JPWO2018123627A1 - Cell pharmaceutical composition, disease treatment kit and cell suspension solution - Google Patents

Abstract

An object of the present invention is to provide a cell pharmaceutical composition capable of maintaining a good cell state and maintaining a high survival rate over a long period of time. The present invention includes (A) a cell, and (B) a cell suspension solution containing an electrolyte and a carbohydrate, and (B) the electrolyte in the cell suspension solution contains Na + and Cl−, and K + It is a cell pharmaceutical composition which is substantially free. (B) It is preferable that the electrolyte in the cell suspension solution is substantially only Na + and Cl− or substantially only Na +, Cl− and Lac−.

Description

  The present invention relates to a cell pharmaceutical composition, a disease treatment kit, and a cell suspension solution.

  Techniques for using pharmaceuticals containing cells for disease treatment are improving year by year. In particular, stem cells such as iPS cells, hematopoietic stem cells, mesenchymal stem cells, skin cells, cardiomyocytes, etc. have moved from the basic research stage to the development stage, and are currently used in clinical settings. There are also. In the treatment of diseases by cells, the functions of cells themselves are used directly or indirectly for disease treatment, and the functions of cells and tissues of damaged patients are newly differentiated from stem cells and It is expected to be supplemented by organs.

  For example, mesenchymal stem cells are pluripotent progenitor cells isolated from bone marrow for the first time by Friedenstein (1982) (Non-patent Document 1). These mesenchymal stem cells have been revealed to exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases ( Patent Documents 1 to 4). Recently, it is known that cells having an equivalent function exist in stromal cells of fetal appendages such as adipose tissue, placenta, umbilical cord, and egg membrane. Therefore, the mesenchymal stem cell may be referred to as a stromal cell (Mesenchymal Stoma Cell).

Japanese translation of PCT publication No. 2002-506831 Special table 2000-508911 gazette JP 2012-157263 A Special table 2012-508733 gazette

Pittenger F.M. M.M. et al. , Science, 1999, 284, pp. 143-147

  In pharmaceuticals containing cells such as mesenchymal stem cells, the cells are sometimes suspended in a solution for the purpose of ensuring safety and facilitating administration of the cells. When cells such as mesenchymal stem cells are transferred into a living body by infusion or injection as a suspension, the survival rate of the cells in the suspension is gradually reduced and sufficient pharmacological action is achieved. There are concerns that problems such as formation of emboli in the patient's pulmonary veins and the like that may not be obtained, cells may aggregate and clog in the cannula, and the like. Then, this invention makes it a subject to provide the cell pharmaceutical composition which can maintain the survival rate of a cell high over a long time.

As a result of diligent research to solve the above-mentioned problems, the present inventors suspended the cells for disease treatment in a solution containing Na ⁺ and Cl ⁻ and substantially not containing K ⁺ , and performed instillation or the like. And the present inventors have found that the decrease in the survival rate of cells during administration is remarkably suppressed. According to the present invention, since a pharmaceutical composition containing cells can maintain a good state of cells and maintain a high survival rate over a long period of time, it has excellent therapeutic effects on various diseases. be able to. That is, the gist of the present invention is as follows.

[1] containing (A) cells, and (B) a cell suspension solution containing an electrolyte and a carbohydrate,
(B) The cell pharmaceutical composition, wherein the electrolyte in the cell suspension solution contains Na ⁺ and Cl ⁻ and substantially does not contain K ⁺ .
[2] (B) The cell medicine according to [1], wherein the electrolyte in the cell suspension solution is substantially only Na ⁺ and Cl ⁻ or substantially only Na ⁺ , Cl ⁻ and Lac ^−. Composition.
[3] The cell pharmaceutical composition according to [1] or [2], wherein the concentration of Na ⁺ and Cl ⁻ in the cell suspension solution (B) is 30.0 mEq / L or more.
[4] The cell pharmaceutical composition according to any one of [1] to [3], wherein the carbohydrate in the (B) cell suspension solution contains glucose.
[5] The cell pharmaceutical composition according to any one of [1] to [4], wherein the sugar concentration in the solution for cell suspension (B) is 1% to 5%.
[6] The cell pharmaceutical composition according to any one of [1] to [5], wherein (A) the cells are mesenchymal stem cells or peripheral blood mononuclear cells.
[7] The cell pharmaceutical composition according to [6], wherein the mesenchymal stem cells are derived from fat, umbilical cord or bone marrow.
[8] containing a cell suspension solution containing (A) cells, and (B) an electrolyte and a carbohydrate,
(B) The disease treatment kit, wherein the electrolyte in the cell suspension solution contains Na ⁺ and Cl ⁻ and does not substantially contain K ⁺ .
[9] A cell suspension solution for a cell pharmaceutical composition comprising an electrolyte and a saccharide, wherein the electrolyte contains Na ⁺ and Cl ⁻ and substantially does not contain K ⁺ .

  Since the cell pharmaceutical composition of the present invention can maintain a good cell state and maintain its survival rate in a high state for a long time, it can be expected to have an excellent therapeutic effect on various diseases.

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

<Cellular pharmaceutical composition>
The cell pharmaceutical composition of the present invention contains (A) a cell, and (B) a cell suspension solution containing an electrolyte and a carbohydrate, and (B) the electrolyte in the cell suspension solution contains Na ⁺ and Cl. ^- it includes a free of ^{K +} substantially. In the present invention, the “cell pharmaceutical composition” refers to a pharmaceutical composition containing cells, which exhibits a therapeutic effect on diseases due to the functions of the cells. The cell pharmaceutical composition of the present invention is excellent for various diseases because the cell viability can be maintained in a high state for a long time by suspending the cells in the specific solution. A therapeutic effect can be achieved. In addition to the essential components (A) cells and (B) cell suspension solution, the cell pharmaceutical composition of the present invention may contain other drugs having a therapeutic effect on diseases. Furthermore, as long as the effects of the present invention are not impaired, other components may be contained. Hereinafter, (A) cells, (B) cell suspension solutions, other drugs, and other components contained in the cell pharmaceutical composition of the present invention will be described in detail.

[(A) Cell]
In the present invention, the (A) cell 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 (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, Examples include hepatocytes, nerve cells, skin cells, fat cells, and other cells constituting each tissue. Among these, mesenchymal stem cells, peripheral blood mononuclear cells, and bone marrow cells are preferable from the viewpoint that (B) the cell suspension solution described later is excellent in viability maintenance effect.

(Mesenchymal stem cells)
In the present invention, the mesenchymal stem cell refers to one or more cells belonging to the mesenchymal system, preferably two or more cells, more preferably three or more cells (bone cells, cardiomyocytes, chondrocytes, tendon cells, fat cells, etc.). It means a cell that has differentiation ability and can proliferate while maintaining the ability. The term mesenchymal stem cell used in the present invention means the same cell as the stromal cell, and does not particularly distinguish them. Moreover, it may be simply described as a mesenchymal cell. Examples of tissues containing 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, Examples include dental pulp and tooth germ. For example, an adipose tissue-derived mesenchymal stem cell means a mesenchymal stem cell contained in an adipose tissue, and may be referred to as an adipose-derived mesenchymal stem cell. Among these, from the viewpoint of effectiveness for treatment of various diseases, from the viewpoint of availability, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, 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.

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

  Since mesenchymal stem cells are unlikely to cause rejection even for allogeneic subjects, cells prepared by pre-expanding donor cells that have been expanded and cryopreserved are used in the cell pharmaceutical composition of the present invention (A ) It can be used as a mesenchymal stem cell as a cell. Therefore, compared to the case of preparing and using autologous mesenchymal stem cells, commercialization is easy, and from the viewpoint that a certain effect can be easily obtained stably, the mesenchymal stem cells in the present invention, More preferably, it is allogeneic.

  In the present invention, the mesenchymal stem cell means any cell population containing mesenchymal stem cells. The cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98 % Or 99% are mesenchymal stem cells.

  The adipose tissue in the present invention means a tissue containing adipocytes and stromal cells including microvascular cells and the like, for example, a tissue obtained by surgical excision or aspiration of mammalian subcutaneous fat. Adipose tissue can be obtained from subcutaneous fat. It is preferably obtained from the same animal as the subject of administration of the adipose-derived mesenchymal stem cells described later, and more preferably human subcutaneous fat in consideration of administration to humans. An individual supplying 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. In the case of collecting from an individual, liposuction is exemplified by PAL (power assist) liposuction, Erconia laser liposuction, or body jet liposuction. From the viewpoint of maintaining the state of cells, ultrasound is used. It is preferable not to use.

  In the present invention, the umbilical cord is a white tubular tissue connecting the fetus and the placenta, and is composed of umbilical vein, umbilical artery, collagenous tissue (Wharton's Jelly), umbilical matrix itself, etc., and mesenchymal stem cells Including many. The umbilical cord is preferably obtained from the same animal as the subject (administration subject) using the cell pharmaceutical composition of the present invention, and more preferably in consideration of administering the cell pharmaceutical composition of the present invention to a human. The human umbilical cord.

  In the present invention, bone marrow refers to soft tissue filling the bone lumen, and is a hematopoietic organ. Bone marrow fluid is present in the bone marrow, and the cells present therein are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes and the like, as well as mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells, and the like. Bone marrow cells can be collected, for example, from human iliac bone, long bone, or other bone.

  In the present invention, each tissue-derived mesenchymal stem cell such as adipose-derived mesenchymal stem cell, umbilical cord-derived mesenchymal stem cell, bone marrow-derived mesenchymal stem cell is a fat-derived mesenchymal stem cell, umbilical cord-derived mesenchymal stem cell, It means any cell population containing mesenchymal stem cells derived from each tissue such as bone marrow-derived mesenchymal stem cells. The cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98 % Or 99% are 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.

  Mesenchymal stem cells according to the present invention have growth characteristics (eg, population doubling ability from aging to aging, doubling time), karyotype analysis (eg, normal karyotype, maternal line or neonatal line), flow cytometry ( For example, surface marker expression by FACS analysis, immunohistochemistry and / or immunocytochemistry (eg, epitope detection), gene expression profiling (eg, gene chip array; polymerase chain such as reverse transcription PCR, real-time PCR, conventional PCR, etc. Reaction), miRNA expression profiling, protein array, protein secretion such as cytokines (eg, plasma coagulation analysis, ELISA, cytokine array), metabolites (metabolome analysis), other methods known in the art, etc. May be.

(Method for preparing mesenchymal stem cells)
Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. Below, the preparation method of a fat-derived mesenchymal stem cell is demonstrated as an example. The adipose-derived mesenchymal stem cells may be obtained, for example, by the production method described in US Pat. No. 6,777,231, and can be produced by a method including the following steps (i) to (iii), for example. :
(I) obtaining a cell suspension by digesting adipose tissue with an enzyme;
(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 show binding to the solid surface.

  The adipose tissue used in step (i) is preferably washed. Washing may be performed by sedimentation with vigorous stirring using a physiologically compatible saline solution (eg, phosphate buffered saline (PBS)). This is because impurities (also called debris, such as damaged tissue, blood, and red blood cells) contained in the adipose tissue are removed from the tissue. Accordingly, washing and sedimentation are generally repeated until the debris is totally removed from the supernatant. Since the remaining cells exist as lumps of various sizes, in order to dissociate them while minimizing damage to the cells themselves, the washed cell lumps are made to have an enzyme (eg, an enzyme that weakens or breaks cell-cell junctions). It is preferable to treat with collagenase, dispase or trypsin. The amount of such enzyme and the duration of treatment vary depending on the conditions used, but are known in the art. Instead of or in combination with such enzyme treatment, the cell mass can be broken down by other treatment methods such as mechanical agitation, ultrasonic energy, thermal energy, etc., but with minimal cell damage In order to suppress it, it is preferable to carry out only by enzyme treatment. When an enzyme is used, in order to minimize harmful effects on cells, it is desirable to deactivate the enzyme using a medium or the like after a suitable period of time.

  The cell suspension obtained by the step (i) includes a slurry or suspension of aggregated cells and various contaminated cells such as erythrocytes, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, the cells in the aggregated state and these contaminated cells may be separated and removed, but they can be removed by adhesion and washing in step (iii) to be described later. May be. When contaminating cells are separated and removed, this can be achieved by centrifugation that forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing contaminating cells is suspended in a physiologically compatible solvent. Suspended cells may contain erythrocytes, but erythrocytes are excluded by selection by adhesion to the individual surface described later, and thus a lysis step is not always necessary. As a method for selectively lysing erythrocytes, for example, a method 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.

  In step (ii), in order to increase the purity of the mesenchymal stem cells in the suspended cells, the cells may be washed once or continuously several times, centrifuged, and resuspended in the medium. Alternatively, cells may be separated based on cell surface marker profile or based on cell size and granularity.

  The medium used in the resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, but such a medium includes basal medium added with serum and / or albumin, transferrin, fatty acid, One or more serum substitutes such as insulin, sodium selenite, cholesterol, collagen precursor, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, and the like may be added. In addition to these media, substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, etc. are added as necessary. May be.

  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, Ham's F16 medium, Examples include a medium, Fischer's medium, MCDB201 medium, and a mixed medium thereof.

  Examples of the 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 and the like. . When using serum, 5 v / v% to 15 v / v%, preferably 10 v / v% may be added to the basal medium.

  Examples of the fatty acid include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Examples of the lipid include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like. Amino acids include, but are not limited to, for example, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine and the like. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, and β2-microglobulin. Examples of the polysaccharide include glycosaminoglycans, and among the glycosaminoglycans, hyaluronic acid, heparan sulfate and the like are particularly exemplified, but the polysaccharide is not limited thereto. Growth factors include, for example, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), epidermal growth factor (EGF) Examples include, but are not limited to, connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and the like. 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 (xenofree) medium that does not contain components derived from different species such as serum. Such a medium is provided as a medium prepared in advance for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R & D Systems, Corning, and Roto. Has been.

  Subsequently, in step (iii), the appropriate cell density and the appropriate cell medium are used on the solid surface without differentiating the cells in the cell suspension obtained in step (ii). Culture under culture conditions. In the present invention, the “solid surface” means any material capable of binding / adhering the adipose-derived mesenchymal stem cells in the present invention. In a particular embodiment, such a material is a plastic material that has been treated to promote the attachment 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. The cells are washed after incubation to remove unbound cells and cell debris.

  In the present invention, the cells that finally remain attached and adhered to the solid surface can be selected as a cell population of adipose-derived mesenchymal stem cells.

  In order to confirm that the selected cells are the adipose-derived mesenchymal stem cells in the present invention, the surface antigen may be analyzed by a conventional method using flow cytometry or the like. Further, the ability to differentiate into each cell line may be examined, and such differentiation can be performed by conventional methods.

Mesenchymal stem cells in the present invention can be prepared as described above, but may be defined as cells having the following characteristics;
(1) It exhibits adhesiveness to plastic under the culture conditions in a standard medium.
(2) Surface antigens CD44, CD73, CD90 are positive, CD31, CD45 are negative, and (3) Differentiated into bone cells, adipocytes, and chondrocytes under culture conditions.

(Peripheral blood mononuclear cells)
In the present invention, peripheral blood mononuclear cells refer to fractions containing lymphocytes, neutrophils, eosinophils, basophils, and monocytes, which are 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 cells (A) in the present invention may be cells separated from peripheral blood, or they may be cultured with various factors, low molecular compounds, antibodies, etc. as necessary. It may be proliferated and activated.

((A) Cryopreservation of cells)
The (A) cell in the present invention may be a cell that has been appropriately cryopreserved and thawed as long as it has a therapeutic effect on various diseases. In the present invention, cryopreservation can be performed by suspending (A) cells in a cryopreservation solution well known to those skilled in the art and cooling. Suspension can be performed by detaching the cells with a release agent such as trypsin as necessary, transferring them to a cryopreservation container, treating them appropriately, and adding a cryopreservation solution.

  The cryopreservation solution may contain DMSO (dimethylsulfoxide) as a frost damage protective agent. However, since DMSO has cytotoxicity, it is preferable to reduce the DMSO content. DMSO is also known to have differentiation-inducing properties for mesenchymal stem cells. As an alternative to DMSO, glycerol, propylene glycol or polysaccharides are exemplified. When DMSO is used, it contains a concentration of 5% to 20%, preferably a concentration of 5% to 10%, more preferably a concentration of 10%. In addition, additives described in WO2007 / 058308 may be included. As such a cryopreservation solution, for example, cryopreservation provided by Bioverde, Nippon Genetics, Reprocell, Xenoac, Cosmo Bio, Kojin Bio, Thermo Fisher Scientific, etc. A liquid may be used.

  When the above-mentioned suspended cells are cryopreserved, they may be stored at a temperature between −80 ° C. and −100 ° C. (eg, −80 ° C.), and any freezer that can achieve the temperature is used. obtain. Although not particularly limited, in order to avoid a sudden temperature change, the cooling rate may be appropriately controlled using a program 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.

  The upper limit of the storage period is not particularly limited as long as the cells cryopreserved under the above conditions are thawed and retain the same properties as before freezing, 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 it at a lower temperature, it may be stored by transferring to a gas phase (about −150 ° C. or lower to −180 ° C. or lower) on liquid nitrogen. When storing in a gas phase on liquid nitrogen, it can be performed 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 a gas phase on liquid nitrogen.

The thawed (A) cells may be appropriately cultured before the next cryopreservation. For example, the mesenchymal stem cells are cultured using the above-described medium in which mesenchymal stem cells can be cultured. Although not particularly limited, the medium contains CO ₂ at a culture temperature of about 30 to 40 ° C., preferably about 37 ° C. It may be performed in an air atmosphere. CO ₂ concentration is about 2-5%, preferably about 5%. In culture, after reaching the appropriate confluency for the culture container (for example, cells may occupy 50% to 80% of the culture container), the cells are detached with a release agent such as trypsin. Alternatively, the culture may be continued by seeding in a separately prepared culture vessel at an appropriate cell density. When seeding cells, typical cell densities are 100 cells / cm ^{2 to} 100,000 cells / cm ² , 500 cells / cm ^{2 to} 50,000 cells / cm ² , 1,000 to 10,000 cells. / ^cm 2, such as 2,000 to 10,000 cells / cm ² is exemplified. In a particular embodiment, the cell density is 2,000 to 10,000 cells / cm ^2. It is preferable to adjust the period until reaching appropriate confluency to 3 to 7 days. During culture, the medium may be changed as necessary.

  Thawing cryopreserved cells can be performed by methods well known to those skilled in the art. For example, the method performed by standing or shaking in a 37 degreeC thermostat or a hot water bath is illustrated.

((A) Cell morphology)
The cell (A) contained in the cell pharmaceutical composition of the present invention may be a cell in any state, for example, a cell recovered by detaching a cell in culture, or frozen in a cryopreservation solution It may be a cell in a state of being formed. Use of the same lot of cells obtained by expanding and culturing in the same lot is preferable in that the same action and effect can be stably obtained and the handling property is excellent.

  The (A) cells in the cryopreservation state may be thawed immediately before use and directly mixed in the cell suspension solution (B) described later while being 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.

The dose (dosage) of (A) cells of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.), dosage form, etc., but from the standpoint of sufficient therapeutic effect, the amount From the viewpoint of suppressing the occurrence of side effects, a smaller amount tends to be preferable. Usually, when administered to an adult, the number of cells is 1 × 10 ^{3 to} 1 × 10 ¹² cells / time, preferably 1 × 10 ^{4 to} 1 × 10 ¹¹ cells / time, more preferably 1 × 10 ^{5 to} 1 × ^{10 10} cells / time, particularly preferably 5 × ¹⁰ 5. ^{6 to 1} × 10 ⁹ pieces / time. In addition, this dose may be administered once as a single dose, or the dose may be divided into multiple doses.

The dose (dosage) of (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 of the composition of the present invention, but is usually administered to an adult. In this case, the number of cells is 1 × 10 to ⁵ × ^{10 10} cells / kg, preferably 1 × 10 ^{2 to} 5 × 10 ⁹ cells / kg, more preferably 1 × 10 ^{3 to} 5 × 10 ⁸ cells / kg, and particularly preferably 1 × 10 ^{4 to} 5 × 10 ⁷ cells / kg. It is. In addition, this dose may be administered once as a single dose, or the dose may be divided into multiple doses.

[(B) Cell suspension solution]
The solution for cell suspension (B) of the present invention contains an electrolyte and a carbohydrate, contains Na ⁺ and Cl ⁻ as the electrolyte, and is substantially free of K ⁺ . By adopting the (B) cell suspension solution having such a composition, in the cell pharmaceutical composition of the present invention, (A) the state of the cells is kept good, and the survival rate is high over a long period of time. Can be maintained.

(B) The cell suspension solution contains Na ⁺ and Cl ⁻ as the electrolyte, and does not substantially contain K ⁺ . The concentration of Na ⁺ and Cl ⁻ is lower than that of physiological saline and higher than that of postoperative recovery liquid (No. 4 infusion) and the like. Specifically, it is 30 mEq / L or more and 130 mEq / L or less, preferably 50 mEq / L or more and 120 mEq / L or less, and more preferably 60 mEq / L or more and 100 mEq / L or less. More preferably, the Na ⁺ concentration is 70 mEq / L or more and 100 mEq / L or less, and the Cl ⁻ concentration is 70 mEq / L or more and 80 mEq / L or less. The concentration of Na ^{+ and} the concentration of Cl ⁻ may be the same or different.
(B) Since the electrolyte concentration is low as described above, the cell suspension solution is prepared to be isotonic by adding a saccharide described below. Note that “substantially free of K ^+” means that (B) does not contain an amount of K ⁺ that reduces the above effect by the cell suspension solution, and is preferably below the detection limit.

(B) as the electrolyte in the cell suspension solution is substantially Na ⁺ and Cl ^- which is preferably only, Na ⁺ and Cl ^- in addition to a certain amount of Lac ^- may contain . The concentration of Lac ⁻ is 0 to 30 mEq / L, and preferably 0 to 20 mEq / L.

(B) Examples of the sugar contained in the cell suspension solution include glucose, dextrin, maltodextrin, oligosaccharide, sucrose, and the like, and one or more of these may be used in combination. Can do. Among these, glucose is preferable as the saccharide. The sugar concentration can be adjusted according to the concentrations of Na ⁺ and Cl ⁻ so that (B) the cell suspension solution is isotonic. Specifically, it is 0.1 w / v% to 10 w / v%, and preferably 1.0 w / v% to 5.0 w / v%.

  The (B) cell suspension solution of the present invention can be referred to as an electrolyte infusion preparation, and what is known by the name of the starting solution or No. 1 infusion is the above-described (B) cell suspension solution. It satisfies the conditions and is preferable. Specifically, as (B) cell suspension solution, Solita (registered trademark) -T1 infusion (Ai Pharma Co., Ltd.), YD Solita (registered trademark) -T1 infusion (Hyoshindo Co., Ltd.) , KN1 infusion (Otsuka Pharmaceutical Factory), Denosaline (registered trademark) 1 infusion (Terumo Corporation), Soldem (registered trademark) 1 infusion (Terumo Corporation), Riplus (registered trademark) 1 infusion (Fuso Pharmaceutical) Commercial products such as Co., Ltd. can also be used. In addition, 2.5% Dextose and 0.45% Sodium Chloride Injection, USP and 5% Dextose and 0.45% Sodium Chloride Injection, USP are listed in US Pharmacopoeia (USP). Infusions sold from BAXTER HEALTHARE LIMITED, etc. in the United States, Canada, etc. can be used. Furthermore, as Sodium Chloride 0.45% w / v & Glucose 2.5% w / v Solution for Infusion BP, Sodium Chloride 0.45% w / v and Glucose 5.0% w / v Solution for Infusion BP, etc. Infusions listed in the British Pharmacopoeia (BP) and marketed by BAXTER HEALTHARE LIMITED etc. in the UK, Belgium, Ireland, Luxembourg, Malta, etc. can be used. As an example, Sodium Chloride 0.45% w / v and Glucose 2.5% w / v Intravenous Infusion BP or the like, an infusion solution that is listed in BP and sold by Braun Melsungen AG or the like can be used.

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

  Examples of liver disease therapeutic agents include hepatitis B therapeutic agents (lamivudine, adefovir, entecavir, tenofovir, etc.), interferon preparations (interferon α, interferon α-2b, interferon β, peginterferon α-2a, peginterferon α-2b, etc. Etc.), hepatitis C drugs (ribavirin, terapyrevir, simeprevir, vaniprevir, daclatasvir, asunaprevir, sofosbuvir, etc.), corticosteroids (prednisolone, methylprednisolone sodium succinate, etc.), anticoagulants (dry concentrated human antithrombin III) , Gabexate mesylate, thrombomodulin α, etc.), antidote (calcium edetate disodium hydrate, glutathione, dimethicaprol, sodium thiosulfate hydrate, sugamama desnato ), 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, vitamins (tocopherol, flavin adenine dinucleotide, thiamine phosphate) Disulfide, pyridoxine, cyanocobalamin and their salts), antibiotics (sulbactam sodium, cefoperazone sodium, meropenem hydrate, vancomycin hydrochloride, etc.).

  Examples of therapeutic agents for heart diseases include ACE inhibitors, angiotensin II receptor antagonists, β-blockers, antiplatelet agents, warfarin, calcium antagonists, nitrates, diuretics, HMG-CoA reductase inhibitors, ancalons, etc. Is mentioned.

  Examples of the therapeutic agent for inflammatory bowel disease include salazosulfapyridine and mesalazine.

  Examples of respiratory drugs include dimorpholine, doxapram hydrochloride hydrate, cyberestat sodium hydrate, pirfenidone, pulmonary surfactant, and Dornase Alpha.

  Examples of the drugs for nervous system include edaravone, interferon beta-1a, interferon beta-1b, fingolimod hydrochloride, riluzole, tartilelin hydrate and the like.

  Examples of cardiovascular drugs include hepronicart, midodrine hydrochloride, amedinium methylmethyl sulfate, ethylephrine hydrochloride, phenylephrine hydrochloride, and the like.

  Examples of the cerebral circulation improving drug include ifenprodil tartrate, nicergoline, ipdilast, dihydroergotoxin mesylate, nizophenone fumarate, and fasudil hydrochloride hydrate.

  Examples of the immunosuppressant include cyclosporine, azathioprine, mizoribine, basiliximab, tacrolimus hydrate, gusperimus hydrochloride, mycophenolate mofetil, everolimus and the like.

  When the cell pharmaceutical composition of the present invention contains the above-mentioned other drug, at the time of storage, it may be stored in a container different from (A) the cell and (B) the cell suspension solution. It may be contained in a form blended with. Depending on the type of disease, treatment method, patient condition, etc., other drugs and (A) cells and (B) cell suspension solutions may be administered simultaneously, or at regular intervals. Also good.

  If the cell pharmaceutical composition of the present invention is within a range not impairing the effects of the present invention, in addition to the above (A) cell and (B) cell suspension solution, according to its use and form, according to a conventional method, Other components such as pharmaceutically acceptable carriers and additives may be included. 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 isotonic agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH regulators, stabilizers, chelating agents. , Oily base, gel base, surfactant, suspending agent, binder, excipient, lubricant, disintegrant, foaming agent, fluidizer, dispersant, emulsifier, buffer, solubilizer , Antioxidants, sweeteners, sour agents, colorants, flavoring agents, fragrances or refreshing agents, but are not limited thereto.

  The cell pharmaceutical composition of the present invention can be used in various forms depending on the purpose, for example, injections (including infusions, implantable injections, continuous injections, injections prepared at the time of use), dialysis agents, patches. It can be used in the form of a poultice. 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 the form of gelation or sheeting at the affected area after spraying. The cell pharmaceutical composition of the present invention can also be applied to the affected area after the (A) cell is made into a sheet or a three-dimensional structure.

  In the cell pharmaceutical composition of the present invention, (A) cells, (B) cell suspension solutions, other drugs, and other components are sealed and stored in separate containers, and are used by mixing them at the time of use. Also good. In addition, at the time of storage, the above (A) cells, (B) cell suspension solution, other drugs, and other components may be stored under conditions suitable for each, for example, freezing conditions, refrigerated conditions, room temperature Any of conditions etc. may be sufficient.

  The pH of the cell pharmaceutical composition of the present invention is not particularly limited as long as it is within a pharmaceutically, pharmacologically (pharmaceutical) or physiologically acceptable range. The range which becomes 9.0, Preferably it is 2.5-8.5, More preferably, it is 3.0-8.0 is mentioned.

  The osmotic pressure of the cell pharmaceutical composition of the present invention is not particularly limited as long as it is within a range acceptable for a living body. As an example of the osmotic pressure ratio of the cell pharmaceutical composition of the present invention, a range of preferably 0.7 to 5.0, more preferably 0.8 to 3.0, and still more preferably 0.9 to 1.4. It is done. The adjustment of the osmotic pressure can be performed by a method known in the art using the above-described electrolyte, sugar 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. Measure by referring to the freezing point method. The standard solution for measuring the osmotic pressure ratio (0.9 w / v% sodium chloride aqueous solution) was dried in sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650 ° C. for 40 to 50 minutes, and then in a desiccator (silica gel). The mixture is allowed to cool and 0.900 g is accurately weighed and dissolved in purified water to make exactly 100 mL, or a commercially available standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) is used.

The concentration of (A) cells in the cell pharmaceutical composition of the present invention, that is, the concentration when (A) the cells are prepared by suspending them in the (B) cell suspension solution and used for administration is the cell type, cell suspension Usually, it is 1 × 10 ^{2 to} 2.5 × 10 ⁸ cells / mL, preferably 1 × 10 ^{3 to} 2.5 × 10 ⁷ cells / mL, more preferably 1 × 10 ^{4 to} 2.5 × 10 ⁶ cells / mL.

As a preferred form of the cell pharmaceutical composition of the present invention, human mesenchymal stem cells are contained in a cell suspension solution at a density of 1 × 10 ^{4 to} 2.5 × 10 ⁶ cells / mL, The turbid solution contains 70-100 mEq / L of sodium ions and 70-80 mEq / L of chlorine ions, is substantially free of potassium ions, and has an osmotic pressure ratio of 0.9-1.4 with respect to physiological saline. Yes, with a pH of 3.0-8.0.

  The administration route to the subject of the cell pharmaceutical composition of the present invention includes subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intraperitoneal administration, rectal administration, vaginal administration, and transdermal administration. From the viewpoint of the effectiveness of the cell pharmaceutical composition of the present invention, implant, intraarterial administration, intravenous administration, and direct administration to an organ are more preferable. Are intravenous and direct administration to the organ.

  The administration rate of the cell pharmaceutical composition of the present invention to the subject may vary depending on the patient's condition (weight, age, symptoms, physical condition, etc.) and the administration route of the non-alcoholic steatohepatitis therapeutic agent of the present invention. 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.

  The administration temperature of the cell pharmaceutical composition of the present invention to the subject may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.), the administration route of the cell pharmaceutical composition of the present invention, etc. It is -45 degreeC, It is preferable that it is 15 degreeC-37 degreeC, and it is more preferable that it is room temperature-37 degreeC.

  The cell pharmaceutical composition of the present invention can be administered to a subject using an infusion set. Specifically, as an infusion set, a wand disposable infusion tube set (manufactured by Yoshida Seisakusho Co., Ltd.), an infusion solution set (manufactured by Fortegro Medical Co., Ltd.), a terfusion (R) infusion set (manufactured by Terumo Corporation), a JMS infusion set (Manufactured by JMS Co., Ltd.), Sure plug 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) Commercial products such as Toray Medical Co., Ltd. can also be used.

  The cell pharmaceutical composition of the present invention can be administered to a subject using an infusion tube. Specifically, as an infusion tube, Lectro Cass (manufactured by Samick International Co., Ltd.), JMS extension tube (manufactured by JMS Co., Ltd.), Saffy extension tube (manufactured by Terumo Corporation), extension tube (manufactured by Co., Ltd.) Top product), connecting tube (chuatsu) (Medicos Hirata Co., Ltd.), safty AP tube (manufactured by Kawasumi Chemical Industry Co., Ltd.), Bioconnector 2 with extension tube (Toray Medical Co., Ltd.), Medicut extension tube set B (Nippon Sherwood) Commercially available products such as Wand Disposable Infusion Tube Set (manufactured by Yoshida Seisakusho Co., Ltd.), Infusion Tube (manufactured by Forte Grow Medical Co., Ltd.), etc.

  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), plasticizer PVC, and mixtures thereof can be used.

  The cell pharmaceutical composition of the present invention can be suitably used for the treatment of various diseases. For example, visceral diseases, specifically heart disease, stomach / duodenal disease, small intestine / colon disease, liver disease, biliary disease, pancreatic disease, kidney disease, lung disease, mediastinal disease, diaphragm disease, pleural disease, peritoneal disease It is preferably used for neurological diseases, central nervous system (CNS) disorders, peripheral arterial diseases, and peripheral venous diseases.

  Specific diseases include, for example, autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (Nonalcoholic steatohepatitis (NASH)), nonalcoholic fatty liver (NAFL), liver fibrosis, cirrhosis, liver cancer, fatty liver, drug allergic liver disorder, hemochromatosis, hemosiderosis, Wilson disease, primary Hepatic diseases such as biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver abscess, chronic active hepatitis, chronic persistent hepatitis; myocardial infarction, heart failure, arrhythmia, palpitations, cardiomyopathy, false Bloody myocardium Heart disease such as angina pectoris, congenital heart disease, valvular heart disease, myocarditis, familial hypertrophic cardiomyopathy, dilated cardiomyopathy, acute coronary syndrome, atherothrombosis, restenosis; acute gastritis, chronic gastritis, Gastric / duodenal diseases such as gastric / duodenal ulcer, gastric cancer, duodenal cancer; ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, simple ulcer, intestinal Behcet's disease, small intestine cancer, colon cancer, etc. Colorectal diseases; acute cholecystitis, acute cholangitis, chronic cholecystitis, cholangiocarcinoma, gallbladder cancer, etc .; pancreatic diseases such as acute pancreatitis, chronic pancreatitis, pancreatic cancer; acute nephritis, chronic nephritis, acute renal failure, chronic renal failure Kidney disease such as: pneumonia, emphysema, pulmonary fibrosis, interstitial pneumonia, idiopathic interstitial pneumonia, exfoliative interstitial pneumonia, acute interstitial pneumonia, nonspecific interstitial pneumonia, drug-induced lung Disease, eosinophilic lung disease, pulmonary hypertension, pulmonary tuberculosis, pulmonary tuberculosis sequelae, acute respiratory distress Syndrome, cystic fibrosis, chronic obstructive pulmonary disease, pulmonary embolism, pneumonia, pneumoconiosis, swallowing pneumonia pulmonary fibrosis, acute upper respiratory tract infection, chronic lower respiratory tract infection, pneumothorax, alveolar epithelium damaged Diseases seen, pulmonary diseases such as lymphatic smooth muscle species, lymphoid interstitial pneumonia, alveolar proteinosis, pulmonary Langerhans cell granulomatosis; mediastinal tumor, mediastinal cystic disease, mediastinum such as mediastinitis Disease: Diaphragmatic diseases such as diaphragmatic hernia; Pleural diseases such as pleurisy, empyema, pleural tumor, cancerous pleurisy, pleural mesothelioma; Peritoneal diseases such as peritonitis, peritoneal tumor; Cerebral palsy syndrome including pediatric cerebral palsy Meningitis, Guillain-Barre syndrome, amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, polyneuropathy, spinal muscular atrophy, spinal disorder, acute transverse myelitis, spinal cord infarction (ischemic spinal cord) Disability), intracranial tumor, spinal tumor, etc. CNS disorders such as neurological diseases; Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, Parkinson's disease; fibromuscular dysplasia, peripheral arterial disease (PAD), obstructive thromboangiitis (Burger's disease), Kawasaki disease (KD) Peripheral vein diseases such as deep vein thrombosis, chronic venous insufficiency, post-phlebitis syndrome, superficial venous thrombosis, etc .; graft-versus-host disease (GVHD), secondary immunodeficiency, primary Immunodeficiency disease, B cell deficiency, T cell deficiency, B and T cell complex deficiency, phagocytic deficiency, complement deficiency in classical pathway, complement deficiency in MBL pathway, complement deficiency in alternative pathway, complement regulatory protein deficiency And immunodeficiency diseases such as complement receptor deficiency.

  Among these, liver diseases, heart diseases, lung diseases, neurological diseases, peripheral arterial diseases, immunodeficiency diseases that have been confirmed to have sufficient therapeutic effects by mesenchymal stem cells are preferred, among which liver fibrosis, Suitable for the treatment of cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, interstitial pneumonia, childhood cerebral palsy, amyotrophic lateral sclerosis (ALS), peripheral arterial disease (PAD), graft-versus-host disease (GVHD) And can be used more suitably for liver fibrosis, cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, and interstitial pneumonia. Moreover, it can use suitably for the cancer of each structure | tissue where it is confirmed that the therapeutic effect by a peripheral blood mononuclear cell is fully acquired.

<Disease treatment kit>
The present invention includes (A) a cell, and (B) a cell suspension solution containing an electrolyte and a carbohydrate. (B) the electrolyte in the cell suspension solution contains Na ⁺ and Cl ⁻ , ^Also included are disease treatment kits that are substantially free of ⁺ . The disease treatment kit of the present invention is a kit containing the above-described cell pharmaceutical composition of the present invention, and may contain (A) cells, (B) cell suspension solutions, and other cell pharmaceutical compositions of the present invention. The description in the section of the cell pharmaceutical composition can be applied to the components. According to the disease treatment kit of the present invention, since the state of cells can be kept good and the survival rate can be maintained high over a long period of time, an excellent therapeutic effect can be obtained for various diseases. .

  The disease treatment kit of the present invention can also be expressed as including the cell pharmaceutical composition, container and label of the present invention. Suitable containers included in the disease treatment kit of the present invention are not particularly limited, and examples include cryotubes for freezing cells, bottles for cell suspension solutions, vials, test tubes, and dialysis bags. These containers may be formed from a variety of materials such as glass, metal, plastic, or combinations thereof. The contents on the labels on these containers describe the contents of the cells, the solution for cell suspension, and the like.

  The disease treatment kit of the present invention includes other additives, other drugs, diluents, filters, needles, syringes, and other packages that are desirable from a commercial and user standpoint, including package inserts that describe usage. Material can be included.

<Cell suspension solution>
A cell suspension solution for a cell pharmaceutical composition that includes an electrolyte and a carbohydrate, wherein the electrolyte includes Na ⁺ and Cl ⁻ and is substantially free of K ⁺ is also within the scope of the present invention. By adopting a cell suspension solution for cell medicine injection / infusion containing an electrolyte and a carbohydrate, the electrolyte containing Na ⁺ and Cl ⁻ and substantially free of K ⁺ It is a finding newly found by the present inventors that the survival rate can be maintained high for a long time. For 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.

<Disease treatment method>
The present invention provides a method for treating a disease in which (A) cells are suspended in a cell suspension solution containing (B) an electrolyte and a carbohydrate and then administered to a patient. Also included is a method of treatment characterized in that the electrolyte comprises Na ⁺ and Cl ⁻ and is substantially free of K ⁺ . According to the treatment method of the present invention, it is possible to maintain a high cell survival rate over a long period of time, and therefore, an excellent therapeutic effect 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 may contain (A) cells, (B) a cell suspension solution, and other cell pharmaceutical compositions of the present invention. The description in the section of the cell pharmaceutical composition can be applied to the components.

  Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited to these Examples and the like.

[Example 1]
(Preparation of adipose-derived mesenchymal stem cells)
After obtaining consent from a human donor, the subcutaneous adipose tissue obtained by the liposuction method was washed with physiological saline. In order to achieve destruction of the extracellular matrix and isolation of the cells, collagenase (Roche diagnostics) (solvent was physiological saline) was added, shaken at 37 ° C. for 90 minutes, and dispersed. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of stromal vascular cell groups. A serum-free medium for mesenchymal stem cells (Rohto) is added to the cell precipitate, the cell suspension is centrifuged at 400 g for 5 minutes, and after removal of the supernatant, a serum-free medium for mesenchymal stem cells (Rohto) The cells were seeded in a flask. Cells were cultured at 37 ° C. for several days in 5% CO ₂ . Several days later, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture solution, and mesenchymal stem cells adhered to a plastic container were obtained.

  The obtained fat-derived mesenchymal stem cells were dispensed into a centrifuge tube and centrifuged at 400 g for 5 minutes to obtain cell precipitates. After removing the supernatant, an appropriate amount of a cell cryopreservation solution (STEM-CELLBANKER (Zenoac)) was added and suspended. The cell suspension was dispensed into a cryotube, stored at −80 ° C. in a freezer, transferred to a gas phase on liquid nitrogen, and stored.

Remove fat-derived mesenchymal stem cells stored in liquid nitrogen gas phase from liquid nitrogen, put glass vials in a storage set at 15 ° C or 30 ° C, let stand for 1 hour, thaw cells, The vial was moved to homogenize the internal cell suspension. To a 50 mL centrifuge tube (Sumitomo Bakelite Co., Ltd., product number: MS-56500), Otsuka raw food injection 500 mL (Japanese Pharmacopoeia physiological saline; Otsuka Pharmaceutical Factory, Lot: 5A81N), Physio (registered trademark) 140 infusion (extracellular) Solution replenisher, 1% glucose-acetated Ringer solution; Otsuka Pharmaceutical Factory, Lot: M5C73), Otsuka Sugar Solution 5% (Japanese Pharmacopoeia Glucose Injection; Otsuka Pharmaceutical Factory, Lot: 4L83S), KN1 Infusion ( After dispensing 30 mL each of No. 1 solution (starting solution); Otsuka Pharmaceutical Factory, Lot: M5K81), 375 μL of the KN1 infusion solution and 300 μL of the cell suspension were added to the other infusion solutions. suspension density of the suspension solution was used KN1 No. infusion in suspension using a 1.25x10 ⁵ cells / mL, other solutions It was prepared so as to 1.0x10 ⁵ cells / mL. After suspending by inversion mixing, store in a storage room at 15 ° C or 30 ° C, and immediately after preparation, take 1 mL from the middle layer with a 5 mL syringe with a 21 G needle after 2 hours, 3 hours, and 4 hours, Transfer 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) is added to distinguish between live cells and dead cells, and a phase contrast microscope (OLYMPUS, product number: Measurement was performed with CKX41SF). In addition, using a disposable cell counting board (WAKEN, product number: WC2-100) for cell counting, 18-compartment counting is performed 5 times, and the average value of 3 measurement values excluding the maximum value and the minimum value is calculated. The cell viability was calculated by the following formula. 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 living cells / total number of cells × 100

  When fat-derived mesenchymal stem cells were suspended in Otsuka raw diet, the cell viability was 70% or more only after storage at 15 ° C and 30 ° C. When fat-derived mesenchymal stem cells were suspended in Physio (registered trademark) 140 infusion, the cell viability was 70% or more when stored at 15 ° C, immediately after preparation, after 2 hours, and when stored at 30 ° C. It was only immediately after. When fat-derived mesenchymal stem cells were suspended in 5% Otsuka sugar solution, the cell viability was 70% or more only immediately after preparation at 30 ° C. storage. On the other hand, when fat-derived mesenchymal stem cells were suspended in KN1 infusion, the cell viability was 70% or more at all storage times at 15 ° C. and 30 ° C. It was revealed that the cell viability was significantly increased by suspending fat-derived mesenchymal cells in KN1 infusion.

[Example 2]
Into a 15 mL centrifuge tube (Sumitomo Bakelite Co., Ltd., product number: MS-56150), KN1 infusion (No.1 solution (starting solution); Otsuka Pharmaceutical Factory, Lot: M6C77), KN2 infusion (No.2 solution (dehydration supplement) Solution); Otsuka Pharmaceutical Factory, Lot: K6E92), KN3 Infusion (No.3 Liquid (Maintenance Solution); Otsuka Pharmaceutical Factory, Lot: K6D96), KN4 Infusion (No.4 Liquid (Postoperative Recovery Solution)) Otsuka Pharmaceutical Factory, Lot: K6D80), Physio (registered trademark) 70 infusion (Otsuka Pharmaceutical Factory, Lot: M6D91), 1% glucose-containing No. 1 liquid (hereinafter referred to as “Glu1%”) and 5% After dispensing 5 mL each of the glucose-containing No. 1 solution (hereinafter referred to as “Glu5%”), the cryopreserved fat-derived mesenchymal stem cells were rapidly thawed in a hot water bath (37 ± 1 ° C.), Adding the cell suspension肪由Klima mesenchymal stem cells by 62.5MyuL, the concentration of the suspension of the cell was adjusted to be 1.23x10 ⁵ cells / mL. In addition, Glu1% and Glu5% are Otsuka raw food injection 500mL (Japanese Pharmacopoeia physiological saline; Otsuka Pharmaceutical factory, Lot: 5A81N), Otsuka sugar solution 50% (Otsuka Pharmaceutical factory, Lot: M5G83) and injection. It was prepared using water (Otsuka Pharmaceutical Factory, Lot: 6098). The cells were suspended by inversion and stored at room temperature. Immediately after the preparation, live cells and dead cells after 2 hours, 4 hours, and 7 hours were measured in the same manner as in Example 1. Similarly, the cell viability was calculated. For cells whose cell viability fell below 70% after 4 hours, measurement after 7 hours was not performed. The composition of each infusion solution is shown in Table 3 below, and the results of cell viability are shown in Table 4 below.

When fat-derived mesenchymal stem cells were suspended in KN2 infusion and Physio (registered trademark) 70 infusion, the cell viability after 4 hours was less than 70%. When fat-derived mesenchymal stem cells were suspended in KN3 infusion and KN4 infusion, cell viability fell below 70% after 7 hours. On the other hand, when fat-derived mesenchymal stem cells are suspended in KN1 infusion, Glu1% and Glu5%, high cell viability can be maintained in all storage times, and about 80% even after 7 hours. there were. As described above, the KN1 infusion solution containing glucose and containing only Na ⁺ and Cl ⁻ as electrolytes, Glu1% and Glu5%, have a significantly high cell viability of fat-derived mesenchymal stem cells over a long period of time. It was found that it can be maintained.

[Example 3]
Using Soldem (registered trademark) 1 infusion solution (No. 1 solution (starting solution); Terumo Corporation, Lot: 160519TA), the concentration in the cell suspension is 1.23 × 10 ⁵ cells / mL. The test was conducted in the same manner as in Example 2, and the cell viability was calculated immediately after preparation, after 2 hours and after 4 hours. The composition of Soldem (registered trademark) 1 infusion is shown in Table 5 below, and the results of cell viability are shown in Table 6 below.

When fat-derived mesenchymal stem cells were suspended in Soldem (registered trademark) 1 infusion, the cell viability was about 90% even after 4 hours. Therefore, the Soldem (registered trademark) 1 infusion solution containing glucose and containing only Na ⁺ , Cl ⁻ , and Lac ⁻ as electrolytes has a high cell viability of fat-derived mesenchymal stem cells over a long period of time. It was found that it can be maintained.

[Example 4]
The fat-derived mesenchymal stem cells of Example 2 were replaced with bone marrow-derived mesenchymal stem cells (Lonza), and KN1 infusion (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory, Lot: M6C77) was used. Then, the concentration in the cell suspension was adjusted to 1.1 × 10 ⁵ cells / mL, and the test was performed in the same manner as in Example 2. In the same manner as in Example 2, immediately after the preparation, 2 hours, 4 hours and 7 hours later, the live cells and dead cells were measured, and the cell viability was calculated. The results are shown in Table 7 below.

Even when bone marrow-derived mesenchymal stem cells were suspended in KN1 infusion, the cell viability was 90% or more after 7 hours. Therefore, the KN1 infusion solution containing glucose and containing only Na ⁺ and Cl ⁻ as electrolytes has a significantly high cell viability for bone marrow-derived mesenchymal stem cells as well as fat-derived mesenchymal stem cells. It was found that it can be maintained for a long time.

[Example 5]
Instead of the fat-derived mesenchymal stem cells of Example 2 with umbilical cord-derived mesenchymal stem cells (Lifeline Cell Technology, LifeLine (registered trademark) _UCMSC, Lot. 160907), KN1 infusion (No. 1 solution (starting solution); Using Otsuka Pharmaceutical Factory, Lot: M6C77), the concentration in the cell suspension was adjusted to 1.23 × 10 ⁵ cells / mL, and the test was performed in the same manner as in Example 2. In the same manner as in Example 2, live cells and dead cells immediately after preparation, 2 hours, 4 hours, and 6 hours were measured, and cell viability was calculated. The results are shown in Table 8 below.

Even when umbilical cord-derived mesenchymal stem cells were suspended in KN1 infusion, the cell viability was 90% or more after 6 hours. Therefore, the KN1 infusion solution containing glucose and containing only Na ⁺ and Cl ⁻ as electrolytes has a significantly high cell viability for the umbilical cord-derived mesenchymal stem cells as well as the fat-derived mesenchymal stem cells. It was found that it can be maintained for a long time.

[Example 6]
The fat-derived mesenchymal stem cells of Example 2 were replaced with peripheral blood mononuclear cells (ACCUCELL (registered trademark) normal donor-derived PBMC, Precision Bioservices (PRECISION FOR MEDICINE), Lot. 13134-10), KN1 Example 2 was prepared using an infusion solution (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory, Lot: M6C77) so that the concentration in the cell suspension was 1.23 × 10 ⁵ cells / mL. The test was conducted in the same manner as above. In the same manner as in Example 2, live cells and dead cells immediately after preparation, 2 hours, 4 hours, and 6 hours were measured, and cell viability was calculated. The results are shown in Table 9 below.

Even when peripheral blood mononuclear cells were suspended in KN1 infusion, the cell viability was about 80% after 6 hours. Therefore, the KN1 infusion solution containing glucose and containing only Na ⁺ and Cl ⁻ as electrolytes has a significantly high cell viability for peripheral blood mononuclear cells as well as fat-derived mesenchymal stem cells. It was found that it can be maintained for a long time.

[Example 7]
Adipose-derived mesenchymal stem cells were prepared in the same manner as in Example 1, and the adipose-derived mesenchymal stem cells stored in the liquid nitrogen gas phase were removed from the liquid nitrogen, and the cells were thawed using a thermostatic chamber (37 ° C.). did. After melting and standing at room temperature for 30 minutes, the solution was added to KN1 infusion (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory), and then the infusion bag was mixed by inversion. The cell viability (%) was calculated immediately after preparation, 2 hours, 3 hours, and 4 hours after the infusion bag was suspended on an infusion stand at room temperature. The mixed solution was bent and mixed once every 30 minutes. In addition, the total cell concentration immediately after production was 1.8 × 10 ^{5 to} 1.9 × 10 ⁵ for Samples 1 to ³ , and 6.3 × 10 ^{5 to} 7.3 × 10 ⁵ for Samples 4 to ⁶ . . The results are shown in Table 10 below.

  When fat-derived mesenchymal stem cells are suspended in KN1 infusion, high cell viability can be maintained during all storage times, and KN1 infusion significantly increases the cell viability of adipose-derived mesenchymal stem cells It was found that it can be maintained for a long time.

[Example 8]
Adipose-derived mesenchymal stem cells were prepared in the same manner as in Example 1, and the adipose-derived mesenchymal stem cells stored in the liquid nitrogen gas phase were removed from the liquid nitrogen, and the cells were thawed using a thermostatic chamber (37 ° C.). did. KN1 infusion solution (No. 1 solution (starting solution); Otsuka Pharmaceutical Co., Ltd.) so as to be 1.25 × 10 ⁵ cells / mL and 5 × 10 ⁵ cells / mL after standing at room temperature for 30 minutes after melting. After addition to the factory, the infusion bag was mixed by inversion. The infusion bag is hung on an infusion stand at room temperature and left to stand for 3 hours. After that, the infusion tube (polyvinyl chloride (plasticizer: tri (2-ethylhexyl) trimellitic acid), polybutadiene) is connected to the infusion bag and mixed. The liquid was added dropwise. The cell viability (%) of the mixed solution was calculated 30 minutes, 60 minutes, 120 minutes, or 111 minutes after the start of dropping. The mixed solution was bent and mixed once every 30 minutes. The results are shown in Table 11 below.

ＫＮ１号輸液に脂肪由来間葉系幹細胞を懸濁した場合に、輸液チューブを通しても、高い細胞生存率を維持できることが明らかとなった。

When fat-derived mesenchymal stem cells were suspended in KN1 infusion, it was revealed that high cell viability could be maintained even through the infusion tube.

[Example 9]
Adipose-derived mesenchymal stem cells were prepared in the same manner as in Example 1, and the adipose-derived mesenchymal stem cells stored in the liquid nitrogen gas phase were removed from the liquid nitrogen, and the cells were thawed using a thermostatic chamber (37 ° C.). did. After thawing the cells, each was added to KN1 infusion (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory), serum-free medium (Rohto Pharmaceutical Co., Ltd.) and physiological saline (Otsuka Pharmaceutical Factory, Inc.). The cells were stored at 4 ° C., and the cell viability (%) immediately after preparation, 16 hours, 24 hours, and 99 hours was calculated. The results are shown in Table 12 below.

  When fat-derived mesenchymal stem cells were suspended in physiological saline and stored at 4 ° C., the cell viability after 16 hours was less than 50%, and after 99 hours, the cell viability was less than 25%. On the other hand, when fat-derived mesenchymal stem cells are suspended in KN1 infusion, high cell viability can be maintained during all storage times, and the viability is equivalent to the serum-free medium used for cell culture. there were. From the above, it was found that KN1 infusion can maintain the cell viability of adipose-derived mesenchymal stem cells over a long period of time even when stored at 4 ° C.

  Since the cell pharmaceutical composition of the present invention can maintain a good cell state and maintain its survival rate in a high state for a long time, it can be expected to have an excellent therapeutic effect on various diseases.

Claims (9)

(A) a cell, and (B) a cell suspension solution containing an electrolyte and a carbohydrate,
(B) The cell pharmaceutical composition, wherein the electrolyte in the cell suspension solution contains Na ⁺ and Cl ⁻ and substantially does not contain K ⁺ . (B) The cell pharmaceutical composition according to claim 1, wherein the electrolyte in the solution for cell suspension is substantially only Na ⁺ and Cl ⁻ or substantially only Na ⁺ , Cl ⁻ and Lac ⁻ . (B) Na ⁺ and Cl in the cell suspension solution ^- the concentration of, at each 30.0mEq / L or more, cells pharmaceutical composition according to claim 1 or 2.   (B) The cell pharmaceutical composition according to any one of claims 1 to 3, wherein the carbohydrate in the cell suspension solution contains glucose.   (B) The cell pharmaceutical composition according to any one of claims 1 to 4, wherein the sugar concentration in the cell suspension solution is 1% to 5%.   (A) The cell pharmaceutical composition according to any one of claims 1 to 5, wherein the cells are mesenchymal stem cells or peripheral blood mononuclear cells.   The cell medicinal composition according to claim 6, wherein the mesenchymal stem cells are derived from fat, umbilical cord or bone marrow. (A) a cell, and (B) a cell suspension solution containing an electrolyte and a carbohydrate,
(B) The disease treatment kit, wherein the electrolyte in the cell suspension solution contains Na ⁺ and Cl ⁻ and does not substantially contain K ⁺ . A cell suspension solution for a cell pharmaceutical composition, comprising an electrolyte and a saccharide, wherein the electrolyte contains Na ⁺ and Cl ⁻ and substantially does not contain K ⁺ .