WO2018225673A1 - Method for reducing cell aggregation and therapeutic composition with reduced cell aggregation - Google Patents

Method for reducing cell aggregation and therapeutic composition with reduced cell aggregation Download PDF

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WO2018225673A1
WO2018225673A1 PCT/JP2018/021333 JP2018021333W WO2018225673A1 WO 2018225673 A1 WO2018225673 A1 WO 2018225673A1 JP 2018021333 W JP2018021333 W JP 2018021333W WO 2018225673 A1 WO2018225673 A1 WO 2018225673A1
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cell
cells
solution
cell preparation
ringer
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PCT/JP2018/021333
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Japanese (ja)
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孝洋 木原
明美 黒宮
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Ｄｓファーマアニマルヘルス株式会社
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Priority to JP2019523518A priority Critical patent/JP7133549B2/en
Publication of WO2018225673A1 publication Critical patent/WO2018225673A1/en

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    • A—HUMAN NECESSITIES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
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    • 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
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Definitions

  • the present disclosure relates to a method for reducing cell aggregation in the field of regenerative medicine / cell medicine, a therapeutic composition with reduced cell aggregation, and the like.
  • Regenerative and cell therapy that treats diseases by administering cells to the living body has been actively developed.
  • the cell preparation when cells to be administered adhere to each other to form an aggregate, it may cause embolization or prevent quantitative administration when administered into blood vessels. Therefore, in the field of regenerative medicine / cell medicine, development of a method for reducing cell aggregation is strongly desired.
  • An object of the present invention is to provide a method for reducing cell aggregation upon administration of a cell preparation, a therapeutic composition with reduced cell aggregation, a method for producing the same, and the like.
  • the present invention provides, in one embodiment, a method for producing a therapeutic composition comprising mammalian cells, comprising dispersing the mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution. To do.
  • the present invention provides a therapeutic composition comprising mammalian cells in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution, or in a mixture of the aqueous glucose solution or Ringer's acetate solution and a cell preservation solution. provide.
  • the present invention provides a cell preparation comprising mammalian cells, wherein the cells are dispersed in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution and administered to a mammal.
  • Cell preparations are used.
  • the invention in a further aspect, is a method of treating a disease in a mammal, comprising Dispersing mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution to provide a therapeutic composition, and administering the resulting therapeutic composition to a mammal .
  • Glucose and Ringer's acetate solution have low toxicity to the living body and can be used at low cost. According to the present invention, it has become possible to produce a therapeutic composition in which cell aggregation at the time of administration of a cell preparation is reduced safely and inexpensively, and in which cell aggregation is reduced.
  • FIG. 1 shows the survival rate of beagle adipose tissue-derived stem cells dispersed in a 5% aqueous glucose solution.
  • FIG. 2 shows the survival rate of stem cells derived from beagle adipose tissue dispersed with 5% aqueous glucose solution or Ringer's acetate solution.
  • % relating to concentration means “w / v%” unless otherwise specified.
  • the cell preparation of the present invention includes mammalian cells.
  • mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, goats, horses, sheep, monkeys, humans, and the like.
  • the mammal is a non-human mammal.
  • the mammal is a dog, cat, pig or horse, more preferably a dog or cat.
  • the mammal is a dog, particularly a beagle or a golden retriever.
  • the cells in this specification include stem cells, fibroblasts, epithelial cells, white blood cells and the like.
  • stem cells include embryonic stem cells (ES cells), embryonic germ stem cells (EG cells), iPS cells, and somatic stem cells.
  • somatic stem cells include mesenchymal stem cells, neural stem cells, and hematopoietic stem cells.
  • the stem cell may be a cell produced by any method.
  • the stem cell is a mesenchymal stem cell.
  • Mesenchymal stem cells can be obtained by known methods from adipose tissue, bone marrow, umbilical cord blood and the like.
  • the mesenchymal stem cells are derived from adipose tissue, such as subcutaneous adipose tissue.
  • Cell preparations include, but are not limited to, typically 10 4 to 10 8 cells per preparation. In certain embodiments, the cell preparation comprises 10 5 to 10 7 or 2.5 ⁇ 10 6 to 5 ⁇ 10 6 cells.
  • the cell preparation may be frozen or non-frozen.
  • the cell preparation usually contains a cell preservation solution in addition to mammalian cells. Examples of the components of the cell preservation solution include phosphoric acid, DMSO, sodium salt (for example, sodium chloride), potassium salt (for example, potassium chloride), serum, glycerol and the like.
  • the cell preservation solution is phosphate buffered saline (PBS) containing about 5% DMSO (eg, Dulbecco's PBS (D-PBS)), and the cell preparation is a mammal in said solution.
  • PBS phosphate buffered saline
  • D-PBS usually contains 0.02% potassium chloride, 0.02% sodium dihydrogen phosphate, 0.8% sodium chloride, and 0.115% disodium hydrogen phosphate.
  • a mammalian cell in a cell preparation When a mammalian cell in a cell preparation is dispersed with an aqueous glucose solution or Ringer's acetate solution, a therapeutic composition for administration to a mammal is obtained.
  • the cells may be dispersed by a normal method in the technical field. For example, an appropriate amount of an aqueous glucose solution or Ringer's acetate solution may be added to the cells and mixed by pipetting or the like, and the obtained cell suspension may be further diluted with an aqueous glucose solution or Ringer's acetate solution to a desired cell concentration.
  • the cells and the aqueous glucose solution or Ringer's acetate solution may be mixed, or the cell preparation containing the cell and the cell preservation solution and the aqueous glucose solution or Ringer's acetate solution may be directly mixed.
  • the cells are dispersed by mixing the cell preparation with an aqueous glucose solution or Ringer's acetate solution.
  • 0.5 ⁇ 10 6 to 1 ⁇ 10 6 cells are administered per kg body weight of the mammal.
  • the number of cells administered per individual is 0.5 ⁇ 10 6 to 4 ⁇ 10 7 , and when 2 kg to 20 kg, 1 ⁇ 10 6 to 2 ⁇ 10 7 pieces.
  • the total amount of the therapeutic composition is not limited, but is, for example, 30 ml to 50 ml, and can be appropriately changed according to the number of cells administered.
  • a therapeutic composition is obtained by mixing a cell preparation with an aqueous glucose solution or Ringer's acetate solution
  • the amount of the aqueous glucose solution or Ringer's acetate solution is appropriately changed according to the number of cells administered and the cell concentration of the cell preparation. For example, 1 to 8 ml of cell preparation and 29 to 22 ml of glucose aqueous solution or Ringer's acetate solution are mixed to make a total volume of 30 ml, or 9 to 16 ml of cell preparation and 41 to 34 ml of glucose aqueous solution or Ringer's acetate solution are mixed to make the total volume. The case where it makes 50 ml is mentioned.
  • the amount of aqueous glucose solution or Ringer's acetate solution is such that the cell preparation is diluted about 2.5 to 100 times, about 5 to 100 times, or about 2.5 to 75 times.
  • the cell concentration of the therapeutic composition is not limited, but is usually 10 3 cells / ml to 10 8 cells / ml, preferably 10 3 cells / ml to 10 6 cells / ml, more preferably 10 4 cells / ml. ⁇ 10 6 pieces / ml.
  • aqueous glucose solution an aqueous glucose solution of about 3% to about 10%, preferably about 5% to about 10%, more preferably about 5% is used.
  • the aqueous glucose solution may be commercially available as an injection solution, for example, Otsuka Sugar Solution 5% (Otsuka Pharmaceutical Factory), 5% Sugar Solution Kit H (Nipro Corporation), Photo Sugar Solution 5% (Light Pharmaceutical Co., Ltd.).
  • the acetate Ringer solution is an infusion solution in which acetate ions are added to Ringer solution obtained by adding K + and Ca 2+ to physiological saline.
  • Ringer's acetate solution usually contains sodium chloride, potassium chloride, sodium acetate hydrate, and sodium acetate hydrate.
  • the Ringer's acetate solution preferably contains 0.6% sodium chloride, 0.03% potassium chloride, 0.02% calcium chloride hydrate, and 0.38% sodium acetate hydrate.
  • What is marketed as an injection solution can be used as a Ringer's acetate solution, for example, Solaceto F (Nipro Corporation), Solugen F injection (Hikari Pharmaceutical Co., Ltd.), etc. are illustrated.
  • the therapeutic composition comprises mammalian cells in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution. In another embodiment, the therapeutic composition comprises mammalian cells in a mixture of about 3% to about 10% glucose or Ringer's acetate and cell preservation solution.
  • the therapeutic composition may contain mammalian cells in a mixture of about 3% to about 10% glucose or acetate Ringer's solution and phosphate buffered saline containing about 5% DMSO, eg, 99% to 60% mixture of about 3% to about 10% aqueous glucose solution or Ringer's acetate solution and 1% to 40% phosphate buffered saline (eg, D-PBS) containing about 5% DMSO Or phosphate buffered saline containing 99% to 80%, about 3% to about 10% aqueous glucose or Ringer's acetate solution and 1% to 20% about 5% DMSO (eg, D- PBS).
  • the therapeutic composition includes, in addition to the cells and components of glucose or acetate Ringer's solution, phosphate, DMSO, potassium salt, and sodium salt, and no other components.
  • the therapeutic composition can be administered to a living body by a known method.
  • it can be administered intravascularly (intravenous or intraarterial) or intrathecally, or directly transplanted into a target tissue.
  • the therapeutic composition is administered intravenously.
  • Intravenous administration can be by injection or infusion.
  • the therapeutic composition can be filled into, for example, an infusion bag or infusion bottle, preferably a plastic infusion bottle, such as PLABOTTLE® (Otsuka Pharmaceutical Factory, Inc.). ).
  • Cell preparations and therapeutic compositions of the present invention include, for example, intervertebral disc herniation, autoimmune diseases (eg systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, aplastic anemia, systemic sclerosis, multiple sclerosis, type 1 Diabetes, autoimmune hepatitis, Crohn's disease (including fistula), ulcerative colitis, etc., cirrhosis, liver failure, chronic hepatitis, hypercholesterolemia, dilated cardiomyopathy, ischemic heart disease, chronic renal failure, acute Nephropathy, bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, emphysema, pulmonary artery hypertension, acute respiratory distress syndrome, chronic obstructive pulmonary disease, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, central nerve injury, Multiple system atrophy, cerebral infarction, fracture fusion failure, bilateral limb dispar
  • a method for producing a therapeutic composition comprising mammalian cells, comprising dispersing the mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
  • the cell is a stem cell.
  • the stem cell is a mesenchymal stem cell. 4).
  • the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells. 5).
  • the method according to any one of 1 to 4 above, wherein the mammalian cell is a dog or cat cell. 6). 6. The method according to 5 above, wherein the mammalian cell is a canine cell. 7). 7.
  • the dog is a beagle or a golden retriever. 8).
  • the mammalian cell is derived from adipose tissue.
  • the adipose tissue is subcutaneous adipose tissue.
  • the aqueous glucose solution is about 5% aqueous glucose solution.
  • the total amount of the therapeutic composition is 30 to 50 ml. 12 12.
  • the cell preparation comprises cells at about 2.5 ⁇ 10 6 cells / ml. 17. 17.
  • the cell preparation comprises mammalian cells in phosphate buffered saline containing about 5% DMSO. 18.
  • the cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
  • the therapeutic composition comprises cells at 10 4 cells / ml to 10 6 cells / ml. 20.
  • the therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO. 20. The method according to any one of 1 to 19 above, wherein the mixed solution contains mammalian cells. 21. 21. The method according to any one of 1 to 20 above, wherein the therapeutic composition is for treating a disease in a mammal.
  • a therapeutic composition comprising mammalian cells in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution, or a mixture of said aqueous glucose solution or Ringer's acetate solution and cell preservation solution.
  • 2 ' The therapeutic composition according to 1 'above, wherein the cell is a stem cell.
  • 3 ' The therapeutic composition according to 2 'above, wherein the stem cells are mesenchymal stem cells.
  • the therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO.
  • a cell preparation comprising mammalian cells, the cell preparation being used to obtain a therapeutic composition for dispersing the cells with about 3% to about 10% glucose aqueous solution or Ringer's acetate solution to administer to mammals.
  • 2 '' The cell preparation according to 1 '' above, wherein the cell is a stem cell.
  • 3 '' The cell preparation according to 2 '' above, wherein the stem cells are mesenchymal stem cells.
  • 4 ''. The cell preparation according to 3 '' above, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells. 5 ''. 5.
  • the cell preparation according to any one of the above 1 ′′ to 17 ′′, wherein the therapeutic composition comprises cells at 10 4 cells / ml to 10 6 cells / ml. 19 ''.
  • the therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO.
  • a method of treating a disease in a mammal comprising: Dispersing mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution to obtain a therapeutic composition, and administering the resulting therapeutic composition to a mammal.
  • 2 ''' The method according to 1 '''above, wherein the cell is a stem cell. 3 '''. The method according to 2 ′ ′′ above, wherein the stem cell is a mesenchymal stem cell. 4 '''. The method according to 3 ''above, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells. 5 '''.
  • Method. 14 '''. The method of 13 ′ ′′ above, wherein the cell preparation comprises cells at 2.5 ⁇ 10 6 to 5 ⁇ 10 6 cells / ml. 15 '''.
  • the cell preparation comprises cells in phosphate buffered saline containing about 5% DMSO.
  • the therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO.
  • the method according to any one of 1 ′ ′′ to 18 ′ ′′, wherein mammalian cells are contained in the mixed solution.
  • the same frozen cells (5 ⁇ 10 6 cells / ml) as described above were thawed, and 1 ml of this cell solution was prepared using 20% aqueous glucose solution, 20% aqueous glucose solution and distilled water for injection. 5%, 5%, 7%, 10% aqueous glucose solution or 4 ml of physiological saline solution, and 5 ml of the resulting cell suspension (1 ⁇ 10 6 cells / ml) is filled into an infusion bag and mixed occasionally. While standing for 30 to 40 minutes, cell aggregation was visually confirmed. In addition, cell aggregation was also confirmed when 5 ⁇ 10 6 cells were dispersed in 5 ml of 5% aqueous glucose solution after removing the cell preservation solution.
  • the survival rate of the cells dispersed with 5% glucose aqueous solution (product name: Otsuka sugar solution 5%) or acetate Ringer's solution (product name: Solaceto F infusion solution) was evaluated.
  • the same frozen cells as described above were thawed, the cell preservation solution was removed, and 5 ⁇ 10 6 cells were suspended in the solutions 1 to 9 (5 ml each) in Table 6, and the cell viability after 3 hours was evaluated. As a result, good cell viability was obtained in solutions 1 to 5, 7, and 9 (FIG. 2).

Abstract

Disclosed are: a method for manufacturing a therapeutic composition containing mammalian cells, said method comprising dispersing the mammalian cells in an aqueous glucose solution having a concentration of about 3-10% or Ringer's acetate solution; a cell preparation usable for manufacturing the same; a therapeutic method using the therapeutic composition, etc.

Description

細胞凝集低減方法および細胞凝集が低減された治療用組成物Cell aggregation reduction method and therapeutic composition with reduced cell aggregation
　本出願は、日本国特許出願第２０１７－１１１８５２号について優先権を主張するものであり、ここに参照することによって、その全体が本明細書中へ組み込まれるものとする。
　本開示は、再生・細胞医療分野における細胞凝集低減方法および細胞凝集が低減された治療用組成物等に関する。 This application claims priority with respect to Japanese Patent Application No. 2017-111852, which is hereby incorporated by reference in its entirety.
The present disclosure relates to a method for reducing cell aggregation in the field of regenerative medicine / cell medicine, a therapeutic composition with reduced cell aggregation, and the like.
　細胞を生体に投与して疾患を治療する再生・細胞医療が盛んに開発されている。細胞製剤では、投与する細胞同士が接着して凝集塊を形成すると、血管内に投与する際に塞栓の原因となったり、定量的な投与の妨げとなったりする可能性がある。そのため、再生・細胞医療の分野では、細胞の凝集を低減する方法の開発が強く希望されている。 Regenerative and cell therapy that treats diseases by administering cells to the living body has been actively developed. In the cell preparation, when cells to be administered adhere to each other to form an aggregate, it may cause embolization or prevent quantitative administration when administered into blood vessels. Therefore, in the field of regenerative medicine / cell medicine, development of a method for reducing cell aggregation is strongly desired.
　細胞の凝集を低減する方法として、トレハロースを添加する方法が報告されている。しかしながら、トレハロースは比較的高価であり、再生・細胞医療製品の広い実用化のため、安全性が高く、かつより安価な方法が求められている。 As a method for reducing cell aggregation, a method of adding trehalose has been reported. However, trehalose is relatively expensive, and there is a need for a safer and cheaper method for wide practical application of regenerative / cell medical products.
特開２０１２－１１５２５３号公報JP 2012-115253 A 国際出願公開第２０１３／１６８４０３号公報International Application Publication No. 2013/168403
　本発明は、細胞製剤の投与時の細胞凝集を低減する方法、並びに細胞凝集が低減された治療用組成物およびその製造方法等を提供することを目的とする。 An object of the present invention is to provide a method for reducing cell aggregation upon administration of a cell preparation, a therapeutic composition with reduced cell aggregation, a method for producing the same, and the like.
　本発明は、ある態様において、哺乳動物細胞を含む治療用組成物の製造方法であって、哺乳動物細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散させることを含む方法を提供する。
　本発明は、さらなる態様において、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液中、または前記ブドウ糖水溶液または酢酸リンゲル液と細胞保存液との混合液中に哺乳動物細胞を含む治療用組成物を提供する。
　本発明は、さらなる態様において、哺乳動物細胞を含む細胞製剤であって、前記細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散して哺乳動物に投与する治療用組成物を得るよう用いられる細胞製剤を提供する。
　本発明は、さらなる態様において、哺乳動物において疾患を治療する方法であって、
　哺乳動物細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散させ治療用組成物を得ること、および
　得られた治療用組成物を哺乳動物に投与すること
　を含む、方法を提供する。 The present invention provides, in one embodiment, a method for producing a therapeutic composition comprising mammalian cells, comprising dispersing the mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution. To do.
In a further aspect, the present invention provides a therapeutic composition comprising mammalian cells in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution, or in a mixture of the aqueous glucose solution or Ringer's acetate solution and a cell preservation solution. provide.
In a further aspect, the present invention provides a cell preparation comprising mammalian cells, wherein the cells are dispersed in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution and administered to a mammal. Cell preparations are used.
The invention, in a further aspect, is a method of treating a disease in a mammal, comprising
Dispersing mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution to provide a therapeutic composition, and administering the resulting therapeutic composition to a mammal .
　ブドウ糖や酢酸リンゲル液は生体への毒性が低く、安価に利用可能である。本発明により、安全かつ安価に細胞製剤の投与時の細胞凝集を低減し、また細胞凝集が低減された治療用組成物を製造することが可能となった。 Glucose and Ringer's acetate solution have low toxicity to the living body and can be used at low cost. According to the present invention, it has become possible to produce a therapeutic composition in which cell aggregation at the time of administration of a cell preparation is reduced safely and inexpensively, and in which cell aggregation is reduced.
図1は、５％ブドウ糖水溶液で分散させたビーグル犬脂肪組織由来幹細胞の生存率を示す。FIG. 1 shows the survival rate of beagle adipose tissue-derived stem cells dispersed in a 5% aqueous glucose solution. 図２は、５％ブドウ糖水溶液または酢酸リンゲル液で分散させたビーグル犬脂肪組織由来幹細胞の生存率を示す。FIG. 2 shows the survival rate of stem cells derived from beagle adipose tissue dispersed with 5% aqueous glucose solution or Ringer's acetate solution.
　特に具体的な定めのない限り、本明細書で使用される用語は、有機化学、医学、獣医学、薬学、分子生物学、微生物学等の分野における当業者に一般に理解されるとおりの意味を有する。以下にいくつかの本明細書で使用される用語についての定義を記載するが、これらの定義は、本明細書において、一般的な理解に優先する。 Unless otherwise specified, the terms used herein have meanings as commonly understood by one of ordinary skill in the fields of organic chemistry, medicine, veterinary medicine, pharmacy, molecular biology, microbiology, etc. Have. The following are definitions for some of the terms used in this specification, but these definitions take precedence over the general understanding herein.
　本明細書では、数値が「約」の用語を伴う場合、その値の±１０％の範囲を含むことを意図する。数値の範囲は、両端点の間の全ての数値および両端点の数値を含む。範囲に関する「約」は、その範囲の両端点に適用される。従って、例えば、「約２０～３０」は、「２０±１０％～３０±１０％」を含むものとする。 In this specification, when a numerical value is accompanied by the term “about”, it is intended to include a range of ± 10% of the value. The numerical range includes all numerical values between the end points and numerical values of the end points. “About” a range applies to the endpoints of the range. Therefore, for example, “about 20 to 30” includes “20 ± 10% to 30 ± 10%”.
　本明細書において、濃度に関する「％」は、特に記載しない限り「ｗ／ｖ％」を意味する。 In the present specification, “%” relating to concentration means “w / v%” unless otherwise specified.
　本発明の細胞製剤は、哺乳動物細胞を含む。哺乳動物としては、マウス、ラット、ハムスター、モルモット、ウサギ、イヌ、ネコ、ブタ、ウシ、ヤギ、ウマ、ヒツジ、サル、ヒトなどが例示される。ある実施形態において、哺乳動物は非ヒト哺乳動物である。好ましい実施形態において、哺乳動物は、イヌ、ネコ、ブタまたはウマ、より好ましくはイヌまたはネコである。ある好ましい実施形態において、哺乳動物は、イヌ、特にビーグルまたはゴールデンレトリバーである。 The cell preparation of the present invention includes mammalian cells. Examples of mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, goats, horses, sheep, monkeys, humans, and the like. In certain embodiments, the mammal is a non-human mammal. In a preferred embodiment, the mammal is a dog, cat, pig or horse, more preferably a dog or cat. In certain preferred embodiments, the mammal is a dog, particularly a beagle or a golden retriever.
　本明細書における細胞には、幹細胞、線維芽細胞、上皮細胞、白血球細胞等が含まれる。 The cells in this specification include stem cells, fibroblasts, epithelial cells, white blood cells and the like.
　幹細胞としては、胚性幹細胞（ＥＳ細胞）、胚性生殖幹細胞（ＥＧ細胞）、ｉＰＳ細胞、体性幹細胞が例示される。体性幹細胞としては、間葉系幹細胞、神経幹細胞、造血幹細胞などが挙げられる。幹細胞は、如何なる方法により製造された細胞であってもよい。ある実施形態において、幹細胞は間葉系幹細胞である。間葉系幹細胞は、脂肪組織、骨髄、臍帯血等から公知の方法により得ることができる。好ましい実施形態において、間葉系幹細胞は、脂肪組織由来、例えば皮下脂肪組織由来である。 Examples of stem cells include embryonic stem cells (ES cells), embryonic germ stem cells (EG cells), iPS cells, and somatic stem cells. Examples of somatic stem cells include mesenchymal stem cells, neural stem cells, and hematopoietic stem cells. The stem cell may be a cell produced by any method. In certain embodiments, the stem cell is a mesenchymal stem cell. Mesenchymal stem cells can be obtained by known methods from adipose tissue, bone marrow, umbilical cord blood and the like. In a preferred embodiment, the mesenchymal stem cells are derived from adipose tissue, such as subcutaneous adipose tissue.
　細胞製剤は、限定はされないが、通常、１製剤あたり、１０４～１０８個の細胞を含む。ある実施形態において、細胞製剤は、１０５個～１０７個または２．５×１０６個～５×１０６個の細胞を含む。細胞製剤は、凍結状態であっても、非凍結状態であってもよい。細胞製剤は、通常、哺乳動物細胞に加えて細胞保存液を含む。細胞保存液の成分としては、リン酸、ＤＭＳＯ、ナトリウム塩（例えば塩化ナトリウム）、カリウム塩（例えば塩化カリウム）、血清、グリセロールなどが例示される。ある実施形態において、細胞保存液は、約５％のＤＭＳＯを含むリン酸緩衝生理食塩水（ＰＢＳ）（例えば、ダルベッコＰＢＳ（Ｄ－ＰＢＳ））であり、細胞製剤は、前記溶液中に哺乳動物細胞を含む。Ｄ－ＰＢＳは、通常、塩化カリウム０．０２％、りん酸二水素ナトリウム０．０２％、塩化ナトリウム０．８％、りん酸水素二ナトリウム０．１１５％を含む。ある実施形態において、細胞製剤は、細胞保存液中に、１０４～１０８個／ｍｌ、１０５個～１０７個／ｍｌ、２．５×１０６個～５×１０６個／ｍｌ、または約２．５×１０６個／ｍｌで細胞を含む。 Cell preparations include, but are not limited to, typically 10 4 to 10 8 cells per preparation. In certain embodiments, the cell preparation comprises 10 5 to 10 7 or 2.5 × 10 6 to 5 × 10 6 cells. The cell preparation may be frozen or non-frozen. The cell preparation usually contains a cell preservation solution in addition to mammalian cells. Examples of the components of the cell preservation solution include phosphoric acid, DMSO, sodium salt (for example, sodium chloride), potassium salt (for example, potassium chloride), serum, glycerol and the like. In certain embodiments, the cell preservation solution is phosphate buffered saline (PBS) containing about 5% DMSO (eg, Dulbecco's PBS (D-PBS)), and the cell preparation is a mammal in said solution. Contains cells. D-PBS usually contains 0.02% potassium chloride, 0.02% sodium dihydrogen phosphate, 0.8% sodium chloride, and 0.115% disodium hydrogen phosphate. In certain embodiments, the cell preparations, the cell storage solution, 10 4 to 10 8 / ml, 10 5 to 10 7 /Ml,2.5×10 6 nucleotides to 5 × 10 6 cells / ml, Or it contains cells at about 2.5 × 10 6 cells / ml.
　細胞製剤中の哺乳動物細胞をブドウ糖水溶液または酢酸リンゲル液で分散すると、哺乳動物に投与するための治療用組成物が得られる。細胞の分散は、当該技術分野の通常の方法で行えばよい。例えば、細胞に適量のブドウ糖水溶液または酢酸リンゲル液を添加してピペッティング等により混合すればよく、得られた細胞懸濁液をさらにブドウ糖水溶液または酢酸リンゲル液で所望の細胞濃度まで希釈してもよい。細胞製剤の細胞保存液を取り除いた後、細胞とブドウ糖水溶液または酢酸リンゲル液とを混合してもよく、細胞および細胞保存液を含む細胞製剤とブドウ糖水溶液または酢酸リンゲル液とを直接混合してもよい。好ましい実施形態において、細胞の分散は、細胞製剤とブドウ糖水溶液または酢酸リンゲル液とを混合することにより行う。 When a mammalian cell in a cell preparation is dispersed with an aqueous glucose solution or Ringer's acetate solution, a therapeutic composition for administration to a mammal is obtained. The cells may be dispersed by a normal method in the technical field. For example, an appropriate amount of an aqueous glucose solution or Ringer's acetate solution may be added to the cells and mixed by pipetting or the like, and the obtained cell suspension may be further diluted with an aqueous glucose solution or Ringer's acetate solution to a desired cell concentration. After removing the cell preservation solution of the cell preparation, the cells and the aqueous glucose solution or Ringer's acetate solution may be mixed, or the cell preparation containing the cell and the cell preservation solution and the aqueous glucose solution or Ringer's acetate solution may be directly mixed. In a preferred embodiment, the cells are dispersed by mixing the cell preparation with an aqueous glucose solution or Ringer's acetate solution.
　通常、哺乳動物の体重１ｋｇあたり０．５×１０６個～１×１０６個の細胞が投与される。例えば、投与対象の体重が１ｋｇ～４０ｋｇの場合、１個体あたりの投与細胞数は０．５×１０６個～４×１０７個であり、２ｋｇ～２０ｋｇの場合、1×１０６個～２×１０７個である。治療用組成物の全量は、限定はされないが、例えば３０ｍｌ～５０ｍｌであり、投与細胞数に応じて適宜変更されうる。 Usually, 0.5 × 10 6 to 1 × 10 6 cells are administered per kg body weight of the mammal. For example, when the subject's body weight is 1 kg to 40 kg, the number of cells administered per individual is 0.5 × 10 6 to 4 × 10 7 , and when 2 kg to 20 kg, 1 × 10 6 to 2 × 10 7 pieces. The total amount of the therapeutic composition is not limited, but is, for example, 30 ml to 50 ml, and can be appropriately changed according to the number of cells administered.
　細胞製剤とブドウ糖水溶液または酢酸リンゲル液とを混合して治療用組成物を得る場合、ブドウ糖水溶液または酢酸リンゲル液の液量は、投与細胞数および細胞製剤の細胞濃度に応じて適宜変更される。例えば、１～８ｍｌの細胞製剤と２９ｍＬ～２２ｍｌのブドウ糖水溶液または酢酸リンゲル液を混合して全量３０ｍｌとする、あるいは９～１６ｍｌの細胞製剤と４１～３４ｍｌのブドウ糖水溶液または酢酸リンゲル液とを混合して全量５０ｍｌとする場合などが挙げられる。ある実施形態において、ブドウ糖水溶液または酢酸リンゲル液の液量は、細胞製剤が約２．５倍～１００倍、約５倍～１００倍、または約２．５倍～７５倍希釈される量である。治療用組成物の細胞濃度は、限定はされないが、通常、１０３個／ｍｌ～１０８個／ｍｌ、好ましくは１０３個／ｍｌ～１０６個／ｍｌ、より好ましくは１０４個／ｍｌ～１０６個／ｍｌである。 When a therapeutic composition is obtained by mixing a cell preparation with an aqueous glucose solution or Ringer's acetate solution, the amount of the aqueous glucose solution or Ringer's acetate solution is appropriately changed according to the number of cells administered and the cell concentration of the cell preparation. For example, 1 to 8 ml of cell preparation and 29 to 22 ml of glucose aqueous solution or Ringer's acetate solution are mixed to make a total volume of 30 ml, or 9 to 16 ml of cell preparation and 41 to 34 ml of glucose aqueous solution or Ringer's acetate solution are mixed to make the total volume. The case where it makes 50 ml is mentioned. In certain embodiments, the amount of aqueous glucose solution or Ringer's acetate solution is such that the cell preparation is diluted about 2.5 to 100 times, about 5 to 100 times, or about 2.5 to 75 times. The cell concentration of the therapeutic composition is not limited, but is usually 10 3 cells / ml to 10 8 cells / ml, preferably 10 3 cells / ml to 10 6 cells / ml, more preferably 10 4 cells / ml. ~ 10 6 pieces / ml.
　ブドウ糖水溶液としては、約３％～約１０％、好ましくは約５％～約１０％、より好ましくは約５％のブドウ糖水溶液を用いる。ブドウ糖水溶液は、注射液として市販されているものであってもよく、例えば、大塚糖液５％（大塚製薬工場）、５％糖液キットＨ（ニプロ株式会社）、光糖液５％（光製薬株式会社）などが例示される。 As the aqueous glucose solution, an aqueous glucose solution of about 3% to about 10%, preferably about 5% to about 10%, more preferably about 5% is used. The aqueous glucose solution may be commercially available as an injection solution, for example, Otsuka Sugar Solution 5% (Otsuka Pharmaceutical Factory), 5% Sugar Solution Kit H (Nipro Corporation), Photo Sugar Solution 5% (Light Pharmaceutical Co., Ltd.).
　酢酸リンゲル液とは、生理食塩水にＫ＋とＣａ２＋とを加えたリンゲル液に酢酸イオンを添加した輸液である。酢酸リンゲル液は、通常、塩化ナトリウム、塩化カリウム、酢酸ナトリウム水和物、および酢酸ナトリウム水和物を含む。酢酸リンゲル液は、好ましくは、塩化ナトリウム０．６％、塩化カリウム０．０３％、塩化カルシウム水和物０．０２％、酢酸ナトリウム水和物０．３８％を含むものである。酢酸リンゲル液としては、注射液として市販されているものを使用することができ、例えば、ソルアセトＦ（ニプロ株式会社）、ソリューゲンＦ注（光製薬株式会社）などが例示される。 The acetate Ringer solution is an infusion solution in which acetate ions are added to Ringer solution obtained by adding K + and Ca 2+ to physiological saline. Ringer's acetate solution usually contains sodium chloride, potassium chloride, sodium acetate hydrate, and sodium acetate hydrate. The Ringer's acetate solution preferably contains 0.6% sodium chloride, 0.03% potassium chloride, 0.02% calcium chloride hydrate, and 0.38% sodium acetate hydrate. What is marketed as an injection solution can be used as a Ringer's acetate solution, for example, Solaceto F (Nipro Corporation), Solugen F injection (Hikari Pharmaceutical Co., Ltd.), etc. are illustrated.
　ある実施形態において、治療用組成物は、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液中に哺乳動物細胞を含む。別の実施形態において、治療用組成物は、約３％～約１０％のブドウ糖または酢酸リンゲル液と細胞保存液との混合液中に哺乳動物細胞を含む。例えば、治療用組成物は、哺乳動物細胞を、約３％～約１０％のブドウ糖または酢酸リンゲル液と約５％のＤＭＳＯを含むリン酸緩衝生理食塩水との混合液中、例えば、９９％～６０％の、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液と、１％～４０％の、約５％のＤＭＳＯを含むリン酸緩衝生理食塩水（例えば、Ｄ－ＰＢＳ）との混合液；または、９９％～８０％の、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液と、１％～２０％の、約５％のＤＭＳＯを含むリン酸緩衝生理食塩水（例えば、Ｄ－ＰＢＳ）との混合液中に含む。ある実施形態において、治療用組成物は、細胞およびブドウ糖または酢酸リンゲル液の成分に加えて、リン酸、ＤＭＳＯ、カリウム塩、およびナトリム塩を含み、これら以外の成分を含まない。 In certain embodiments, the therapeutic composition comprises mammalian cells in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution. In another embodiment, the therapeutic composition comprises mammalian cells in a mixture of about 3% to about 10% glucose or Ringer's acetate and cell preservation solution. For example, the therapeutic composition may contain mammalian cells in a mixture of about 3% to about 10% glucose or acetate Ringer's solution and phosphate buffered saline containing about 5% DMSO, eg, 99% to 60% mixture of about 3% to about 10% aqueous glucose solution or Ringer's acetate solution and 1% to 40% phosphate buffered saline (eg, D-PBS) containing about 5% DMSO Or phosphate buffered saline containing 99% to 80%, about 3% to about 10% aqueous glucose or Ringer's acetate solution and 1% to 20% about 5% DMSO (eg, D- PBS). In certain embodiments, the therapeutic composition includes, in addition to the cells and components of glucose or acetate Ringer's solution, phosphate, DMSO, potassium salt, and sodium salt, and no other components.
　治療用組成物は、公知の方法により生体に投与することができ、例えば、血管内（静脈内または動脈内）または髄腔内へ投与してもよく、標的組織へ直接移植してもよい。ある実施形態において、治療用組成物は、静脈内投与される。静脈内投与は、注射または点滴により行うことができる。治療用組成物を点滴により投与する場合、治療用組成物は、例えば輸液バッグまたは輸液ボトルに充填することができ、好ましくは、プラスチック製輸液ボトル、例えばPLABOTTLE（登録商標）（株式会社大塚製薬工場）に充填される。 The therapeutic composition can be administered to a living body by a known method. For example, it can be administered intravascularly (intravenous or intraarterial) or intrathecally, or directly transplanted into a target tissue. In certain embodiments, the therapeutic composition is administered intravenously. Intravenous administration can be by injection or infusion. When the therapeutic composition is administered by infusion, the therapeutic composition can be filled into, for example, an infusion bag or infusion bottle, preferably a plastic infusion bottle, such as PLABOTTLE® (Otsuka Pharmaceutical Factory, Inc.). ).
　本発明の細胞製剤および治療用組成物は、例えば、椎間板ヘルニア、自己免疫疾患（例えば全身性エリテマトーデス、シェーグレン症候群、関節リウマチ、再生不良性貧血、全身性強皮症、多発性硬化症、１型糖尿病、自己免疫性肝炎、クローン病（瘻孔性含む）、潰瘍性大腸炎など）、肝硬変、肝不全、慢性肝炎、高コレステロール血症、拡張型心筋症、虚血性心疾患、慢性腎不全、急性腎障害、気管支肺異形成症、突発性肺線維化症、肺気腫、肺動脈高血圧、急性呼吸窮迫症候群、慢性閉塞性肺疾患、アルツハイマー病、筋萎縮性側索硬化症、パーキンソン病、中枢神経損傷、多系統萎縮症、脳梗塞、骨折癒合不全，両肢長不均等症、骨嚢胞、骨異形成症、骨形成不全、強直性脊椎炎、椎間板変性、脊椎変性，脊椎すべり症、脊椎損傷、変形性関節症、多発性関節炎、大腿骨骨頭壊死、重症虚血性肢、肛門周囲瘻孔、骨髄異形成症候群、筋ジストロフィー、アトピー皮膚炎、歯周病、ドライアイ、網膜色素変性症、角膜輪部機能不全症候群、２型糖尿病、移植片対宿主病などの治療に用いることができる。細胞製剤および治療用組成物は、限定はされないが、通常、その細胞と同じ種の哺乳動物の治療に用いられる。 Cell preparations and therapeutic compositions of the present invention include, for example, intervertebral disc herniation, autoimmune diseases (eg systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, aplastic anemia, systemic sclerosis, multiple sclerosis, type 1 Diabetes, autoimmune hepatitis, Crohn's disease (including fistula), ulcerative colitis, etc., cirrhosis, liver failure, chronic hepatitis, hypercholesterolemia, dilated cardiomyopathy, ischemic heart disease, chronic renal failure, acute Nephropathy, bronchopulmonary dysplasia, idiopathic pulmonary fibrosis, emphysema, pulmonary artery hypertension, acute respiratory distress syndrome, chronic obstructive pulmonary disease, Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, central nerve injury, Multiple system atrophy, cerebral infarction, fracture fusion failure, bilateral limb disparity, bone cyst, osteodysplasia, bone dysplasia, ankylosing spondylitis, disc degeneration, spinal degeneration, spondylolisthesis, spinal injury Osteoarthritis, polyarthritis, femoral head necrosis, severe ischemic limb, perianal fistula, myelodysplastic syndrome, muscular dystrophy, atopic dermatitis, periodontal disease, dry eye, retinitis pigmentosa, limbal function It can be used to treat failure syndrome, type 2 diabetes, graft-versus-host disease, and the like. Cell preparations and therapeutic compositions are not limited, but are usually used to treat mammals of the same species as the cells.
　本発明の例示的実施形態を以下に記載する。 Exemplary embodiments of the present invention are described below.
１．哺乳動物細胞を含む治療用組成物の製造方法であって、哺乳動物細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散させることを含む方法。
２．細胞が幹細胞である、前記１に記載の方法。
３．幹細胞が間葉系幹細胞である、前記２に記載の方法。
４．間葉系幹細胞が脂肪組織由来間葉系幹細胞である、前記３に記載の方法。
５．哺乳動物細胞がイヌまたはネコの細胞である、前記１～４のいずれかに記載の方法。
６．哺乳動物細胞がイヌの細胞である、前記５に記載の方法。
７．イヌがビーグルまたはゴールデンレトリバーである、前記６に記載の方法。
８．哺乳動物細胞が脂肪組織由来である、前記１～７のいずれかに記載の方法。
９．脂肪組織が皮下脂肪組織である、前記８に記載の方法。
１０．ブドウ糖水溶液が約５％のブドウ糖水溶液である、前記１～９のいずれかに記載の方法。
１１．治療用組成物の全量が３０～５０ｍｌである、前記１～１０のいずれかに記載の方法。
１２．投与対象の哺乳動物の体重１ｋｇあたり０．５×１０６個～１×１０６個の細胞を分散させる、前記１～１１のいずれかに記載の方法。
１３．０．５×１０６個～４×１０７個の細胞を分散させる、前記１～１２のいずれかに記載の方法。
１４．細胞の分散が、哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することを含む、前記１～１３のいずれかに記載の方法。
１５．細胞製剤が２．５×１０６個～５×１０６個／ｍｌで細胞を含む、前記１４に記載の方法。
１６．細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、前記１５に記載の方法。
１７．細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に哺乳動物細胞を含む、前記１４～１６のいずれかに記載の方法。
１８．細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、前記１４～１７のいずれかに記載の方法。
１９．治療用組成物が１０４個／ｍｌ～１０６個／ｍｌで細胞を含む、前記１～１８のいずれかに記載の方法。
２０．治療用組成物が、９９％～６０％の、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液と、１％～４０％の、約５％のＤＭＳＯを含むリン酸緩衝生理食塩水との混合液中に哺乳動物細胞を含む、前記１～１９のいずれかに記載の方法。
２１．治療用組成物が哺乳動物において疾患を治療するためのものである、前記１～２０のいずれかに記載の方法。 1. A method for producing a therapeutic composition comprising mammalian cells, comprising dispersing the mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
2. 2. The method according to 1 above, wherein the cell is a stem cell.
3. 3. The method according to 2 above, wherein the stem cell is a mesenchymal stem cell.
4). 4. The method according to 3 above, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells.
5). 5. The method according to any one of 1 to 4 above, wherein the mammalian cell is a dog or cat cell.
6). 6. The method according to 5 above, wherein the mammalian cell is a canine cell.
7). 7. The method according to 6 above, wherein the dog is a beagle or a golden retriever.
8). 8. The method according to any one of 1 to 7 above, wherein the mammalian cell is derived from adipose tissue.
9. 9. The method according to 8 above, wherein the adipose tissue is subcutaneous adipose tissue.
10. 10. The method according to any one of 1 to 9 above, wherein the aqueous glucose solution is about 5% aqueous glucose solution.
11. 11. The method according to any one of 1 to 10 above, wherein the total amount of the therapeutic composition is 30 to 50 ml.
12 12. The method according to any one of 1 to 11 above, wherein 0.5 × 10 6 to 1 × 10 6 cells are dispersed per kg body weight of the mammal to be administered.
13. The method according to any one of 1 to 12, wherein 0.5 × 10 6 to 4 × 10 7 cells are dispersed.
14 14. The method according to any one of 1 to 13, wherein the cell dispersion comprises mixing a cell preparation containing mammalian cells and about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
15. 15. The method according to 14 above, wherein the cell preparation contains cells at 2.5 × 10 6 to 5 × 10 6 cells / ml.
16. 16. The method according to 15 above, wherein the cell preparation comprises cells at about 2.5 × 10 6 cells / ml.
17. 17. The method according to any one of 14 to 16, wherein the cell preparation comprises mammalian cells in phosphate buffered saline containing about 5% DMSO.
18. 18. The method according to any one of 14 to 17, wherein the cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
19. The method according to any one of 1 to 18, wherein the therapeutic composition comprises cells at 10 4 cells / ml to 10 6 cells / ml.
20. The therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO. 20. The method according to any one of 1 to 19 above, wherein the mixed solution contains mammalian cells.
21. 21. The method according to any one of 1 to 20 above, wherein the therapeutic composition is for treating a disease in a mammal.
１’．約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液中、または前記ブドウ糖水溶液または酢酸リンゲル液と細胞保存液との混合液中に哺乳動物細胞を含む、治療用組成物。
２’．細胞が幹細胞である、前記１’に記載の治療用組成物。
３’．幹細胞が間葉系幹細胞である、前記２’に記載の治療用組成物。
４’．間葉系幹細胞が脂肪組織由来間葉系幹細胞である、前記３’に記載の治療用組成物。
５’．哺乳動物細胞がイヌまたはネコの細胞である、前記１’～４’のいずれかに記載の治療用組成物。
６’．哺乳動物細胞がイヌの細胞である、前記５’に記載の治療用組成物。
７’．イヌがビーグルまたはゴールデンレトリバーである、前記６’に記載の治療用組成物。
８’．哺乳動物細胞が脂肪組織由来である、前記１’～７’のいずれかに記載の治療用組成物。
９’．脂肪組織が皮下脂肪組織である、前記８’に記載の方法。
１０’．ブドウ糖水溶液が約５％のブドウ糖水溶液である、前記１’～９’のいずれかに記載の治療用組成物。
１１’．治療用組成物の全量が３０～５０ｍｌである、前記１’～１０’のいずれかに記載の治療用組成物。
１２’．治療用組成物が投与対象の哺乳動物の体重１ｋｇあたり０．５×１０６個～１×１０６個の細胞を含む、前記１’～１１’のいずれかに記載の治療用組成物。
１３’．治療用組成物が０．５×１０６個～４×１０７個の細胞を含む、前記１’～１２’のいずれかに記載の治療用組成物。
１４’．治療用組成物が哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することにより得られる、前記１’～１３’のいずれかに記載の治療用組成物。
１５’．細胞製剤が２．５×１０６個～５×１０６個／ｍｌで細胞を含む、前記１４’に記載の治療用組成物。
１６’．細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、前記１５’に記載の治療用組成物。
１７’．細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に細胞を含む、前記１４’～１６’のいずれかに記載の治療用組成物。
１８’細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、前記１４’～１７’のいずれかに記載の治療用組成物。
１９’．治療用組成物が１０４個／ｍｌ～１０６個／ｍｌで細胞を含む、前記１’～１８’のいずれかに記載の治療用組成物。
２０’．治療用組成物が、９９％～６０％の、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液と、１％～４０％の、約５％のＤＭＳＯを含むリン酸緩衝生理食塩水との混合液中に哺乳動物細胞を含む、前記１’～１９’のいずれかに記載の治療用組成物。
２１’．哺乳動物において疾患を治療するための、前記１’～２０’のいずれかに記載の治療用組成物。 1 '. A therapeutic composition comprising mammalian cells in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution, or a mixture of said aqueous glucose solution or Ringer's acetate solution and cell preservation solution.
2 '. The therapeutic composition according to 1 'above, wherein the cell is a stem cell.
3 '. The therapeutic composition according to 2 'above, wherein the stem cells are mesenchymal stem cells.
4 '. The therapeutic composition according to 3 'above, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells.
5 '. The therapeutic composition according to any one of 1 'to 4' above, wherein the mammalian cell is a canine or feline cell.
6 '. The therapeutic composition according to 5 ′ above, wherein the mammalian cell is a canine cell.
7 '. The therapeutic composition according to 6 'above, wherein the dog is a beagle or a golden retriever.
8 '. The therapeutic composition according to any one of 1 'to 7' above, wherein the mammalian cells are derived from adipose tissue.
9 '. The method according to 8 ′ above, wherein the adipose tissue is subcutaneous adipose tissue.
10 '. The therapeutic composition according to any one of 1 'to 9' above, wherein the aqueous glucose solution is about 5% aqueous glucose solution.
11 '. The therapeutic composition according to any one of 1 'to 10' above, wherein the total amount of the therapeutic composition is 30 to 50 ml.
12 '. The therapeutic composition according to any one of 1 'to 11' above, wherein the therapeutic composition comprises 0.5 × 10 6 to 1 × 10 6 cells per kg body weight of the mammal to be administered.
13 '. The therapeutic composition according to any one of 1 ′ to 12 ′ above, wherein the therapeutic composition comprises 0.5 × 10 6 to 4 × 10 7 cells.
14 '. The therapeutic composition according to any one of 1 'to 13' above, wherein the therapeutic composition is obtained by mixing a cell preparation containing mammalian cells with about 3% to about 10% glucose aqueous solution or Ringer's acetate solution. object.
15 '. The therapeutic composition according to 14 'above, wherein the cell preparation comprises cells at 2.5 × 10 6 to 5 × 10 6 cells / ml.
16 '. The therapeutic composition according to 15 ′ above, wherein the cell preparation comprises cells at about 2.5 × 10 6 cells / ml.
17 '. The therapeutic composition according to any one of 14 'to 16' above, wherein the cell preparation comprises cells in phosphate buffered saline containing about 5% DMSO.
The therapeutic composition according to any one of the above 14 'to 17', wherein the 18 'cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
19 '. The therapeutic composition according to any one of 1 'to 18' above, wherein the therapeutic composition comprises cells at 10 4 cells / ml to 10 6 cells / ml.
20 '. The therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO. The therapeutic composition according to any one of 1 'to 19' above, comprising mammalian cells in the mixed solution.
21 '. The therapeutic composition according to any one of 1 'to 20' above, for treating a disease in a mammal.
１’’．哺乳動物細胞を含む細胞製剤であって、前記細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散して哺乳動物に投与する治療用組成物を得るよう用いられる、細胞製剤。
２’’．細胞が幹細胞である、前記１’’に記載の細胞製剤。
３’’．幹細胞が間葉系幹細胞である、前記２’’に記載の細胞製剤。
４’’．間葉系幹細胞が脂肪組織由来間葉系幹細胞である、前記３’’に記載の細胞製剤。
５’’．哺乳動物細胞がイヌまたはネコの細胞である、前記１’’～４’’のいずれかに記載の細胞製剤。
６’’．哺乳動物細胞がイヌの細胞である、前記５’’に記載の細胞製剤。
７’’．イヌがビーグルまたはゴールデンレトリバーである、前記６’’に記載の細胞製剤。
８’’．哺乳動物細胞が脂肪組織由来である、前記１’’～７’’のいずれかに記載の細胞製剤。
９’’．脂肪組織が皮下脂肪組織である、前記８’’に記載の細胞製剤。
１０’’．ブドウ糖水溶液が約５％のブドウ糖水溶液である、前記１’’～９’’のいずれかに記載の細胞製剤。
１１’’．治療用組成物の全量が３０～５０ｍｌである、前記１’’～１０’’のいずれかに記載の細胞製剤。
１２’’．哺乳動物の体重１ｋｇあたり０．５×１０６個～１×１０６個の細胞が投与されるよう用いられる、前記１’’～１１’’のいずれかに記載の細胞製剤。
１３’’．細胞の分散が、哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することを含む、前記１’’～１２’’のいずれかに記載の細胞製剤。
１４’’．細胞製剤が２．５×１０６個～５×１０６個／ｍｌで細胞を含む、前記１’’～１３’’のいずれかに記載の細胞製剤。
１５’’．細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、前記１４’’に記載の細胞製剤。
１６’’．細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に細胞を含む、前記１’’～１５’’のいずれかに記載の細胞製剤。
１７’’細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、前記１’’～１６’’のいずれかに記載の細胞製剤。
１８’’．治療用組成物が１０４個／ｍｌ～１０６個／ｍｌで細胞を含む、前記１’’～１７’’のいずれかに記載の細胞製剤。
１９’’．治療用組成物が、９９％～６０％の、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液と、１％～４０％の、約５％のＤＭＳＯを含むリン酸緩衝生理食塩水との混合液中に哺乳動物細胞を含む、前記１’’～１８’’のいずれかに記載の細胞製剤。
２０’’．哺乳動物において疾患を治療するための、前記１’’～１９’’のいずれかに記載の細胞製剤。 1 ''. A cell preparation comprising mammalian cells, the cell preparation being used to obtain a therapeutic composition for dispersing the cells with about 3% to about 10% glucose aqueous solution or Ringer's acetate solution to administer to mammals.
2 ''. The cell preparation according to 1 '' above, wherein the cell is a stem cell.
3 ''. The cell preparation according to 2 '' above, wherein the stem cells are mesenchymal stem cells.
4 ''. The cell preparation according to 3 '' above, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells.
5 ''. 5. The cell preparation according to any one of the above 1 ″ to 4 ″, wherein the mammalian cell is a dog or cat cell.
6 ''. 5. The cell preparation according to 5 ″ above, wherein the mammalian cell is a canine cell.
7 ''. The cell preparation according to 6 '' above, wherein the dog is a beagle or a golden retriever.
8 ''. The cell preparation according to any one of 1 '' to 7 '', wherein the mammalian cells are derived from adipose tissue.
9 ''. The cell preparation according to 8 ″ above, wherein the adipose tissue is subcutaneous adipose tissue.
10 ''. The cell preparation according to any one of the above 1 ″ to 9 ″, wherein the aqueous glucose solution is about 5% aqueous glucose solution.
11 ''. The cell preparation according to any one of the above 1 ″ to 10 ″, wherein the total amount of the therapeutic composition is 30 to 50 ml.
12 ''. The cell preparation according to any one of the above 1 ″ to 11 ″, which is used so that 0.5 × 10 6 to 1 × 10 6 cells are administered per 1 kg body weight of a mammal.
13 ''. The cell preparation according to any of 1 '' to 12 '' above, wherein the cell dispersion comprises mixing a cell preparation containing mammalian cells and about 3% to about 10% aqueous glucose solution or Ringer's acetate solution. .
14 ''. The cell preparation according to any one of the above 1 ″ to 13 ″, wherein the cell preparation contains cells at 2.5 × 10 6 to 5 × 10 6 cells / ml.
15 ''. The cell preparation of 14 ″ above, wherein the cell preparation comprises cells at about 2.5 × 10 6 cells / ml.
16 ''. The cell preparation according to any one of the above 1 ″ to 15 ″, wherein the cell preparation comprises cells in phosphate buffered saline containing about 5% DMSO.
The cell preparation according to any one of 1 '' to 16 '' above, wherein the 17 '' cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
18 ''. The cell preparation according to any one of the above 1 ″ to 17 ″, wherein the therapeutic composition comprises cells at 10 4 cells / ml to 10 6 cells / ml.
19 ''. The therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO. The cell preparation according to any one of the above 1 ″ to 18 ″, which contains mammalian cells in the mixed solution.
20 ''. 20. The cell preparation according to any one of the above 1 ″ to 19 ″ for treating a disease in a mammal.
１’’’．哺乳動物において疾患を治療する方法であって、
　哺乳動物細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散させ治療用組成物を得ること、および
　得られた治療用組成物を哺乳動物に投与すること
　を含む、方法。
２’’’．細胞が幹細胞である、前記１’’’に記載の方法。
３’’’．幹細胞が間葉系幹細胞である、前記２’’’に記載の方法。
４’’’．間葉系幹細胞が脂肪組織由来間葉系幹細胞である、前記３’’’に記載の方法。
５’’’．哺乳動物細胞がイヌまたはネコの細胞である、前記１’’’～４’’’のいずれかに記載の方法。
６’’’．哺乳動物細胞がイヌの細胞である、前記５’’’に記載の方法。
７’’’．イヌがビーグルまたはゴールデンレトリバーである、前記６’’’に記載の方法。
８’’’．哺乳動物細胞が脂肪組織由来である、前記１’’’～７’’’のいずれかに記載の方法。
９’’’．脂肪組織が皮下脂肪組織である、前記８に記載の方法。
１０’’’．ブドウ糖水溶液が約５％のブドウ糖水溶液である、前記１’’’～９’’’のいずれかに記載の方法。
１１’’’．治療用組成物の全量が３０～５０ｍｌである、前記１’’’～１０’’’のいずれかに記載の方法。
１２’’’．哺乳動物の体重１ｋｇあたり０．５×１０６個～１×１０６個の細胞が投与される、前記１’’’～１１’’’のいずれかに記載の方法。
１３’’’．細胞の分散が、哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することを含む、前記１’’’～１２’’’のいずれかに記載の方法。
１４’’’．細胞製剤が２．５×１０６個～５×１０６個／ｍｌで細胞を含む、前記１３’’’に記載の方法。
１５’’’．細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、前記１４’’’に記載の方法。
１６’’’．細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に細胞を含む、前記１３’’’～１５’’’のいずれかに記載の方法。
１７’’’ 細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、前記１３’’’～１６’’’のいずれかに記載の方法。
１８’’’．治療用組成物が１０４個／ｍｌ～１０６個／ｍｌで細胞を含む、前記１’’’～１７’’’のいずれかに記載の方法。
１９’’’．治療用組成物が、９９％～６０％の、約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液と、１％～４０％の、約５％のＤＭＳＯを含むリン酸緩衝生理食塩水との混合液中に哺乳動物細胞を含む、前記１’’’～１８’’’のいずれかに記載の方法。 1 '''. A method of treating a disease in a mammal, comprising:
Dispersing mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution to obtain a therapeutic composition, and administering the resulting therapeutic composition to a mammal.
2 '''. The method according to 1 '''above, wherein the cell is a stem cell.
3 '''. The method according to 2 ′ ″ above, wherein the stem cell is a mesenchymal stem cell.
4 '''. The method according to 3 '''above, wherein the mesenchymal stem cells are adipose tissue-derived mesenchymal stem cells.
5 '''. The method according to any one of 1 '''to4''', wherein the mammalian cell is a dog or cat cell.
6 '''. The method according to 5 ′ '' above, wherein the mammalian cell is a canine cell.
7 '''. The method of 6 '''above, wherein the dog is a beagle or a golden retriever.
8 '''. The method according to any one of 1 ′ ″ to 7 ′ ″, wherein the mammalian cell is derived from adipose tissue.
9 '''. 9. The method according to 8 above, wherein the adipose tissue is subcutaneous adipose tissue.
10 '''. The method according to any one of 1 '''to9''', wherein the aqueous glucose solution is about 5% aqueous glucose solution.
11 '''. The method according to any one of 1 ′ ″ to 10 ′ ″, wherein the total amount of the therapeutic composition is 30 to 50 ml.
12 '''. The method according to any one of 1 ′ ″ to 11 ′ ″, wherein 0.5 × 10 6 to 1 × 10 6 cells are administered per kg body weight of the mammal.
13 '''. The cell dispersion according to any one of 1 '''to12''' above, wherein the cell dispersion comprises mixing a cell preparation comprising mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution. Method.
14 '''. The method of 13 ′ ″ above, wherein the cell preparation comprises cells at 2.5 × 10 6 to 5 × 10 6 cells / ml.
15 '''. The method of 14 ′ ″ above, wherein the cell preparation comprises cells at about 2.5 × 10 6 cells / ml.
16 '''. The method according to any one of 13 ′ ″ to 15 ′ ″, wherein the cell preparation comprises cells in phosphate buffered saline containing about 5% DMSO.
The method according to any one of 13 ′ ″ to 16 ′ ″, wherein the cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
18 '''. The method according to any one of 1 ′ ″ to 17 ′ ″, wherein the therapeutic composition comprises cells at 10 4 cells / ml to 10 6 cells / ml.
19 '''. The therapeutic composition comprises 99% to 60%, about 3% to about 10% glucose aqueous solution or Ringer's acetate solution, and 1% to 40% phosphate buffered saline containing about 5% DMSO. The method according to any one of 1 ′ ″ to 18 ′ ″, wherein mammalian cells are contained in the mixed solution.
　以下、実施例により本発明を説明するが、如何なる意味においても本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples in any way.
　各種輸液およびＤＭＥＭ培地で細胞を分散させた際の細胞凝集を評価した。
　凍結細胞（ビーグル犬の脂肪組織由来幹細胞）（５×１０６細胞／ｍｌ、５％ＤＭＳＯ含有Ｄ－ＰＢＳ中）を解凍し、表１に示すＡ）～Ｇ）の輸液またはＤＭＥＭ培地と混合して細胞を懸濁して、表２の３種類の濃度の細胞溶液を作成した。この細胞懸濁液を、時々混和しつつ３０～４０分間放置後、凝集の有無を目視で確認した。この段階での目視での凝集結果を表３にまとめた。本実験を２回実施（表３の１回目、２回目）し、再現性を確認した。 Cell aggregation was evaluated when the cells were dispersed in various infusions and DMEM medium.
Frozen cells (beagle dog adipose tissue-derived stem cells) (5 × 10 6 cells / ml in D-PBS containing 5% DMSO) are thawed and mixed with the infusion solutions of A) to G) shown in Table 1 or DMEM medium. The cells were suspended to prepare cell solutions having three concentrations shown in Table 2. The cell suspension was allowed to stand for 30 to 40 minutes with occasional mixing, and the presence or absence of aggregation was visually confirmed. The results of visual agglomeration at this stage are summarized in Table 3. This experiment was performed twice (first time and second time in Table 3), and reproducibility was confirmed.
　同様に、前記と同じ凍結細胞（５×１０６細胞／ｍｌ）を解凍し、この細胞液１ｍｌを、２０％ブドウ糖水溶液、２０％ブドウ糖水溶液と注射用蒸留水を用いて調製した１％、３％、５％、７％、１０％のブドウ糖水溶液、または生理食塩液４ｍｌで希釈し、得られた細胞懸濁液（１×１０６細胞／ｍｌ）５ｍｌを点滴バックに充填し、時々混和しつつ３０～４０分間放置し、細胞凝集を目視で確認した。また、細胞保存液を除去後、５×１０６細胞を５％ブドウ糖水溶液５ｍｌで分散させた場合の細胞凝集も確認した。 Similarly, the same frozen cells (5 × 10 6 cells / ml) as described above were thawed, and 1 ml of this cell solution was prepared using 20% aqueous glucose solution, 20% aqueous glucose solution and distilled water for injection. 5%, 5%, 7%, 10% aqueous glucose solution or 4 ml of physiological saline solution, and 5 ml of the resulting cell suspension (1 × 10 6 cells / ml) is filled into an infusion bag and mixed occasionally. While standing for 30 to 40 minutes, cell aggregation was visually confirmed. In addition, cell aggregation was also confirmed when 5 × 10 6 cells were dispersed in 5 ml of 5% aqueous glucose solution after removing the cell preservation solution.
　その結果、一般に獣医療で用いられるＡ）～Ｆ）の輸液、ＤＭＥＭ、各種濃度のぶどう糖水溶液、および生理食塩水のうち、３～２０％ブドウ糖水溶液および酢酸リンゲル液（製品名：ソルアセトＦ輸液）では顕著に細胞の凝集が抑制された（表３、表４）。細胞保存液を除去し５％ブドウ糖水溶液で分散させた場合も、細胞の凝集は認められなかった。 As a result, among the infusions of A) to F), DMEM, various aqueous glucose solutions, and physiological saline used in veterinary medicine, 3-20% glucose aqueous solution and Ringer's acetate solution (product name: Solacet F infusion) Cell aggregation was remarkably suppressed (Tables 3 and 4). When the cell preservation solution was removed and dispersed with a 5% aqueous glucose solution, no cell aggregation was observed.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　次いで、５％ブドウ糖水溶液（製品名：大塚糖液５％）で分散させた細胞の生存率を評価した。前記と同じ凍結細胞を解凍し、細胞保存液を除去後、２．５×１０６細胞を表５の１～６の溶液（各５ｍｌ）で懸濁し、３時間後の細胞生存率を評価した。その結果、溶液１、３、および５において、良好な細胞生存率が得られた（図１）。
Figure JPOXMLDOC01-appb-T000005
 Subsequently, the survival rate of the cells dispersed in a 5% glucose aqueous solution (product name: Otsuka sugar solution 5%) was evaluated. After thawing the same frozen cells as above and removing the cell preservation solution, 2.5 × 10 6 cells were suspended in the solutions 1 to 6 in Table 5 (5 ml each), and the cell viability after 3 hours was evaluated. . As a result, good cell viability was obtained in solutions 1, 3, and 5 (FIG. 1).
Figure JPOXMLDOC01-appb-T000005
　同様に、５％ブドウ糖水溶液（製品名：大塚糖液５％）または酢酸リンゲル液（製品名：ソルアセトＦ輸液）で分散させた細胞の生存率を評価した。前記と同じ凍結細胞を解凍し、細胞保存液を除去後、５×１０６細胞を表６の１～９の溶液（各５ｍｌ）で懸濁し、３時間後の細胞生存率を評価した。その結果、溶液１～５、７、および９において、良好な細胞生存率が得られた（図２）。
Figure JPOXMLDOC01-appb-T000006
 Similarly, the survival rate of the cells dispersed with 5% glucose aqueous solution (product name: Otsuka sugar solution 5%) or acetate Ringer's solution (product name: Solaceto F infusion solution) was evaluated. The same frozen cells as described above were thawed, the cell preservation solution was removed, and 5 × 10 6 cells were suspended in the solutions 1 to 9 (5 ml each) in Table 6, and the cell viability after 3 hours was evaluated. As a result, good cell viability was obtained in solutions 1 to 5, 7, and 9 (FIG. 2).
Figure JPOXMLDOC01-appb-T000006
　これらの結果から、細胞製剤とブドウ糖水溶液または酢酸リンゲル液とを混合して細胞を分散する場合、細胞製剤の少なくとも１００倍希釈までは細胞生存率が維持されることが確認された。また、細胞製剤中の細胞保存液を除去するより、細胞製剤とブドウ糖水溶液または酢酸リンゲル液とを直接混合する方が、細胞生存率が良好であることがわかった。 From these results, it was confirmed that when the cell preparation was mixed with an aqueous glucose solution or Ringer's acetate solution to disperse the cells, the cell viability was maintained until at least 100-fold dilution of the cell preparation. It was also found that the cell viability was better when the cell preparation was directly mixed with the aqueous glucose solution or Ringer's acetate solution than the cell preservation solution in the cell preparation was removed.

Claims (28)

  1. 　哺乳動物細胞を含む細胞製剤であって、前記細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散して哺乳動物に投与する治療用組成物を得るよう用いられる、細胞製剤。 A cell preparation containing mammalian cells, which is used to obtain a therapeutic composition for dispersing the cells in about 3% to about 10% aqueous glucose solution or Ringer's acetate solution to administer to mammals.
  2. 　哺乳動物細胞が脂肪組織由来間葉系幹細胞である、請求項１に記載の細胞製剤。 The cell preparation according to claim 1, wherein the mammalian cells are adipose tissue-derived mesenchymal stem cells.
  3. 　哺乳動物細胞がイヌの細胞である、請求項１または２に記載の細胞製剤。 The cell preparation according to claim 1 or 2, wherein the mammalian cell is a dog cell.
  4. 　細胞の分散が、哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することを含む、請求項１～３のいずれかに記載の細胞製剤。 The cell preparation according to any one of claims 1 to 3, wherein the cell dispersion comprises mixing a cell preparation containing mammalian cells and about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
  5. 　細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、請求項１～４のいずれかに記載の細胞製剤。 The cell preparation according to any one of claims 1 to 4, wherein the cell preparation comprises cells at about 2.5 × 10 6 cells / ml.
  6. 　細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に細胞を含む、請求項１～５のいずれかに記載の細胞製剤。 The cell preparation according to any one of claims 1 to 5, wherein the cell preparation comprises cells in phosphate buffered saline containing about 5% DMSO.
  7. 　細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、請求項１～６のいずれかに記載の細胞製剤。 The cell preparation according to any one of claims 1 to 6, wherein the cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
  8. 　哺乳動物細胞を含む治療用組成物の製造方法であって、哺乳動物細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散させることを含む方法。 A method for producing a therapeutic composition containing mammalian cells, comprising dispersing the mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
  9. 　哺乳動物細胞が脂肪組織由来間葉系幹細胞である、請求項８に記載の方法。 The method according to claim 8, wherein the mammalian cells are adipose tissue-derived mesenchymal stem cells.
  10. 　哺乳動物細胞がイヌの細胞である、請求項８または９に記載の方法。 10. The method according to claim 8 or 9, wherein the mammalian cell is a canine cell.
  11. 　細胞の分散が、哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することを含む、請求項８～１０のいずれかに記載の方法。 The method according to any one of claims 8 to 10, wherein the cell dispersion comprises mixing a cell preparation containing mammalian cells and about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
  12. 　細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、請求項１１に記載の方法。 12. The method of claim 11, wherein the cell preparation comprises cells at about 2.5 × 10 6 cells / ml.
  13. 　細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に哺乳動物細胞を含む、請求項１１または１２に記載の方法。 The method according to claim 11 or 12, wherein the cell preparation comprises mammalian cells in phosphate buffered saline containing about 5% DMSO.
  14. 　細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、請求項８～１３のいずれかに記載の方法。 The method according to any one of claims 8 to 13, wherein the cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
  15. 　哺乳動物において疾患を治療する方法であって、
    　哺乳動物細胞を約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液で分散させ治療用組成物を得ること、および
    　得られた治療用組成物を哺乳動物に投与すること
    　を含む、方法。     A method of treating a disease in a mammal, comprising:
    Dispersing mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution to obtain a therapeutic composition, and administering the resulting therapeutic composition to a mammal.
  16. 　哺乳動物細胞が脂肪組織由来間葉系幹細胞である、請求項１５に記載の方法。 The method according to claim 15, wherein the mammalian cells are adipose tissue-derived mesenchymal stem cells.
  17. 　哺乳動物細胞がイヌの細胞である、請求項１５または１６に記載の方法。 The method according to claim 15 or 16, wherein the mammalian cell is a canine cell.
  18. 　細胞の分散が、哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することを含む、請求項１５～１７のいずれかに記載の方法。 The method according to any one of claims 15 to 17, wherein the cell dispersion comprises mixing a cell preparation containing mammalian cells and about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
  19. 　細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、請求項１８に記載の方法。 19. The method of claim 18, wherein the cell preparation comprises cells at about 2.5 x 10 < 6 > / ml.
  20. 　細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に細胞を含む、請求項１８または１９に記載の方法。 20. The method of claim 18 or 19, wherein the cell preparation comprises cells in phosphate buffered saline containing about 5% DMSO.
  21. 　細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、請求項１８～２０のいずれかに記載の方法。 The method according to any one of claims 18 to 20, wherein the cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
  22. 　約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液、または前記ブドウ糖水溶液または酢酸リンゲル液と細胞保存液との混合液中に哺乳動物細胞を含む、治療用組成物。 A therapeutic composition comprising mammalian cells in about 3% to about 10% of an aqueous glucose solution or Ringer's acetate solution, or a mixture of the aqueous glucose solution or Ringer's acetate solution and a cell preservation solution.
  23. 　哺乳動物細胞が脂肪組織由来間葉系幹細胞である、請求項２２に記載の治療用組成物。 The therapeutic composition according to claim 22, wherein the mammalian cells are adipose tissue-derived mesenchymal stem cells.
  24. 　哺乳動物細胞がイヌの細胞である、請求項２２または２３に記載の方法。 24. The method of claim 22 or 23, wherein the mammalian cell is a canine cell.
  25. 　哺乳動物細胞を含む細胞製剤と約３％～約１０％のブドウ糖水溶液または酢酸リンゲル液とを混合することにより得られる、請求項２２～２４のいずれかに記載の治療用組成物。 The therapeutic composition according to any one of claims 22 to 24, which is obtained by mixing a cell preparation containing mammalian cells with about 3% to about 10% aqueous glucose solution or Ringer's acetate solution.
  26. 　細胞製剤が約２．５×１０６個／ｍｌで細胞を含む、請求項２５に記載の治療用組成物。 26. The therapeutic composition of claim 25, wherein the cell preparation comprises cells at about 2.5 × 10 6 cells / ml.
  27. 　細胞製剤が約５％のＤＭＳＯを含むリン酸緩衝生理食塩水中に哺乳動物細胞を含む、請求項２５または２６に記載の治療用組成物。 27. The therapeutic composition according to claim 25 or 26, wherein the cell preparation comprises mammalian cells in phosphate buffered saline containing about 5% DMSO.
  28. 　細胞製剤がブドウ糖水溶液または酢酸リンゲル液により２．５～１００倍希釈される、請求項２５～２７のいずれかに記載の治療用組成物。 The therapeutic composition according to any one of claims 25 to 27, wherein the cell preparation is diluted 2.5 to 100 times with an aqueous glucose solution or Ringer's acetate solution.
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JP2009242265A (en) * 2008-03-28 2009-10-22 Jcr Pharmaceuticals Co Ltd Therapeutic agent for atopic dermatitis
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