JP7644564B2 - Cellular pharmaceutical composition, disease treatment kit, and cell suspension solution - Google Patents

Description

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

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

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

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

Pittenger F. 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 or injected into a living body as a suspension by drip infusion or injection, there are concerns that the viability of the cells in the suspension will gradually decrease, resulting in insufficient pharmacological action, that the cells will aggregate and clog the cannula, and that embolism will form in the patient's pulmonary vein, etc. Therefore, the objective of the present invention is to provide a cell pharmaceutical composition that can maintain high cell viability for a long period of time.

As a result of intensive research to solve the above problems, the present inventors have found that when cells for disease treatment are suspended in an isotonic electrolyte solution, such as Ringer's solution, acetate Ringer's solution, or bicarbonate Ringer's solution, which contains Na ⁺ , Cl ^- , K ⁺ , and Ca ²⁺ as electrolytes and does not contain carbohydrates, and then administered by drip infusion, the decrease in cell viability during administration is significantly suppressed, and the present invention has been completed. According to the present invention, a pharmaceutical composition containing cells can maintain the cell condition in good condition and maintain the cell viability at a high level for a long period of time, thereby providing excellent therapeutic effects against various diseases. That is, the gist of the present invention is as follows.

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

The cell pharmaceutical composition of the present invention can maintain the cell condition in good condition and keep the cell viability at a high level for a long period of time, and is therefore expected to have excellent therapeutic effects against a variety of diseases.

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

<Cell medicine composition>
The cell pharmaceutical composition of the present invention contains (A) cells and (B) a cell suspension solution, and is characterized in that (B) the cell suspension solution is an isotonic electrolyte solution containing Na ⁺ , Cl ^- , K ⁺ and Ca ²⁺ as electrolytes and not containing carbohydrates. In the present invention, the "cell pharmaceutical composition" refers to a pharmaceutical composition containing cells, which exerts a therapeutic effect against a disease due to the function of the cells. The cell pharmaceutical composition of the present invention can maintain the cell viability at a high level for a long period of time by suspending the cells in the specific solution, and therefore can exert an excellent therapeutic effect against various diseases. The cell pharmaceutical composition of the present invention may contain other drugs having a therapeutic effect against a disease in addition to the essential components (A) cells and (B) cell suspension solution. Furthermore, other components may be contained as long as they do not impair the effects of the present invention. Hereinafter, the (A) cells, (B) cell suspension solution, other drugs, and other components contained in the cell pharmaceutical composition of the present invention will be described in detail.

(A) Cells
In the present invention, the (A) cells are not particularly limited as long as they are effective in treating a disease, and examples thereof include mesenchymal stem cells, peripheral blood mononuclear cells (including neutrophils, eosinophils, basophils, lymphocytes, monocytes, etc.), 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, cardiac muscle cells, hepatic cells, nerve cells, skin cells, adipocytes, and other cells that constitute various tissues. Among these, mesenchymal stem cells, peripheral blood mononuclear cells, and bone marrow cells are preferred from the viewpoint of excellent viability maintenance effect by the (B) cell suspension solution described below.

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

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

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

In the present invention, mesenchymal stem cells refer to any cell population that contains 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% mesenchymal stem cells.

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

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

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

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

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

(Method of 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 may be obtained by the production method described in U.S. Patent No. 6,777,231, for example, and can be produced by a method including the following steps (i) to (iii):
(i) obtaining a cell suspension by enzymatic digestion of adipose tissue;
(ii) sedimenting the cells and resuspending the cells in an appropriate medium; and (iii) culturing the cells on a solid surface and removing cells that do not exhibit binding to the solid surface.

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

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

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

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

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

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

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

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

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

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

The mesenchymal stem cells of the present invention can be prepared as described above, but may also be defined as cells having the following characteristics:
(1) Under standard culture conditions, it exhibits adhesion to plastic.
(2) They are positive for the surface antigens CD44, CD73, and CD90, and negative for CD31 and CD45, and (3) can differentiate into osteocytes, adipocytes, and chondrocytes under certain culture conditions.

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

(A) Cryopreservation of cells
The (A) cells of the present invention may be cells that have been repeatedly frozen and thawed as appropriate, so long as they have a therapeutic effect against various diseases. In the present invention, cryopreservation can be performed by suspending the (A) cells in a cryopreservation solution known to those skilled in the art and cooling them. The suspension can be performed by detaching the cells with a detaching agent such as trypsin as necessary, transferring them to a cryopreservation container, treating them appropriately, and then adding the 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. Examples of substitutes for DMSO include glycerol, propylene glycol, and polysaccharides. When DMSO is used, it contains a concentration of 5% to 20%, preferably a concentration of 5% to 10%, and more preferably a concentration of 10%. In addition, it may contain additives described in WO2007/058308. As such a cryopreservation solution, for example, cryopreservation solutions provided by Bio Verde, Nippon Genetics, ReproCell, Zenoac, Cosmo Bio, Kohjin Bio, Thermo Fisher Scientific, etc. 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), and any freezer capable of achieving said temperature may be used. Although not particularly limited, in order to avoid sudden temperature changes, the cooling rate may be appropriately controlled using a programmable freezer. The cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the manufacturer's instructions for the cryopreservation solution.

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

The thawed (A) cells may be appropriately cultured before the next cryopreservation. For example, the culture of mesenchymal stem cells is performed using a medium capable of culturing the above-mentioned mesenchymal stem cells, and may be performed at a culture temperature of about 30 to 40°C, preferably about 37°C, under an atmosphere of _CO2 -containing air, although not particularly limited thereto. The _CO2 concentration is about 2 to 5%, preferably about 5%. In the culture, after the cells reach an appropriate confluency for the culture vessel (for example, the cells occupy 50% to 80% of the culture vessel), the cells may be detached with a detaching agent such as trypsin, and seeded at an appropriate cell density in a separately prepared culture vessel to continue the culture. When the cells are seeded, typical cell densities include 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 , and 2,000 to 10,000 cells/cm ² . In a specific embodiment, the cell density is 2,000 to 10,000 cells/cm ² . It is preferable to adjust the period until the cells reach an appropriate confluency to 3 to 7 days. During the culture, the medium may be replaced as needed.

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

(A) Cell morphology
The (A) cells contained in the cell pharmaceutical composition of the present invention may be in any state, for example, cells recovered by detaching cells during culture, or cells frozen in a cryopreservation solution. The use of cells obtained by expansion culture and then dividing them into small portions and cryopreserving them is preferred in terms of the same effects being stably obtained and ease of handling.

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

The dose (administration amount) of the (A) cells of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and dosage form, but from the viewpoint of exerting a sufficient therapeutic effect, the amount tends to be more preferable, while from the viewpoint of suppressing the occurrence of side effects, the amount tends to be less preferable. Usually, when administered to an adult, the number of cells is 1x10 ³ to 1x10 ¹² cells/time, preferably 1x10 ⁴ to 1x10 ¹¹ cells/time, more preferably 1x10 ⁵ to 1x10 ¹⁰ cells/time, and particularly preferably 5x10 ⁶ to 1x10 ⁹ cells/time. This dose may be administered multiple times as a single dose, or this dose may be administered in multiple divided doses.

The dose (administration amount) of the (A) cells of the present invention may vary depending on the condition of the patient (body weight, age, symptoms, physical condition, etc.) and the dosage form of the composition of the present invention, but typically, when administered to an adult, the number of cells is 1x10 to ^5x10 /kg, preferably ^1x10 to ^5x10 /kg, more preferably ^1x10 to ^5x10 /kg, and particularly preferably ^1x10 to ^5x10 /kg. This dose may be administered multiple times as a single dose, or this dose may be administered in multiple divided doses.

(B) Cell suspension solution
The (B) cell suspension solution of the present invention is an isotonic electrolyte solution containing Na ⁺ , Cl ^- , K ⁺ and Ca ²⁺ as electrolytes and not containing carbohydrates. The (B) cell suspension solution may further contain at least one electrolyte selected from the group consisting of CH ₃ COO ^- , HCO ₃ ^- , citrate ion and lactate ion. The (B) cell suspension solution can be said to be an isotonic electrolyte infusion preparation having an extracellular fluid replacement effect in a living body, and specifically, at least one selected from the group consisting of Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution and lactate Ringer's solution is preferably mentioned. By adopting the (B) cell suspension solution having such a composition, the cell pharmaceutical composition of the present invention can maintain the state of the (A) cells in a good condition and maintain their survival rate at a high level for a long period of time.

(B) As a solution for cell suspension, Ringer's solution containing no carbohydrate and containing Na ⁺ , K ⁺ , Ca ²⁺ , and Cl ^- as electrolytes, with Na ⁺ of 140 mEq/L to 160 mEq/L, K ⁺ of 1 mEq/L to 10 mEq/L, Ca ²⁺ of 1 mEq/L to 10 mEq/L, and Cl ^- of 140 mEq/L to 170 mEq/L, can be mentioned as a preferred example. The Ringer's solution can be prepared so that each electrolyte has the above-mentioned concentration range, but commercially available products such as Ringer's solution "Otsuka" (Japanese Pharmacopoeia Ringer's solution, Otsuka Pharmaceutical Factory, Inc.) and Ringer's solution "Fuso" (Japanese Pharmacopoeia Ringer's solution, Fuso Pharmaceutical Industries, Ltd.) can also be used.

(B) As the cell suspension solution, an acetate Ringer's solution that does not contain carbohydrates and contains Na ⁺ , K ⁺ , Ca ²⁺ , Cl ^- , and CH ₃ COO ^- as electrolytes, with Na ⁺ of 120 mEq/L to 140 mEq/L, 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, can be mentioned as a preferred example. The acetate Ringer's solution can be prepared so that each electrolyte has the above concentration range, but commercially available products such as Veen (registered trademark) F infusion (Kowa Co., Ltd.), Solugen (registered trademark) F injection (Kyowa Critical Care Co., Ltd.), and Solaceto (registered trademark) F infusion (Terumo Corporation) can also be used.

(B) The cell suspension solution does not contain carbohydrates and contains the electrolytes Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cl ^- , HCO ₃ ^- , and citrate ions (Citrate ³⁺ ), with the respective electrolyte concentrations being as follows: Na ⁺ 120 mEq/L to 145 mEq/L, K ⁺ 1 mEq/L to 10 mEq/L, Ca ²⁺ 1 mEq/L to 10 mEq/L, Mg ²⁺ 0.1 mEq/L to 10 mEq/L, Cl ^- 95 mEq/L to 135 mEq/L, HCO ₃ ^- 10 mEq/L to 40 mEq/L, and citrate ions (Citrate ^{3+ )} . A preferred example is a bicarbonate Ringer's solution having an electrolyte concentration of 1 mEq/L to 10 mEq/L. The bicarbonate Ringer's solution can be prepared so that each electrolyte falls within the above-mentioned concentration range, but commercially available products such as Bicanate (registered trademark) Infusion (Otsuka Pharmaceutical Factory, Inc.) and Bicarbon (registered trademark) Infusion (AY Pharmaceuticals, Inc.) can also be used.

(B) A preferred example of a cell suspension solution is lactated Ringer's solution which does not contain carbohydrates and contains the electrolytes Na ⁺ , K ⁺ , Ca ²⁺ , Cl ^- , and lactate ions (lactate ^-) , with respective electrolyte concentrations of Na ⁺ 120 mEq/L to 140 mEq/L, K ⁺ 1 mEq/L to 10 mEq/L, Ca ²⁺ 1 mEq/L to 10 mEq/L, Cl ^- 90 mEq/L to 130 mEq/L, and lactate ^- 15 mEq/L to 45 mEq/L. The lactated Ringer's solution can be prepared with each electrolyte in the above-mentioned concentration range, but commercially available products such as Sollact (registered trademark) Infusion (Terumo Corporation), Nisori (registered trademark) Infusion (Mylan Pharmaceutical Co., Ltd.), Hartmann's Solution "Kobayashi" (Kyowa Critical Care Co., Ltd.), Hartmann's Infusion "NP" (Nipro Corporation), Hartmann's Infusion pH8 "NP" (Nipro Corporation), Lactec (registered trademark) Injection (Otsuka Pharmaceutical Factory, Inc.), and Lactated Ringer's Solution "Fuso" (Fuso Pharmaceutical Industries, Ltd.) can also be used.

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

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.), drugs for treating hepatitis C (ribavirin, telapirevir, simeprevir, vaniprevir, daclatasvir, asunaprevir, sofosbuvir, etc.), corticosteroids (prednisolone, methylprednisolone sodium succinate, etc.), anticoagulants (dried human antithrombin III concentrate, gabexate mesilate, thrombomodulin α, etc.), antidotes (calcium disodium hydrate, glutathione, dimethycaprol, sodium thiosulfate, etc.), hydrate, sugamadesc sodium, etc.), human serum albumin, liver extract, ursodeoxycholic acid, glycyrrhizic acid, azathioprine, bezafibrate, amino acids (glycine, L-cysteine, L-isoleucine, L-leucine, L-valine, L-threonine, L-serine, L-alanine, L-methionine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine, and salts thereof, etc.), vitamins (tocopherol, flavin adenine dinucleotide, thiamine disulfide phosphate, pyridoxine, cyanocobalamin, and salts thereof, etc.), antibiotics (sulbactam sodium, cefoperazone sodium, meropenem hydrate, vancomycin hydrochloride, etc.).

Examples of drugs to treat heart disease include ACE inhibitors, angiotensin II receptor antagonists, beta-blockers, antiplatelet drugs, warfarin, calcium channel blockers, nitrates, diuretics, HMG-CoA reductase inhibitors, and ancalon.

Examples of drugs for treating inflammatory bowel disease include salazosulfapyridine and mesalazine.

Examples of respiratory medications include dimorpholamine, doxapram hydrochloride hydrate, sivelestat sodium hydrate, pirfenidone, pulmonary surfactant, dornase alfa, etc.

Examples of nervous system medications include edaravone, interferon beta-1a, interferon beta-1b, fingolimod hydrochloride, riluzole, taltirelin hydrate, etc.

Examples of cardiovascular medications include hepronicate, midodrine hydrochloride, amezinium methyl methylsulfate, etilefrine hydrochloride, phenylephrine hydrochloride, etc.

Examples of drugs that improve cerebral circulation include ifenprodil tartrate, nicergoline, ipdilast, dihydroergotoxine mesylate, nizofenone fumarate, and fasudil hydrochloride hydrate.

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

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

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

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

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

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

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

The concentration of the (A) cells in the cell pharmaceutical composition of the present invention, i.e., the concentration when the (A) cells are suspended in the (B) cell suspension solution to be prepared and used for administration, may vary depending on the type of cells and the cell suspension solution, but is usually 1 x ¹⁰ to 2.5 x ¹⁰ cells/mL, preferably 1 x ¹⁰ to 2.5 x ¹⁰ cells/mL, and more preferably 1 x ¹⁰ to 2.5 x ¹⁰ cells/mL.

In one preferred embodiment 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 x ¹⁰ to 2.5 x ¹⁰ cells/mL, the cell suspension solution contains 125 to 150 mEq/L sodium ions, 100 to 160 mEq/L chloride ions, 3 to 5 mEq/L potassium ions, and 2 to 6 mEq/L calcium ions, has an osmotic pressure ratio of 0.8 to 1.0 relative to physiological saline, and has a pH of 4.0 to 8.0.

Routes of administration of the cell pharmaceutical composition of the present invention to a subject include subcutaneous administration, intramuscular administration, intravenous administration, intra-arterial administration, intrathecal administration, intraperitoneal administration, rectal administration, vaginal administration, transdermal administration, implant, and direct administration to an organ, but from the viewpoint of the effectiveness of the cell pharmaceutical composition of the present invention, implant, intra-arterial administration, intravenous administration, and direct administration to an organ are preferred, and intravenous administration and direct administration to an organ are more preferred.

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

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

The cell pharmaceutical composition of the present invention can be administered to a subject using an infusion set. Specifically, commercially available infusion sets such as Wand Disposable Infusion Tube Set (Yoshida Manufacturing Co., Ltd.), Infusion Set (Forte Grow Medical Co., Ltd.), Terufusion (registered trademark) Infusion Set (Terumo Corporation), JMS Infusion Set (JMS Co., Ltd.), SurePlug Infusion Set (Terumo Corporation), Infusion Set (Nipro Corporation), Top Infusion Set NP (Top Corporation), and Infusion Set with Filter (EX Type) (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, commercially available infusion tubes such as LectroCath (manufactured by Samick International Co., Ltd.), JMS Extension Tube (manufactured by JMS Co., Ltd.), Safeed Extension Tube (manufactured by Terumo Corporation), Extension Tube (manufactured by Top Co., Ltd.), Connecting Tube (Chuatsu) (Medicos Hirata Co., Ltd.), Safty AP Tube (manufactured by Kawasumi Chemical Industries Co., Ltd.), BioNectar 2 with Extension Tube (Toray Medical Co., Ltd.), Medicut Extension Tube Set B (manufactured by Japan Sherwood Co., Ltd.), Wand Disposable Infusion Tube Set (manufactured by Yoshida Manufacturing Co., Ltd.), and Infusion Tube (manufactured by Forte Grow Medical Co., Ltd.) can also be used.

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

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

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

Among these, liver disease, heart disease, lung disease, neurological disease, peripheral arterial disease, and immunodeficiency disease are preferred, for which it has been confirmed that mesenchymal stem cells can provide sufficient therapeutic effects. In particular, it is more suitable for use in the treatment of liver fibrosis, liver cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, interstitial pneumonia, pediatric cerebral palsy, amyotrophic lateral sclerosis (ALS), peripheral arterial disease (PAD), and graft-versus-host disease (GVHD), and is even more suitable for use in the treatment of liver fibrosis, liver cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, and interstitial pneumonia. It is also suitable for use in the treatment of cancers of various tissues for which it has been confirmed that peripheral blood mononuclear cells can provide sufficient therapeutic effects.

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

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

The disease treatment kits of the present invention may include other materials desirable from a commercial and user standpoint, including other additives, other medications, diluents, filters, needles, syringes, and package inserts providing instructions for use.

<Cell suspension solution>
The present invention also includes a cell suspension solution for a cell pharmaceutical composition, which is an isotonic electrolyte solution containing Na ⁺ , Cl ^- , K ⁺ and Ca ²⁺ as electrolytes and containing no carbohydrates. The present inventors have newly discovered that by adopting an isotonic electrolyte solution containing Na ⁺ , Cl ^- , K ⁺ and Ca ²⁺ and containing no carbohydrates as a cell suspension solution for injection and infusion of a cell pharmaceutical, the cell viability can be maintained high for a long period of time. For details of the cell suspension solution, the explanation of (B) Cell Suspension Solution in the section on Cell Pharmaceutical Composition can be applied.

<Disease treatment method>
The present invention also includes a method for treating a disease in which (A) cells are suspended in (B) a cell suspension solution and administered to a patient, characterized in that (B) the cell suspension solution is an isotonic electrolyte solution containing Na ⁺ , Cl ^- , K ⁺ and Ca ²⁺ as electrolytes and containing no carbohydrates. According to the treatment method of the present invention, the cell viability can be maintained high for a long period of time, and therefore excellent therapeutic effects can be achieved against various diseases. The disease treatment method of the present invention is a treatment method using the above-mentioned cellular pharmaceutical composition of the present invention, and the explanation in the section on the cellular pharmaceutical composition can be applied to the components that may be contained in (A) cells, (B) the cell suspension solution, and the cellular pharmaceutical composition of the present invention.

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 consent from the human donor, the subcutaneous adipose tissue obtained by liposuction was washed with physiological saline. To achieve the destruction of the extracellular matrix and the isolation of the cells, collagenase (Roche diagnostics) (solvent: physiological saline) was added, and the cells were shaken and dispersed at 37°C for 90 minutes. The suspension was then centrifuged at 800g for 5 minutes to obtain a precipitate of stromal vascular cells. Serum-free medium for mesenchymal stem cells (Rohto) was added to the precipitate of the cells, the cell suspension was centrifuged at 400g for 5 minutes, and after removing the supernatant, the cells were resuspended in serum-free medium for mesenchymal stem cells (Rohto), and the cells were seeded in a flask. The cells were cultured at 37°C for several days in 5% _CO2 . After several days, the culture was washed with PBS to remove blood cells and residual adipose tissue contained in the culture solution, and mesenchymal stem cells attached to a plastic container were obtained.

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

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

Cell viability (%) = number of live cells/total number of cells x 100

When adipose-derived mesenchymal stem cells were suspended in KN No. 2 infusion solution and Physio (registered trademark) 70, the cell viability after 4 hours was below 70%. When adipose-derived mesenchymal stem cells were suspended in KN No. 3 infusion solution and KN No. 4 infusion solution, the cell viability after 7 hours was below 70%. In contrast, when adipose-derived mesenchymal stem cells were suspended in Ringer's solution, the cell viability was 80% or higher even after 7 hours. From these results, it was found that by suspending adipose-derived mesenchymal stem cells in Ringer's solution, the cell viability can be maintained at a significantly high level for a long period of time.

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

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

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

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

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

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

[Example 5]
A similar test was performed using Ringer's solution (Otsuka Ringer's solution, Otsuka Pharmaceutical Factory, Inc., Lot: K4K73) instead of peripheral blood mononuclear cells (ACCUCELL (registered trademark) normal donor-derived PBMC, Precision Bioservices, Inc. (Precision For Medicine), Lot. 13134-10) instead of the adipose-derived mesenchymal stem cells of Example 1, and the number of live cells and the number of dead cells were measured immediately after preparation, and after 2 hours, 4 hours, and 6 hours to calculate the cell survival rate. The results are shown in Table 7 below.

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

The cell pharmaceutical composition of the present invention can maintain the cell condition in good condition and keep the cell viability at a high level for a long period of time, and is therefore expected to have excellent therapeutic effects against a variety of diseases.

Claims (3)

(A) cells; and (B) a cell suspension solution,
(A) the cell is a human mesenchymal stem cell or a human peripheral blood mononuclear cell;
(B) A cell pharmaceutical composition in which the cell suspension solution is Ringer's solution, acetate Ringer's solution, or bicarbonate Ringer's solution (excluding those containing carbohydrates , and excluding those containing at least one selected from the group consisting of dimethyl sulfoxide, serum albumin, and hyaluronic acid). The cell pharmaceutical composition according to claim 1, wherein the mesenchymal stem cells are derived from adipose tissue, umbilical cord tissue, or bone marrow. (A) cells; and (B) a cell suspension solution,
(A) the cell is a human mesenchymal stem cell or a human peripheral blood mononuclear cell;
(B) A disease treatment kit in which the cell suspension solution is Ringer's solution, acetate Ringer's solution, or bicarbonate Ringer's solution (excluding those containing carbohydrates , and excluding those containing at least one selected from the group consisting of dimethyl sulfoxide, serum albumin, and hyaluronic acid).