WO2020218461A1 - トレハロースを含む哺乳動物細胞保存用液 - Google Patents
トレハロースを含む哺乳動物細胞保存用液 Download PDFInfo
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- WO2020218461A1 WO2020218461A1 PCT/JP2020/017586 JP2020017586W WO2020218461A1 WO 2020218461 A1 WO2020218461 A1 WO 2020218461A1 JP 2020017586 W JP2020017586 W JP 2020017586W WO 2020218461 A1 WO2020218461 A1 WO 2020218461A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
Definitions
- the present invention contains trehalose or a derivative thereof or a salt thereof (hereinafter, these may be collectively referred to as “trehaloses”), and has a hydrogen ion index (pH) of 6.5 to 8.5.
- the present invention relates to a mammalian cell preservation solution (hereinafter, may be referred to as “Mammalian cell preservation solution”), a method for preserving mammalian cells using the Mammalian cell preservation solution, and the like.
- Regenerative medicine using stem cells aims to restore the functions of cells and tissues damaged by various diseases by utilizing the self-renewal ability and pluripotency of stem cells and the factors secreted by stem cells. It is medical treatment.
- bone marrow transplantation is performed on patients with intractable blood diseases such as leukemia and aplastic anemia, hematopoietic stem cells engraft in the patient's body, and hematopoietic ability can be maintained for almost a lifetime.
- Non-Patent Documents 1 to 3 cancer immuno-cell therapy is a state-of-the-art cell medicine in which immune cells that have the function of attacking cancer are taken out of the body, strengthened, and then returned to the body.
- Dendritic cell vaccine therapy, alpha -Therapies using T cells such as beta T cell therapy ( ⁇ T cell therapy), gamma delta T cell therapy ( ⁇ T cell therapy), CTL therapy, and natural killer cell therapy (NK cell therapy) are practiced.
- cryopreservatives such as DMSO and glycerol are usually added to the cryopreservation solution, it is necessary to remove the cryopreservative after thawing the cryopreserved stem cells and T cells and before performing transplantation treatment. There was a problem that it took time and effort. In addition, even if a cryopreservative is added to the cryopreservation solution, the cytoskeleton is significantly damaged by the crystallization of water during freezing, and the cell viability after freezing and thawing is lowered. Therefore, there is an urgent need to develop a cell preservation solution that is excellent in convenience and can suppress a decrease in cell viability.
- trehalose has an effect of suppressing a decrease in cell viability that occurs when mammalian cells are stored in a liquid (Patent Documents 1 and 2).
- Patent Documents 1 and 2 it has not been known that when the pH of these solutions is adjusted, the effect of suppressing the decrease in cell viability is further enhanced and the effect of suppressing the decrease in self-renewal ability of mammalian stem cells is further enhanced.
- An object of the present invention is that the decrease in cell viability that occurs when mammalian cells are stored in a liquid and the decrease in self-renewal ability that occurs when a mammalian stem cell is stored in a liquid can be effectively suppressed, and the mammal is fed.
- An object of the present invention is to provide a mammalian cell preservation solution or the like which has a low possibility of adversely affecting the ecology of a mammal when the animal cell is administered into the living body of the mammal.
- the present inventors are continuing diligent research to solve the above problems.
- the solution buffers the pH. It has been found that cell death can be effectively suppressed and the proportion of living cells can be increased even when it has no effect. It was also confirmed that when mammalian stem cells are stored in the solution, the decrease in self-renewal ability of the mammalian stem cells is effectively suppressed even when the solution does not have a pH buffering action.
- the present invention has been completed based on these findings.
- the present invention is as follows.
- [1] A mammalian cell preservation solution containing trehalose or a derivative thereof or a salt thereof, and a hydrogen carbonate as a pH adjuster, and having a pH of 6.5 to 8.5.
- [2] The storage solution according to the above [1], wherein the hydrogen carbonate is sodium hydrogen carbonate.
- [3] The storage solution according to the above [1] or [2], further containing a polysaccharide or a derivative thereof or a salt thereof.
- [4] The storage solution according to any one of the above [1] to [3], which is an isotonic solution.
- [5] The storage solution according to [4] above, wherein the isotonic solution is a lactated ringer solution.
- [15] The storage solution according to [14] above, wherein the mammalian stem cells are mammalian mesenchymal stem cells.
- the hydrogen carbonate is sodium hydrogen carbonate
- Trehalose or a derivative thereof or a salt thereof, and a hydrogen carbonate as a pH adjuster and the pH is A method for preserving mammalian cells, which comprises a step of preserving the mammalian cells in a storage solution having a value of 6.5 to 8.5.
- the Mammalian Cell Preservation Solution containing Mammalian Cells is used for subjects requiring transplantation of Mammalian cells (for example, patients with traumatic diseases, patients with tissue degenerative diseases, cancer).
- a method of transplanting mammalian cells which comprises the step of administering to the patient); or adding mammalian cells to a solution containing trehaloses (preferably isotonic solution), or a solution containing mammalian cells (preferably).
- the step of preparing the Mammalian Cell Preservation Solution and the prepared Mammalian Cell by adding trehaloses to the isotonic solution) and further adjusting the pH of the solution to 6.5 to 8.5.
- a step of preserving mammalian cells in a preservative solution and a subject requiring transplantation of mammalian cells in the Mammalian Cell Preservation Solution containing the preserved mammalian cells.
- Method of transplanting mammalian cells including administration to degenerative disease patients, cancer patients); use of trehaloses in the production of the present mammalian cell preservation solution, and survival of mammalian cells in the liquid.
- the Mammalian Cell Preservation Solution Containing or a solution containing trehaloses (preferably an isotonic solution) to which the mammalian cells are added, or a solution containing the mammalian cells (preferably an isotonic solution).
- trehaloses preferably an isotonic solution
- a solution containing the mammalian cells preferably an isotonic solution.
- Examples thereof include a method for preparing a solution; and a solution for preserving mammalian cells containing mammalian cells.
- the mammalian cell preservation solution containing the mammalian cells is preserved under temperature conditions in which the preservation solution exists in a liquid state.
- This does not include a step of storing the preservation solution in a solid state (for example, a step of storing mammalian cells in a dormant state such as a step of cryopreserving and a step of cryodrying).
- the cell viability that occurs when mammalian cells are stored in the liquid is reduced.
- the decrease in self-renewal ability that occurs when mammalian stem cells are stored in a liquid can be effectively suppressed.
- trehaloses are disaccharides that are unlikely to adversely affect the ecology of mammals when administered in vivo into mammals, the mammalian cells are stored in the mammalian cell preservation solution. After that, it can be directly administered into the living body of a mammal without being replaced with a new transplant solution.
- Examples 1 to 3 and Comparative Examples 1 to 3 human adipose-derived mesenchymal stem cells (hAD-MSC; Human Adipose tissue-derived Mesenchymal Stem Cell) were stored for each storage period (1 day, 2 days, 4 days, 7 days). It is a figure which shows the result of having measured the cell viability (FIG. 1A) and the viable cell recovery rate (FIG. 1B) when stored in the trehalose-containing preservation solution CSP-01 (day and 14 days).
- hAD-MSC Human Adipose tissue-derived Mesenchymal Stem Cell
- hBM-MSC Human Bone Marrow Mesenchymal Stem Cell
- the mammalian cell preservation solution of the present invention contains trehaloses and has a pH of 6.5 to 8.5, which is specified for the purpose of "preserving mammalian cells" (that is, the present invention).
- Mammalian cell preservation solution In Patent Document 2, the Lactated Ringer's solution containing trehalose and the Lactated Ringer's solution containing trehalose and dextran used for the preservation of mammalian cells are the CSP-01 solution of this example described later (Comparative Examples 3 and 4), respectively. And CSP-11 solution (Comparative Examples 5 and 7) having a pH of less than 6.5, which is different from the mammalian cell preservation solution of the present case.
- the pH of the solution for preserving mammalian cells may be in the range of 6.5 to 8.5, for example, 6.5 to 8.4; 6.5 to 8.3; 6.5 to 8. .2; 6.5-8.1; 6.5-8.0; 6.5-7.9; 6.5-7.8; 6.5-7.7; 6.5-7.6 6.5-7.5; 6.5-7.4; 6.5-7.3; 6.5-7.2; 6.5-7.1; 6.5-7.0; 6 5.5 to 6.9; 6.5 to 6.8; 6.6 to 8.5; 6.7 to 8.5; 6.8 to 8.5; 6.9 to 8.5; 7.0 ⁇ 8.5; 7.1 to 8.5; 7.2 to 8.5; 7.3 to 8.5; 7.4 to 8.5; 7.5 to 8.5; 7.6 to 8 .5; 7.7 to 8.5; 7.8 to 8.5; 7.9 to 8.5; 8.0 to 8.5; 8.1 to 8.5; 8.2 to 8.5 6.6 to 8.4; 6.6 to 8.3; 6.6 to 8.2; 6.6 to 8.1; 6.6 to 8.0;
- the mammalian cell preservation solution may be any solution that can preserve mammalian cells (for example, isotonic solution, hypotonic solution, hypertonic solution), and isotonic solution can be preferably exemplified.
- isotonic fluid means a fluid having an osmotic pressure substantially the same as the osmotic pressure of body fluid or extracellular fluid, and specifically, a fluid having an osmotic pressure in the range of 250 to 380 mOsm / L. Means.
- the “hypotonic fluid” means a fluid having an osmotic pressure lower than the osmotic pressure of a body fluid or an extracellular fluid, and specifically, a fluid having an osmotic pressure of less than 250 mOsm / L.
- a hypotonic solution a hypotonic solution that does not cause cell rupture (specifically, a solution having an osmotic pressure within the range of 100 to 250 mOsm / L) is preferable.
- the “hypertonic fluid” means a fluid having an osmotic pressure higher than the osmotic pressure of body fluid or extracellular fluid, and specifically, the osmotic pressure is more than 380 mOsm / L (preferably 380 mOsm / L). It means (within the range of super to 1000 mOsm / L).
- the isotonic solution is not particularly limited as long as it is an isotonic solution in which the salt concentration, sugar concentration, etc. are adjusted by sodium ion, potassium ion, calcium ion, etc. so as to have almost the same osmotic pressure as body fluid or cell fluid.
- physiological saline physiological saline having a buffering effect
- a buffering effect for example, PBS, Tris Buffered Saline (TBS), HEPES buffered saline
- Ringer's solution Lactobacillus, Ringer's acetate, Ringer's bicarbonate solution, 5% glucose aqueous solution, basal medium for animal cell culture (eg, DMEM, EMEM, RPMI-1640, ⁇ -MEM, F-12, F-10, M-199), isotonic agent (eg, glucose) , D-sorbitol, D-mannitol, lactose, sodium chloride) and the like, and among these, lactated Ringer's solution is preferable.
- PBS Tris Buffered Saline
- HEPES buffered saline Ringer's solution
- Lactobacillus Ringer's acetate
- Ringer's bicarbonate solution 5% glucose aqueous solution
- the isotonic solution may be a commercially available product or a self-prepared product.
- Commercially available products include Otsuka Raw Food Injection (manufactured by Otsuka Pharmaceutical Factory) (physiological saline), Ringer's solution "Otsuka” (manufactured by Otsuka Pharmaceutical Factory) (Ringer's solution), and Lactec (registered trademark) Note (manufactured by Otsuka Pharmaceutical Factory).
- the mammalian cell preservation solution has a pH of 6.5 to 8 by adding a hydrogen carbonate as a pH adjuster to a solution containing trehalose or a powder containing trehalose. It can be adjusted and obtained to be .5.
- a hydrogen carbonate as a pH adjuster
- Examples of the above-mentioned hydrogen carbonate include ammonium hydrogen carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, calcium hydrogen carbonate and the like, and sodium hydrogen carbonate is preferable.
- the mammalian cell preservation solution whose pH has been adjusted with the above-mentioned hydrogen carbonate does not have to have a pH buffering action.
- ⁇ and ⁇ -trehalose which are disaccharides in which two ⁇ -glucoses are 1,1-glycosidated
- ⁇ -glucose and ⁇ -glucose are 1,1-glycosidated.
- examples thereof include ⁇ and ⁇ -trehalose, which are disaccharides, and ⁇ , ⁇ -trehalose, which is a disaccharide in which two ⁇ -glucoses are 1,1-glycosidated, and among these, ⁇ and ⁇ -trehalose are preferable. ..
- trehalose can be produced by any known method such as chemical synthesis, production by microorganisms, production by enzymes, etc., but commercially available products can also be used.
- commercially available products such as ⁇ , ⁇ -trehalose (manufactured by Hayashibara Co., Ltd. or Fuji Film Wako Pure Chemical Industries, Ltd.) can be mentioned.
- the trehalose derivative in the above trehalose is not particularly limited as long as it is a glycosyl trehalose in which one or a plurality of sugar units are bound to the disaccharide trehalose, and the glycosyl trehalose includes glucosyltrehalose, maltosyltrehalose, and maltotriosyl. Includes trehalose and the like.
- salts of trehalose and its derivatives in the above trehaloses include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, and the like.
- Acid addition salts such as succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc.
- Metal salts such as sodium salt, potassium salt, calcium salt, ammonium salt, alkylammonium salt and the like.
- these salts are used as a solution at the time of use, and it is preferable that the action is the same as that of trehalose.
- These salts may form a hydrate or a solvate, and either one may be used alone or two or more thereof may be used in combination as appropriate.
- the concentration of trehalose in the mammalian cell preservation solution may be any concentration as long as it exerts the effect of suppressing the decrease in cell viability due to trehalose, for example, 0.1 (w / v)% in terms of trehalose. As described above, preferably 0.3 (w / v)% or more, more preferably 0.6 (w / v)% or more, still more preferably 1.0 (w / v)% or more, and most preferably 2.0 ( w / v)% or more.
- the concentration of trehalose in the mammalian cell preservation solution is, for example, in the range of 0.1 to 40 (w / v)% in terms of trehalose, preferably 0.3 to 20 (w / v). %, More preferably 0.6 to 15 (w / v)%, still more preferably 1.0 to 10% (w / v)%, most preferably 2.0 to 6.0 (w / v)%. is there.
- the Mammalian Cell Preservation Solution is used to store mammalian cells for an arbitrary period under temperature conditions in which the Mammalian Cell Preservation Solution containing mammalian cells exists in a liquid state.
- the Mammalian Cell Preservation Solution effectively suppresses the decrease in cell viability that occurs when mammalian cells are stored in liquid and the decrease in self-renewal ability that occurs when mammalian stem cells are stored in liquid. It is a liquid that has a low possibility of adversely affecting the ecology of mammals when it is administered into the living body of a mammal.
- the Mammalian Cell Preservation Solution is further used for "to suppress a decrease in the viability of a mammalian cell”; “to suppress a decrease in the self-renewal ability of a mammalian cell”; and / or Those specified for “for transplanting mammalian cells”; are preferred.
- the mammalian cell preservation solution may be a solution containing trehalose alone, a solution containing two or more types selected from trehalose, or a solution containing any component in addition to trehalose. You may.
- optional component includes, for example, isotonic agents (for example, glucose, sorbitol, mannitol, lactose, sodium chloride), chelating agents (for example, EDTA, EGTA, citric acid, salicylate), solubilizing agents, and the like.
- PH adjusters other than preservatives, antioxidants, amino acids (eg, proline, glutamine), polymers (eg, polyether), phospholipids (eg, Lysophosphatidic acid [LPA]), hydrogen carbonates (eg, Lysophosphatidic acid) , Acids such as hydroxides, acetates, carbonates; acids such as citric acid, succinic acid, acetic acid, lactic acid, glacial acetic acid, hydrochloric acid).
- the term "arbitrary component" means a component that may or may not be contained.
- the present invention includes a powder formulation for preparing the present mammalian cell preservation solution containing trehalose.
- the powder formulation may contain the above optional ingredients.
- trehalose alone exerts an effect of suppressing a decrease in the viability of mammalian cells. Therefore, in addition to trehalose, a component having an effect of suppressing a decrease in the viability of mammalian cells (for example, acarbose).
- a component having an effect of suppressing a decrease in the viability of mammalian cells for example, acarbose.
- Stakiose dextran
- polysaccharides such as hydroxyethyl starch [HES] or derivatives thereof or salts thereof; monosaccharides such as glucose or derivatives thereof or salts thereof
- monosaccharides such as glucose or derivatives thereof or salts thereof
- the above component specifically, a polysaccharide or a derivative thereof or a salt thereof is further contained, and the effect is demonstrated in this example described later. Therefore, those further containing dextran or a derivative thereof or salts thereof (hereinafter, these may be collectively referred to as "dextrans”) can be preferably exemplified.
- dextran 40 manufactured by Tokyo Kasei Kogyo Co., Ltd.
- dextran 70 manufactured by Tokyo Kasei Kogyo Co., Ltd.
- low-molecular-weight dextran L injection 10 [w / v]% dextran-containing lactated ringer solution) (manufactured by Otsuka Pharmaceutical Factory), etc. You can list the items.
- the dextran derivative in the above-mentioned dextran species includes dextran sulfate, carboxylated dextran, diethylaminoethyl (DEAE) -dextran and the like.
- salts of dextran and its derivatives in the above dextrans include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, and the like.
- Acid addition salts such as succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc.
- Metal salts such as sodium salt, potassium salt, calcium salt, ammonium salt, alkylammonium salt and the like.
- these salts are used as a solution at the time of use, and it is preferable that the action is the same as that of dextran.
- These salts may form a hydrate or a solvate, and either one may be used alone or two or more thereof may be used in combination as appropriate.
- the concentration of dextran in the mammalian cell preservation solution may be any concentration as long as it can suppress the decrease in cell viability due to dextran, for example, 0.1 (w / v)% in terms of dextran. As described above, preferably 0.3 (w / v)% or more, more preferably 0.6 (w / v)% or more, still more preferably 1.0 (w / v)% or more, still more preferably 2.0. It is (w / v)% or more, most preferably 4.0 (w / v)% or more.
- the concentration of dextrans in the mammalian cell preservation solution is, for example, in the range of 0.1 to 50 (w / v)% in terms of dextran, preferably 0.3 to 20 (w / v).
- % More preferably 0.6 to 15 (w / v)%, even more preferably 1.0 to 12% (w / v)%, even more preferably 2.0 to 9.0% (w / v). %, Most preferably 4.0 to 7.0 (w / v)%.
- the mammalian cell preservation solution when the mammalian cell preservation solution containing mammalian cells is used as it is for transplantation, a solution suitable for mammalian cell transplantation is preferable, and a solution suitable for such mammalian cell transplantation is preferable.
- a solution suitable for mammalian cell transplantation eg, biological components (eg, serum or serum-derived components [eg, albumin]); or mammalian cells when mammalian cells are cryopreserved or cryopreserved.
- DMSO dimethylsulfoxide
- glycerin ethylene glycol, trimethylene glycol, dimethylacetamide
- PEG polyethylene glycol
- polyvinylpyrrolidone serum or serum-derived components (eg, , Albumin) and other cryoprotectants or cryoprotectants; preferably not contained.
- the method for preserving mammalian cells of the present invention includes a step of preserving mammalian cells for an arbitrary period in a solution containing trehalose and having a pH of 6.5 to 8.5 (hereinafter, "" This is sometimes called “the storage method”).
- the storage method is usually to store the mammalian cell storage solution containing mammalian cells under temperature conditions in which the storage solution exists in a liquid state, and the storage solution is in a solid state. It does not include a step of storing under the temperature conditions existing in (for example, a step of storing mammalian cells in a dormant state such as a step of cryopreserving and a step of cryodrying).
- the density of mammalian cells in the mammalian cell preservation solution is, for example, in the range of 10 3 to 10 10 cells / mL.
- the pH is 6.5 to 8 by further adding a hydrogen carbonate as the pH adjuster to a solution containing trehaloses or a solution containing powdered trehalose-containing substances.
- the cell survival of the mammalian cells in the preservation solution is preferable, for example, 6 hours or more, 12 hours or more, 1 day (24 hours) or more, 1.5 days (36 hours) or more, 2 days ( 48 hours) or more, 3 days (72 hours) or more, 4 days (96 hours) or more, 7 days (168 hours) or more, and if the storage period of mammalian cells is too long, the survival of the cells is adversely affected.
- the storage period is, for example, 6 hours to 21 days; 12 hours to 21 days; 1 to 21 days; 1.5 to 21 days; 2 to 21 days; 3 to 21 days; 4 to 21 days; 7 ⁇ 21 days; 6 hours ⁇ 16 days; 6 hours ⁇ 14 days; 6 hours ⁇ 10 days; 6 hours ⁇ 7 days; 6 hours ⁇ 4 days; 12 hours ⁇ 16 days; 12 hours ⁇ 14 days; 12 hours ⁇ 10 Days; 12 hours to 7 days; 12 hours to 4 days; 1 to 16 days; 1 to 14 days; 1 to 10 days; 1 to 7 days; 1 to 4 days; 1.5 to 16 days; 1.5 to 14 days; 1.5-10 days; 1.5-7 days; 1.5-4 days; 2-16 days; 2-14 days; 2-10 days; 2-7 days; 2-4 days; 3 ⁇ 16 days; 3-14 days; 3-10 days; 3-7
- cell death was suppressed in mammalian cells stored in the mammalian cell preservation solution is known to be able to detect cell death such as trypan blue staining method, TUNEL method, Nexin method, and FLICA method. It can be confirmed by using the method of.
- the "temperature at which the mammalian cell storage solution containing mammalian cells exists in a liquid state” is defined as the state in which the mammalian cell storage solution containing mammalian cells is in a liquid state without freezing. It suffices as long as it exists at a temperature at which the mammalian cells in the storage solution can grow, and is usually in the range of 0 to 40 ° C., preferably in the range of 0 to 30 ° C. (room temperature).
- the mammalian cells are administered via blood vessels, for example, in regenerative medicine for diseases requiring mammalian cell transplantation treatment (cancer; type I diabetes; organ diseases such as liver disease; etc.).
- Mammalian cells specifically, stem cells; pancreatic islet cells; hepatocytes; dendritic cells; etc. can be exemplified, and stem cells and hepatocytes are preferable.
- These cells can be isolated by known common methods. For example, a fluorescence activated cell sorter (FACS) using antibodies against various cell surface markers, an antibody against the above cell surface marker labeled with a fluorescent substance or a labeling substance such as biotin or avidin, and an antibody against such labeling substance and MACS beads.
- FACS fluorescence activated cell sorter
- fluorescent substance examples include allophycocyanin (APC), phycocyanin (PE), FITC (fluorescein isothiocyanate), Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, PE-Texas Red, PE-Cy5, PE-Cy7 and the like. be able to.
- stem cell means an immature cell having self-renewal ability and differentiation / proliferation ability.
- Stem cells include subpopulations such as pluripotent stem cells (pluripotent stem ce11), multipotent stem cells (multipotent stem ce11), and monopotent stem cells (unipotent stem ce11), depending on their ability to differentiate.
- Pluripotent stem cells mean cells that cannot become individuals by themselves, but have the ability to differentiate into all the tissues and cells that make up the living body.
- a multipotent stem cell means a cell capable of differentiating into multiple types of tissues and cells, but not all types.
- a monopoly stem cell means a cell having the ability to differentiate into a specific tissue or cell.
- pluripotent stem cells examples include embryonic stem cells (ES cells), EG cells (Embryonic germ cells), induced pluripotent stem cells (iPS cells), and the like.
- ES cells can be produced by culturing the inner cell mass on feeder cells or in a medium containing LIF. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 10157, US5,843,780, US6,200,806, US6,280,718 and the like.
- EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF and bFGF (Ce11, 70: 841-847, 1992).
- iPS cells are produced by introducing reprogramming factors such as Oct3 / 4, Sox2 and Klf4 (further c-Myc or n-Myc as needed) into somatic cells (eg, fibroblasts, skin cells, etc.).
- somatic cells eg, fibroblasts, skin cells, etc.
- Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (Nature, 385,810 (1997); Science, 280,1256 (1998); Nature. Biotechnology, 17,456 (1999); Nature, 394,369 (1998); Nature Genetics, 22,127 (1999); Proc.Nat1. Acad.Sci.USA, 96,14984 (1999), Rideout III et al. (Nature Genetics, 24,109 (2000)) ).
- pluripotent stem cells examples include mesenchymal stem cells capable of differentiating into cells such as adipocytes, bone cells, chondrocytes, and adipocytes, and neural stem cells capable of differentiating into cells such as neurons, astrosites, and oligodendrocytes. Examples thereof include somatic stem cells such as bone marrow stem cells and reproductive stem cells. Multipotent stem cells are preferably mesenchymal stem cells. Mesenchymal stem cells mean stem cells capable of differentiating into all or some of osteoblasts, chondroblasts and lipoblasts. Multipotent stem cells can be isolated from living organisms by methods known per se.
- mesenchymal stem cells can be collected from mammalian bone marrow, adipose tissue, peripheral blood, cord blood and the like by a known general method.
- human mesenchymal stem cells can be isolated by culturing and subculturing hematopoietic stem cells after bone marrow puncture (Journal of Autoimmunity, 30 (2008) 163-171).
- Pluripotent stem cells can also be obtained by culturing the pluripotent stem cells under appropriate inducing conditions.
- mammals include rodents such as mice, rats, hamsters and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, horses and sheep, and cats such as dogs and cats.
- rodents such as mice, rats, hamsters and guinea pigs
- lagomorphs such as rabbits
- ungulates such as pigs, cows, goats, horses and sheep
- cats such as dogs and cats.
- Humans, monkeys, cynomolgus monkeys, cynomolgus monkeys, marmosets, orangutans, primates such as orangutans, and the like, and among them, mice, pigs, and humans can be preferably exemplified.
- an adhesive cell (also referred to as “adhesive”) can be exemplified.
- adheresive cells mean scaffold-dependent cells that can survive, proliferate, and produce substances by adhering to the scaffold.
- adherent stem cells include pluripotent stem cells, mesenchymal stem cells, neural stem cells, bone marrow stem cells, reproductive stem cells, and the like, and mesenchymal stem cells can be preferably exemplified.
- the mammalian cells may be isolated from the living body or subcultured in vitro, but are preferably isolated or purified.
- isolated or purification means that an operation for removing a component other than the target component has been performed.
- the purity of isolated or purified mammalian cells is usually 30% or more, preferably 50% or more, more preferably 70% or more. More preferably, it is 90% or more (for example, 100%).
- the mammalian cells (population) stored in the mammalian cell preservation solution may be in the state of a single cell (single cell).
- single cell state means that it does not gather together with other cells to form a mass (that is, a state in which it does not aggregate).
- Mammalian cells in the single cell state can be prepared by enzymatically treating mammalian cells cultured in vitro with trypsin / EDTA or the like.
- the proportion of mammalian cells in a single cell state contained in the mammalian cells is, for example, 70% or more, preferably 90% or more, more preferably 95% or more, still more preferably 99% or more (for example, 100%). is there.
- the proportion of cells in the single cell state is the presence or absence of aggregation of mammalian cells dispersed in PBS and observed under a microscope for a plurality of randomly selected cells (eg, 1000). It can be determined by examining.
- the mammalian cells (population) stored in the mammalian cell preservation solution may be suspended.
- floating means that mammalian cells are retained in the liquid without contacting the inner wall of the container containing the storage liquid.
- the mammalian cells stored in the Mammalian Cell Preservation Solution When the mammalian cells stored in the Mammalian Cell Preservation Solution are aggregated or precipitated, the mammalian cells can be suspended by a well-known method in the art such as pipetting or tapping before transplantation. preferable.
- the lactated Ringer's solution containing 3 (w / v)% trehalose and 5 (w / v)% dextran 40 may be referred to as “CSP-01 solution” for convenience, and 3 (w / v).
- the lactated Ringer solution containing% trehalose may be referred to as "CSP-11 solution” for convenience.
- CSP-01 solution The CSP-01 solution is ⁇ , ⁇ -trehalose (manufactured by Hayashibara Co., Ltd. or Fuji Film Wako Pure Chemical Industries, Ltd.) and low molecular weight dextran L injection (10 [w / v]% dextran-containing lactate ringer solution) (Otsuka Pharmaceutical Factory). To the lactate Ringer's solution (Lactec infusion; manufactured by Otsuka Pharmaceutical Factory) so that the final concentrations of trehalose and dextran are 3 (w / v)% and 5 (w / v)%, respectively. (See Patent Document 2).
- CSP-11 solution The CSP-11 solution is ⁇ , ⁇ -trehalose (manufactured by Hayashibara Co., Ltd. or Fuji Film Wako Pure Chemical Industries, Ltd.), and lactated Ringer's solution (Lactec infusion) so that the final concentration of trehalose is 3 (w / v)%.
- ⁇ , ⁇ -trehalose manufactured by Hayashibara Co., Ltd. or Fuji Film Wako Pure Chemical Industries, Ltd.
- lactated Ringer's solution Lactec infusion
- hAD-MSC The hAD-MSCs were cultured according to a conventional method. That is, hAD-MSC is contained in ADSC-BM (Adipose Derived Stem Cell Basal Medium) (Lonza Walkersville, PT-3273) (hereinafter, PT-3273) containing a human adipose-derived stem cell addition factor set (Lonza Walkersville, PT-4503).
- ADSC-BM Adipose Derived Stem Cell Basal Medium
- PT-3273 containing a human adipose-derived stem cell addition factor set (Lonza Walkersville, PT-4503).
- the cells were placed in 75 cm 2 flasks to which (simply referred to as “culture solution”) was added, and subcultured in a CO 2 incubator (under 37 ° C. conditions). In addition, the culture solution was exchanged every 3 days.
- hAD-MSC-containing liquid was prepared according to the following procedures [1] to [10]. [1] The personal incubator was heated to 37 ⁇ 2 ° C. [2] A 75 cm 2 flask in which hAD-MSC was cultured was taken out from the CO 2 incubator. [3] The state of the cells was observed under an inverted microscope, and those having about 80% confluence were used. [4] The culture solution was aspirated, and 8 mL of PBS ( ⁇ ) was added to each 75 cm 2 flask.
- trypsin / EDTA (CC-5012, manufactured by Lonza Walkersville) was added to the flask in an amount of 4 mL each, and the mixture was incubated in a personal incubator under 37 ⁇ 2 ° C. conditions for 5 minutes.
- the cells were slowly shaken while observing under an inverted microscope until about 90% of the cells were detached.
- 8 mL each of trypsin neutralized solution (TNS; CC-5002, manufactured by Lonza Walkersville) was added, cells were detached by pipetting, and the cells were transferred to a 50 mL conical tube.
- the cell viability and viable cell recovery rate during each storage period were calculated by measuring the total number of cells and the number of trypan blue-positive cells (dead cells) using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). ..
- [CFU assay] The colony forming ability when mammalian mesenchymal stem cells were stored in the test solution was measured according to the following procedures [1] to [4]. [1] Using a fin pipette (100-1000 ⁇ L), 1 mL each of the prepared mammalian cell-containing solution is dispensed into a 15 mL clarifine polypropylene conical tube, and after centrifugation (210 ⁇ g, 5 minutes, 25 ° C.). , Ice-cooled.
- Example 3 CSP-01 solution [pH 7.29] was higher than Example 2 (CSP-01 solution [pH 6.95]) (CSP-01 solution [pH 7.29]). See Table 4 and FIG. 1A). In addition, a similar tendency was observed for the recovery rate of living cells (see Table 5 and FIG. 1B).
- Example 5 CSP-11 solution [pH 6.35]
- the cell viability at 48 hours (2 days) after the start of cell preservation decreased to about 45%, and at 96 hours (4 days).
- the cell viability at 48 hours (2 days) after the start of cell preservation exceeded 75%, and at 96 hours (4 days).
- Over 45% see Table 7
- a similar tendency was observed for the recovery rate of living cells (see Table 8).
- Examples 6 to 10 had higher cell viability and viable cell recovery rate than Comparative Example 6 (Lactated Ringer) in any storage period. It was particularly high, and the cell viability and viable cell recovery rate of Example 8 (CSP-01 solution [pH 7.35]) and Example 9 (CSP-01 solution [pH 7.68]) tended to be the highest (Table). 10 and 11 and FIG. 2).
- Examples 11 to 14 (CSP-11 liquid [pH 7.04 to 8.00]) are compared with Comparative Example 6 (Lactated Ringer) and Comparative Example 7 (CSP-11 liquid [pH 6.44]).
- the cell viability and viable cell recovery rate are high even during the storage period of, and in particular, the cell viability and viable cell recovery rate of Examples 12 to 14 (CSP-11 solution [pH 7.16 to 8.00]) tend to be the highest. (See Tables 12 and 13 and FIG. 3).
- CSP-01 solution that is, Lactated Ringer's solution containing trehalose and dextran
- trehalose and dextran are contained and It is shown that the decrease in colony-forming ability can be suppressed as compared with the case where mammalian mesenchymal stem cells are stored in a lactated Ringer's solution having a pH of around 6.16.
- CSP-11 solution that is, Lactated Ringer's solution containing trehalose (without dextran)
- trehalose is contained and the pH is adjusted.
- the decrease in colony forming ability that is, self-renewal ability
- hBM-MSCs Human bone marrow-derived mesenchymal stem cells
- the CSP-01 solution contains ⁇ , ⁇ -trehalose (manufactured by Hayashihara Co., Ltd. or Fuji Film Wako Junyaku Co., Ltd.) and low-molecular-weight dextran L injection (10 [w / v]% dextran) as in Example 1. Lactobacillus (Lactec Ringer's solution) (manufactured by Otsuka Pharmaceutical Factory) so that the final concentrations of trehalose and dextran are 3 (w / v)% and 5 (w / v)%, respectively. ) To obtain CSP-01 solution (5.6 unadjusted).
- a 0.5 mol / L sodium hydroxide solution was added so that the pH became 7.300 to obtain a CSP-01 solution (7.3 NaOH).
- a sodium hydrogen carbonate solution was added as a pH adjuster to prepare a CSP-01 solution (7.2 NaHCO 3 ).
- the pH of CSP-01 solution (7.2 NaHCO 3 ) was 7.158.
- the pH of each test solution is a value measured at room temperature (21.2 to 24.0 ° C.).
- Lactated Ringer's solution containing hBM-MSC was prepared according to the following procedures [1] to [8]. [1] Add 4 ⁇ 10 5 hBM-MSCs to a 75 cm 2 flask, and in the presence of a medium kit for human mesenchymal stem cells (manufactured by Lonza) (hereinafter referred to as “MSC medium”), 37 ° C., 5%. The cells were cultured in a CO 2 incubator. The state of the cells was observed under a microscope, and the cells were cultured until they became about 80% confluent.
- MSC medium a medium kit for human mesenchymal stem cells
- hBM-MSCs were stored in various test isotonic solutions according to the following procedures [1] and [2]. [1] 0.5 mL of the prepared lactated Ringer solution containing hBM-MSC was dispensed into each tube, and centrifugation (600 ⁇ g, 5 minutes) was performed. [2] The supernatant (lactated Ringer's solution) was removed, and the precipitate (hBM-MSC) was suspended in 0.5 mL of various test isotonic solutions and stored at 5 ° C. for 3 days.
- the addition of trehaloses in a liquid reduces the cell viability that occurs when mammalian cells are stored in a liquid and the decrease in self-renewal ability that occurs when mammalian stem cells are stored in a liquid. It can be effectively suppressed. Furthermore, since trehaloses are disaccharides that are unlikely to adversely affect the ecology of mammals when administered in vivo into mammals, the mammalian cells are stored in the mammalian cell preservation solution. After that, it can be directly administered into the living body of a mammal without being replaced with a new transplant solution.
Abstract
Description
〔1〕トレハロース若しくはその誘導体又はこれらの塩、及び、pH調整剤としての炭酸水素塩を含み、かつ、pHが6.5~8.5である哺乳動物細胞保存用液。
〔2〕炭酸水素塩が炭酸水素ナトリウムである、上記〔1〕に記載の保存用液。
〔3〕さらに、多糖類若しくはその誘導体又はこれらの塩を含む、上記〔1〕又は〔2〕に記載の保存用液。
〔4〕等張液である、上記〔1〕~〔3〕のいずれか1項に記載の保存用液。
〔5〕等張液が、乳酸リンゲル液である、上記〔4〕に記載の保存用液。
〔6〕多糖類が、デキストランである、上記〔3〕~〔5〕のいずれか1項に記載の保存用液。
〔7〕トレハロース若しくはその誘導体又はこれらの塩の濃度が、2.0~6.0(w/v)%である、上記〔1〕~〔6〕のいずれか1項に記載の保存用液。
〔8〕デキストラン若しくはその誘導体又はこれらの塩の濃度が、4.0~7.0(w/v)%である、上記〔6〕又は〔7〕に記載の保存用液。
〔9〕哺乳動物細胞を0~40℃で保存するための、上記〔1〕~〔8〕のいずれか1項に記載の保存用液。
〔10〕哺乳動物細胞を6時間~14日間保存するための、上記〔1〕~〔9〕のいずれか1項に記載の保存用液。
〔11〕哺乳動物細胞の生存率低下を抑制するために用いられる、上記〔1〕~〔10〕のいずれか1項に記載の保存用液。
〔12〕哺乳動物細胞の自己複製能低下を抑制するために用いられる、上記〔1〕~〔11〕のいずれか1項に記載の保存用液。
〔13〕哺乳動物細胞の移植に用いられる、上記〔1〕~〔12〕のいずれか1項に記載の保存用液。
〔14〕哺乳動物細胞が、哺乳動物幹細胞である、上記〔1〕~〔13〕のいずれか1項に記載の保存用液。
〔15〕哺乳動物幹細胞が、哺乳動物間葉系幹細胞である、上記〔14〕に記載の保存用液。
〔16〕トレハロース若しくはその誘導体又はこれらの塩、及び、pH調整剤としての炭酸水素塩を含む、上記〔1〕~〔15〕のいずれか1項に記載の保存用液を調製するための粉末製剤。
[17]炭酸水素塩が炭酸水素ナトリウムである、上記[16]に記載の粉末製剤
〔18〕トレハロース若しくはその誘導体又はこれらの塩、及び、pH調整剤としての炭酸水素塩を含み、かつ、pHが6.5~8.5である保存用液中で、哺乳動物細胞を保存する工程を含む、哺乳動物細胞の保存方法。
〔19〕炭酸水素塩が炭酸水素ナトリウムである、上記〔18〕に記載の保存方法。
〔20〕保存用液が、さらに、多糖類若しくはその誘導体又はこれらの塩を含む、上記〔18〕又は〔19〕に記載の保存方法。
〔21〕保存用液が等張液である、上記〔18〕~〔20〕のいずれか1項に記載の保存方法。
〔22〕等張液が乳酸リンゲル液である、上記〔21〕に記載の保存方法。
〔23〕多糖類が、デキストランである、上記〔20〕~〔22〕のいずれか1項に記載の保存方法。
〔24〕保存用液中で、哺乳動物細胞を6時間~14日保存することを特徴とする上記〔18〕~〔23〕のいずれか1項に記載の保存方法。
また、本発明には、トレハロース類を含む、本件哺乳動物細胞保存用液を調製するための粉末製剤が含まれる。当該粉末製剤には上記の任意成分を含んでいてもよい。
細胞(multipotent stem ce11)、単能性幹細胞(unipotent stem ce11)等の亜集団が含まれる。多能性幹細胞とは、それ自体では個体になることができないが、生体を構成する全ての組織や細胞へ分化し得る能力を有する細胞を意味する。複能性幹細胞とは、全ての種類ではないが、複数種の組織や細胞へ分化し得る能力を有する細胞を意味する。単能性幹細胞とは、特定の組織や細胞へ分化し得る能力を有する細胞を意味する。
1.材料及び方法
[哺乳動物細胞]
以下の実験1~4には、以下の表1に記載のヒト脂肪由来間葉系幹細胞(hAD-MSC)を用いた。
CSP-01液は、α,α-トレハロース(株式会社林原社製又は富士フィルム和光純薬社製)と、低分子デキストランL注(10[w/v]%デキストラン含有乳酸リンゲル液)(大塚製薬工場社製)とを、トレハロース及びデキストランの終濃度がそれぞれ3(w/v)%及び5(w/v)%となるように乳酸リンゲル液(ラクテック輸液;大塚製薬工場社製)へ添加し、調製した(特許文献2参照)。
CSP-11液は、α,α-トレハロース(株式会社林原社製又は富士フィルム和光純薬社製)を、トレハロースの終濃度が3(w/v)%となるように乳酸リンゲル液(ラクテック輸液;大塚製薬工場社製)へ添加し、調製した(特許文献2参照)。CSP-01液及びCSP-11液の組成を、以下の表2に示す。
hAD-MSCは定法にしたがって培養した。すなわち、hAD-MSCを、ヒト脂肪由来幹細胞添加因子セット(Lonza Walkersville社製、PT-4503)を含むADSC-BM(Adipose Derived Stem Cell Basal Medium)(Lonza Walkersville社製、PT-3273)(以下、単に「培養液」という)を添加した75cm2フラスコに入れ、CO2インキュベーター(37℃条件下)内で継代培養した。また、培養液の交換は3日毎行った。
hAD-MSC含有液を、以下の〔1〕~〔10〕の手順に従って調製した。
〔1〕パーソナルインキュベーターを37±2℃に加温しておいた。
〔2〕hAD-MSCを培養している75cm2フラスコを、CO2インキュベーターから取り出した。
〔3〕倒立顕微鏡下で細胞の状態を観察し、約80%コンフルエントのものを使用した。〔4〕培養液を吸引し、PBS(-)を各75cm2フラスコに8mLずつ添加した。
〔5〕PBS(-)を吸引後、トリプシン/EDTA(CC-5012、Lonza Walkersville社製)をフラスコに4mLずつ添加し、パーソナルインキュベーターにて37±2℃条件下で5分間インキュベートした。
〔6〕細胞が90%程度剥離するまで倒立顕微鏡下で観察しながら、ゆっくりと揺らした。
〔7〕トリプシン反応を停止させるために、トリプシン中和液(TNS;CC-5002、Lonza Walkersville社製)を8mLずつ加えて、ピペッティングにより細胞を剥離し、50mLのコニカルチューブに移した。
〔8〕遠心処理(210×g、5分間、20℃)後、上清を除去し、一定量(各75cm2フラスコ当たり1.5mL)のPBS(-)を添加し、細胞を懸濁した。
〔9〕細胞懸濁液から一部(20μL)分取し、20μLのトリパンブルー染色液(Gibco社製)と混合しワンセルカウンター(細胞計数盤;バイオメディカルサイエンス社製)で全細胞数(生細胞数及び死細胞数)を計測した。なお、細胞数は、ワンセルカウンターにおける9ヵ所のエリアのうち、4隅のエリア内にある細胞を計測し、以降の計測も同様に行った。
〔10〕全細胞数が5.0×105細胞/mLとなるようにPBS(-)を添加し、氷冷し、哺乳動物細胞含有液を調製した。
被験液中に哺乳動物細胞を保存したときの細胞生存率及び生細胞回収率を、以下の〔1〕~〔4〕の手順に従って測定した。
〔1〕調製した哺乳動物細胞含有液を、フィンピペット(100-1000μL)を用いて、15mLクラリファインドポリプロピレンコニカルチューブに1mLずつ分注し、遠心処理(210×g、5分間、25℃)後、氷冷した。
〔2〕上清を除去し、フィンピペット(100-1000μL)を用いて、1mLの各被験液に細胞を懸濁した後、その一部(20μL)を、予めトリパンブルー染色液(Gibco社製)20μLを添加した1.5mLマイクロチューブに分取した。トリパンブルー染色液と混和した細胞懸濁液を、ワンセルカウンターに分取し、光学顕微鏡(ECLIPSE TS100、ニコン社製)を用いて全細胞数及びトリパンブルー陽性細胞(死細胞)数を計測することにより、保存開始直後の細胞生存率を算出した。
〔3〕残りの細胞懸濁液は、薬用冷蔵ショーケース(5℃に設定)にて各保存期間まで静置保存した。
〔4〕各保存期間が経過した時点において、チップの先を、細胞を含む液が入ったチューブの底から目視で5mm程度の位置まで挿入し、緩やかに攪拌(500μLの液量でのピペッティングを5回)することにより細胞が懸濁した状態で、その一部(20μL)を1.5mLマイクロチューブに採取し、トリパンブルー染色液(Gibco社製)20μLと混和後、ワンセルカウンターに分取し、光学顕微鏡(ECLIPSE TS100、ニコン社製)を用いて全細胞数及びトリパンブルー陽性細胞(死細胞)数を計測することにより、各保存期間における細胞生存率及び生細胞回収率を算出した。なお、細胞生存率は、式「(全生細胞数/全細胞数)×100=([全細胞数-死細胞数]/全細胞数)×100=細胞生存率(%)」を用いて算出し、また、生細胞回収率は、式「(各保存期間の全生細胞数/保存開始直後の全生細胞数)×100=([各保存期間の全細胞数-各保存期間の死細胞数]/[保存開始直後の全細胞数-保存開始直後の死細胞数])×100=生細胞回収率(%)」を用いて算出した。
被験液中に哺乳動物間葉系幹細胞を保存したときのコロニー形成能を、以下の〔1〕~〔4〕の手順に従って測定した。
〔1〕調製した哺乳動物細胞含有液を、フィンピペット(100-1000μL)を用いて、15mLクラリファインドポリプロピレンコニカルチューブに1mLずつ分注し、遠心処理(210×g、5分間、25℃)後、氷冷した。
〔2〕上清を除去し、フィンピペット(100-1000μL)を用いて、1mLの各被験液に細胞を懸濁した後、その一部(20μL)を、60mmディッシュ(面積が21cm2)に、15細胞/cm2となるように播種し、約8日後に形成されたコロニー数を測定し、保存開始直後のコロニー形成単位(CFU)、すなわち、播種した細胞数に対するコロニー数の比率を算出した。
〔3〕残りの細胞懸濁液は、薬用冷蔵ショーケース(5℃に設定)にて各保存期間まで静置保存した。
〔4〕各保存期間が経過した時点において、チップの先を、細胞を含む液が入ったチューブの底から目視で5mm程度の位置まで挿入し、緩やかに攪拌(500μLの液量でのピペッティングを5回)することにより細胞が懸濁した状態で、60mmディッシュ(面積が21cm2)に、15細胞/cm2となるように播種し、約8日後に形成されたコロニー数を測定し、保存開始直後のCFUを算出した。
[実験1]
hAD-MSCを、以下の表3に示す6種類の被験液中に各保存期間(1日、2日間、4日間、7日間、及び14日間)保存したときの細胞生存率(表4及び図1A参照)及び生細胞回収率(表5及び図1B参照)を、上記[細胞生存率及び生細胞回収率の測定]の項目に記載の方法に従って測定した。
hAD-MSCを、以下の表6に示す4種類の被験液中に各保存期間(6時間、24時間、48時間、及び96時間)保存したときの細胞生存率(表7参照)及び生細胞回収率(表8参照)を、上記[細胞生存率及び生細胞回収率の測定]の項目に記載の方法に従って測定した。
hAD-MSCを、以下の表9に示す11種類の被験液中に各保存期間(24時間、48時間、96時間、及び168時間)保存したときの細胞生存率(表10及び12参照)及び生細胞回収率(表11及び13参照)を、上記[細胞生存率及び生細胞回収率の測定]の項目に記載の方法に従って測定した。
hAD-MSCを、以下の表14に示す4種類の被験液中に各保存期間(6時間、24時間、及び48時間)保存したときのコロニー形成能(表15参照)を、上記[CFUアッセイ]の項目に記載の方法に従って測定した。
1.材料及び方法
[哺乳動物細胞]
以下の実験には、以下の表16に記載のヒト骨髄由来間葉系幹細胞(hBM-MSC)を用いた。
CSP-01液は、実施例1と同様に、α,α-トレハロース(株式会社林原社製又は富士フィルム和光純薬社製)と、低分子デキストランL注(10[w/v]%デキストラン含有乳酸リンゲル液)(大塚製薬工場社製)を、トレハロース及びデキストランの終濃度がそれぞれ3(w/v)%及び5(w/v)%となるように乳酸リンゲル液(ラクテック輸液;大塚製薬工場社製)へ添加し、CSP-01液(5.6未調整)を得た。0.5mol/Lの水酸化ナトリウム溶液を、pHが7.300になるように添加し、CSP-01液(7.3NaOH)を得た。pH調整剤として炭酸水素ナトリウム溶液を添加し、CSP-01液(7.2NaHCO3) を調製した。 CSP-01液(7.2NaHCO3) のpHは7.158であった。なお、各被験液におけるpHは、室温(21.2~24.0℃)で測定した際の値である。
hBM-MSCを含む乳酸リンゲル液は、以下の手順〔1〕~〔8〕に従って調製した。
〔1〕4×105個のhBM-MSCを、75cm2フラスコに加え、ヒト間葉系幹細胞専用培地キット(Lonza社製)(以下、「MSC培地」という)存在下で37℃、5%CO2インキュベーターにて培養を行った。顕微鏡下で細胞の状態を観察し、約80%程度コンフルエントになるまで培養した。
〔2〕MSC培地を除き、10mLのPBS(-)でhBM-MSCをリンスした。
〔3〕PBS(-)を除き、4mLのトリプシン-EDTA(CC-3232、Lonza社製)を加え、室温で5分間静置した。
〔4〕hBM-MSCが90%程度剥離するまで顕微鏡下で観察しながら、ゆっくりと揺らした。
〔5〕5mLのMSC培地を加え、トリプシン反応を停止させ、ピペッティングによりhBM-MSCを回収し、50mL遠心チューブに移した。
〔6〕600×g、5分間、室温で遠心分離を行った。
〔7〕上清(MSC培地)を除き、9mLの乳酸リンゲル液を加え、沈殿(hBM-MSC)を懸濁した。
〔8〕細胞数を計測し、5×105cells/mLとなるように乳酸リンゲル液で調整し、hBM-MSCを含む乳酸リンゲル液を調製した。
各種被験用等張液中でのhBM-MSCの保存は、以下の手順〔1〕~〔2〕に従って行った。
〔1〕調製したhBM-MSCを含む乳酸リンゲル液を、各チューブに0.5mLずつ分注し、遠心分離(600×g、5分間)を行った。
〔2〕上清(乳酸リンゲル液)を除き、沈殿(hBM-MSC)を、0.5mLの各種被験用等張液で懸濁し、5℃で3日間保存した。
トリパンブルー染色法によるhBM-MSCの細胞生存率の解析は、以下の手順〔1〕~〔2〕に従って行った。
〔1〕5℃で3日間保存後のhBM-MSCを含む各種被験用等張液のそれぞれから、20μLを採取し、チューブに移した後、トリパンブルー染色液(Gibco社製)20μLを混合した。なお、比較対照として、各種被験用等張液に懸濁する前(5℃で保存前)のhBM-MSCを含む乳酸リンゲル液のそれぞれから、20μLを採取し、チューブに移した後、トリパンブルー染色液20μLを混合した。
〔2〕顕微鏡下にてワンセルカウンター(バイオメディカルサイエンス社製)を用いて全細胞数とトリパンブルー陽性細胞(死細胞)数の測定を行い、全細胞数に対するトリパンブルー陰性細胞の割合、すなわち、細胞生存率を算出した。細胞生存率(%)を以下の式1を用いて算出した。
[式1]
細胞生存率(%)=(全細胞数-死細胞数)/全細胞数×100
[実験]
hBM-MSCを、以下の表17に示す3種類の被験中に5℃で3日間保存したときの細胞生存率(図4参照)を、上記[細胞生存率の測定]の項目に記載の方法に従って測定した。結果を、表18及び図4に示す。
Claims (24)
- トレハロース若しくはその誘導体又はこれらの塩、及び、pH調整剤としての炭酸水素塩を含み、かつ、pHが6.5~8.5である哺乳動物細胞保存用液。
- 炭酸水素塩が炭酸水素ナトリウムである、請求項1に記載の保存用液。
- さらに、多糖類若しくはその誘導体又はこれらの塩を含む、請求項1又は2に記載の保存用液。
- 等張液である、請求項1~3のいずれかに記載の保存用液。
- 等張液が乳酸リンゲル液である、請求項4に記載の保存用液。
- 多糖類がデキストランである、請求項3~5のいずれかに記載の保存用液。
- トレハロース若しくはその誘導体又はこれらの塩の濃度が、2.0~6.0(w/v)%である、請求項1~6のいずれかに記載の保存用液。
- デキストラン若しくはその誘導体又はこれらの塩の濃度が、4.0~7.0(w/v)%である、請求項6又は7に記載の保存用液。
- 哺乳動物細胞を0~40℃で保存するための、請求項1~8のいずれかに記載の保存用液。
- 哺乳動物細胞を6時間~14日間保存するための、請求項1~9のいずれかに記載の保存用液。
- 哺乳動物細胞の生存率低下を抑制するために用いられる、請求項1~10のいずれかに記載の保存用液。
- 哺乳動物細胞の自己複製能低下を抑制するために用いられる、請求項1~11のいずれかに記載の保存用液。
- 哺乳動物細胞の移植に用いられる、請求項1~12のいずれかに記載の保存用液。
- 哺乳動物細胞が、哺乳動物幹細胞である、請求項1~13のいずれかに記載の保存用液。
- 哺乳動物幹細胞が、哺乳動物間葉系幹細胞である、請求項14に記載の保存用液。
- トレハロース若しくはその誘導体又はこれらの塩、及び、pH調整剤としての炭酸水素塩を含む、請求項1~15のいずれかに記載の保存用液を調製するための粉末製剤。
- 炭酸水素塩が炭酸水素ナトリウムである、請求項16に記載の粉末製剤。
- トレハロース若しくはその誘導体又はこれらの塩、及び、pH調整剤としての炭酸水素塩を含み、かつ、pHが6.5~8.5である保存用液中で、哺乳動物細胞を保存する工程を含む、哺乳動物細胞の保存方法。
- 炭酸水素塩が炭酸水素ナトリウムである、請求項18に記載の保存方法。
- 保存用液が、さらに、多糖類若しくはその誘導体又はこれらの塩を含む、請求項18又は19に記載の保存方法。
- 保存用液が等張液である、請求項18~20のいずれかに記載の保存方法。
- 等張液が乳酸リンゲル液である、請求項21に記載の保存方法。
- 多糖類がデキストランである、請求項20~22のいずれかに記載の保存方法。
- 保存用液中で、哺乳動物細胞を6時間~14日保存する、請求項18~23のいずれかに記載の保存方法。
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CN202080029578.3A CN113728089A (zh) | 2019-04-26 | 2020-04-24 | 含海藻糖的哺乳动物细胞保存用液 |
AU2020263769A AU2020263769B2 (en) | 2019-04-26 | 2020-04-24 | Trehalose-containing liquid for mammalian cell preservation |
CA3133779A CA3133779A1 (en) | 2019-04-26 | 2020-04-24 | Trehalose-containing liquid for mammalian cell preservation |
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