WO2018123627A1 - Composition pharmaceutique de cellules, kit pour traitement de maladie, et solution pour suspension de cellules - Google Patents

Composition pharmaceutique de cellules, kit pour traitement de maladie, et solution pour suspension de cellules Download PDF

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Publication number
WO2018123627A1
WO2018123627A1 PCT/JP2017/044992 JP2017044992W WO2018123627A1 WO 2018123627 A1 WO2018123627 A1 WO 2018123627A1 JP 2017044992 W JP2017044992 W JP 2017044992W WO 2018123627 A1 WO2018123627 A1 WO 2018123627A1
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Prior art keywords
cell
cells
mesenchymal stem
pharmaceutical composition
solution
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English (en)
Japanese (ja)
Inventor
里枝 武田
くみ子 長岡
陽子 堀内
輝 長谷川
琴絵 玉田
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Rohto Pharmaceutical Co Ltd
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Rohto Pharmaceutical Co Ltd
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Priority to CN201780072935.2A priority Critical patent/CN109996535B/zh
Priority to JP2018559039A priority patent/JP7136700B2/ja
Publication of WO2018123627A1 publication Critical patent/WO2018123627A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions

Definitions

  • the present invention relates to a cell pharmaceutical composition, a disease treatment kit and a cell suspension solution.
  • stem cells such as iPS cells, hematopoietic stem cells, mesenchymal stem cells, skin cells, cardiomyocytes, etc. have moved from the basic research stage to the development stage, and are currently used in clinical settings. There are also.
  • the functions of cells themselves are used directly or indirectly for disease treatment, and the functions of cells and tissues of damaged patients are newly differentiated from stem cells and It is expected to be supplemented by organs.
  • mesenchymal stem cells are pluripotent progenitor cells isolated from bone marrow for the first time by Friedenstein (1982) (Non-patent Document 1). These mesenchymal stem cells have been revealed to exist in various tissues such as bone marrow, umbilical cord, and fat, and mesenchymal stem cell transplantation is expected as a new treatment method for various intractable diseases ( Patent Documents 1 to 4). Recently, it is known that cells having an equivalent function exist in stromal cells of fetal appendages such as adipose tissue, placenta, umbilical cord, and egg membrane. Therefore, the mesenchymal stem cell may be referred to as a stromal cell (Mesenchymal Stroma Cell).
  • the cells may be suspended in a solution for the purpose of ensuring safety and facilitating administration of the cells.
  • a solution for the purpose of ensuring safety and facilitating administration of the cells.
  • the survival rate of the cells in the suspension is gradually reduced and sufficient pharmacological action is achieved.
  • problems such as formation of emboli in the patient's pulmonary veins and the like that may not be obtained, cells may aggregate and clog in the cannula, and the like. Then, this invention makes it a subject to provide the cell pharmaceutical composition which can maintain the survival rate of a cell high over a long time.
  • the present inventors suspended the cells for disease treatment in a solution containing Na + and Cl ⁇ and substantially not containing K + , and performed instillation or the like. And the present inventors have found that the decrease in the survival rate of cells during administration is remarkably suppressed. According to the present invention, since a pharmaceutical composition containing cells can maintain a good state of cells and maintain a high survival rate over a long period of time, it has excellent therapeutic effects on various diseases. be able to. That is, the gist of the present invention is as follows.
  • the cell pharmaceutical composition according to [6], wherein the mesenchymal stem cells are derived from fat, umbilical cord or bone marrow.
  • a cell suspension solution for a cell pharmaceutical composition comprising an electrolyte and a saccharide, wherein the electrolyte contains Na + and Cl ⁇ and substantially does not contain K + .
  • the cell pharmaceutical composition of the present invention can maintain a good cell state and maintain its survival rate in a high state for a long time, it can be expected to have an excellent therapeutic effect on various diseases.
  • the cell pharmaceutical composition, disease treatment kit, and cell suspension solution for injection of the present invention will be described in detail.
  • the cell pharmaceutical composition of the present invention contains (A) a cell, and (B) a cell suspension solution containing an electrolyte and a carbohydrate, and (B) the electrolyte in the cell suspension solution contains Na + and Cl. - it includes a free of K + substantially.
  • the “cell pharmaceutical composition” refers to a pharmaceutical composition containing cells, which exhibits a therapeutic effect on diseases due to the functions of the cells.
  • the cell pharmaceutical composition of the present invention is excellent for various diseases because the cell viability can be maintained in a high state for a long time by suspending the cells in the specific solution. A therapeutic effect can be achieved.
  • the cell pharmaceutical composition of the present invention may contain other drugs having a therapeutic effect on diseases. Furthermore, as long as the effects of the present invention are not impaired, other components may be contained.
  • (A) cells, (B) cell suspension solutions, other drugs, and other components contained in the cell pharmaceutical composition of the present invention will be described in detail.
  • the (A) cell is not particularly limited as long as it has an effect on the treatment of diseases.
  • mesenchymal stem cells peripheral blood mononuclear cells (neutrophils, eosinophils, basophils, Lymphocytes, monocytes, etc.), red blood cells, T cells, NK cells, NKT cells, NKM cells, LAK cells, dendritic cells, fibroblasts, hematopoietic stem cells, iPS cells, ES cells, bone marrow cells, cardiomyocytes
  • mesenchymal stem cells, peripheral blood mononuclear cells, and bone marrow cells are preferable from the viewpoint that (B) the cell suspension solution described later is excellent in viability maintenance effect.
  • the mesenchymal stem cell refers to one or more cells belonging to the mesenchymal system, preferably two or more cells, more preferably three or more cells (bone cells, cardiomyocytes, chondrocytes, tendon cells, fat cells, etc.). It means a cell that has differentiation ability and can proliferate while maintaining the ability.
  • the term mesenchymal stem cell used in the present invention means the same cell as the stromal cell, and does not particularly distinguish them. Moreover, it may be simply described as a mesenchymal cell.
  • tissues containing mesenchymal stem cells include adipose tissue, umbilical cord, bone marrow, umbilical cord blood, endometrium, placenta, amniotic membrane, chorion, decidua, dermis, skeletal muscle, periosteum, dental follicle, periodontal ligament, Examples include dental pulp and tooth germ.
  • an adipose tissue-derived mesenchymal stem cell means a mesenchymal stem cell contained in an adipose tissue, and may be referred to as an adipose-derived mesenchymal stem cell.
  • adipose-derived mesenchymal stem cells from the viewpoint of effectiveness for treatment of various diseases, from the viewpoint of availability, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, Dental pulp-derived mesenchymal stem cells are preferred, and adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells are more preferred.
  • the mesenchymal stem cells in the present invention may be derived from the same species as the subject to be treated (subject) or from a different species.
  • examples of the mesenchymal stem cell species in the present invention include humans, horses, cows, sheep, pigs, dogs, cats, rabbits, mice, and rats, preferably cells derived from the same species as the subject to be treated (subject).
  • the mesenchymal stem cells in the present invention may be derived from the subject (subject) to be treated, that is, autologous cells (allogeneic), or derived from another subject of the same species, ie, allogeneic cells (allogeneic). ). Preferred are allogeneic cells (allogeneic).
  • mesenchymal stem cells are unlikely to cause rejection even for allogeneic subjects, cells prepared by pre-expanding donor cells that have been expanded and cryopreserved are used in the cell pharmaceutical composition of the present invention (A ) It can be used as a mesenchymal stem cell as a cell. Therefore, compared to the case of preparing and using autologous mesenchymal stem cells, commercialization is easy, and from the viewpoint that a certain effect can be easily obtained stably, the mesenchymal stem cells in the present invention, More preferably, it is allogeneic.
  • the mesenchymal stem cell means an arbitrary cell population including the mesenchymal stem cell.
  • the cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98 % Or 99% are mesenchymal stem cells.
  • an adipose tissue means a tissue containing stromal cells including adipocytes and 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. It is preferably obtained from the same animal as the subject of administration of the adipose-derived mesenchymal stem cells described later, and more preferably human subcutaneous fat in consideration of administration to humans.
  • An individual supplying subcutaneous fat may be alive or dead, but the adipose tissue used in the present invention is preferably a tissue collected from a living individual.
  • liposuction is exemplified by PAL (power assist) liposuction, Erconia laser liposuction, or body jet liposuction. From the viewpoint of maintaining the state of cells, ultrasound is used. It is preferable not to use.
  • the umbilical cord is a white tubular tissue that connects the fetus and the placenta, and is composed of umbilical vein, umbilical artery, collagenous tissue (Wharton's Jelly), umbilical matrix itself, etc., and mesenchymal stem cells Including many.
  • the umbilical cord is preferably obtained from the same animal as the subject (administration subject) using the cell pharmaceutical composition of the present invention, and more preferably in consideration of administering the cell pharmaceutical composition of the present invention to a human.
  • the human umbilical cord is preferably obtained from the same animal as the subject (administration subject) using the cell pharmaceutical composition of the present invention, and more preferably in consideration of administering the cell pharmaceutical composition of the present invention to a human.
  • the bone marrow refers to a soft tissue filling the bone lumen, and is a hematopoietic organ.
  • Bone marrow fluid is present in the bone marrow, and the cells present therein are called bone marrow cells.
  • Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes and the like, as well as mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells, and the like.
  • Bone marrow cells can be collected, for example, from human iliac bone, long bone, or other bone.
  • each tissue-derived mesenchymal stem cell such as adipose-derived mesenchymal stem cell, umbilical cord-derived mesenchymal stem cell, bone marrow-derived mesenchymal stem cell is a fat-derived mesenchymal stem cell, umbilical cord-derived mesenchymal stem cell, It means any cell population containing mesenchymal stem cells derived from each tissue such as bone marrow-derived mesenchymal stem cells.
  • the cell population is at least 20% or more, preferably 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, 97%, 98 % Or 99% are mesenchymal stem cells derived from various tissues such as adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.
  • Mesenchymal stem cells have growth characteristics (eg, population doubling ability from aging to aging, doubling time), karyotype analysis (eg, normal karyotype, maternal line or neonatal line), flow cytometry ( For example, surface marker expression by FACS analysis, immunohistochemistry and / or immunocytochemistry (eg, epitope detection), gene expression profiling (eg, gene chip array; polymerase chain such as reverse transcription PCR, real-time PCR, conventional PCR, etc. Reaction), miRNA expression profiling, protein array, protein secretion such as cytokines (eg, plasma coagulation analysis, ELISA, cytokine array), metabolites (metabolome analysis), other methods known in the art, etc. May be.
  • growth characteristics eg, population doubling ability from aging to aging, doubling time
  • karyotype analysis eg, normal karyotype, maternal line or neonatal line
  • flow cytometry For example, surface marker expression by FACS analysis, immunohisto
  • Mesenchymal stem cells can be prepared by methods well known to those skilled in the art. Below, the preparation method of a fat-derived mesenchymal stem cell is demonstrated as an example. Adipose-derived mesenchymal stem cells may be obtained by the production method described in, for example, US Pat. No. 6,777,231, and can be produced, for example, by a method including the following steps (i) to (iii).
  • washing may be performed by sedimentation with vigorous stirring using a physiologically compatible saline solution (eg, phosphate buffered saline (PBS)).
  • PBS physiologically compatible saline solution
  • impurities also called debris, such as damaged tissue, blood, and red blood cells
  • washing and sedimentation are generally repeated until the debris is totally removed from the supernatant. Since the remaining cells exist as lumps of various sizes, in order to dissociate them while minimizing damage to the cells themselves, the washed cell lumps are made to have an enzyme (eg, an enzyme that weakens or breaks cell-cell junctions).
  • collagenase dispase or trypsin.
  • the amount of such enzyme and the duration of treatment vary depending on the conditions used, but are known in the art.
  • the cell mass can be broken down by other treatment methods such as mechanical agitation, ultrasonic energy, thermal energy, etc., but with minimal cell damage
  • an enzyme in order to minimize harmful effects on cells, it is desirable to deactivate the enzyme using a medium or the like after a suitable period of time.
  • the cell suspension obtained by the step (i) includes a slurry or suspension of aggregated cells, and various kinds of contaminated cells such as erythrocytes, smooth muscle cells, endothelial cells, and fibroblasts. Therefore, the cells in the aggregated state and these contaminated cells may be separated and removed, but they can be removed by adhesion and washing in step (iii) to be described later. May be. When contaminating cells are separated and removed, this can be achieved by centrifugation that forcibly separates the cells into a supernatant and a precipitate. The resulting precipitate containing contaminating cells is suspended in a physiologically compatible solvent.
  • Suspended cells may contain erythrocytes, but erythrocytes are excluded by selection by adhesion to the individual surface described later, and thus a lysis step is not always necessary.
  • a method for selectively lysing erythrocytes for example, a method well known in the art such as incubation in a hypertonic medium or a hypotonic medium by lysis with ammonium chloride can be used. After lysis, the lysate may be separated from the desired cells, for example, by filtration, centrifugation, or density fractionation.
  • the cells in step (ii), in order to increase the purity of the mesenchymal stem cells in the suspended cells, the cells may be washed once or continuously several times, centrifuged, and resuspended in the medium. Alternatively, cells may be separated based on cell surface marker profile or based on cell size and granularity.
  • the medium used in the resuspension is not particularly limited as long as it is a medium capable of culturing mesenchymal stem cells, but such a medium includes basal medium added with serum and / or albumin, transferrin, fatty acid, One or more serum substitutes such as insulin, sodium selenite, cholesterol, collagen precursor, trace elements, 2-mercaptoethanol, 3′-thiolglycerol, and the like may be added.
  • substances such as lipids, amino acids, proteins, polysaccharides, vitamins, growth factors, low molecular compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, etc. are added as necessary. May be.
  • basal medium examples include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, Ham's F16 medium.
  • EMEM Eagle's Minimum Essential Medium
  • DMEM Dulbecco's modified Eagle's Medium
  • Ham's F12 medium Ham's F16 medium.
  • Examples include a medium, Fischer's medium, MCDB201 medium, and a mixed medium thereof.
  • the serum examples 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.
  • FBS fetal bovine serum
  • bovine serum bovine serum
  • calf serum goat serum
  • horse serum horse serum
  • pig serum sheep serum
  • rabbit serum rat serum
  • 5 v / v% to 15 v / v% preferably 10 v / v% may be added to the basal medium.
  • fatty acid examples include, but are not limited to, linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid.
  • lipid examples include, but are not limited to, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and the like.
  • Amino acids include, but are not limited to, for example, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine and the like.
  • proteins include, but are not limited to, ecotin, reduced glutathione, fibronectin and ⁇ 2-microglobulin.
  • polysaccharide examples include glycosaminoglycans, and among the glycosaminoglycans, hyaluronic acid, heparan sulfate and the like are particularly exemplified, but the polysaccharide is not limited thereto.
  • Growth factors include, for example, platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF- ⁇ ), hepatocyte growth factor (HGF), epidermal growth factor (EGF) Examples include, but are not limited to, connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and the like. From the viewpoint of using the adipose-derived mesenchymal stem cells obtained in the present invention for cell transplantation, it is preferable to use a (xenofree) medium that does not contain components derived from different species such as serum.
  • a (xenofree) medium that does not contain components derived from different species such as serum.
  • Such a medium is provided as a medium prepared in advance for mesenchymal stem cells (stromal cells) from, for example, PromoCell, Lonza, Biological Industries, Veritas, R & D Systems, Corning, and Roto. Has been.
  • the appropriate cell density and the appropriate cell medium are used on the solid surface without differentiating the cells in the cell suspension obtained in step (ii).
  • Culture under culture conditions the “solid surface” means any material capable of binding / adhering the adipose-derived mesenchymal stem cells in the present invention.
  • such a material is a plastic material that has been treated to promote the attachment and adhesion of mammalian cells to its surface.
  • the shape of the culture vessel having a solid surface is not particularly limited, but a petri dish or a flask is preferably used. The cells are washed after incubation to remove unbound cells and cell debris.
  • cells that finally remain attached and adhered to the solid surface can be selected as a cell population of adipose-derived mesenchymal stem cells.
  • the surface antigen may be analyzed by a conventional method using flow cytometry or the like. Further, the ability to differentiate into each cell line may be examined, and such differentiation can be performed by conventional methods.
  • Mesenchymal stem cells in the present invention can be prepared as described above, but may be defined as cells having the following characteristics; (1) It exhibits adhesiveness to plastic under the culture conditions in a standard medium. (2) Surface antigens CD44, CD73, CD90 are positive, CD31, CD45 are negative, and (3) Differentiated into bone cells, adipocytes, and chondrocytes under culture conditions.
  • peripheral blood mononuclear cells refer to fractions containing lymphocytes, neutrophils, eosinophils, basophils, and monocytes, which are obtained from human or animal peripheral blood.
  • Peripheral blood mononuclear cells can be separated from peripheral blood by density gradient centrifugation using Ficoll-hypaque (registered trademark) or the like.
  • Peripheral blood mononuclear cells as cells (A) in the present invention may be cells separated from peripheral blood, or they may be cultured with various factors, low molecular compounds, antibodies, etc. as necessary. It may be proliferated and activated.
  • the (A) cell in the present invention may be a cell that has been appropriately cryopreserved and thawed as long as it has a therapeutic effect on various diseases.
  • cryopreservation can be performed by suspending (A) cells in a cryopreservation solution well known to those skilled in the art and cooling. Suspension can be performed by detaching the cells with a release agent such as trypsin as necessary, transferring them to a cryopreservation container, treating them appropriately, and adding a cryopreservation solution.
  • the cryopreservation solution may contain DMSO (Dimethylsulfoxide) as a frost damage protective agent.
  • DMSO Dimethylsulfoxide
  • DMSO is also known to have differentiation-inducing properties for mesenchymal stem cells.
  • glycerol, propylene glycol or polysaccharides are exemplified.
  • 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%.
  • additives described in WO2007 / 058308 may be included.
  • cryopreservation solution for example, cryopreservation provided by Bioverde, Nippon Genetics, Reprocell, Xenoac, Cosmo Bio, Kojin Bio, Thermo Fisher Scientific, etc.
  • a liquid may be used.
  • the cooling rate may be appropriately controlled using a program freezer.
  • the cooling rate may be appropriately selected depending on the components of the cryopreservation solution, and may be performed according to the manufacturer's instructions for the cryopreservation solution.
  • the upper limit of the storage period is not particularly limited as long as the cells cryopreserved under the above conditions are thawed and retain the same properties as before freezing, for example, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, 1 year or more, or more. Since cell damage can be suppressed by storing at a lower temperature, it may be transferred to a gas phase on liquid nitrogen (from about ⁇ 150 ° C. to ⁇ 180 ° C.). When storing in a gas phase on liquid nitrogen, it can be performed using a storage container well known to those skilled in the art. Although not particularly limited, for example, when storing for 2 weeks or more, it is preferable to store in a gas phase on liquid nitrogen.
  • the thawed (A) cells may be appropriately cultured before the next cryopreservation.
  • the mesenchymal stem cells are cultured using the above-described medium capable of culturing mesenchymal stem cells, and are not particularly limited, but contain CO 2 at a culture temperature of about 30 to 40 ° C., preferably about 37 ° C. It may be performed in an air atmosphere.
  • the CO 2 concentration is about 2-5%, preferably about 5%.
  • the culture after reaching the appropriate confluency for the culture container (for example, cells may occupy 50% to 80% of the culture container), the cells are detached with a release agent such as trypsin.
  • the culture may be continued by seeding in a separately prepared culture vessel at an appropriate cell density.
  • 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, etc. are exemplified. In a particular embodiment, the cell density is between 2,000 and 10,000 cells / cm 2 . It is preferable to adjust the period until reaching appropriate confluency from 3 days to 7 days. During culture, the medium may be changed as necessary.
  • Thawing of cryopreserved cells can be performed by methods well known to those skilled in the art. For example, the method performed by standing or shaking in a 37 degreeC thermostat or a hot water bath is illustrated.
  • the cell (A) contained in the cell pharmaceutical composition of the present invention may be a cell in any state, for example, a cell recovered by detaching a cell in culture, or frozen in a cryopreservation solution It may be a cell in a state of being formed.
  • Use of the same lot of cells obtained by expanding and culturing in the same lot is preferable in that the same action and effect can be stably obtained and the handling property is excellent.
  • the (A) cells in the cryopreservation state may be thawed immediately before use and directly mixed with the cell suspension solution (B) described later while being suspended in the cryopreservation solution.
  • the cryopreservation solution may be removed by a method such as centrifugation and then suspended in the (B) cell suspension solution.
  • the dose (dosage) of (A) cells of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.), dosage form, etc., but from the standpoint of sufficient therapeutic effect, the amount From the viewpoint of suppressing the occurrence of side effects, a smaller amount tends to be preferable.
  • the number of cells is 1 ⁇ 10 3 to 1 ⁇ 10 12 cells / time, preferably 1 ⁇ 10 4 to 1 ⁇ 10 11 cells / time, more preferably 1 ⁇ 10 5 to 1 ⁇ 10 10 cells / time, particularly preferably 5 ⁇ 10 5. 6 to 1 ⁇ 10 9 pieces / time.
  • this dose may be administered once as a single dose, or the dose may be divided into multiple doses.
  • the dose (dosage) of the cell (A) of the present invention may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.) and the dosage form of the composition of the present invention, but is usually administered to an adult.
  • the number of cells is 1 ⁇ 10 to 5 ⁇ 10 10 cells / kg, preferably 1 ⁇ 10 2 to 5 ⁇ 10 9 cells / kg, more preferably 1 ⁇ 10 3 to 5 ⁇ 10 8 cells / kg, particularly preferably 1 ⁇ 10 4 to 5 ⁇ 10 7 cells / kg. It is.
  • this dose may be administered once as a single dose, or the dose may be divided into multiple doses.
  • the (B) cell suspension solution of the present invention comprises an electrolyte and a carbohydrate, contains Na + and Cl ⁇ as the electrolyte, and is substantially free of K + .
  • the cell suspension solution contains Na + and Cl ⁇ as the electrolyte and substantially does not contain K + .
  • concentrations of Na + and Cl ⁇ are lower than that of physiological saline and higher than that of postoperative recovery fluid (No. 4 infusion) and the like. Specifically, it is 30 mEq / L or more and 130 mEq / L or less, preferably 50 mEq / L or more and 120 mEq / L or less, and more preferably 60 mEq / L or more and 100 mEq / L or less.
  • the Na + concentration is 70 mEq / L or more and 100 mEq / L or less
  • the Cl ⁇ concentration is 70 mEq / L or more and 80 mEq / L or less.
  • the Na + concentration and the Cl ⁇ concentration may be the same or different.
  • (B) as the electrolyte in the cell suspension solution is substantially Na + and Cl - which is preferably only, Na + and Cl - in addition to a certain amount of Lac - may contain .
  • the concentration of Lac ⁇ is 0 to 30 mEq / L, preferably 0 to 20 mEq / L.
  • sugar contained in the cell suspension solution examples include glucose, dextrin, maltodextrin, oligosaccharide, sucrose, and the like, and one or more of these may be used in combination. Can do. Among these, glucose is preferable as the saccharide.
  • the sugar concentration can be adjusted according to the concentrations of Na + and Cl ⁇ so that (B) the cell suspension solution is isotonic. Specifically, it is 0.1 w / v% to 10 w / v%, and preferably 1.0 w / v% to 5.0 w / v%.
  • the (B) cell suspension solution of the present invention can be referred to as an electrolyte infusion preparation, and what is known by the name of the starting solution or No. 1 infusion is the above-described (B) cell suspension solution. It satisfies the conditions and is preferable.
  • (B) cell suspension solution Solita (registered trademark) -T1 infusion (Ai Pharma Co., Ltd.), YD Solita (registered trademark) -T1 infusion (Yosindo) , KN1 infusion (Otsuka Pharmaceutical Factory), Denosaline (registered trademark) 1 infusion (Terumo Corporation), Soldem (registered trademark) 1 infusion (Terumo Corporation), Riplus (registered trademark) 1 infusion (Fuso Pharmaceutical) Commercial products such as Co., Ltd. can also be used.
  • the cell pharmaceutical composition of the present invention may contain one or more other drugs having a therapeutic effect on a disease.
  • Other drugs that can be used as liver disease drugs, heart disease drugs, inflammatory bowel disease drugs, respiratory drugs, nervous system drugs, cardiovascular drugs, cerebral circulation improving drugs, immunosuppressive drugs Of these drugs.
  • liver diseases examples include hepatitis B therapeutic agents (lamivudine, adefovir, entecavir, tenofovir, etc.), interferon preparations (interferon ⁇ , interferon ⁇ -2b, interferon ⁇ , peginterferon ⁇ -2a, peginterferon ⁇ -2b, etc.
  • hepatitis C drugs ribavirin, terapyrevir, simeprevir, vaniprevir, daclatasvir, asunaprevir, sofosbuvir, etc.
  • corticosteroids prednisolone, methylprednisolone sodium succinate, etc.
  • anticoagulants dry concentrated human antithrombin III
  • antidote calcium edetate disodium hydrate, glutathione, dimethicaprol, sodium thiosulfate hydrate, Sugamasekuna 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,
  • therapeutic agents for heart diseases include ACE inhibitors, angiotensin II receptor antagonists, ⁇ -blockers, antiplatelet agents, warfarin, calcium antagonists, nitrates, diuretics, HMG-CoA reductase inhibitors, ancalons, etc. Is mentioned.
  • Examples of the therapeutic agent for inflammatory bowel disease include salazosulfapyridine and mesalazine.
  • Examples of the respiratory medicine include dimorpholine, doxapram hydrochloride hydrate, cyberestat sodium hydrate, pirfenidone, pulmonary surfactant, and Dornase alpha.
  • Examples of the nervous system drug include edaravone, interferon beta-1a, interferon beta-1b, fingolimod hydrochloride, riluzole, tartilelin hydrate and the like.
  • circulatory drugs examples include hepronicart, midodrine hydrochloride, amedinium methylmethyl sulfate, ethylephrine hydrochloride, phenylephrine hydrochloride, and the like.
  • cerebral circulation improving drug examples include ifenprodil tartrate, nicergoline, ipdilast, dihydroergotoxin mesylate, nizophenone fumarate, fasudil hydrochloride hydrate and the like.
  • immunosuppressant examples include cyclosporine, azathioprine, mizoribine, basiliximab, tacrolimus hydrate, gusperimus hydrochloride, mycophenolate mofetil, everolimus and the like.
  • the cell pharmaceutical composition of the present invention contains the above-mentioned other drug, at the time of storage, it may be stored in a container different from (A) the cell and (B) the cell suspension solution. It may be contained in a form blended with. Depending on the type of disease, treatment method, patient condition, etc., other drugs and (A) cells and (B) cell suspension solutions may be administered simultaneously, or at regular intervals. Also good.
  • the cell pharmaceutical composition of the present invention is within a range not impairing the effects of the present invention, in addition to the above (A) cell and (B) cell suspension solution, according to its use and form, according to a conventional method, Other components such as pharmaceutically acceptable carriers and additives may be included.
  • Such carriers and additives may be contained in the (B) cell suspension solution, or may be contained separately from the (B) cell suspension solution.
  • Examples of such carriers and additives include isotonic agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH regulators, stabilizers, chelating agents.
  • Oily base Oily base, gel base, surfactant, suspending agent, binder, excipient, lubricant, disintegrant, foaming agent, fluidizer, dispersant, emulsifier, buffer, solubilizer , Antioxidants, sweeteners, sour agents, colorants, flavoring agents, fragrances or refreshing agents, but are not limited thereto.
  • the cell pharmaceutical composition of the present invention can be used in various forms depending on the purpose, for example, injections (including infusions, implantable injections, continuous injections, injections prepared at the time of use), dialysis agents, patches. It can be used in the form of a poultice.
  • the cell pharmaceutical composition of the present invention can also be applied to the affected area by spraying, and the cell pharmaceutical composition of the present invention can also be used in the form of gelation or sheeting at the affected area after spraying.
  • the cell pharmaceutical composition of the present invention can also be applied to the affected area after the (A) cell is made into a sheet or a three-dimensional structure.
  • (A) cells, (B) cell suspension solutions, other drugs, and other components are sealed and stored in separate containers, and are used by mixing them at the time of use. Also good.
  • the above (A) cells, (B) cell suspension solution, other drugs, and other components may be stored under conditions suitable for each, for example, freezing conditions, refrigerated conditions, room temperature Any of conditions etc. may be sufficient.
  • the pH of the cell pharmaceutical composition of the present invention is not particularly limited as long as it is within a pharmaceutically, pharmacologically (pharmaceutical) or physiologically acceptable range.
  • the range is 9.0, preferably 2.5 to 8.5, more preferably 3.0 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 the range acceptable to the living body.
  • An example of the osmotic pressure ratio of the cell pharmaceutical composition of the present invention is preferably in the range of 0.7 to 5.0, more preferably 0.8 to 3.0, and still more preferably 0.9 to 1.4. It is done.
  • the adjustment of the osmotic pressure can be performed by a method known in the art using the above-described electrolyte, sugar and the like.
  • the osmotic pressure ratio is the ratio of the osmotic pressure of the sample to the osmotic pressure of 286 mOsm (0.9 w / v% sodium chloride aqueous solution) based on the 15th revised Japanese Pharmacopoeia. Measure by referring to the freezing point method.
  • the standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) was prepared by drying sodium chloride (Japanese Pharmacopoeia standard reagent) at 500 to 650 ° C. for 40 to 50 minutes, and then in a desiccator (silica gel).
  • the mixture is allowed to cool and 0.900 g is accurately weighed and dissolved in purified water to make exactly 100 mL, or a commercially available standard solution for osmotic pressure ratio measurement (0.9 w / v% sodium chloride aqueous solution) is used.
  • the concentration of (A) cells in the cell pharmaceutical composition of the present invention is the cell type, cell suspension although it may vary depending on the solution used, 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.
  • human mesenchymal stem cells are contained in a cell suspension solution at a density of 1 ⁇ 10 4 to 2.5 ⁇ 10 6 cells / mL,
  • the turbid solution contains 70 to 100 mEq / L of sodium ions and 70 to 80 mEq / L of chloride ions, is substantially free of potassium ions, and has an osmotic pressure ratio of 0.9 to 1.4 with respect to physiological saline. Yes, and has a pH of 3.0 to 8.0.
  • the administration route to the subject of the cell pharmaceutical composition of the present invention includes subcutaneous administration, intramuscular administration, intravenous administration, intraarterial administration, intrathecal administration, intraperitoneal administration, rectal administration, vaginal administration, and transdermal administration.
  • implant, intraarterial administration, intravenous administration, and direct administration to an organ are more preferable. Are intravenous and direct administration to the organ.
  • the administration rate of the cell pharmaceutical composition of the present invention to the subject may vary depending on the patient's condition (weight, age, symptoms, physical condition, etc.) and the administration route of the non-alcoholic steatohepatitis therapeutic agent of the present invention.
  • it When administered to an adult, it is 50 mL / h to 1,000 mL / h, preferably 75 mL / h to 500 mL / h, and more preferably 100 mL / h to 250 mL / h.
  • the administration temperature of the cell pharmaceutical composition of the present invention to the subject may vary depending on the patient's condition (body weight, age, symptoms, physical condition, etc.), the administration route of the cell pharmaceutical composition of the present invention, etc. ⁇ 45 ° C., preferably 15 ° C. to 37 ° C., 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.
  • an infusion set a wand disposable infusion tube set (manufactured by Yoshida Seisakusho Co., Ltd.), an infusion solution set (manufactured by Fortegro Medical Co., Ltd.), a terfusion (R) infusion set (manufactured by Terumo Corporation), a JMS infusion set (Manufactured by JMS Co., Ltd.), Sure plug infusion set (manufactured by Terumo Corporation), infusion set (manufactured by Nipro Corporation), top infusion set NP (manufactured by Top Co., Ltd.), infusion set with filter (EX type) Commercial products such as Toray Medical Co., Ltd. can also be used.
  • the cell pharmaceutical composition of the present invention can be administered to a subject using an infusion tube.
  • an infusion tube Lectro Cass (manufactured by Samick International Co., Ltd.), JMS extension tube (manufactured by JMS Co., Ltd.), Saffy extension tube (manufactured by Terumo Corporation), extension tube (manufactured by Co., Ltd.) Top product), Connecting tube (Chuatsu) (Medicos Hirata Co., Ltd.), SAFTY AP tube (manufactured by Kawasumi Chemical Industry Co., Ltd.), Bioconnector 2 with extension tube (Toray Medical Co., Ltd.), Medicut Extension Tube Set B (Nippon Sherwood) Commercially available products such as Wand Disposable Infusion Tube Set (manufactured by Yoshida Seisakusho Co., Ltd.), Infusion Tube (manufactured by Forte Grow Medical Co., Ltd.), and the like can also be used.
  • the material of the infusion tube used when administering the cell pharmaceutical composition of the present invention to a subject includes polyvinyl chloride, thermoplastic elastomer, TPE thermoplastic elastomer, silicone, silicone rubber, polyethylene, polybutadiene, Teflon (registered trademark), Polyurethane, polypropylene, natural rubber, polyolefin, PVC (plasticizer: TOTM, DOA), plasticizer PVC, and mixtures thereof can be used.
  • the cell pharmaceutical composition of the present invention can be suitably used for the treatment of various diseases.
  • visceral diseases specifically heart disease, stomach / duodenal disease, small intestine / colon disease, liver disease, biliary disease, pancreatic disease, kidney disease, lung disease, mediastinal disease, diaphragm disease, pleural disease, peritoneal disease
  • CNS central nervous system
  • peripheral arterial diseases peripheral venous diseases.
  • Specific diseases include, for example, autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (Nonalcoholic steatohepatitis (NASH)), non-alcoholic fatty liver (NAFL), liver fibrosis, cirrhosis, liver cancer, fatty liver, drug allergic liver disorder, hemochromatosis, hemosiderosis, Wilson's disease, primary Hepatic diseases such as biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver abscess, chronic active hepatitis, chronic persistent hepatitis; myocardial infarction, heart failure, arrhythmia, palpitations, cardiomyopathy, false blood Cardiomyopathy, angina, congenital heart disease, valvular heart disease, myocarditis,
  • biliary tract diseases such as acute cholecystitis, acute cholangitis, chronic cholecystitis, cholangiocarcinoma, gallbladder cancer
  • pancreatic diseases such as acute pancreatitis, chronic pancreatitis, pancreatic cancer
  • acute nephritis, chronic nephritis, acute renal failure chronic Renal diseases such as renal failure
  • Diaphragmatic diseases such as diaphragmatic hernia
  • Pleural diseases such as pleurisy, pyothorax, pleural tumor, cancerous pleurisy, pleural mesothelioma
  • Peritoneal diseases such as peritonitis, peritoneal tumor
  • Aseptic meningitis Guillain-Barre syndrome, amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, polyneuropathy, spinal muscular atrophy, spinal disorder, acute transverse myelitis, spinal cord infarction (false) Hematologic spinal cord disorder), intracranial tumor, spine
  • Neurological diseases such as tumors; CNS disorders such as Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, Parkinson's disease; fibromyalplasia, peripheral arterial disease (PAD), obstructive thromboangitis (Burger's disease), Kawasaki Peripheral arterial disease such as KD disease; deep vein
  • liver diseases, heart diseases, lung diseases, neurological diseases, peripheral arterial diseases, immunodeficiency diseases that have been confirmed to have sufficient therapeutic effects by mesenchymal stem cells are preferred, among which liver fibrosis, Suitable for the treatment of cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, interstitial pneumonia, childhood cerebral palsy, amyotrophic lateral sclerosis (ALS), peripheral arterial disease (PAD), graft-versus-host disease (GVHD) And can be used more suitably for liver fibrosis, cirrhosis, myocardial infarction, heart failure, pulmonary fibrosis, and interstitial pneumonia. Moreover, it can use suitably for the cancer of each structure
  • the present invention includes (A) a cell, and (B) a cell suspension solution containing an electrolyte and a carbohydrate. (B) the electrolyte in the cell suspension solution contains Na + and Cl ⁇ , Also included are disease treatment kits that are substantially free of + .
  • the disease treatment kit of the present invention is a kit containing the above-described cell pharmaceutical composition of the present invention, and may contain (A) cells, (B) cell suspension solutions, and other cell pharmaceutical compositions of the present invention. The description in the section of the cell pharmaceutical composition can be applied to the components. According to the disease treatment kit of the present invention, since the state of cells can be kept good and the survival rate can be maintained high over a long period of time, an excellent therapeutic effect can be obtained for various diseases. .
  • the disease treatment kit of the present invention can also be expressed as comprising the cell pharmaceutical composition, container and label of the present invention.
  • Suitable containers included in the disease treatment kit of the present invention are not particularly limited, and examples include cryotubes for freezing cells, bottles for cell suspension solutions, vials, test tubes, and dialysis bags. These containers may be formed from a variety of materials such as glass, metal, plastic, or combinations thereof.
  • the contents on the labels on these containers describe the contents of the cells, the solution for cell suspension, and the like.
  • the disease treatment kit of the present invention includes other additives, other drugs, diluents, filters, needles, syringes, and other packages that are desirable from a commercial and user standpoint, including package inserts that describe usage. Material can be included.
  • ⁇ Cell suspension solution> Also within the scope of the present invention is a cell suspension solution for a cell pharmaceutical composition comprising an electrolyte and a carbohydrate, wherein the electrolyte comprises Na + and Cl ⁇ and is substantially free of K + .
  • the electrolyte comprises Na + and Cl ⁇ and is substantially free of K + .
  • the present invention provides a method for treating a disease in which (A) cells are suspended in a cell suspension solution containing (B) an electrolyte and a carbohydrate and then administered to a patient. Also included is a therapeutic method wherein the electrolyte comprises Na + and Cl ⁇ and is substantially free of K + . According to the treatment method of the present invention, it is possible to maintain a high cell survival rate over a long period of time, and therefore, an excellent therapeutic effect can be achieved for various diseases.
  • the disease method of the present invention is a treatment method using the above-described cell pharmaceutical composition of the present invention, and may contain (A) cells, (B) a cell suspension solution, and other cell pharmaceutical compositions of the present invention. The description in the section of the cell pharmaceutical composition can be applied to the components.
  • Example 1 Preparation of adipose-derived mesenchymal stem cells
  • the subcutaneous adipose tissue obtained by the liposuction method was washed with physiological saline.
  • collagenase (Roche diagnostics) (solvent was physiological saline) was added, shaken at 37 ° C. for 90 minutes, and dispersed. Subsequently, the suspension was centrifuged at 800 g for 5 minutes to obtain a precipitate of stromal vascular cell groups.
  • a serum-free medium for mesenchymal stem cells (Rohto) is added to the cell precipitate, the cell suspension is centrifuged at 400 g for 5 minutes, and after removal of the supernatant, a serum-free medium for mesenchymal stem cells (Rohto)
  • the cells were seeded in a flask. Cells were cultured at 37 ° C. for several days in 5% CO 2 . Several days later, the culture was washed with PBS to remove residual blood cells and adipose tissue contained in the culture solution, and mesenchymal stem cells adhered to a plastic container were obtained.
  • adipose-derived mesenchymal stem cells were dispensed into a centrifuge tube and centrifuged at 400 g for 5 minutes to obtain cell precipitates. After removing the supernatant, an appropriate amount of a cell cryopreservation solution (STEM-CELLBANKER (Zenoac)) was added and suspended. The cell suspension was dispensed into a cryotube, stored at ⁇ 80 ° C. in a freezer, transferred to a gas phase on liquid nitrogen, and storage was continued.
  • a cell cryopreservation solution STEM-CELLBANKER (Zenoac)
  • the cell viability was 70% or more only after storage at 15 ° C and 30 ° C.
  • Physio registered trademark 140 infusion
  • the cell viability was 70% or more when stored at 15 ° C, immediately after preparation, after 2 hours, and when stored at 30 ° C. It was only immediately after.
  • fat-derived mesenchymal stem cells were suspended in 5% Otsuka sugar solution, the cell viability was 70% or more only immediately after preparation at 30 ° C. storage.
  • Example 2 Into a 15 mL centrifuge tube (Sumitomo Bakelite Co., Ltd., product number: MS-56150), KN1 infusion (No.1 solution (starting solution); Otsuka Pharmaceutical Factory, Lot: M6C77), KN2 infusion (No.2 solution (dehydration supplement) Solution); Otsuka Pharmaceutical Factory, Lot: K6E92), KN3 Infusion (No.3 Liquid (Maintenance Solution); Otsuka Pharmaceutical Factory, Lot: K6D96), KN4 Infusion (No.4 Liquid (Postoperative Recovery Solution)) Otsuka Pharmaceutical Factory, Lot: K6D80), Physio (registered trademark) 70 infusion (Otsuka Pharmaceutical Factory, Lot: M6D91), 1% glucose-containing No.
  • Glu1% glucose-containing No. 1 solution
  • Glu5% glucose-containing No. 1 solution
  • Glu1% and Glu5% are Otsuka raw food injection 500mL (Japanese Pharmacopoeia physiological saline; Otsuka Pharmaceutical factory, Lot: 5A81N), Otsuka sugar solution 50% (Otsuka Pharmaceutical factory, Lot: M5G83) and injection. It was prepared using water (Otsuka Pharmaceutical Factory, Lot: 6098). The cells were suspended by inversion and stored at room temperature. Immediately after the preparation, live cells and dead cells after 2 hours, 4 hours, and 7 hours were measured in the same manner as in Example 1. Similarly, the cell viability was calculated. For cells whose cell viability fell below 70% after 4 hours, measurement after 7 hours was not performed. The composition of each infusion solution is shown in Table 3 below, and the results of cell viability are shown in Table 4 below.
  • KN1 infusion solution containing glucose and containing only Na + and Cl ⁇ as electrolytes, Glu 1% and Glu 5% have a significantly high cell viability of fat-derived mesenchymal stem cells over a long period of time. It was found that it can be maintained.
  • Example 3 Using Soldem (registered trademark) 1 infusion solution (No. 1 solution (starting solution); Terumo Corporation, Lot: 160519TA), the concentration in the cell suspension is 1.23 ⁇ 10 5 cells / mL. The test was conducted in the same manner as in Example 2, and the cell viability was calculated immediately after preparation, after 2 hours and after 4 hours. The composition of Soldem (registered trademark) 1 infusion is shown in Table 5 below, and the results of cell viability are shown in Table 6 below.
  • Soldem registered trademark 1 infusion solution
  • Example 4 The fat-derived mesenchymal stem cells of Example 2 were replaced with bone marrow-derived mesenchymal stem cells (Lonza), and KN1 infusion (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory, Lot: M6C77) was used. Then, the concentration in the cell suspension was adjusted to 1.1 ⁇ 10 5 cells / mL, and the test was performed in the same manner as in Example 2. In the same manner as in Example 2, immediately after the preparation, 2 hours, 4 hours and 7 hours later, the viable cells and dead cells were measured, and the cell viability was calculated. The results are shown in Table 7 below.
  • the KN1 infusion solution containing glucose and containing only Na + and Cl ⁇ as electrolytes has a significantly high cell viability for bone marrow-derived mesenchymal stem cells as well as fat-derived mesenchymal stem cells. It was found that it can be maintained for a long time.
  • Example 5 instead of the fat-derived mesenchymal stem cells of Example 2 with umbilical cord-derived mesenchymal stem cells (Lifeline Cell Technology, LifeLine (registered trademark) _UCMSC, Lot. 160907), KN1 infusion (No. 1 solution (starting solution); Using Otsuka Pharmaceutical Factory, Lot: M6C77), the concentration in the cell suspension was adjusted to 1.23 ⁇ 10 5 cells / mL, and the test was performed in the same manner as in Example 2. In the same manner as in Example 2, live cells and dead cells immediately after preparation, 2 hours, 4 hours, and 6 hours were measured, and cell viability was calculated. The results are shown in Table 8 below.
  • the KN1 infusion solution containing glucose and containing only Na + and Cl ⁇ as electrolytes has a significantly high cell viability for the umbilical cord-derived mesenchymal stem cells as well as the fat-derived mesenchymal stem cells. It was found that it can be maintained for a long time.
  • Example 6 The fat-derived mesenchymal stem cells of Example 2 were replaced with peripheral blood mononuclear cells (ACCUCELL (registered trademark) normal donor-derived PBMC, Precision Bioservices (PRECISION FOR MEDICINE), Lot. 13134-10), KN1 Example 2 was prepared using an infusion solution (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory, Lot: M6C77) so that the concentration in the cell suspension was 1.23 ⁇ 10 5 cells / mL. The test was conducted in the same manner as above. In the same manner as in Example 2, live cells and dead cells immediately after preparation, 2 hours, 4 hours, and 6 hours were measured, and cell viability was calculated. The results are shown in Table 9 below.
  • ACCUCELL registered trademark
  • Otsuka Pharmaceutical Factory Lot: M6C77
  • KN1 infusion solution containing glucose and containing only Na + and Cl ⁇ as electrolytes has a significantly high cell viability for peripheral blood mononuclear cells as well as adipose-derived mesenchymal stem cells. It was found that it can be maintained for a long time.
  • Adipose-derived mesenchymal stem cells were prepared in the same manner as in Example 1, and the adipose-derived mesenchymal stem cells stored in the liquid nitrogen gas phase were removed from the liquid nitrogen, and the cells were thawed using a thermostatic chamber (37 ° C.). did. After melting and standing at room temperature for 30 minutes, the solution was added to KN1 infusion (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory), and then the infusion bag was mixed by inversion. The cell viability (%) was calculated immediately after preparation, 2 hours, 3 hours, and 4 hours after the infusion bag was suspended on an infusion stand at room temperature. The mixed solution was bent and mixed once every 30 minutes. The total cell concentration immediately after production was 1.8 ⁇ 10 5 to 1.9 ⁇ 10 5 for Samples 1 to 3, and 6.3 ⁇ 10 5 to 7.3 ⁇ 10 5 for Samples 4 to 6. . The results are shown in Table 10 below.
  • KN1 infusion When fat-derived mesenchymal stem cells are suspended in KN1 infusion, high cell viability can be maintained during all storage times, and KN1 infusion significantly increases the cell viability of adipose-derived mesenchymal stem cells It was found that it can be maintained for a long time.
  • Adipose-derived mesenchymal stem cells were prepared in the same manner as in Example 1, and the adipose-derived mesenchymal stem cells stored in the liquid nitrogen gas phase were removed from the liquid nitrogen, and the cells were thawed using a thermostatic chamber (37 ° C.). did. KN1 infusion solution (No. 1 solution (starting solution); Otsuka Pharmaceutical Co., Ltd.) so as to be 1.25 ⁇ 10 5 cells / mL and 5 ⁇ 10 5 cells / mL after standing at room temperature for 30 minutes after melting. After addition to the factory, the infusion bag was mixed by inversion. The infusion bag is hung on an infusion stand at room temperature and allowed to stand for 3 hours.
  • KN1 infusion solution No. 1 solution (starting solution); Otsuka Pharmaceutical Co., Ltd.
  • the infusion tube polyvinyl chloride (plasticizer: tri (2-ethylhexyl) trimellitic acid), polybutadiene
  • the liquid was added dropwise.
  • the cell viability (%) of the mixed solution was calculated 30 minutes, 60 minutes, 120 minutes, or 111 minutes after the start of dropping.
  • the mixed solution was bent and mixed once every 30 minutes. The results are shown in Table 11 below.
  • Adipose-derived mesenchymal stem cells were prepared in the same manner as in Example 1, and the adipose-derived mesenchymal stem cells stored in the liquid nitrogen gas phase were removed from the liquid nitrogen, and the cells were thawed using a thermostatic chamber (37 ° C.). did. After thawing the cells, each was added to KN1 infusion (No. 1 solution (starting solution); Otsuka Pharmaceutical Factory), serum-free medium (Rohto Pharmaceutical Co., Ltd.) and physiological saline (Otsuka Pharmaceutical Factory, Inc.). The cells were stored at 4 ° C., and the cell viability (%) immediately after preparation, 16 hours, 24 hours, and 99 hours was calculated. The results are shown in Table 12 below.
  • the cell pharmaceutical composition of the present invention can maintain a good cell state and maintain its survival rate in a high state for a long time, it can be expected to have an excellent therapeutic effect on various diseases.

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Abstract

L'invention a pour objet de fournir une composition pharmaceutique de cellules qui préserve de manière satisfaisante l'état de cellules, et qui permet de maintenir un taux de survie élevé sur une longue durée. Plus précisément, l'invention concerne une composition pharmaceutique de cellules qui comprend des cellules (A), et une solution pour suspension de cellules (B) contenant un électrolyte et un glucide. Ledit électrolyte de la solution pour suspension de cellules (B), contient Ｎａ＋ et Ｃｌ－, mais ne contient pas de manière pratique de Ｋ＋. En outre, de préférence, ledit électrolyte de la solution pour suspension de cellules (B), de manière pratique consiste en Ｎａ＋ et Ｃｌ－ uniquement, ou en Ｎａ＋, Ｃｌ－ et － uniquement.
PCT/JP2017/044992 2016-12-28 2017-12-14 Composition pharmaceutique de cellules, kit pour traitement de maladie, et solution pour suspension de cellules Ceased WO2018123627A1 (fr)

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