JP2022179749A - Cell pharmaceutical composition, disease treatment kit, and cell suspension solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: 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

COPYRIGHT: (C)2023,JPO&INPIT

Description

TECHNICAL FIELD The present invention relates to cell pharmaceutical compositions, disease treatment kits, and cell suspension solutions.

Techniques for using pharmaceuticals containing cells to treat diseases are advancing year by year. In particular, iPS cells, hematopoietic stem cells, stem cells such as mesenchymal stem cells, skin cells, cardiomyocytes, etc. have moved from the basic research stage to the development stage, and are currently being used in clinical settings. Some exist. In the treatment of diseases with cells, the functions of the cells themselves are used directly or indirectly for the treatment of diseases, and the functions of damaged patient cells and tissues are replaced with cells newly differentiated from stem cells. It is expected that it will be supplemented by organs.

For example, mesenchymal stem cells are multipotent progenitor cells that were first isolated from bone marrow by Friedenstein (1982) (Non-Patent Document 1). Mesenchymal stem cells have been found to exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected to be a new therapeutic method for various intractable diseases ( Patent Documents 1 to 4). Recently, it is known that stromal cells of fetal appendages such as adipose tissue, placenta, umbilical cord, and egg membranes have cells with similar functions. Therefore, mesenchymal stem cells are sometimes called stromal cells.

Japanese Patent Publication No. 2002-506831 Japanese Patent Publication No. 2000-508911 JP 2012-157263 A Japanese Patent Publication No. 2012-508733

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

In medicines containing cells such as mesenchymal stem cells, the cells are sometimes suspended in a solution for the purpose of ensuring safety, facilitating administration of the cells, and the like. When cells such as mesenchymal stem cells are used as a suspension and transferred or injected into the body by methods such as drip infusion or injection, the viability of the cells in the suspension gradually decreases, resulting in insufficient pharmacological action. It is feared that the cells may not be obtained, the cells may clump together to clog the cannula, and the patient's pulmonary veins may be embolized. Accordingly, an object of the present invention is to provide a cell pharmaceutical composition capable of maintaining high cell viability over a long period of time.

As a result of intensive research to solve the above problems, the present inventors have found that cells for treating diseases, such as Ringer's solution, Ringer's acetate or Ringer's bicarbonate, contain Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes, In addition, the present inventors have found that when the cells are suspended in an isotonic electrolytic solution that does not contain carbohydrates and are then instilled, the drop in viability of cells during administration is remarkably suppressed, and the present invention has been accomplished. INDUSTRIAL APPLICABILITY According to the present invention, a pharmaceutical composition containing cells can keep the cells in good condition and maintain a high survival rate for a long period of time, thereby exhibiting excellent therapeutic effects on various diseases. be able to. That is, the gist of the present invention is as follows.

[1] (A) containing cells and (B) a cell suspension solution,
(B) A cellular pharmaceutical composition, wherein the cell-suspending solution is an isotonic electrolytic solution containing Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes and containing no sugar.
[2] (B) The cell suspension solution according to [1], further comprising at least one electrolyte selected from the group consisting of CH ₃ COO ⁻ , HCO ₃ ⁻ , citrate ions and lactate ions. Cell pharmaceutical composition.
[3] (B) The cellular pharmaceutical composition of [1] or [2], wherein the cell suspension solution is at least one selected from the group consisting of Ringer's solution, Ringer's acetate, Ringer's bicarbonate and Ringer's lactate. thing.
[4] The cell pharmaceutical composition of any one of [1] to [3], wherein (A) the cells are mesenchymal stem cells or peripheral blood mononuclear cells.
[5] The cell pharmaceutical composition of [4], wherein the mesenchymal stem cells are fat-derived, umbilical cord-derived, or bone marrow-derived.
[6] (A) containing cells and (B) a cell suspension solution,
(B) A disease treatment kit, wherein the cell suspension solution is an isotonic electrolytic solution containing Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes and containing no sugar.
[7] A cell-suspending solution for a cell pharmaceutical composition, which is an isotonic electrolytic solution containing Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes and containing no sugar.

INDUSTRIAL APPLICABILITY The cell pharmaceutical composition of the present invention can keep cells in good condition and maintain a high survival rate for a long period of time, and thus can be expected to have excellent therapeutic effects 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.

<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 contains Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes. It is characterized by being an isotonic electrolytic solution containing and not containing sugar. In the present invention, the term "cell pharmaceutical composition" refers to a pharmaceutical composition containing cells, which exerts therapeutic effects on diseases by means of the functions of the cells. The cell pharmaceutical composition of the present invention can maintain a high cell viability for a long period of time by suspending cells in the above specific solution, and is therefore excellent for various diseases. It becomes possible to exhibit a therapeutic effect. The cell pharmaceutical composition of the present invention may contain, in addition to the essential components (A) cells and (B) cell suspension solution, other agents having a therapeutic effect on diseases. Furthermore, other ingredients may be contained as long as the effects of the present invention are not impaired. Hereinafter, (A) cells, (B) cell suspension solution, other agents, and other components contained in the cell pharmaceutical composition of the present invention will be described in detail.

[(A) cells]
In the present invention, (A) cells are not particularly limited as long as they are cells that are effective in treating diseases. For example, mesenchymal stem cells, peripheral blood mononuclear cells (neutrophils, eosinophils, basophils, including 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 that constitute each tissue. Among these cells, mesenchymal stem cells, peripheral blood mononuclear cells, and bone marrow cells are preferable from the viewpoint of the viability maintenance effect of (B) the cell suspension solution described later.

(mesenchymal stem cells)
In the present invention, mesenchymal stem cells refer to one or more, preferably two or more, more preferably three or more cells belonging to the mesenchymal system (osteocytes, myocardial cells, chondrocytes, tenocytes, adipocytes, etc.). It means a cell that has differentiation ability and can proliferate while maintaining the ability. The term mesenchymal stem cells used in the present invention means the same cells as stromal cells, and there is no particular distinction between the two. They are also sometimes simply referred to as mesenchymal cells. Tissues containing mesenchymal stem cells include, for example, 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, adipose tissue-derived mesenchymal stem cells mean mesenchymal stem cells contained in adipose tissue, and may also be referred to as adipose-derived mesenchymal stem cells. Among them, 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 a different species. Species of mesenchymal stem cells 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 (subject) to be treated. . 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 type, that is, allogeneic cells (allogeneic). ). Allogeneic cells (allogeneic) are preferred.

Mesenchymal stem cells are less likely to cause rejection even in allogeneic subjects. ) can be used as mesenchymal stem cells as cells. Therefore, compared to the case of preparing and using autologous mesenchymal stem cells, the mesenchymal stem cells of the present invention are easier to commercialize and more likely to stably obtain a certain effect. Allogeneic is more preferred.

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

In the present invention, adipose tissue means a tissue containing adipocytes and interstitial cells including microvascular cells, and is, for example, a tissue obtained by surgical excision or aspiration of mammalian subcutaneous fat. Adipose tissue can be obtained from subcutaneous fat. They are preferably obtained from animals of the same species as the target of administration of the adipose-derived mesenchymal stem cells described later, and more preferably human subcutaneous fat in consideration of administration to humans. The subcutaneous fat-supplying individual may be alive or dead, but the adipose tissue used in the present invention is preferably tissue collected from a living individual. When collecting from an individual, liposuction is exemplified by, for example, PAL (power assisted) liposuction, Erconia laser liposuction, or body jet liposuction, and from the viewpoint of maintaining the state of cells, ultrasonic waves It is preferred not to use

In the present invention, the umbilical cord is a white tubular tissue that connects the fetus and placenta, and is composed of umbilical vein, umbilical cord artery, collagenous tissue (Wharton's Jelly), umbilical cord matrix itself, etc., and mesenchymal stem cells. contains a lot of The umbilical cord is preferably obtained from an animal of the same species as the subject (administration subject) using the cell pharmaceutical composition of the present invention, and more preferably, considering administration of the cell pharmaceutical composition of the present invention to humans. , the human umbilical cord.

In the present invention, the bone marrow refers to the soft tissue that fills the lumen of bones, and is a hematopoietic organ. Bone marrow contains bone marrow fluid, and cells existing therein are called bone marrow cells. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes, mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells and the like. Bone marrow cells can be harvested, for example, from human ilium, long bones, or other bones.

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

Mesenchymal stem cells in the present invention are characterized by growth characteristics (e.g. population doubling capacity from passage to senescence, doubling time), karyotype analysis (e.g. normal karyotype, maternal or neonatal lineage), flow cytometry ( surface marker expression by, e.g., FACS analysis), immunohistochemistry and/or immunocytochemistry (e.g., epitope detection), gene expression profiling (e.g., gene chip arrays; polymerase chaining such as reverse transcription PCR, real-time PCR, conventional PCR, etc.) response), miRNA expression profiling, protein arrays, protein secretion such as cytokines (e.g., plasma coagulation analysis, ELISA, cytokine arrays), metabolites (metabolomic 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. As an example, a method for preparing adipose-derived mesenchymal stem cells is described below. Adipose-derived mesenchymal stem cells can be obtained, for example, by the production method described in US Pat. No. 6,777,231, and can be produced, for example, by a method comprising the following steps (i) to (iii). :
(i) enzymatic digestion of adipose tissue to obtain a cell suspension;
(ii) sedimenting the cells and resuspending the cells in a suitable medium; and (iii) culturing the cells on a solid surface and removing cells that do not exhibit binding to the solid surface.

The adipose tissue used in step (i) is preferably washed. Washing may be performed by sedimentation with vigorous agitation using a physiologically compatible saline solution (eg, phosphate-buffered saline (PBS)). This is to remove contaminants (also called debris, such as damaged tissue, blood, red blood cells, etc.) contained in adipose tissue from the tissue. Therefore, washing and sedimentation are generally repeated until the supernatant is totally free of debris. Since the remaining cells exist as clumps of various sizes, in order to dissociate while minimizing damage to the cells themselves, the washed cell clumps are treated with an enzyme that weakens or breaks intercellular junctions (e.g., collagenase, dispase, trypsin, etc.). Amounts of such enzymes and duration of treatment vary depending on the conditions used, but are known in the art. Alternatively, or in combination with such enzymatic treatments, cell clumps can be broken up by other treatments such as mechanical agitation, ultrasonic energy, thermal energy, etc., while minimizing cell damage. In order to suppress it, it is preferable to carry out only with enzyme treatment. When an enzyme is used, it is desirable to deactivate the enzyme using a medium or the like after an appropriate period of time in order to minimize harmful effects on cells.

The cell suspension obtained by step (i) contains a slurry or suspension of aggregated cells as well as various contaminant cells such as red blood cells, smooth muscle cells, endothelial cells and fibroblasts. Therefore, the aggregated cells and these contaminant cells may be subsequently separated and removed, but since they can be removed by adhesion and washing in step (iii) described later, the separation and removal are omitted. may Separation and removal of contaminating cells can be achieved by centrifugation, which forces the cells to separate into supernatant and precipitate. The resulting pellet containing contaminating cells is suspended in a physiologically compatible solvent. Cells in suspension may contain erythrocytes, but erythrocytes are excluded by selection based on adhesion to a solid surface, which will be described later. Therefore, the step of lysing is not necessarily required. Methods well known in the art can be used for selectively lysing erythrocytes, such as incubation in hypertonic or hypotonic media with lysis with ammonium chloride. After lysis, the lysate may be separated from the desired cells by, for example, filtration, centrifugation, or density fractionation.

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

The medium used for resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells. One or more serum replacements such as insulin, sodium selenite, cholesterol, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol may be added to make it possible. Substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts are added to these media as necessary. may

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, and RPMI 1640. medium, Fischer's medium, MCDB201 medium, mixed medium thereof, and the like.

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, linoleic acid, arachidonic acid, myristic acid, palmitoic acid, palmitic acid, and stearic acid. Examples of lipids include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like. Examples of 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, L-glycine, and the like. Examples of proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin, β2-microglobulin, and the like. Polysaccharides are exemplified by glycosaminoglycans, and among glycosaminoglycans, hyaluronic acid, heparan sulfate and the like are particularly exemplified, but 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) , connective tissue growth factor (CTGF), vascular endothelial cell growth factor (VEGF) and the like, but are not limited thereto. From the viewpoint of using the adipose-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a (xeno-free) medium that does not contain heterologous components such as serum. Such media are provided as pre-prepared media for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R&D Systems, Corning and Rohto. It is

Subsequently, in step (iii), the cells in the cell suspension obtained in step (ii) are treated on a solid surface without differentiation, using a suitable cell culture medium as described above, at a suitable cell density and Incubate in culture conditions. In the present invention, the term "solid surface" means any material that enables binding and adhesion of adipose-derived mesenchymal stem cells in the present invention. In certain embodiments, such material is a plastic material that has been treated to promote binding and adhesion of mammalian cells to its surface. The shape of the culture vessel having a solid surface is not particularly limited, but petri dishes, flasks, and the like are preferably used. Cells are washed after incubation to remove unbound cells and cell debris.

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

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

Mesenchymal stem cells in the present invention can be prepared as described above, and may be defined as cells with the following characteristics;
(1) shows adhesion to plastic under culture conditions in a standard medium;
(2) positive for surface antigens CD44, CD73 and CD90, negative for CD31 and CD45; and (3) capable of differentiating into osteocytes, adipocytes and chondrocytes under culture conditions.

(peripheral blood mononuclear cells)
In the present invention, peripheral blood mononuclear cells refer to a fraction containing lymphocytes, neutrophils, eosinophils, basophils, and monocytes 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. Peripheral blood mononuclear cells as (A) cells in the present invention may be cells isolated from peripheral blood, and if necessary, by culturing them with various factors, low-molecular-weight compounds, antibodies, etc. , 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 repeatedly 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 them. Suspension can be performed by detaching the cells with a detachment agent such as trypsin as necessary, transferring them to a cryopreservation container, treating them appropriately, and then adding a cryopreservation solution.

The cryopreservation solution may contain DMSO (Dimethyl sulfoxide) as a cryoprotectant, but since DMSO has cytotoxicity, it is preferable to reduce the DMSO content. It is also known that DMSO has the property of inducing differentiation of mesenchymal stem cells. Glycerol, propylene glycol or polysaccharides are exemplified as alternatives to DMSO. 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. Examples of such cryopreservation solutions include cryopreservation provided by Bioverde, Nippon Genetics, Reprocell, Zenoac, Cosmo Bio, Kohjin 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 (e.g., -80°C), using any freezer that can achieve that temperature. obtain. Although not particularly limited, a program freezer may be used to appropriately control the cooling rate in order to avoid sudden temperature changes. 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 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. 4 weeks or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, 6 months or longer, 1 year or longer, or longer. Since cytotoxicity can be suppressed by storage at a lower temperature, the cells may be stored by transferring to a vapor phase (approximately −150° C. or less to −180° C. or less) on liquid nitrogen. Storage in the vapor phase over liquid nitrogen can be carried out using storage containers well known to those skilled in the art. Although not particularly limited, for example, when storing for two weeks or more, it is preferable to store in the gas phase above liquid nitrogen.

The thawed (A) cells may be appropriately cultured before the next cryopreservation. For example, mesenchymal stem cells are cultured using the medium capable of culturing the mesenchymal stem cells described above, although not particularly limited, at a culture temperature of about 30 to 40° C., preferably about 37° C., containing CO ₂ . It may be performed in an atmosphere of air. The CO ₂ concentration is about 2-5%, preferably about 5%. In culture, after reaching an appropriate confluency for the culture vessel (for example, cells occupy 50% to 80% of the culture vessel), the cells are detached with a detachment agent such as trypsin. Then, the cells may be seeded at an appropriate cell density in a separately prepared culture vessel and the culture may be continued. 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 , 2,000 to 10,000 cells/cm ² and the like are exemplified. In certain aspects, the cell density is 2,000-10,000 cells/cm ² . It is preferable to adjust the period to reach appropriate confluency to be 3 to 7 days. During culturing, the medium may be changed as appropriate, if necessary.

Thawing of cryopreserved cells can be performed by methods well known to those skilled in the art. For example, a method of standing or shaking in a 37° C. constant temperature bath or hot water bath is exemplified.

((A) cell morphology)
The (A) cells contained in the cell pharmaceutical composition of the present invention may be cells in any state, for example, cells collected by exfoliating cells in culture, or cells frozen in a cryopreservation medium. The cells may be in a state where the It is preferable to subdivide the cells of the same lot obtained by expansion culture and use the cryopreserved cells in terms of stably obtaining the same effects and excellent handleability.

(A) cells in a cryopreserved state may be thawed immediately before use, suspended in a cryopreservation solution, and directly mixed with a cell suspension solution (B) described below. Alternatively, the cells may be suspended in (B) the cell suspension solution after removing the cryopreservation medium by a method such as centrifugation.

The dose (administration amount) of the (A) cells of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.), dosage form, etc., but from the viewpoint of exhibiting a sufficient therapeutic effect, the amount A larger amount tends to be preferable, while a smaller amount tends to be preferable from the viewpoint of suppressing the occurrence of side effects. Generally, 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 cells/time. ⁶ to 1×10 ⁹ pieces/time. In addition, this dose may be treated as a single dose, and may be administered in multiple doses, or may be administered in multiple doses.

The dose (dosage) of the (A) cells of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the dosage form of the composition of the present invention, but is usually administered to adults. In some cases, the number of cells is 1×10 to 5× ^{10 10} /kg, preferably 1×10 ² to 5×10 ⁹ /kg, more preferably 1×10 ³ to 5×10 ⁸ /kg, particularly preferably 1×10 ⁴ to 5×10 ⁷ /kg. is. In addition, this dose may be treated as a single dose, and may be administered in multiple doses, or may be administered in multiple doses.

[(B) Cell suspension solution]
The (B) cell suspension solution of the present invention is an isotonic electrolytic solution containing Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes and containing no sugar. (B) The cell suspension solution may further contain at least one electrolyte selected from the group consisting of CH ₃ COO ⁻ , HCO ₃ ⁻ , citrate ions and lactate ions. (B) The cell suspension solution can also be said to be an isotonic electrolyte infusion preparation having an extracellular fluid rehydration effect in a living body, and is specifically selected from the group consisting of Ringer's solution, Ringer's acetate, Ringer's bicarbonate, and Ringer's lactate. At least one is preferred. By adopting the (B) cell suspension solution having such a composition, the cell pharmaceutical composition of the present invention (A) keeps the cells in good condition and maintains a high survival rate for a long period of time. can be maintained at

(B) The cell suspension solution does not contain carbohydrates and contains Na ⁺ , K ⁺ , Ca ²⁺ , and Cl ⁻ as electrolytes, with Na ⁺ of 140 mEq/L to 160 mEq/L and K ⁺ of 1 mEq/L. Ringer's solution with L to 10 mEq/L, Ca ²⁺ from 1 mEq/L to 10 mEq/L, and Cl 2 ⁻ from 140 mEq/L to 170 mEq/L can be preferably mentioned. The above Ringer's solution can be prepared with each electrolyte within the above concentration range. A commercial item can also be used.

(B) the cell suspension solution does not contain carbohydrates and contains Na ⁺ , K ⁺ , Ca ²⁺ , Cl ⁻ , and CH ₃ COO ⁻ as electrolytes, and Na ⁺ is 120 mEq/L to 140 mEq/L; Ringer's acetate having K ⁺ of 1 mEq/L to 10 mEq/L, Ca ²⁺ of 1 mEq/L to 10 mEq/L, Cl ⁻ of 90 mEq/L to 130 mEq/L, and CH ₃ COO ⁻ of 10 mEq/L to 40 mEq/L. It can be mentioned as a preferable one. The above acetate Ringer's solution can be prepared with each electrolyte in the above concentration range. Commercially available products such as F infusion (Terumo Corporation) can also be used.

(B) The cell suspension solution does not contain carbohydrates and contains Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cl ⁻ , HCO ₃ ⁻ , and citrate ions (Citrate ³⁺ ) as electrolytes. The electrolyte concentration of 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, and 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 ion (Citrate ³⁺ ) is 1 mEq/L to 10 mEq/L. can be done. The above-mentioned bicarbonate Ringer's solution can be prepared with each electrolyte within the above concentration range. can also be used.

(B) The cell suspension solution does not contain carbohydrates and contains Na ⁺ , K ⁺ , Ca ²⁺ , Cl ⁻ , and lactate ⁻ as electrolytes ^. 120 mEq/L to 140 mEq/L, K ⁺ from 1 mEq/L to 10 mEq/L, Ca ²⁺ from 1 mEq/L to 10 mEq/L, Cl ^- from 90 mEq/L to 130 mEq/L, Lactate ^- from 15 mEq/L to 45 mEq/L Ringer's lactate L can be mentioned as a preferable one. The lactated Ringer's solution can be prepared with each electrolyte in the above concentration range. Criti Care Co., Ltd.), Hartmann's Injection "NP" (Nipro Co., Ltd.), Hartmann's Injection pH 8 "NP" (Nipro Co., Ltd.), Lactec (trademark registered) Note (Otsuka Pharmaceutical Factory Co., Ltd.), Lactoringel solution "Fuso" (Fuso Pharmaceutical Co., Ltd.) Kogyo Co., Ltd.) can also be used.

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

Examples of drugs for treating liver disease include 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.), hepatitis C drugs (ribavirin, telapirevir, 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 disodium edetate hydrate, glutathione, dimethicaprol, sodium thiosulfate hydrate, sugammadec sodium, etc.), human serum albumin, liver extract, ursodeoxycholic acid, glycyrrhizic acid, azathioprine, bezafibrate, amino acids (glycine, L-cysteine, L-isoleucine, L-leucine, L-valine, L-threonine, L-serine, L-alanine, L -methionine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine and salts thereof, etc.), vitamins (tocopherol, flavin adenine dinucleotide, thiamine disulfide phosphate, pyridoxine, cyanocobalamin and salts thereof etc.), antibiotics (sulbactam sodium, cefoperazone sodium, meropenem hydrate, vancomycin hydrochloride, etc.) and the like.

Cardiac disease therapeutic agents include, for example, ACE inhibitors, angiotensin II receptor antagonists, β-blockers, antiplatelet agents, warfarin, calcium antagonists, nitrates, diuretics, HMG-CoA reductase inhibitors, Ancaron, and the like. is mentioned.

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

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

Nervous system agents include, for example, edaravone, interferon beta-1a, interferon beta-1b, fingolimod hydrochloride, riluzole, tartirelin hydrate and the like.

Cardiovascular drugs include, for example, hepronicate, midodrine hydrochloride, amezinium methylmethylsulfate, etilephrine hydrochloride, phenylephrine hydrochloride and the like.

Cerebral circulation improving agents include, for example, ifenprodil tartrate, nicergoline, ipdilast, dihydroergotoxin mesylate, nizophenone fumarate, fasudil hydrochloride hydrate and the like.

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

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

The cell pharmaceutical composition of the present invention, in addition to the above (A) cells and (B) cell suspension solution, can be prepared according to a conventional method according to its use and form, as long as it does not impair the effects of the present invention. Other ingredients such as pharmaceutically acceptable carriers and additives may be included. Such carriers and additives may be contained in (B) the cell suspension solution, or (B) may be contained separately from the cell suspension solution. Examples of such carriers and additives include tonicity agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents. , oily base, gel base, surfactant, suspending agent, binder, excipient, lubricant, disintegrant, foaming agent, fluidizer, dispersant, emulsifier, buffer, solubilizer , antioxidants, sweeteners, acidulants, coloring agents, flavoring agents, flavoring agents or cooling agents, but are not limited to these.

The cell pharmaceutical composition of the present invention can be in various forms depending on the purpose, such as injections (including infusions, implanted injections, long-acting injections, injections prepared just before use), dialysis agents, and patches. , poultices, 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 the form of being gelled or formed into a sheet on the affected area after being sprayed. The cell pharmaceutical composition of the present invention can also be applied to the affected area after forming the (A) cells into a sheet or three-dimensional structure.

In the cell pharmaceutical composition of the present invention, (A) cells, (B) a cell suspension solution, other drugs, and other components are sealed and stored in separate containers, and these are mixed at the time of use. good too. In addition, during storage, the above (A) cells, (B) cell suspension solution, other drugs, and other components may be stored under conditions suitable for each, such as freezing conditions, refrigeration conditions, and room temperature. Any condition may be used.

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. 9.0, preferably 2.5 to 8.5, more preferably 4.0 to 8.0.

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 to living organisms. An example of the osmotic pressure ratio of the cell pharmaceutical composition of the present invention is preferably 0.6 to 1.5, more preferably 0.7 to 1.2, still more preferably 0.8 to 1.0. be done. The osmotic pressure can be adjusted by a method known in the art using the above electrolytes and the like. The osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w/v% sodium chloride aqueous solution) based on the 15th revision of the Japanese Pharmacopoeia. Freezing point depression method). The standard solution for osmotic pressure ratio measurement (0.9 w/v% sodium chloride aqueous solution) was obtained by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650 ° C. for 40 to 50 minutes and then placing it in a desiccator (silica gel). Allow to cool, accurately weigh 0.900 g, dissolve in purified water to make exactly 100 mL, or use a commercially available standard solution for osmotic pressure ratio measurement (0.9 w/v % sodium chloride aqueous solution).

The concentration of (A) cells in the cell pharmaceutical composition of the present invention, that is, the concentration when (A) cells are prepared by suspending them in (B) a cell suspension solution and used for administration, depends on the type of cells, the cell suspension Although it may vary depending on the solution, it is usually 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.

A preferred form of the cell pharmaceutical composition of the present invention comprises human mesenchymal stem cells in a cell suspension solution at a density of 1×10 ⁴ to 2.5×10 ⁶ cells/mL. The turbidity solution contains 125-150 mEq/L of sodium ions, 100-160 mEq/L of chloride ions, 3-5 mEq/L of potassium ions and 2-6 mEq/L of calcium ions, and has an osmotic pressure ratio to saline of 0. .8 to 1.0 and a pH of 4.0 to 8.0.

Administration routes of the cell pharmaceutical composition of the present invention to a subject include subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intraperitoneal administration, transrectal administration, transvaginal administration, and transdermal administration. , implants, direct administration to organs, and the like, and from the viewpoint of efficacy of the cell pharmaceutical composition of the present invention, implants, intraarterial administration, intravenous administration, and direct administration to organs are preferable, and more preferable. are intravenous administration and direct administration to an organ.

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.), the route of administration of the therapeutic agent for non-alcoholic steatohepatitis of the present invention, etc., but usually When administered to adults, the dosage is 50 mL/h to 1,000 mL/h, preferably 75 mL/h to 500 mL/h, more preferably 100 mL/h to 250 mL/h.

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

The cell pharmaceutical composition of the present invention can be administered to a subject using an infusion set. Specifically, the infusion set includes a wand disposable infusion tube set (manufactured by Yoshida Seisakusho Co., Ltd.), an infusion set (manufactured by Forteglow Medical Co., Ltd.), a Terfusion (registered trademark) infusion set (manufactured by Terumo Corporation), and JMS infusion. Set (manufactured by JMS Co., Ltd.), SurePlug infusion set (manufactured by Terumo Corporation), Infusion set (manufactured by Nipro Corporation), Top infusion set NP (manufactured by Top Co., Ltd.), Infusion set with filter (EX type) ) (manufactured by Toray Medical Co., Ltd.).

The cell pharmaceutical composition of the present invention can be administered to a subject using an infusion tube. Specific examples of infusion tubes include Lectro Cass (manufactured by Sumic International Co., Ltd.), JMS Extension Tube (manufactured by JMS Co., Ltd.), Safide Extension Tube (manufactured by Terumo Corporation), and Extension Tube (manufactured by Terumo Corporation). Top), Connecting Tube (Chuatsu) (Medico Hirata Co., Ltd.), Safty AP Tube (Kawasumi Chemical Industry Co., Ltd.), Bionectar 2 with Extension Tube (Toray Medical Co., Ltd.), Medicut Extension Tube Set B (Nippon Sherwood) Co., Ltd.), wand disposable infusion tube set (manufactured by Yoshida Seisakusho Co., Ltd.), infusion tube (manufactured by Forteglow Medical Co., Ltd.), and other commercially available products can also be used.

Materials for the infusion tube used for administering the cell pharmaceutical composition of the present invention to a subject include polyvinyl chloride, thermoplastic elastomer, TPE thermoplastic elastomer, silicone, silicone rubber, polyethylene, polybutadiene, Teflon (registered trademark), Polyurethanes, polypropylenes, natural rubbers, polyolefins, PVC (plasticizers: TOTM, DOA), plasticizer-free PVC and mixtures thereof can be used.

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

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, liver cirrhosis, liver cancer, fatty liver, drug-allergic liver injury, hemochromatosis, hemosiderosis, Wilson's disease, primary Liver diseases such as biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver abscess, chronic active hepatitis, chronic persistent hepatitis; Heart disease such as hemorrhagic cardiomyopathy, angina pectoris, congenital heart disease, valvular heart disease, myocarditis, familial hypertrophic cardiomyopathy, dilated cardiomyopathy, acute coronary syndrome, atherothrombosis, restenosis; acute gastritis, Gastric/duodenal diseases such as chronic gastritis, gastric/duodenal ulcer, gastric cancer, duodenal cancer; ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, simple ulcer, intestinal Behcet's disease, small intestinal cancer, colon cancer, etc. Small and large intestine 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, Kidney disease such as chronic renal failure; pneumonia, emphysema, pulmonary fibrosis, interstitial pneumonia, idiopathic interstitial pneumonia, exfoliative interstitial pneumonia, acute interstitial pneumonia, non-specific interstitial pneumonia, drugs 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, pulmonary empyema, pneumoconiosis, aspiration pneumonia Pulmonary fibrosis, acute upper respiratory tract infection, chronic lower respiratory tract infection, pneumothorax, disease with damage to alveolar epithelium, lymphatic leiomyoma, lymphatic interstitial pneumonia, alveolar proteinosis, pulmonary Langerhans cell granulation Pulmonary disease such as tumor; mediastinal disease such as mediastinal tumor, mediastinal cystic disease, mediastinitis; diaphragmatic disease such as diaphragmatic hernia; pleuritis, empyema, pleural tumor, carcinomatous pleurisy, pleural mesothelioma, etc. peritoneal diseases such as peritonitis and peritoneal tumor; cerebral palsy syndrome including childhood cerebral palsy, aseptic meningitis, Guillain-Barre syndrome, amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, Polyneuropathy, spinal muscular atrophy, spinal disorders, acute transverse myelitis, spinal cord infarction (ischemic spinal cord injury), cranial Neurological diseases such as internal tumors and spinal tumors; CNS disorders such as Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, Parkinson's disease; fibromuscular dysplasia, peripheral arterial disease (PAD), thromboangiitis obliterans (Buerger disease), Kawasaki disease (KD); Peripheral venous disease such as deep vein thrombosis, chronic venous insufficiency, postphlebitic syndrome, superficial venous thrombosis; Graft-versus-host disease (GVHD), secondary sexual immunodeficiency disorders, primary immunodeficiency disorders, B cell deficiency, T cell deficiency, combined B and T cell deficiency, phagocytic cell deficiency, complement deficiency in the classical pathway, complement deficiency in the MBL pathway, complement in the alternative pathway Immunodeficiency diseases such as deficiencies, complement regulatory protein deficiencies, and complement receptor deficiencies can be mentioned.

Among these, liver diseases, heart diseases, pulmonary diseases, neurological diseases, peripheral artery diseases, and immunodeficiency diseases, for which it has been confirmed that mesenchymal stem cells have sufficient therapeutic effects, are preferred. More suitable for the treatment of liver 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) It can be used for liver fibrosis, liver cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, and interstitial pneumonia. In addition, it can be suitably used for cancers of various tissues for which it has been confirmed that peripheral blood mononuclear cells have sufficient therapeutic effects.

<Disease treatment kit>
The present invention contains (A) cells and (B) a cell suspension solution, and (B) the cell suspension solution contains Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes, and sugar Also included is a disease treatment kit, which is an isotonic, electrolyte-free solution. 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) a cell suspension solution, and other cell pharmaceutical compositions of the present invention. Regarding the components, the description in the section on cell pharmaceutical composition can be applied. According to the kit for treating diseases of the present invention, the state of cells can be maintained in good condition and the survival rate can be maintained high for a long period of time, so that excellent therapeutic effects can be exhibited for various diseases. .

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

The disease treatment kit of the present invention may include other additives, other drugs, diluents, filters, needles, syringes, package inserts describing directions for use, and other materials desirable from a commercial and user standpoint. material can be included.

<Solution for cell suspension>
Also within the scope of the present invention is a cell suspension solution for a cell pharmaceutical composition, which is an isotonic electrolytic solution containing Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ as electrolytes and containing no carbohydrates. be. By adopting an isotonic electrolytic solution containing Na ⁺ , Cl ⁻ , K ⁺ and Ca ²⁺ and not containing carbohydrates as a cell suspension solution for injection/infusion of cell pharmaceuticals, The present inventors have newly discovered that a high survival rate can be maintained over a long period of time. For the details of the cell suspension solution, the description of (B) the cell suspension solution in the cell pharmaceutical composition section can be applied.

<Method for treating disease>
The present invention provides a treatment method for a disease in which (A) cells are suspended in (B) a cell suspension solution and administered to a patient, and (B) the cell suspension solution contains Na ⁺ and Cl as electrolytes. ^- , K ⁺ and Ca ²⁺ and carbohydrate-free, isotonic electrolytes. According to the treatment method of the present invention, a high survival rate of cells can be maintained for a long period of time, and therefore excellent therapeutic effects can be obtained 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) cell suspension solution, and other cell pharmaceutical compositions of the present invention. Regarding the components, the description in the section on cell pharmaceutical composition can be applied.

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

[Example 1]
(Preparation of adipose-derived mesenchymal stem cells)
After obtaining informed consent from human donors, subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. In order to disrupt the extracellular matrix and isolate the cells, collagenase (Roche diagnostics) (solvent: physiological saline) was added and dispersed by shaking at 37°C for 90 minutes. Subsequently, this suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of stromal vascular cell clusters. 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 the supernatant is removed, followed by a serum-free medium for mesenchymal stem cells (Rohto). and seeded the flask with cells. Cells were cultured at 37° C. for several days in 5% CO ₂ . A few days later, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture medium, thereby obtaining mesenchymal stem cells adhering to the plastic container.

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

In a 15 mL centrifuge tube (Sumitomo Bakelite Co., Ltd., product number: MS-56150), Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory Co., Ltd., Lot: K4K73), KN2 infusion solution (No. 2 solution (dehydration replenishing solution)) (stock Company Otsuka Pharmaceutical Factory, Lot: K6E92), KN3 Infusion (No.3 solution (maintenance solution)) (Otsuka Pharmaceutical Factory Co., Ltd., Lot: K6D96), KN4 Infusion (No.4 solution (postoperative recovery solution)) (Stock Company Otsuka Pharmaceutical Factory, Lot: K6D80) and Physio (registered trademark) 70 (2.5% glucose and acetated Ringer's solution) (Otsuka Pharmaceutical Factory Co., Ltd., Lot: M6D91) were each dispensed 5 mL each, and then frozen. After rapidly thawing the adipose tissue-derived mesenchymal stem cells in a hot water bath (37±1° C.), 62.5 μL of a cell suspension of adipose tissue-derived mesenchymal stem cells was added. After being suspended by inversion mixing, it was stored at room temperature, and immediately after preparation, 2 hours, 4 hours, and 7 hours later, 1 mL was taken from the middle layer using a 5 mL syringe with a 21 G injection needle, and transferred to a 1.5 mL tube. . 10 μL of trypan blue (Trypan Blue Stain (0.4%); Life technologies, 15250-061) was added to 10 μL of the cell suspension, and live cells and dead cells were distinguished and examined with a phase contrast microscope (OLYMPUS, product number: CKX41SF) was used for measurement. In addition, a disposable cell counting board (WAKEN, product number: WC2-100) was used to count cells, 18 sections were counted 5 times, and the average value of the 3 measured values excluding the maximum and minimum values was calculated. was used to calculate the cell viability according to the following formula. If the cell viability was less than 70% after 4 hours, the measurement was not performed after 7 hours. The composition of each infusion solution is shown in Table 1 below, and the cell viability results are shown in Table 2 below.

Cell viability (%) = viable cell count/total cell count x 100

When adipose-derived mesenchymal stem cells were suspended in KN2 Infusion and Physio (registered trademark) 70, the cell viability was below 70% after 4 hours. When adipose-derived mesenchymal stem cells were suspended in KN3 and KN4 infusions, the cell viability was below 70% after 7 hours. In contrast, when the adipose-derived mesenchymal stem cells were suspended in Ringer's solution, the cell survival rate 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, it is possible to maintain a remarkably high cell viability for a long period of time.

[Example 2]
Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory Co., Ltd., Lot: K4K73), bicarbonate Ringer's solution (Bikanate (registered trademark) infusion, Otsuka Pharmaceutical Co., Ltd. Factory) were each dispensed 5 mL each, and then, in the same manner as in Example 1, the adipose-derived mesenchymal stem cells that had been cryopreserved were quickly thawed in a hot water bath (37±1° C.), and then cell suspensions of the adipose-derived mesenchymal stem cells were prepared. The suspension was added in 62.5 μL portions. After being suspended by inversion mixing, the suspension was stored at room temperature. Then, the cell survival rate was calculated for immediately after preparation. The composition of each Ringer's solution is shown in Table 3 below, and the cell viability results are shown in Table 4 below.

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% for all storage times including 4 hours. From the above results, it was found that by suspending the adipose-derived mesenchymal stem cells in Ringer's solution and bicarbonate Ringer's solution, a remarkably high cell survival rate can be maintained for a long period of time.

[Example 3]
The adipose-derived mesenchymal stem cells of Example 1 were replaced with bone marrow-derived mesenchymal stem cells (manufactured by Lonza), and Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory Co., Ltd., Lot: K4K73) was used in the same test. Immediately after preparation, after 2 hours, after 3 hours and after 7 hours, the number of viable cells and the number of dead cells were measured to calculate the cell viability. The results are shown in Table 5 below.

Even when bone marrow-derived mesenchymal stem cells were suspended in Ringer's solution, the cell survival rate was 95% or more after 7 hours. From the above results, it was found that by suspending bone marrow-derived stromal cells in Ringer's solution, it was possible to maintain a remarkably high cell survival rate over a long period of time.

[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 Ringer's solution (Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory Co., Ltd., Lot : K4K73), the number of viable cells and the number of dead cells were measured immediately after preparation, 2 hours, 4 hours and 6 hours after preparation, and the cell viability was calculated in the same manner as in Example 1. . The results are shown in Table 6 below.

Even when the umbilical cord-derived stromal 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 of umbilical cord-derived mesenchymal stem cells as well as adipose-derived mesenchymal stem cells in a remarkably high state for a long period of time.

[Example 5]
The adipose-derived mesenchymal stem cells of Example 1 were replaced with peripheral blood mononuclear cells (ACCUCELL (registered trademark) PBMCs derived from normal donors, manufactured by Precision Bioservices (PRECISION FOR MEDICINE), Lot. 13134-10), and Ringer's solution ( Ringer's solution "Otsuka", Otsuka Pharmaceutical Factory Co., Ltd., Lot: K4K73) was similarly tested, and the number of viable cells and the number of dead cells were measured immediately after preparation, 2 hours, 4 hours and 6 hours later. Cell viability was calculated. The results are shown in Table 7 below.

Even when peripheral blood mononuclear cells were suspended in Ringer's solution, the cell viability was about 80% after 6 hours. Therefore, it was found that Ringer's solution can maintain the cell viability of peripheral blood mononuclear cells in a remarkably high state for a long period of time, similarly to adipose-derived mesenchymal stem cells.

INDUSTRIAL APPLICABILITY The cell pharmaceutical composition of the present invention can keep cells in good condition and maintain a high survival rate for a long period of time, and thus can be expected to have excellent therapeutic effects on various diseases.

Claims (1)

The invention described herein.