na + at 140 mEq / L to 160 mEq / L, K + at 1 mEq / L to 10 mEq / L, Ca 2+ at 1 mEq / L to 10 mEq / L, Cl - at 140 mEq / L to 170 mEq / L, Ringer's solution can be preferably mentioned. The above Ringer's solution can be prepared with each electrolyte in the above concentration range, but commercially available products such as Ringer's solution "Otsuka" (Nissho Ringer's solution, Otsuka Pharmaceutical Factory, Inc.), Ringer's solution "Fuso" (Nissho Ringer's solution, Fuso Pharmaceutical Industries, Ltd.) can also be used.

[0047] (B) As the cell suspension solution, it does not contain carbohydrates and contains Na as an electrolyte + 、 K + 、 Ca 2+ 、 Cl - 、 CH 3 COO - and contains Na + at 120 mEq / L to 140 mEq / L, K + at 1 mEq / L to 10 mEq / L, Ca 2+ at 1 mEq / L to 10 mEq / L, Cl - at 90 mEq / L to 130 mEq / L, CH 3 COO - at 10 mEq / L to 40 mEq / L, acetate Ringer's solution can be preferably mentioned. The above acetate Ringer's solution can be prepared with each electrolyte in the above concentration range, but commercially available products such as Vene (registered trademark) F infusion solution (Kowa Company, Ltd.), Solugen (registered trademark) F injection (Kyowa Critical Care Co., Ltd.), Soluacet (registered trademark) F infusion solution (Terumo Corporation) can also be used.

[0048] (B) As the cell suspension solution, it does not contain carbohydrates and contains Na as an electrolyte + 、 K + 、 Ca 2+ 、 Mg 2+ 、 Cl - 、 HCO 3 -, containing citrate ions (Citrate 3+ ), and the concentration of each electrolyte is such that Na + is 120 mEq / L to 145 mEq / L, K + is 1 mEq / L to 10 mEq / L, Ca 2+ is 1 mEq / L to 10 mEq / L, Mg 2+ is 0.1 mEq / L to 10 mEq / L, Cl - is 95 mEq / L to 135 mEq / L, HCO 3 - is 10 mEq / L to 40 mEq / L, and citrate ions (Citrate 3+ ) are 1 mEq / L to 10 mEq / L. A preferred example is sodium bicarbonate Ringer's solution. The above sodium bicarbonate Ringer's solution can be prepared with each electrolyte within the above concentration range, but commercially available products such as Bicainate (registered trademark) infusion (Otsuka Pharmaceutical Factory, Inc.) and Bicarbon (registered trademark) infusion (Awi Pharma Co., Ltd.) can also be used.

[0049] (B) As the cell suspension solution, it does not contain carbohydrates and contains Na + , K + , Ca 2+ , Cl - , and lactate ions (Lactate -) . The concentration of each electrolyte is such that Na + is 120 mEq / L to 140 mEq / L, K + is 1 mEq / L to 10 mEq / L, Ca 2+ is 1 mEq / L to 10 mEq / L, Cl - is 90 mEq / L to 130 mEq / L, and Lactate -Lactated Ringer's solution with a concentration of 15 mEq / L to 45 mEq / L can be preferably cited. The above Lactated Ringer's solution can be prepared with each electrolyte within the above concentration range. However, commercially available products such as Sol-lact (registered trademark) infusion (Terumo Corporation), Nisol (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 (trademark registered) injection (Otsuka Pharmaceutical Factory, Inc.), Lactated Ringer's solution "Fuso" (Fuso Pharmaceutical Industries, Ltd.) can also be used.

[0050] [Other drugs] The cell pharmaceutical composition of the present invention may contain one or more other drugs having a therapeutic effect on a disease. Examples of other drugs include any drugs that can be used as liver disease therapeutic drugs, heart disease therapeutic drugs, inflammatory bowel disease therapeutic drugs, respiratory drugs, nervous system drugs, cardiovascular drugs, cerebral circulation improving drugs, and immunosuppressive drugs.

[0051] Examples of drugs for treating liver diseases include, for example, drugs for treating hepatitis B (lamivudine, adefovir, entecavir, tenofovir, etc.), interferon preparations (interferon α, interferon α-2b, interferon β, peginterferon α-2a, peginterferon α-2b, etc.), drugs for treating hepatitis C (ribavirin, telaprevir, simeprevir, vaniprevir, daclatasvir, asunaprevir, sofosbuvir, etc.), corticosteroids (prednisolone, methylprednisolone sodium succinate, etc.), anticoagulants (dry concentrated human antithrombin III, gabexate mesilate, thrombomodulin α, etc.), antidotes (calcium disodium edetate hydrate, glutathione, dimercaprol, sodium thiosulfate hydrate, sugamadex 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 their salts, etc.), vitamins (tocopherol, flavin adenine dinucleotide, thiamine disulfide phosphate, pyridoxine, cyanocobalamin and their salts, etc.), antibiotics (sulbactam sodium, cefoperazone sodium, meropenem hydrate, vancomycin hydrochloride, etc.), and the like.

[0052] Examples of drugs for treating heart diseases include, for example, ACE inhibitors, angiotensin II receptor antagonists, β-blockers, antiplatelet drugs, warfarin, calcium antagonists, nitrates, diuretics, HMG-CoA reductase inhibitors, amiodarone, and the like.

[0053] Examples of drugs for treating inflammatory bowel diseases include, for example, sulfasalazine, mesalazine, and the like.

[0054] Examples of drugs for respiratory use include, for example, dimorpholamine, doxapram hydrochloride hydrate, sivelestat sodium hydrate, pirfenidone, pulmonary surfactant, dornase alpha, and the like.

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

[0056] Examples of drugs for the circulatory system include, for example, hepronicate, midodrine hydrochloride, amezinium methyl sulfate, etilefrine hydrochloride, phenylephrine hydrochloride, and the like.

[0057] Examples of drugs for improving cerebral circulation include, for example, ifenprodil tartrate, nicergoline, iprazilast, dihydroergotoxine mesylate, nizofenone fumarate, fasudil hydrochloride hydrate, and the like.

[0058] Examples of immunosuppressive drugs include, for example, 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 above other drugs, during storage, (A) the cells and (B) the cell suspension solution may be stored in separate containers, or may be contained in a form blended with either one. Depending on the type of disease, treatment method, patient condition, etc., the other drugs and (A) the cells and (B) the cell suspension solution may be administered simultaneously or at certain intervals.

[0060] The cell pharmaceutical composition of the present invention may contain, in addition to the above (A) cells and (B) cell suspension solution, other components such as pharmaceutically acceptable carriers and additives according to common methods, as long as the effects of the present invention are not impaired, depending on its use and form. 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, but are not limited to, tonicity agents, thickeners, saccharides, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizing agents, antioxidants, sweeteners, acidulants, coloring agents, flavoring agents, fragrances or cooling agents, etc.

[0061] The cell pharmaceutical composition of the present invention can be used in various forms according to the purpose, for example, in the form of injections (including infusion solutions, implanted injections, sustained-release injections, injections prepared at the time of use), agents for dialysis, patches, cataplasms, etc. The cell pharmaceutical composition of the present invention can also be applied to the affected area by spraying, and the cell pharmaceutical composition of the present invention can also be used in a form that gels or forms a sheet at the affected area after spraying. The cell pharmaceutical composition of the present invention can also be applied to the affected area after forming the above (A) cells into a sheet or a three-dimensional structure.

[0062] The (A) cells, (B) cell suspension solution, other drugs, and other components of the cell pharmaceutical composition of the present invention may be stored in separate containers and mixed and used at the time of use. When storing, the above (A) cells, (B) cell suspension solution, other drugs, and other components only need to be stored under conditions suitable for each, and may be any of, for example, freezing conditions, refrigeration conditions, room temperature conditions, etc.

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

[0064] Regarding the osmotic pressure of the cell pharmaceutical composition of the present invention, there is no particular limitation as long as it is within the range acceptable to the living body. As an example of the osmotic pressure ratio of the cell pharmaceutical composition of the present invention, preferably it is 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 adjustment of the osmotic pressure can be carried out by using the above-mentioned electrolytes and the like in a method known in the art. 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 Fifteenth 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. In addition, the standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution) is prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650 °C for 40 to 50 minutes, then cooling it in a desiccator (silica gel), accurately weighing 0.900 g of it, dissolving it in purified water and making it exactly 100 mL, or using a commercially available standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution).

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

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

[0067] The administration routes of the cell 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, direct administration to an organ, etc. From the viewpoint of the effectiveness of the cell pharmaceutical composition of the present invention, implant, intra-arterial administration, intravenous administration, and direct administration to an organ are preferred, and intravenous administration and direct administration to an organ are more preferred.

[0068] The administration rate of the cell 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. Usually, when administered to an adult, it is 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 administration temperature of the cell 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 cell pharmaceutical composition of the present invention. Usually, it is 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, as the infusion set, commercially available products such as a wand disposable infusion tube set (manufactured by Yoshida Seisakusho Co., Ltd.), an infusion set (manufactured by Fortegro Medical Co., Ltd.), a Terufusion (registered trademark) infusion set (manufactured by Terumo Corporation), a JMS infusion set (manufactured by J.M.S. Co., Ltd.), a Sureplug infusion set (manufactured by Terumo Corporation), an infusion set (manufactured by Nipro Corporation), a Top infusion set NP (manufactured by Top Corporation), a filter-equipped infusion set (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, as the infusion tube, commercially available products such as Rectro-Cast (manufactured by Samick International Co., Ltd.), JMS Extension Tube (manufactured by J.M.S. Co., Ltd.), Saffide Extension Tube (manufactured by Terumo Corporation), Extension Tube (manufactured by Top Co., Ltd.), Connecting Tube (Chuwatsu) (manufactured by Medicos Hirata Co., Ltd.), Safety AP Tube (manufactured by Kawasumi Chemical Industry Co., Ltd.), Extension Tube-attached Bioconnector 2 (manufactured by Toray Medical Co., Ltd.), Medcut Extension Tube Set B (manufactured by Nippon Sherwood Co., Ltd.), Wand Disposable Infusion Tube Set (manufactured by Yoshida Seisakusho Co., Ltd.), Infusion Tube (manufactured by Fortegra Medical Co., Ltd.) can also be used.

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

[0073] The cell pharmaceutical composition of the present invention can be suitably used for the treatment of various diseases. For example, it is preferably used for visceral diseases, specifically, heart diseases, gastric and duodenal diseases, small intestine and large intestine diseases, liver diseases, biliary tract diseases, pancreatic diseases, kidney diseases, lung diseases, diaphragm diseases, diaphragm diseases, pleural diseases, peritoneal diseases, nerve diseases, central nervous system (CNS) disorders, peripheral arterial diseases, and peripheral venous diseases.

[0074] Specific diseases include, for example, autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver (NAFL), liver fibrosis, cirrhosis, liver cancer, fatty liver, drug allergic liver injury, hemochromatosis, hemosiderosis, Wilson's disease, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver abscess, chronic active hepatitis, chronic persistent hepatitis, and other liver diseases; myocardial infarction, heart failure, arrhythmia, palpitation, cardiomyopathy, ischemic cardiomyopathy, angina pectoris, congenital heart disease, valvular heart disease, myocarditis, familial hypertrophic cardiomyopathy, dilated cardiomyopathy, acute coronary syndrome, atherosclerotic thrombosis, restenosis, and other heart diseases; acute gastritis, chronic gastritis, gastric and duodenal ulcers, gastric cancer, duodenal cancer, and other gastric and duodenal diseases; ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, simple ulcer, intestinal Behcet's disease, small intestine cancer, colon cancer, and other small intestine and colon diseases; acute cholecystitis, acute cholangitis, chronic cholecystitis, cholangiocarcinoma, gallbladder cancer, and other biliary tract diseases; acute pancreatitis, chronic pancreatitis, pancreatic cancer, and other pancreatic diseases; acute nephritis, chronic nephritis, acute renal failure, chronic renal failure, and other kidney diseases; pneumonia, emphysema, pulmonary fibrosis, interstitial pneumonia, idiopathic interstitial pneumonia, desquamative interstitial pneumonia, acute interstitial pneumonia, nonspecific interstitial pneumonia, drug-induced lung disease, eosinophilic lung disease, pulmonary hypertension, pulmonary tuberculosis, sequelae of pulmonary tuberculosis, acute respiratory distress syndrome, cystic fibrosis, chronic obstructive pulmonary disease, pulmonary embolism, lung abscess, pneumoconiosis, aspiration pneumonia pulmonary fibrosis, acute upper respiratory tract infection, chronic lower respiratory tract infection, pneumothorax, diseases with damage to alveolar epithelium, lymphangioleiomyomatosis, lymphocytic interstitial pneumonia, pulmonary alveolar proteinosis, pulmonary Langerhans cell granulomatosis, and other lung diseases; mediastinal tumors, cystic diseases of the mediastinum, mediastinitis, and other mediastinal diseases; diaphragmatic hernia and other diaphragmatic diseases; pleurisy, empyema, pleural tumors, cancerous pleurisy, pleural mesothelioma, and other pleural diseases; peritonitis, peritoneal tumors, and other peritoneal diseases;Neurological diseases such as cerebral palsy syndrome including pediatric 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 disorder), intracranial tumors, spinal tumors; CNS disorders such as Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, Parkinson's disease; Peripheral arterial diseases such as fibromuscular dysplasia, peripheral arterial disease (PAD), thromboangiitis obliterans (Buerger's disease), Kawasaki disease (KD); Peripheral venous diseases such as deep vein thrombosis, chronic venous insufficiency, postphlebitic syndrome, superficial vein thrombosis; Immunodeficiency diseases such as graft-versus-host disease (GVHD), secondary immunodeficiency, primary immunodeficiency diseases, B cell deficiency, T cell deficiency, combined B and T cell deficiency, phagocyte deficiency, complement deficiency in the classical pathway, complement deficiency in the MBL pathway, complement deficiency in the alternative pathway, complement regulatory protein deficiency, complement receptor deficiency, etc.

[0075] Among these, liver diseases, heart diseases, lung diseases, neurological diseases, peripheral arterial diseases, and immunodeficiency diseases in which the therapeutic effect of mesenchymal stem cells has been confirmed to be sufficiently obtained are preferred. Among them, it can be preferably used for the treatment of liver fibrosis, liver cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, interstitial pneumonia, pediatric cerebral palsy, amyotrophic lateral sclerosis (ALS), peripheral arterial disease (PAD), graft-versus-host disease (GVHD), and can be more preferably used for liver fibrosis, liver cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, interstitial pneumonia. In addition, it can be preferably used for cancers of each tissue in which the therapeutic effect of peripheral blood mononuclear cells has been confirmed to be sufficiently obtained.

[0076] <Kit for treating diseases> The present invention contains (A) cells and (B) a cell suspension solution, and the (B) cell suspension solution contains Na as an electrolyte + , Cl - , K + and Ca 2+Also included is an isotonic electrolyte solution that contains [substance] and does not contain carbohydrates, for use in a kit for treating diseases. The kit for treating diseases of the present invention is a kit that includes the above-described cell-based pharmaceutical composition of the present invention. For (A) cells, (B) the cell suspension solution, and other components that the cell-based pharmaceutical composition of the present invention may contain, the descriptions in the section on the cell-based pharmaceutical composition are applicable. According to the kit for treating diseases of the present invention, the state of the cells can be maintained well and the survival rate can be kept high over a long period of time, so that excellent therapeutic effects can be achieved for various diseases.

[0077] Also, the kit for treating diseases of the present invention can be expressed as including the cell-based pharmaceutical composition of the present invention, a container, and a label. Suitable containers included in the kit for treating diseases of the present invention are not particularly limited, and examples include cryotubes for cell freezing, bottles for cell suspension solutions, vials, test tubes, dialysis bags, etc. These containers may be formed from various materials such as glass, metal, plastic, or combinations thereof. The label on these containers describes the contents such as the cells and the cell suspension solution.

[0078] The kit for treating diseases of the present invention can include other additives, other drugs, diluents, filters, needles, syringes, and other materials that are desirable from a commercial and user perspective, including an attached document describing the usage method.

[0079] <Cell suspension solution> As electrolytes, Na + , Cl - , K + and Ca 2+ is also within the scope of the present invention. As a cell suspension solution for injecting / dripping cell-based pharmaceuticals, Na + , Cl - , K + and Ca 2+By adopting an isotonic electrolyte solution that contains [substance] and does not contain carbohydrates, it is a newly discovered finding by the present inventors that the survival rate of cells can be maintained high over a long period of time. Regarding the details of the cell suspension solution, the description of (B) the cell suspension solution in the section of the cell pharmaceutical composition can be applied.

[0080] <Method for treating diseases> The present invention relates to a method for treating a disease, comprising (A) suspending cells in (B) a cell suspension solution and administering the suspension to a patient, wherein (B) the cell suspension solution is an isotonic electrolyte solution containing Na as an electrolyte + , Cl - , K + and Ca 2+ and does not contain carbohydrates. According to the treatment method of the present invention, since the survival rate of cells can be maintained high over a long period of time, excellent treatment effects can be achieved for various diseases. The disease method of the present invention is a treatment method using the above-described cell pharmaceutical composition of the present invention, and the description in the section of the cell pharmaceutical composition can be applied to (A) cells, (B) the cell suspension solution, and other components that the cell pharmaceutical composition of the present invention may contain.

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 by these examples and the like.

[0082] [Example 1] (Preparation of adipose-derived mesenchymal stem cells) After obtaining consent from a human donor, the subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To achieve extracellular matrix disruption and cell isolation, collagenase (Roche diagnostics) (solvent: physiological saline) was added, and the mixture was shaken and inverted at 37°C for 90 minutes to disperse. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of the stromal vascular cell group. Serum-free medium for mesenchymal stem cells (Rohto) was added to the cell precipitate, and the cell suspension was centrifuged at 400 g for 5 minutes. After removing the supernatant, the cells were resuspended in serum-free medium for mesenchymal stem cells (Rohto) and seeded into a flask. The cells were cultured at 37°C for several days in 5% CO 2 and incubated. After several days, the culture was washed with PBS to remove blood cells and residual adipose tissue contained in the culture medium, and mesenchymal stem cells adhered to the plastic container were obtained.

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

[0084] 15 mL centrifuge tube (Sumitomo Bakelite Co., Ltd., product number: MS-56150) was aliquoted with 5 mL each of Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory, Inc., Lot: K4K73), KN No. 2 infusion solution (No. 2 solution (dehydration and replenishment 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 (postoperative recovery solution)) (Otsuka Pharmaceutical Factory, Inc., Lot: K6D80), and Physio (registered trademark) 70 (2.5% glucose plus Ringer's acetate solution) (Otsuka Pharmaceutical Factory, Inc., Lot: M6D91). After that, adipose tissue-derived mesenchymal stem cells stored frozen were rapidly thawed in a water bath (37 ± 1 °C), and then 62.5 μL of the cell suspension of adipose tissue-derived mesenchymal stem cells was added to each. After suspension by inverting and mixing, it was stored at room temperature. Immediately after preparation, 1 mL was sampled from the middle layer with a 5 mL syringe equipped with a 21G injection needle at 2 hours, 4 hours, and 7 hours after preparation and transferred to a 1.5 mL tube. To 10 μL of the cell suspension, 10 μL of trypan blue (Trypan Blue Stain (0.4%); Life technologies, 15250-061) was added to distinguish live cells and dead cells, and measurement was performed with a phase contrast microscope (OLYMPUS, product number: CKX41SF). For cell counting, a disposable cell counting board (WAKEN, product number: WC2-100) was used. The 18-compartment count was performed 5 times, and the average value of the 3 measurement values excluding the maximum value and the minimum value was used to calculate the cell viability according to the following formula. For those with a cell viability below 70% at 4 hours, measurement at 7 hours was not performed. The composition of each infusion solution is shown in Table 1 below, and the results of cell viability are shown in Table 2 below. Cell viability (%) = Number of live cells / Total number of cells × 100

[0085]

Table 1

[0086]

Table 2

[0087] When adipose-derived mesenchymal stem cells were suspended in KN2 infusion and Physio (registered trademark) 70, the cell viability after 4 hours was less than 70%. When adipose-derived mesenchymal stem cells were suspended in KN3 infusion and KN4 infusion, the cell viability was less than 70% after 7 hours. 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 time.

[0088] [Example 2] 5 mL each of Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory, Inc., Lot: K4K73) and sodium bicarbonate Ringer's solution (Bicarbonate (registered trademark) infusion, Otsuka Pharmaceutical Factory, Inc.) were dispensed into 15 mL centrifuge tubes (Sumitomo Bakelite Co., Ltd., product number: MS-56150). Then, in the same manner 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 to each. After suspending by inverting and mixing, it was stored at room temperature. Immediately after preparation, 1 hour later, 2 hours later, and 4 hours later, the number of live cells and dead cells were measured in the same manner as in Example 1 to calculate the cell viability, and the cell viability relative to immediately after preparation was calculated. The composition of each Ringer's solution is shown in Table 3 below, and the results of 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 as high as about 90% at all storage times including 4 hours later. From the above results, it was found 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 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 the same test was carried out using Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory, Inc., Lot: K4K73). Immediately after preparation, 2 hours later, 3 hours later, and 7 hours later, the number of live cells and dead cells were measured, and the cell viability was calculated. The results are shown in Table 5 below.

[0093]

Table 5

[0094] Even when bone marrow-derived mesenchymal stem cells were suspended in Ringer's solution, the cell viability was 95% or more at 7 hours later. From the above results, it was found 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 time.

[0095] [Example 4] The adipose-derived mesenchymal stem cells of Example 1 were replaced with umbilical cord-derived mesenchymal stem cells (Lifeline Cell Technology, LifeLine (registered trademark)_UCMSC, Lot. 160907), and the same test was carried out using Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory, Inc., Lot: K4K73). Immediately after preparation, 2 hours later, 4 hours later, and 6 hours later, the number of live cells and dead cells were measured, and the cell viability was calculated in the same manner as in Example 1. The results are shown in Table 6 below.

[0096]

Table 6

[0097] Even when the umbilical cord-derived mesenchymal cells were suspended in Ringer's solution, the cell viability was 90% or more after 6 hours. Therefore, it was found that Ringer's solution can maintain the cell viability at a significantly high level for a long time for umbilical cord-derived mesenchymal stem cells as well as for adipose-derived mesenchymal stem cells.

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

[0099]

Table 7

[0100] Even when the 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 at a significantly high level for a long time for peripheral blood mononuclear cells as well as for adipose-derived mesenchymal stem cells.

Industrial Applicability

[0101] Since the cell pharmaceutical composition of the present invention can keep the state of cells good and maintain its viability at a high level for a long time, excellent therapeutic effects can be expected for various diseases.

Claims

[Claim 1] The invention described herein.

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