WO2021145364A1 - Hematopoietic-cell storage solution containing trehalose - Google Patents
Hematopoietic-cell storage solution containing trehalose Download PDFInfo
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- WO2021145364A1 WO2021145364A1 PCT/JP2021/000988 JP2021000988W WO2021145364A1 WO 2021145364 A1 WO2021145364 A1 WO 2021145364A1 JP 2021000988 W JP2021000988 W JP 2021000988W WO 2021145364 A1 WO2021145364 A1 WO 2021145364A1
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- Prior art keywords
- cells
- cell
- solution
- trehalose
- blood
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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 4.5 to 7.2.
- Blood cell cell preservation solution hereinafter, may be referred to as “the blood cell cell preservation solution”
- a solution containing trehalose and having a pH in the range of 4.5 to 7.2 hereinafter, ""
- a method for preserving blood cell lineage cells for an arbitrary period in the trehalose-containing solution etc.
- Cancer immunotherapy is a state-of-the-art cell therapy in which immune cells that have the function of attacking cancer are taken out of the body, strengthened, and then returned to the body.
- T cells such as dendritic cell vaccine therapy, alpha 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 used.
- ⁇ T cell therapy alpha beta T cell therapy
- ⁇ T cell therapy gamma delta T cell therapy
- CTL therapy CTL therapy
- NK cell therapy natural killer cell therapy
- an artificial chimeric protein that is, chimeric in which T cells collected from peripheral blood or the like are fused with a single-stranded antibody that recognizes a cancer cell surface antigen and a signal transduction region that induces T cell activation (that is, chimeric).
- CAR-T cells CAR-expressing T cells
- CAR-T cells CAR-expressing T cells
- CAR-T cells chimeric antigen receptor
- Clinical trials of cancer immunotherapy using T cell are underway all over the world, and results showing its effectiveness in the treatment of hematopoietic malignant tumors such as leukemia and lymphoma have been obtained.
- T cells used for transplantation are stored, the cell viability cannot be maintained well by storage in liquid. Therefore, when T cells for transplantation are stored for a long period of time, they are generally cryopreserved.
- 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.
- Patent Documents 1 and 2 The present inventors have reported a mammalian cell preservation solution such as mesenchymal stem cells containing trehalose (Patent Documents 1 and 2). Based on the findings described in such patent documents, Cellstore® S (registered trademark), which is a cell suspension preservation solution containing 3 (w / v)% trehalose and 5 (w / v)% dextran 40, has recently been introduced. Otsuka Pharmaceutical Factory, Ltd.) and Cell Store (registered trademark) W (manufactured by Otsuka Pharmaceutical Factory, Ltd.), which is a cell washing preservation solution containing 3 (w / v)% trehalose, are manufactured and sold.
- Otsuka Pharmaceutical Factory, Ltd. and Cell Store (registered trademark) W (manufactured by Otsuka Pharmaceutical Factory, Ltd.)
- Otsuka Pharmaceutical Factory, Ltd. and Cell Store (registered trademark) W (manufactured by Otsuka Pharmaceutical Factory,
- the present inventors have found that when blood cell lineage cells are stored in a trehalose-containing cell preservation solution, cell aggregation occurs.
- the object of the present invention is to effectively suppress the decrease in cell viability, cell aggregation, and cell adhesion to the storage container that occur when blood cell lines are stored in a trehalose-containing cell storage solution, and to obtain blood cells.
- An object of the present invention is to provide a blood cell cell preservation solution or the like that has a low possibility of adversely affecting the ecology of a mammal when administered into a living body of a mammal.
- the present invention is as follows.
- [1] A blood cell cell preservation solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
- [3] The solution according to [2] above, wherein the isotonic solution is a lactated ringer solution.
- [4] The solution according to any one of [1] to [3] above, for storing blood cell lineage cells at 0 to 40 ° C.
- [5] The solution according to any one of the above [1] to [4], which is used to suppress a decrease in the survival rate of blood cell lineage cells.
- [6] The solution according to any one of [1] to [5] above, which is used for suppressing cell aggregation of blood cell lineage cells.
- [7] The solution according to any one of [1] to [6] above, which is used for suppressing adhesion of blood cell lineage cells to a storage container.
- [8] The solution according to any one of the above [1] to [7], which is used for transplantation of blood cell lineage cells.
- [9] The solution according to any one of [1] to [8] above, wherein the blood cell lineage cells are T cells.
- a method for preserving blood cells which comprises a step of preserving blood cells in a solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
- the storage method according to the above [12], wherein the isotonic solution is a lactated ringer solution.
- the storage method according to any one of the above [11] to [13], wherein the blood cell line cells are stored at 0 to 40 ° C.
- the storage method according to any one of the above [11] to [14], wherein the blood cell lineage cells are T cells.
- the trehalose-containing solution containing the blood cell lineage cell is administered to a subject (for example, a cancer patient) who requires transplantation of the blood cell lineage cell. How to transplant; A step of preparing a trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the trehalose-containing solution or adding trehaloses to the solution containing blood cell-based cells, and a step of preparing the trehalose-containing solution containing blood cell-based cells, and the prepared solution. If necessary, the pH is adjusted to 4.5 to 7.2 by adding a pH adjuster to obtain the Trehalose-containing solution containing blood cell lines, and the Trehalose-containing solution.
- a method for transplanting blood cells which comprises a step of preserving blood cells in a liquid and a step of administering the stored liquid to a subject (for example, a cancer patient) who requires transplantation of blood cells. ;or, The step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding a pH adjuster to the trehalose-containing liquid as necessary, and the trehalose-containing liquid.
- a step of preparing the trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the solution, a step of preserving the blood cell-based cells in the trehalose-containing solution, and a step of preserving the solution after storage.
- a method for transplanting blood cells Use of trehalose in the production of the trehalose-containing liquid containing blood cell-containing cells, and use of trehalose to suppress a decrease in the survival rate of blood cell-based cells in the liquid; Use of trehalose to suppress cell aggregation of blood cells in fluids; Use of trehalose to prevent adhesion of blood cells in liquid to storage vessels;
- the Trehalose-containing solution containing blood cells for use in the treatment of diseases requiring transplantation of blood cells (eg, cancer);
- a step of preparing a trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the trehalose-containing solution or adding trehaloses to the solution containing blood cell-based cells, and a step of preparing the trehalose-containing solution containing blood cell-based cells, and the prepared solution.
- Blood cell system including the step of adjusting the pH to 4.5 to 7.2 by adding a pH adjuster, if necessary, to obtain the Trehalose-containing solution containing blood cell system cells.
- Method for preparing the trehalose-containing solution containing cells The step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding a pH adjuster to the trehalose-containing liquid as necessary, and the trehalose-containing liquid.
- a method for preparing the trehalose-containing solution containing blood cell-based cells which comprises a step of preparing the trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the solution; Trehalose-containing solution containing blood cells; The Blood Cell Cell Preservation Solution Containing Blood Cells; Can be mentioned.
- the blood cell lineage cells are usually preserved by preserving the trehalose-containing solution containing the blood cell lineage cells under the temperature condition in which the solution exists in a liquid state. It does not include a step of storing in a solid state (for example, a step of storing blood cells in a dormant state such as a step of cryopreserving and a step of freeze-drying).
- the present invention it is possible to effectively suppress the decrease in cell viability that occurs when blood cell lineage cells are stored in a liquid. Therefore, cell transplantation using high-quality blood cell lineage cells can be performed, and improvement in therapeutic effect can be expected.
- the fluid containing the blood cells in the infusion bag is infused into the living body, but during the infusion, the blood cells in the infusion bag aggregate.
- the blood cells in the infusion bag aggregate.
- cell aggregation that occurs when blood cell lineage cells are stored in a liquid can be effectively suppressed, so that these risks can be reduced.
- the blood cell line cells are stored, there is a problem that the blood cell line cells adhere to a plastic material (for example, an infusion bag or a cannula). According to the present invention, adhesion of blood cell lineage cells to a storage container can be effectively suppressed. Furthermore, since trehalose is a disaccharide that has a low possibility of adversely affecting the living body, the blood cell lineage cells are stored in the trehalose-containing solution and then in vivo without being replaced with a new transplantation solution. Can be administered to.
- Comparative Example 4 (“Ratio 4” in the figure) and Comparative Example 5 (“Ratio 5” in the figure)
- Example 3 (“real 3” in the figure)
- Comparative Example 6 (“ratio 6” in the figure) and Comparative Example 2 (“ratio 2” in the figure)
- human peripheral blood-derived CD8 + T cells were stored for 6 hours.
- the blood cell cell preservation solution of the present invention contains trehalose and has a pH in the range of 4.5 to 7.2, which is specified for the purpose of "preserving blood cell cells" ( That is, the blood cell lineage cell preservation solution).
- the blood cell cell preservation solution is a trehalose-containing solution (for example, Cellstore S [manufactured by Otsuka Pharmaceutical Factory], Cellstore W [manufactured by Otsuka Pharmaceutical Factory]) or a powdered trehalose-containing substance (for example, manufactured by Otsuka Pharmaceutical Factory).
- pH adjuster By adding a pH adjuster to the solution containing ⁇ , ⁇ -trehalose dihydrate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] as necessary, the pH becomes 4.5 to 7.2. Can be adjusted and obtained.
- pH adjusters include hydrogen carbonates (eg, ammonium hydrogen carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, calcium hydrogen carbonate, etc.) and hydroxides (eg, ammonium hydroxide, potassium hydroxide, sodium hydroxide, etc.).
- acetates eg, ammonium acetate, potassium acetate, sodium acetate, calcium acetate, etc.
- carbonates eg, ammonium carbonate, potassium carbonate, sodium carbonate, calcium carbonate, etc.
- phosphates eg, calcium carbonate, etc.
- alkalis such as lactate (eg, sodium lactate, etc.); citric acid, succinic acid, acetic acid, lactic acid, Acids such as glacial acetic acid, hydrochloric acid, and phosphoric acid; can be mentioned.
- the method for preserving blood cells of the present invention preserves blood cells in a solution containing trehalose and having a pH in the range of 4.5 to 7.2 (that is, the trehalose-containing solution).
- the storage method is usually to store the trehalose-containing solution containing blood cell lines under the temperature condition in which the solution exists in a liquid state, and under the temperature condition in which the solution exists in a solid state.
- the density of blood cell lines in the solution is, for example, in the range of 10 3 to 10 10 cells / mL.
- trehalose is added to a trehalose-containing solution or a solution to which a powdered trehalose-containing substance is added before the preservation step, or trehalose is added to a solution containing trehalose cells.
- trehalose-containing solution containing blood cell lineage cells By adding the above-mentioned trehalose-containing solution containing blood cell lineage cells, and by adding the above-mentioned pH adjuster to the prepared solution, if necessary, the pH is 4.5 to 7. Further including a step of obtaining the trehalose-containing solution containing blood cell lineage cells by adjusting to 2; or, before the storage step, trehalose-containing solution or powdered trehalose-containing substance is added.
- the step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding the above-mentioned pH adjuster to the added liquid as necessary, and the process of obtaining the trehalose-containing liquid and the trehalose-containing liquid. It may further include a step of preparing the trehalose-containing solution containing the trehalose cells by adding the trehalose cells to the solution;
- the “blood cell lineage cell” means a cell derived from blood, and the blood cell lineage cell includes a blood cell (specifically, a cell in which differentiation is induced from a blood cell hematopoietic stem cell to a specific blood cell. ) And hematopoietic stem cells are included.
- the hematopoietic stem cells even those collected from the bone marrow, are embryonic stem cells (ES cells), EG cells (Embryonic germ cells), and induced pluripotent stem cells (iPS cells).
- ES cells embryonic stem cells
- EG cells Embryonic germ cells
- iPS cells induced pluripotent stem cells
- Etc. may be obtained by inducing differentiation from pluripotent stem cells.
- the blood cells include erythrocytes; lymphocytes (eg, CD8 + T cells, CD4 + T cells, alpha beta [ ⁇ ] T cells, gamma delta [ ⁇ ] T cells, cytotoxic T cells [cytotoxic T lymphocytes; CTL]. ], T cells such as helper T cells; natural killer (NK) cells; B cells); leukocytes (eg, macrophages, neutrophils, eosinophils); platelets; etc. It can be exemplified. These cells can be isolated by known common methods.
- lymphocytes eg, CD8 + T cells, CD4 + T cells, alpha beta [ ⁇ ] T cells, gamma delta [ ⁇ ] T cells, cytotoxic T cells [cytotoxic T lymphocytes; CTL].
- T cells such as helper T cells; natural killer (NK) cells; B cells); leukocytes (eg, macrophages, neutrophils, eosinophils); platelets; etc. It can be exemplified.
- leukocyte cell surface markers CD45
- T cell cell surface markers CD3
- CD8 + T cells and cytotoxic T cell cell surface markers CD8
- CD8 + T cells CD8
- CD8 + T cells CD8
- CD8 + T cells CD8
- CD8 + T cells CD8
- CD8 + T cells CD8
- CD8 + T cells CD8
- CD8 + T cells CD8
- CD8 + T cells CD8
- CD8 + T cells CD8 + T cells
- helper T cell cell surface marker CD4
- ⁇ T cell cell surface marker CD39
- FACS fluorescence activated cell sorter
- These blood cell lineage cells can be isolated by an automatic magnetic cell separator (autoMACS) using an antibody against the above-mentioned cell surface marker, an antibody against such a labeling substance, and a conjugated antibody with MACS beads (magnetic beads).
- autoMACS automatic magnetic cell separator
- the 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.
- the T cells also include genetically modified T cells such as CAR-T cells.
- the blood cell lineage cells are usually derived from mammals, and the mammals include rodents such as mice, rats, hamsters and guinea pigs, lagomorphs such as rabbits, pigs, cows and goats. , Lagomorphs such as horses and sheep, cats such as dogs and cats, humans, monkeys, red-tailed monkeys, crab monkeys, marmosets, orangutans, chimpanzees and other primates, among others, mice, pigs, humans Can be preferably exemplified.
- pH is "4.5 to 7.2", for example, 4.5-7.1; 4.5-7.0; 4.5-6.9; 4.5-6.8; 4.5-6.7; 4.5-6.6; 4. 5 to 6.5; 4.5 to 6.4; 4.5 to 6.3; 4.5 to 6.2; 4.5 to 6.1; 4.5 to 6.0; 4.5 to 5.9; 4.5 to 5.8; 4.5 to 5.7; 4.5 to 5.6; 4.5 to 5.5; 4.5 to 5.4; 4.5 to 5. 3; 4.5-5.2; 4.5-5.1; 4.5-5.0; 4.5-4.9; 4.5-4.8; 4.5-4.7; 4.6-7.2; 4.7-7.2; 4.8-7.2; 4.9-7.2; 5.0-7.2; 5.1-7.2; 5.
- pH of 4.5 to 7.2 is usually under room temperature (specifically, in the range of 20 to 28 ° C., preferably 24-26 ° C., more preferably 25 ° C.). It is a value measured in. Therefore, even if the pH is a trehalose-containing solution outside the range of 4.5 to 7.2 under a temperature other than room temperature (for example, cold temperature [0 to 10 ° C.]), when the trehalose-containing solution is subjected to room temperature conditions.
- a trehalose-containing solution having a pH of 4.5 to 7.2 and used for "preserving blood cell lineage cells" is included in the blood cell lineage cell preservation solution.
- pH of 4.5 to 7.2 is 4.5 to 7.2 when the value of pH measured under room temperature conditions is rounded to the second decimal place.
- the pH to be, specifically, the pH of 4.45 or more and less than 7.25 is also included.
- the blood cell cell preservation solution and the trehalose-containing solution are solutions capable of preserving blood cell cells (for example, isotonic solution, hypotonic solution, hypertonic solution), and isotonic solution is preferable.
- 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. do.
- 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 be substantially the same as the osmotic pressure of body fluid or cell fluid.
- physiological saline physiological saline having a buffering effect
- Ringer's solution 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 eg, glucose
- D-sorbitol D-mannitol
- lactose sodium chloride
- 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).
- ⁇ 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.
- ⁇ , ⁇ -trehalose dihydrate manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
- ⁇ , ⁇ -trehalose dihydrate manufactured by Wako Pure Chemical Industries, Ltd.
- ⁇ , ⁇ -trehalose dihydrate manufactured by Hayashibara Co., Ltd.
- other commercially available products 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 blood cell cell preservation solution and the trehalose-containing solution is as follows: the effect of suppressing the decrease in cell viability by the trehalose, the effect of suppressing cell aggregation, and / or the effect of cell adhesion to the storage container.
- the concentration may be any concentration at which the inhibitory effect is exhibited, for example, 0.1 (w / v)% or more, preferably 0.3 (w / v)% or more, more preferably 0.6 (w / v) in terms of trehalose. v)% or more, more preferably 1.0 (w / v)% or more, and most preferably 2.0 (w / v)% or more.
- the concentration of trehaloses in the blood cell preservation solution and the trehalose-containing solution is, for example, in the range of 0.1 to 40 (w / v)% in terms of trehalose, preferably 0.3 to 0.3. 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)%.
- the Blood Cell Cell Preservation Solution is used to store blood cells for an arbitrary period under temperature conditions in which the trehalose-containing solution containing blood cell cells exists in a liquid state.
- the Blood Cell Cell Preservation Solution has the effect of effectively suppressing cell viability reduction, cell aggregation, and / or cell adhesion to the storage container, which occurs when blood cell cells are stored in the liquid.
- the Blood Cell Cell Preservation Solution is further used for "to suppress a decrease in the viability of blood cells”; “to suppress cell aggregation of blood cells”; “for suppressing blood cell cell aggregation”.
- Those specified for "inhibiting adhesion to storage solutions"; and / or “for transplanting blood cell lineage cells”; are preferred.
- the blood cell cell preservation solution and the trehalose-containing solution may be a solution containing trehalose alone, a solution containing two or more types selected from trehalose, and other than trehalose. It may be a liquid containing an arbitrary component.
- the “arbitrary component” includes, for example, isotonic agents (for example, glucose, sorbitol, mannitol, lactose, sodium chloride), chelating agents (for example, EDTA, EGTA, citric acid, salicylate), lysis aids, and the like. Preservatives, antioxidants, amino acids (eg, proline, glutamine), polymers (eg, polyether), phospholipids (eg, lysophosphatidic acid [LPA]), pH regulators mentioned above can be mentioned. ..
- the term "arbitrary component” means a component that may or may not be contained.
- the present invention includes a powder preparation for preparing the Blood Cell Cell Preservation Solution, which contains trehalose. The powder formulation may contain the above-mentioned optional ingredients.
- the blood cell cell preservation solution and the trehalose-containing solution include components having an action of reducing the viability of blood cell lines and suppressing aggregation (for example, acarbose, stachyose, dextran, hydroxyethyl starch [Hydroxyethyl]. It may not contain polysaccharides such as starch; HES] or derivatives thereof or salts thereof; monosaccharides such as glucose or derivatives thereof or salts thereof).
- the blood cell lineage preservation solution when the blood cell lineage cell preservation solution containing blood cell lineage cells is used as it is for transplantation, a solution suitable for blood cell lineage cell transplantation is preferable, and a solution suitable for such blood cell lineage cell transplantation is suitable.
- Substances unsuitable for blood cell transplantation such as biological components (eg, serum or serum-derived components [eg, albumin]); Ingredients that suppress the decline in viability, such as dimethylsulfoxide [Dimethylsulfoxide; DMSO], glycerin, ethylene glycol, trimethylene glycol, dimethylacetamide, polyethylene glycol [PEG], polyvinylpyrrolidone, serum or serum-derived components (eg, , Albumin) and other cryoprotectants or cryoprotectants; preferably not contained.
- biological components eg, serum or serum-derived components [eg, albumin]
- Ingredients that suppress the decline in viability such as dimethylsulfoxide [Dimethylsulfoxide; DMSO], glycerin, ethylene glycol, trimethylene glycol, dimethylacetamide, polyethylene glycol [PEG], polyvinylpyrrolidone, serum or serum-derived components (eg, , Albumin) and other cryoprotectants or cryoprotect
- the period for storing blood cell lines is such that when the Trehalose-containing solution containing blood cells is stored in a liquid state, the cell viability of the blood cells in the solution decreases, cell aggregation, and the like. And / or a period in which cell adhesion to the storage vessel can be suppressed, the proportion of viable cells can be increased, the number of cell aggregates can be reduced, and / or the recovery rate of cells from the storage container can be increased, 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 If it is more than a day (168 hours) and the storage period of blood cell lineage cells is too long, it may adversely affect the survival of the cells.
- 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 to 10 days; 1.5 to 7 days; 1.5 to 4 days; 2 to 16 days; 2 to 14 days; 2 to 10 days; 2 to 7 days; 2 to 4 days; 3 -16 days; 3-14
- the suppression of cell death in blood cell lines stored in the Trehalose-containing solution is a known method capable of detecting cell death such as trypan blue staining method, TUNEL method, Nexin method, and FLICA method. Can be confirmed using. In addition, it can be confirmed by cell observation with a phase-contrast microscope that cell aggregation was suppressed in the blood cell lineage cells stored in the trehalose-containing solution of the present case.
- the "temperature at which the trehalose-containing solution containing blood cell-based cells exists in a liquid state” is defined as the temperature at which the trehalose-containing solution containing blood cell-based cells exists in a liquid state without freezing.
- the temperature may be such that the blood cell cells in the liquid can grow, and it is usually in the range of 0 to 40 ° C, preferably in the range of 0 to 30 ° C (room temperature).
- the temperature suitable for preserving blood cell lineage cells is 8 to 40 ° C., preferably 10 to 40 ° C., more preferably 12 to 38 ° C., and most preferably 15 to 37 ° C.
- examples of the storage container for blood cell lineage cells include a glass container, a plastic container, and the like.
- a resin container selected from the group consisting of a copolymer, an ethylene / ⁇ -olefin copolymer, and a blended resin of these resins is preferable.
- the storage container also includes a device attached to a cell administration bag such as a cannula.
- the blood cell lineage cells may be those isolated from the living body or those 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 the isolated or purified blood cell line is usually 30% or more, preferably 50% or more, more preferably 70% or more, still more preferably 90% or more (). For example, 100%).
- the blood cell lineage cells (population) to be stored in the blood cell lineage cell preservation solution or the trehalose-containing solution may be in a single cell state.
- 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).
- Blood cell lineage cells in a single cell state can be prepared by enzymatically treating blood cell lineage cells cultured in vitro with trypsin / EDTA or the like.
- the proportion of single-celled blood cells contained in blood 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%). be.
- the percentage of cells in the single cell state is that blood cell lineage cells are dispersed in PBS and observed under a microscope to determine the presence or absence of aggregation in a plurality of randomly selected cells (eg, 1000 cells). It can be determined by examining.
- the blood cell line cells (population) to be stored in the blood cell line cell preservation solution or the trehalose-containing solution may be suspended.
- the term "floating" means that blood cell lineage cells are retained in the liquid without contacting the inner wall of the container containing the storage liquid.
- the blood cell cells stored in the blood cell cell preservation solution or the trehalose-containing solution are aggregated or precipitated, the blood cell cells are collected by a well-known method in the art such as pipetting or tapping before transplantation. Is preferably suspended.
- T cells CD8 + T cells derived from human peripheral blood (hereinafter, simply referred to as “T cells”) shown in Table 1 below were used.
- Lactec injection manufactured by Otsuka Pharmaceutical Factory, see Table 2 (hereinafter also referred to as "LR"), which is a lactated Ringer's solution with a pH of 6.574, and ⁇ , ⁇ -trehalose dihydrate (Fujifilm Wako Pure Chemical Industries, Ltd.) was added so that the final concentration of trehalose was 3 (w / v)%, and LRT-5 (see Table 3) was obtained.
- LR-5 lactated Ringer's solution with a pH of 6.574
- LRT-5 see Table 3
- 0.1 mol / L hydrochloric acid was added to LRT-5 so that the pH was 4.468, 5.021, 5.997, and 6.482, respectively, and LRT-1 to 4 (see Table 3).
- the cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
- T cells were cultured according to the method described in (1) above.
- T cells were cultured according to the method described in (1) above.
- the cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
- the remaining cell suspension, so that T cells becomes 4.5 ⁇ 10 5 cells were transferred to Falcon (TM) 15 mL high transparency polypropylene conical tube (Corning), centrifuged (300 ⁇ g, 10 minutes, room temperature), the supernatant is removed, 0.9 mL of the three test solutions shown in Table 9 (Example 3, Comparative Example 6 and Comparative Example 2) is added, the cell pellet is suspended, and 25 Stored at ° C.
- the cell recovery rate and cell viability rate during each storage period were calculated.
- T cells were cultured according to the method described in (1) above.
- the cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
- Non-blood cell lineage cells human bone marrow-derived mesenchymal stem cells (hBM-MSC) shown in Table 14 below were used.
- a lactated ringer solution containing hBM-MSC was prepared according to the following procedures [1] to [8]. [1] 4 ⁇ 10 5 hBM-MSCs were added to a 75 cm 2 flask, and the temperature was 37 ° C., 5% in the presence of a medium kit for human mesenchymal stem cells (manufactured by Lonza) (hereinafter referred to as “MSC medium”). 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
- the cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
- cell transplantation using high-quality blood cell lineage cells can be performed, and improvement in therapeutic effect for diseases such as cancer can be expected.
- the blood cell cell preservation solution containing blood cell cells can be directly administered into the living body without being replaced with a new transplantation solution.
- it does not contain biological components, suppresses cell adhesion to cell storage containers and administration bags, and can administer an accurate number of cells in vivo. Therefore, the present invention contributes to the field of transplantation medicine using blood cell lineage cells.
Abstract
The present invention addresses the problem of providing a hematopoietic-cell storage solution or the like: with which it is possible to effectively suppress a reduction in cell survival rate, cell aggregation, and cell adhesion to a storage vessel that occur when hematopoietic cells are stored in a trehalose-containing cell storage solution; and that is unlikely to adversely affect the ecology of a mammalian animal when administered to the living body of the mammalian animal. In the present invention, a solution that contains trehalose, a derivative thereof, or a salt of such substances and that has a pH of 4.5-7.2 is employed to store hematopoietic cells.
Description
本発明は、トレハロース若しくはその誘導体又はこれらの塩（以下、これらを総称して「トレハロース類」ということがある）を含み、かつ、水素イオン指数（ｐＨ）が４．５～７．２である血球系細胞保存用液（以下、「本件血球系細胞保存用液」ということがある）；トレハロース類を含み、かつ、ｐＨが４．５～７．２の範囲内である液（以下、「本件トレハロース類含有液」ということがある）中で、任意の期間、血球系細胞を保存する方法；等に関する。
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 4.5 to 7.2. Blood cell cell preservation solution (hereinafter, may be referred to as "the blood cell cell preservation solution"); A solution containing trehalose and having a pH in the range of 4.5 to 7.2 (hereinafter, "" A method for preserving blood cell lineage cells for an arbitrary period in the trehalose-containing solution); etc.
がん免疫細胞療法は、がんを攻撃する働きを有する免疫細胞を体外に取り出し、その働きを強化後、再び体内に戻すという最先端の細胞医療である。例えば、樹状細胞ワクチン療法、アルファ・ベータＴ細胞療法（αβＴ細胞療法）、ガンマ・デルタＴ細胞療法（γδＴ細胞療法）、ＣＴＬ療法、ナチュラルキラー細胞療法（ＮＫ細胞療法）等のＴ細胞を用いた療法が実践されている。近年、末梢血等から採取したＴ細胞に、がん細胞表面抗原を認識する一本鎖抗体と、Ｔ細胞の活性化を誘導するシグナル伝達領域を融合させた人工的なキメラタンパク質（すなわち、キメラ抗原受容体［Chimeric Antigen Receptor；ＣＡＲ］）をコードする遺伝子を導入することにより得られたＣＡＲ発現Ｔ細胞（ＣＡＲ－Ｔ細胞）キメラ抗原受容体（Chimeric Antigen Receptor:以下、「ＣＡＲ」ともいう）を用いたがん免疫療法が世界中で臨床試験が進行しており、白血病やリンパ腫などの造血器悪性腫瘍の治療において、有効性を示す結果が得られている。
Cancer immunotherapy is a state-of-the-art cell therapy in which immune cells that have the function of attacking cancer are taken out of the body, strengthened, and then returned to the body. For example, T cells such as dendritic cell vaccine therapy, alpha beta T cell therapy (αβ T cell therapy), gamma delta T cell therapy (γδ T cell therapy), CTL therapy, and natural killer cell therapy (NK cell therapy) are used. The therapy that was used is being practiced. In recent years, an artificial chimeric protein (that is, chimeric) in which T cells collected from peripheral blood or the like are fused with a single-stranded antibody that recognizes a cancer cell surface antigen and a signal transduction region that induces T cell activation (that is, chimeric). CAR-expressing T cells (CAR-T cells) chimeric antigen receptor (hereinafter also referred to as "CAR") obtained by introducing a gene encoding an antigen receptor [Chimeric Antigen Receptor; CAR]). Clinical trials of cancer immunotherapy using T cell are underway all over the world, and results showing its effectiveness in the treatment of hematopoietic malignant tumors such as leukemia and lymphoma have been obtained.
がん免疫療法において、移植に用いるＴ細胞を保存する場合、液体中での保存では細胞生存率を良好に保つことができない。このため、移植用Ｔ細胞を長期保存する場合、凍結保存することが一般的である。しかし、凍結保存液中には、通常ＤＭＳＯ、グリセロール等の凍結保存剤が添加されているため、凍結保存した幹細胞やＴ細胞を解凍した後、移植治療を行う前に凍結保存剤を除去する必要があり、手間がかかることが問題とされていた。また、凍結保存液に凍結保存剤を添加しても凍結時の水の結晶化による細胞骨格のダメージが大きく、凍結融解後の細胞生存率が低下することも問題とされていた。このため、簡便性に優れ、且つ細胞生存率の低下を抑制できる細胞保存液の開発が急務とされていた。
In cancer immunotherapy, when T cells used for transplantation are stored, the cell viability cannot be maintained well by storage in liquid. Therefore, when T cells for transplantation are stored for a long period of time, they are generally cryopreserved. However, since 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.
本発明者らは、トレハロースを含む、間葉系幹細胞等の哺乳動物細胞保存液を報告している（特許文献１及び２）。かかる特許文献に記載の知見等に基づき、最近、３（ｗ／ｖ）％のトレハロース及び５（ｗ／ｖ）％のデキストラン４０を含む細胞懸濁保存液であるセルストア（登録商標）Ｓ（大塚製薬工場社製）と、３（ｗ／ｖ）％のトレハロースを含む細胞洗浄保存液であるセルストア（登録商標）Ｗ（大塚製薬工場社製）が製造・販売されている。
The present inventors have reported a mammalian cell preservation solution such as mesenchymal stem cells containing trehalose (Patent Documents 1 and 2). Based on the findings described in such patent documents, Cellstore® S (registered trademark), which is a cell suspension preservation solution containing 3 (w / v)% trehalose and 5 (w / v)% dextran 40, has recently been introduced. Otsuka Pharmaceutical Factory, Ltd.) and Cell Store (registered trademark) W (manufactured by Otsuka Pharmaceutical Factory, Ltd.), which is a cell washing preservation solution containing 3 (w / v)% trehalose, are manufactured and sold.
本発明者らは、血球系細胞をトレハロース含有細胞保存液で保存すると、細胞凝集を起こすことを見出した。本発明の課題は、血球系細胞をトレハロース含有細胞保存液中で保存したときに生じる細胞生存率低下、細胞凝集、保存容器への細胞接着を効果的に抑制でき、かつ、血球系細胞を、哺乳動物の生体内に投与したときに、哺乳動物の生態に悪影響を及ぼす可能性の低い血球系細胞保存用液等を提供することにある。
The present inventors have found that when blood cell lineage cells are stored in a trehalose-containing cell preservation solution, cell aggregation occurs. The object of the present invention is to effectively suppress the decrease in cell viability, cell aggregation, and cell adhesion to the storage container that occur when blood cell lines are stored in a trehalose-containing cell storage solution, and to obtain blood cells. An object of the present invention is to provide a blood cell cell preservation solution or the like that has a low possibility of adversely affecting the ecology of a mammal when administered into a living body of a mammal.
本発明者らは、上記課題を解決すべく鋭意研究を続けている。その過程において、ヒト末梢血由来ＣＤ８陽性（＋）Ｔ細胞を、前述したセルストアＷ中に保存したところ、細胞凝集が認められた。そこで、トレハロース含有液について検討を重ねたところ、トレハロース含有液のｐＨを特定の範囲に調整することにより、ヒト末梢血由来ＣＤ８＋Ｔ細胞を保存した場合により生じる細胞凝集を効果的に抑制することに加え、保存容器への細胞接着も効果的に抑制することができることを見出した。また、ｐＨを特定の範囲に調整したトレハロース含有液は、ヒト末梢血由来ＣＤ８＋Ｔ細胞を保存した場合により生じる細胞生存率低下の抑制効果が高いことも確認した。さらに、ｐＨを特定の範囲に調整したトレハロース含有液中に保存した際の細胞凝集の抑制効果は、血球系細胞に特異的であることも確認した。本発明は、これらの知見に基づき、完成するに至ったものである。
The present inventors are continuing diligent research to solve the above problems. In the process, when human peripheral blood-derived CD8-positive (+) T cells were stored in the above-mentioned cell store W, cell aggregation was observed. Therefore, as a result of repeated studies on trehalose-containing solutions, in addition to effectively suppressing cell aggregation caused by storage of human peripheral blood-derived CD8 + T cells by adjusting the pH of the trehalose-containing solution to a specific range. , It was found that cell adhesion to the storage container can also be effectively suppressed. It was also confirmed that the trehalose-containing solution having the pH adjusted to a specific range has a high effect of suppressing the decrease in cell viability caused by the storage of human peripheral blood-derived CD8 + T cells. Furthermore, it was also confirmed that the inhibitory effect on cell aggregation when stored in a trehalose-containing solution having a pH adjusted to a specific range is specific to blood cell lineage cells. The present invention has been completed based on these findings.
すなわち、本発明は以下のとおりである。
〔１〕トレハロース若しくはその誘導体又はこれらの塩を含み、かつ、ｐＨが４．５～７．２である血球系細胞保存用液。
〔２〕等張液である、上記〔１〕に記載の液。
〔３〕等張液が、乳酸リンゲル液である、上記〔２〕に記載の液。
〔４〕血球系細胞を０～４０℃で保存するための、上記〔１〕～〔３〕のいずれかに記載の液。
〔５〕血球系細胞の生存率低下を抑制するために用いられる、上記〔１〕～〔４〕のいずれかに記載の液。
〔６〕血球系細胞の細胞凝集を抑制するために用いられる、上記〔１〕～〔５〕のいずれかに記載の液。
〔７〕血球系細胞の保存容器への接着を抑制するために用いられる、上記〔１〕～〔６〕のいずれかに記載の液。
〔８〕血球系細胞の移植に用いられる、上記〔１〕～〔７〕のいずれかに記載の液。
〔９〕血球系細胞がＴ細胞である、上記〔１〕～〔８〕のいずれかに記載の液。
〔１０〕トレハロース若しくはその誘導体又はこれらの塩を含む、上記〔１〕～〔９〕のいずれかに記載の液を調製するための粉末製剤。
〔１１〕トレハロース若しくはその誘導体又はこれらの塩を含み、かつ、ｐＨが４．５～７．２である液中で、血球系細胞を保存する工程を含む、血球系細胞の保存方法。
〔１２〕液が、等張液である、上記〔１１〕に記載の保存方法。
〔１３〕等張液が、乳酸リンゲル液である、上記〔１２〕に記載の保存方法。
〔１４〕血球系細胞を０～４０℃で保存する、上記〔１１〕～〔１３〕のいずれかに記載の保存方法。
〔１５〕血球系細胞がＴ細胞である、上記〔１１〕～〔１４〕のいずれか１項に記載の保存方法。 That is, the present invention is as follows.
[1] A blood cell cell preservation solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
[2] The liquid according to the above [1], which is an isotonic liquid.
[3] The solution according to [2] above, wherein the isotonic solution is a lactated ringer solution.
[4] The solution according to any one of [1] to [3] above, for storing blood cell lineage cells at 0 to 40 ° C.
[5] The solution according to any one of the above [1] to [4], which is used to suppress a decrease in the survival rate of blood cell lineage cells.
[6] The solution according to any one of [1] to [5] above, which is used for suppressing cell aggregation of blood cell lineage cells.
[7] The solution according to any one of [1] to [6] above, which is used for suppressing adhesion of blood cell lineage cells to a storage container.
[8] The solution according to any one of the above [1] to [7], which is used for transplantation of blood cell lineage cells.
[9] The solution according to any one of [1] to [8] above, wherein the blood cell lineage cells are T cells.
[10] A powder preparation for preparing the solution according to any one of the above [1] to [9], which contains trehalose or a derivative thereof or a salt thereof.
[11] A method for preserving blood cells, which comprises a step of preserving blood cells in a solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
[12] The storage method according to the above [11], wherein the liquid is an isotonic liquid.
[13] The storage method according to the above [12], wherein the isotonic solution is a lactated ringer solution.
[14] The storage method according to any one of the above [11] to [13], wherein the blood cell line cells are stored at 0 to 40 ° C.
[15] The storage method according to any one of the above [11] to [14], wherein the blood cell lineage cells are T cells.
〔１〕トレハロース若しくはその誘導体又はこれらの塩を含み、かつ、ｐＨが４．５～７．２である血球系細胞保存用液。
〔２〕等張液である、上記〔１〕に記載の液。
〔３〕等張液が、乳酸リンゲル液である、上記〔２〕に記載の液。
〔４〕血球系細胞を０～４０℃で保存するための、上記〔１〕～〔３〕のいずれかに記載の液。
〔５〕血球系細胞の生存率低下を抑制するために用いられる、上記〔１〕～〔４〕のいずれかに記載の液。
〔６〕血球系細胞の細胞凝集を抑制するために用いられる、上記〔１〕～〔５〕のいずれかに記載の液。
〔７〕血球系細胞の保存容器への接着を抑制するために用いられる、上記〔１〕～〔６〕のいずれかに記載の液。
〔８〕血球系細胞の移植に用いられる、上記〔１〕～〔７〕のいずれかに記載の液。
〔９〕血球系細胞がＴ細胞である、上記〔１〕～〔８〕のいずれかに記載の液。
〔１０〕トレハロース若しくはその誘導体又はこれらの塩を含む、上記〔１〕～〔９〕のいずれかに記載の液を調製するための粉末製剤。
〔１１〕トレハロース若しくはその誘導体又はこれらの塩を含み、かつ、ｐＨが４．５～７．２である液中で、血球系細胞を保存する工程を含む、血球系細胞の保存方法。
〔１２〕液が、等張液である、上記〔１１〕に記載の保存方法。
〔１３〕等張液が、乳酸リンゲル液である、上記〔１２〕に記載の保存方法。
〔１４〕血球系細胞を０～４０℃で保存する、上記〔１１〕～〔１３〕のいずれかに記載の保存方法。
〔１５〕血球系細胞がＴ細胞である、上記〔１１〕～〔１４〕のいずれか１項に記載の保存方法。 That is, the present invention is as follows.
[1] A blood cell cell preservation solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
[2] The liquid according to the above [1], which is an isotonic liquid.
[3] The solution according to [2] above, wherein the isotonic solution is a lactated ringer solution.
[4] The solution according to any one of [1] to [3] above, for storing blood cell lineage cells at 0 to 40 ° C.
[5] The solution according to any one of the above [1] to [4], which is used to suppress a decrease in the survival rate of blood cell lineage cells.
[6] The solution according to any one of [1] to [5] above, which is used for suppressing cell aggregation of blood cell lineage cells.
[7] The solution according to any one of [1] to [6] above, which is used for suppressing adhesion of blood cell lineage cells to a storage container.
[8] The solution according to any one of the above [1] to [7], which is used for transplantation of blood cell lineage cells.
[9] The solution according to any one of [1] to [8] above, wherein the blood cell lineage cells are T cells.
[10] A powder preparation for preparing the solution according to any one of the above [1] to [9], which contains trehalose or a derivative thereof or a salt thereof.
[11] A method for preserving blood cells, which comprises a step of preserving blood cells in a solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
[12] The storage method according to the above [11], wherein the liquid is an isotonic liquid.
[13] The storage method according to the above [12], wherein the isotonic solution is a lactated ringer solution.
[14] The storage method according to any one of the above [11] to [13], wherein the blood cell line cells are stored at 0 to 40 ° C.
[15] The storage method according to any one of the above [11] to [14], wherein the blood cell lineage cells are T cells.
また本発明の実施の他の形態として、血球系細胞を含む本件トレハロース類含有液を、血球系細胞の移植を必要とする対象（例えば、がん患者）へ投与する工程を含む、血球系細胞の移植方法；や、
トレハロース類含有液に、血球系細胞を加えるか、或いは、血球系細胞を含む液に、トレハロース類を加えることにより、血球系細胞を含むトレハロース類含有液を調製する工程と、調製した当該液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、血球系細胞を含む本件トレハロース類含有液を得る工程と、本件トレハロース類含有液中で血球系細胞を保存する工程と、保存後の当該液を、血球系細胞の移植を必要とする対象（例えば、がん患者）へ投与する工程とを含む、血球系細胞の移植方法；や、
トレハロース類含有液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、本件トレハロース類含有液を得る工程と、本件トレハロース類含有液に、血球系細胞を加えることにより、血球系細胞を含む本件トレハロース類含有液を調製する工程と、本件トレハロース類含有液中で血球系細胞を保存する工程と、保存後の当該液を、血球系細胞の移植を必要とする対象（例えば、がん患者）へ投与する工程とを含む、血球系細胞の移植方法；や、
血球系細胞を含む本件トレハロース類含有液の製造における、トレハロース類の使用や、液体中の血球系細胞の生存率低下を抑制するための、トレハロース類の使用；や、
液体中の血球系細胞の細胞凝集を抑制するための、トレハロース類の使用；や、
液体中の血球系細胞の保存容器への接着を抑制するための、トレハロース類の使用；や、
血球系細胞の移植を必要とする疾患（例えば、がん）の治療における使用のための、血球系細胞を含む本件トレハロース類含有液；や、
トレハロース類含有液に、血球系細胞を加えるか、或いは、血球系細胞を含む液に、トレハロース類を加えることにより、血球系細胞を含むトレハロース類含有液を調製する工程と、調製した当該液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、血球系細胞を含む本件トレハロース類含有液を得る工程とを含む、血球系細胞を含む本件トレハロース類含有液の調製方法；や、
トレハロース類含有液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、本件トレハロース類含有液を得る工程と、本件トレハロース類含有液に、血球系細胞を加えることにより、血球系細胞を含む本件トレハロース類含有液を調製する工程とを含む、血球系細胞を含む本件トレハロース類含有液の調製方法；や、
血球系細胞を含む本件トレハロース類含有液；や、
血球系細胞を含む本件血球系細胞保存用液；
を挙げることができる。なお、上記移植方法において、血球系細胞の保存は、通常、血球系細胞を含む本件トレハロース類含有液を、当該液が液体の状態で存在する温度条件下で保存するものであり、当該液が固体の状態で保存する工程（例えば、凍結保存する工程、凍結乾燥保存する工程等の血球系細胞を休眠状態で保存する工程）を含まない。 Further, as another embodiment of the present invention, the trehalose-containing solution containing the blood cell lineage cell is administered to a subject (for example, a cancer patient) who requires transplantation of the blood cell lineage cell. How to transplant;
A step of preparing a trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the trehalose-containing solution or adding trehaloses to the solution containing blood cell-based cells, and a step of preparing the trehalose-containing solution containing blood cell-based cells, and the prepared solution. If necessary, the pH is adjusted to 4.5 to 7.2 by adding a pH adjuster to obtain the Trehalose-containing solution containing blood cell lines, and the Trehalose-containing solution. A method for transplanting blood cells, which comprises a step of preserving blood cells in a liquid and a step of administering the stored liquid to a subject (for example, a cancer patient) who requires transplantation of blood cells. ;or,
The step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding a pH adjuster to the trehalose-containing liquid as necessary, and the trehalose-containing liquid. A step of preparing the trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the solution, a step of preserving the blood cell-based cells in the trehalose-containing solution, and a step of preserving the solution after storage. A method for transplanting blood cells;
Use of trehalose in the production of the trehalose-containing liquid containing blood cell-containing cells, and use of trehalose to suppress a decrease in the survival rate of blood cell-based cells in the liquid;
Use of trehalose to suppress cell aggregation of blood cells in fluids;
Use of trehalose to prevent adhesion of blood cells in liquid to storage vessels;
The Trehalose-containing solution containing blood cells for use in the treatment of diseases requiring transplantation of blood cells (eg, cancer);
A step of preparing a trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the trehalose-containing solution or adding trehaloses to the solution containing blood cell-based cells, and a step of preparing the trehalose-containing solution containing blood cell-based cells, and the prepared solution. Blood cell system, including the step of adjusting the pH to 4.5 to 7.2 by adding a pH adjuster, if necessary, to obtain the Trehalose-containing solution containing blood cell system cells. Method for preparing the trehalose-containing solution containing cells;
The step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding a pH adjuster to the trehalose-containing liquid as necessary, and the trehalose-containing liquid. A method for preparing the trehalose-containing solution containing blood cell-based cells, which comprises a step of preparing the trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the solution;
Trehalose-containing solution containing blood cells;
The Blood Cell Cell Preservation Solution Containing Blood Cells;
Can be mentioned. In the above transplantation method, the blood cell lineage cells are usually preserved by preserving the trehalose-containing solution containing the blood cell lineage cells under the temperature condition in which the solution exists in a liquid state. It does not include a step of storing in a solid state (for example, a step of storing blood cells in a dormant state such as a step of cryopreserving and a step of freeze-drying).
トレハロース類含有液に、血球系細胞を加えるか、或いは、血球系細胞を含む液に、トレハロース類を加えることにより、血球系細胞を含むトレハロース類含有液を調製する工程と、調製した当該液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、血球系細胞を含む本件トレハロース類含有液を得る工程と、本件トレハロース類含有液中で血球系細胞を保存する工程と、保存後の当該液を、血球系細胞の移植を必要とする対象（例えば、がん患者）へ投与する工程とを含む、血球系細胞の移植方法；や、
トレハロース類含有液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、本件トレハロース類含有液を得る工程と、本件トレハロース類含有液に、血球系細胞を加えることにより、血球系細胞を含む本件トレハロース類含有液を調製する工程と、本件トレハロース類含有液中で血球系細胞を保存する工程と、保存後の当該液を、血球系細胞の移植を必要とする対象（例えば、がん患者）へ投与する工程とを含む、血球系細胞の移植方法；や、
血球系細胞を含む本件トレハロース類含有液の製造における、トレハロース類の使用や、液体中の血球系細胞の生存率低下を抑制するための、トレハロース類の使用；や、
液体中の血球系細胞の細胞凝集を抑制するための、トレハロース類の使用；や、
液体中の血球系細胞の保存容器への接着を抑制するための、トレハロース類の使用；や、
血球系細胞の移植を必要とする疾患（例えば、がん）の治療における使用のための、血球系細胞を含む本件トレハロース類含有液；や、
トレハロース類含有液に、血球系細胞を加えるか、或いは、血球系細胞を含む液に、トレハロース類を加えることにより、血球系細胞を含むトレハロース類含有液を調製する工程と、調製した当該液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、血球系細胞を含む本件トレハロース類含有液を得る工程とを含む、血球系細胞を含む本件トレハロース類含有液の調製方法；や、
トレハロース類含有液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、本件トレハロース類含有液を得る工程と、本件トレハロース類含有液に、血球系細胞を加えることにより、血球系細胞を含む本件トレハロース類含有液を調製する工程とを含む、血球系細胞を含む本件トレハロース類含有液の調製方法；や、
血球系細胞を含む本件トレハロース類含有液；や、
血球系細胞を含む本件血球系細胞保存用液；
を挙げることができる。なお、上記移植方法において、血球系細胞の保存は、通常、血球系細胞を含む本件トレハロース類含有液を、当該液が液体の状態で存在する温度条件下で保存するものであり、当該液が固体の状態で保存する工程（例えば、凍結保存する工程、凍結乾燥保存する工程等の血球系細胞を休眠状態で保存する工程）を含まない。 Further, as another embodiment of the present invention, the trehalose-containing solution containing the blood cell lineage cell is administered to a subject (for example, a cancer patient) who requires transplantation of the blood cell lineage cell. How to transplant;
A step of preparing a trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the trehalose-containing solution or adding trehaloses to the solution containing blood cell-based cells, and a step of preparing the trehalose-containing solution containing blood cell-based cells, and the prepared solution. If necessary, the pH is adjusted to 4.5 to 7.2 by adding a pH adjuster to obtain the Trehalose-containing solution containing blood cell lines, and the Trehalose-containing solution. A method for transplanting blood cells, which comprises a step of preserving blood cells in a liquid and a step of administering the stored liquid to a subject (for example, a cancer patient) who requires transplantation of blood cells. ;or,
The step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding a pH adjuster to the trehalose-containing liquid as necessary, and the trehalose-containing liquid. A step of preparing the trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the solution, a step of preserving the blood cell-based cells in the trehalose-containing solution, and a step of preserving the solution after storage. A method for transplanting blood cells;
Use of trehalose in the production of the trehalose-containing liquid containing blood cell-containing cells, and use of trehalose to suppress a decrease in the survival rate of blood cell-based cells in the liquid;
Use of trehalose to suppress cell aggregation of blood cells in fluids;
Use of trehalose to prevent adhesion of blood cells in liquid to storage vessels;
The Trehalose-containing solution containing blood cells for use in the treatment of diseases requiring transplantation of blood cells (eg, cancer);
A step of preparing a trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the trehalose-containing solution or adding trehaloses to the solution containing blood cell-based cells, and a step of preparing the trehalose-containing solution containing blood cell-based cells, and the prepared solution. Blood cell system, including the step of adjusting the pH to 4.5 to 7.2 by adding a pH adjuster, if necessary, to obtain the Trehalose-containing solution containing blood cell system cells. Method for preparing the trehalose-containing solution containing cells;
The step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding a pH adjuster to the trehalose-containing liquid as necessary, and the trehalose-containing liquid. A method for preparing the trehalose-containing solution containing blood cell-based cells, which comprises a step of preparing the trehalose-containing solution containing blood cell-based cells by adding blood cell-based cells to the solution;
Trehalose-containing solution containing blood cells;
The Blood Cell Cell Preservation Solution Containing Blood Cells;
Can be mentioned. In the above transplantation method, the blood cell lineage cells are usually preserved by preserving the trehalose-containing solution containing the blood cell lineage cells under the temperature condition in which the solution exists in a liquid state. It does not include a step of storing in a solid state (for example, a step of storing blood cells in a dormant state such as a step of cryopreserving and a step of freeze-drying).
本発明によると、血球系細胞を液体中に保存したときに生じる細胞生存率低下を効果的に抑制することができる。このため、良質な血球系細胞による細胞移植を実施することができ、治療効果の向上が期待できる。また、血球系細胞を移植する際、多くの場合、輸液バッグ内の血球系細胞を含む液を生体内へ点滴注入するが、点滴している間に、輸液バッグ内の血球系細胞同士が凝集し、カニューレ中に詰まったり、肺静脈等の細い血管中に塞栓を形成してしまうリスクがある。本発明によると、血球系細胞を液体中で保存したときに生じる細胞凝集を効果的に抑制することもできるため、これらリスクを低減することができる。また、血球系細胞を保存した際、特にプラスチック素材（例えば、輸液バッグやカニューレ）に血球系細胞が接着するという問題がある。本発明によると、血球系細胞の保存容器への接着を効果的に抑制することができる。さらに、トレハロース類は、生体に悪影響を及ぼす可能性の低い二糖類であることから、血球系細胞を本件トレハロース類含有液中で保存した後、新しい移植用液に置換することなく、そのまま生体内へ投与することができる。
According to the present invention, it is possible to effectively suppress the decrease in cell viability that occurs when blood cell lineage cells are stored in a liquid. Therefore, cell transplantation using high-quality blood cell lineage cells can be performed, and improvement in therapeutic effect can be expected. In addition, when transplanting blood cells, in many cases, the fluid containing the blood cells in the infusion bag is infused into the living body, but during the infusion, the blood cells in the infusion bag aggregate. However, there is a risk of clogging in the cannula or forming an embolus in a small blood vessel such as a pulmonary vein. According to the present invention, cell aggregation that occurs when blood cell lineage cells are stored in a liquid can be effectively suppressed, so that these risks can be reduced. Further, when the blood cell line cells are stored, there is a problem that the blood cell line cells adhere to a plastic material (for example, an infusion bag or a cannula). According to the present invention, adhesion of blood cell lineage cells to a storage container can be effectively suppressed. Furthermore, since trehalose is a disaccharide that has a low possibility of adversely affecting the living body, the blood cell lineage cells are stored in the trehalose-containing solution and then in vivo without being replaced with a new transplantation solution. Can be administered to.
本発明の血球系細胞保存用液は、「血球系細胞を保存するため」という用途に特定された、トレハロース類を含み、かつ、ｐＨが４．５～７．２の範囲内である液（すなわち、本件血球系細胞保存用液）である。本件血球系細胞保存用液は、トレハロース類含有液（例えば、セルストアＳ［大塚製薬工場社製］、セルストアＷ［大塚製薬工場社製］）、又は粉体のトレハロース類含有物（例えば、α，α－トレハロース二水和物［富士フイルム和光純薬社製］）を添加した液に、必要に応じて、ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、得ることができる。かかるｐＨ調整剤としては、例えば、炭酸水素塩（例えば、炭酸水素アンモニウム、炭酸水素カリウム、炭酸水素ナトリウム、炭酸水素カルシウム等）、水酸化物（例えば、水酸化アンモニウム、水酸化カリウム、水酸化ナトリウム、水酸化カルシウム等）、酢酸塩（例えば、酢酸アンモニウム、酢酸カリウム、酢酸ナトリウム、酢酸カルシウム等）、炭酸塩（例えば、炭酸アンモニウム、炭酸カリウム、炭酸ナトリウム、炭酸カルシウム等）、リン酸塩（例えば、リン酸水素２ナトリウム、リン酸水素１ナトリウム、リン酸水素２カリウム、リン酸水素１カリウム等）、乳酸塩（例えば、乳酸ナトリウム等）等のアルカリ；クエン酸、コハク酸、酢酸、乳酸、氷酢酸、塩酸、リン酸等の酸；を挙げることができる。
The blood cell cell preservation solution of the present invention contains trehalose and has a pH in the range of 4.5 to 7.2, which is specified for the purpose of "preserving blood cell cells" ( That is, the blood cell lineage cell preservation solution). The blood cell cell preservation solution is a trehalose-containing solution (for example, Cellstore S [manufactured by Otsuka Pharmaceutical Factory], Cellstore W [manufactured by Otsuka Pharmaceutical Factory]) or a powdered trehalose-containing substance (for example, manufactured by Otsuka Pharmaceutical Factory). By adding a pH adjuster to the solution containing α, α-trehalose dihydrate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] as necessary, the pH becomes 4.5 to 7.2. Can be adjusted and obtained. Examples of such pH adjusters include hydrogen carbonates (eg, ammonium hydrogen carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate, calcium hydrogen carbonate, etc.) and hydroxides (eg, ammonium hydroxide, potassium hydroxide, sodium hydroxide, etc.). , Calcium hydroxide, etc.), acetates (eg, ammonium acetate, potassium acetate, sodium acetate, calcium acetate, etc.), carbonates (eg, ammonium carbonate, potassium carbonate, sodium carbonate, calcium carbonate, etc.), phosphates (eg, calcium carbonate, etc.) , 2 sodium hydrogen phosphate, 1 sodium hydrogen phosphate, 2 potassium hydrogen phosphate, 1 potassium hydrogen phosphate, etc.), alkalis such as lactate (eg, sodium lactate, etc.); citric acid, succinic acid, acetic acid, lactic acid, Acids such as glacial acetic acid, hydrochloric acid, and phosphoric acid; can be mentioned.
本発明の血球系細胞の保存方法は、トレハロース類を含み、かつ、ｐＨが４．５～７．２の範囲内である液（すなわち、本件トレハロース類含有液）中で、血球系細胞を保存する工程（以下、「本件保存工程」ということがある）を含むもの（以下、「本件保存方法」ということがある）である。本件保存方法は、通常、血球系細胞を含む本件トレハロース類含有液を、当該液が液体の状態で存在する温度条件下で保存するものであり、当該液が固体の状態で存在する温度条件下で保存する工程（例えば、凍結保存する工程、凍結乾燥保存する工程等の血球系細胞を休眠状態で保存する工程）を含まない。また、当該液中の血球系細胞の密度は、例えば、１０３～１０１０個／ｍＬの範囲内である。
The method for preserving blood cells of the present invention preserves blood cells in a solution containing trehalose and having a pH in the range of 4.5 to 7.2 (that is, the trehalose-containing solution). This includes a process (hereinafter, sometimes referred to as "the present preservation process") (hereinafter, may be referred to as "the present preservation method"). The storage method is usually to store the trehalose-containing solution containing blood cell lines under the temperature condition in which the solution exists in a liquid state, and under the temperature condition in which the solution exists in a solid state. Does not include a step of preserving in a dormant state (for example, a step of preserving blood cells in a dormant state such as a step of cryopreserving and a step of lyophilizing). The density of blood cell lines in the solution is, for example, in the range of 10 3 to 10 10 cells / mL.
本件保存方法としては、本件保存工程の前に、トレハロース類含有液、又は粉体のトレハロース類含有物を添加した液に、血球系細胞を加えるか、或いは、血球系細胞を含む液に、トレハロース類を加えることにより、血球系細胞を含むトレハロース類含有液を調製する工程と、調製した当該液に、必要に応じて、上記ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、血球系細胞を含む本件トレハロース類含有液を得る工程とをさらに含むもの；や、本件保存工程の前に、トレハロース類含有液、又は粉体のトレハロース類含有物を添加した液に、必要に応じて、上記ｐＨ調整剤を添加することにより、ｐＨが４．５～７．２となるように調整し、本件トレハロース類含有液を得る工程と、本件トレハロース類含有液に、血球系細胞を加えることにより、血球系細胞を含む本件トレハロース類含有液を調製する工程とをさらに含むもの；であってもよい。
As the preservation method, trehalose is added to a trehalose-containing solution or a solution to which a powdered trehalose-containing substance is added before the preservation step, or trehalose is added to a solution containing trehalose cells. By adding the above-mentioned trehalose-containing solution containing blood cell lineage cells, and by adding the above-mentioned pH adjuster to the prepared solution, if necessary, the pH is 4.5 to 7. Further including a step of obtaining the trehalose-containing solution containing blood cell lineage cells by adjusting to 2; or, before the storage step, trehalose-containing solution or powdered trehalose-containing substance is added. The step of obtaining the trehalose-containing liquid by adjusting the pH to 4.5 to 7.2 by adding the above-mentioned pH adjuster to the added liquid as necessary, and the process of obtaining the trehalose-containing liquid and the trehalose-containing liquid. It may further include a step of preparing the trehalose-containing solution containing the trehalose cells by adding the trehalose cells to the solution;
本明細書において、「血球系細胞」とは、血液由来の細胞を意味し、血球系細胞には、血液細胞（具体的には、血液細胞造血幹細胞から特定の血液細胞へ分化誘導された細胞）や、造血幹細胞が包含される。ここで造血幹細胞は、骨髄から採取されたものであっても、胚性幹細胞（Embryonic stem cell；ＥＳ細胞）、ＥＧ細胞（Embryonic germ cell）、誘導多能性幹細胞（Induced pluripotent stem cell；ｉＰＳ細胞）等の多能性幹細胞から分化誘導することにより得られたものであってもよい。上記血液細胞としては、例えば、赤血球；リンパ球（例えば、ＣＤ８＋Ｔ細胞、ＣＤ４＋Ｔ細胞、アルファ・ベータ［αβ］Ｔ細胞、ガンマ・デルタ［γδ］Ｔ細胞、細胞傷害性Ｔ細胞［cytotoxic T lymphocyte；ＣＴＬ］、ヘルパーＴ細胞等のＴ細胞；ナチュラルキラー（ＮＫ）細胞；Ｂ細胞）；白血球（例えば、マクロファージ、好中球、好酸球）；血小板；などを挙げることができ、Ｔ細胞を好適に例示することができる。これら細胞は、公知の一般的な方法で単離することができる。例えば、溶血処理した末梢血又は臍帯血試料から、白血球の細胞表面マーカー（ＣＤ４５）；Ｔ細胞の細胞表面マーカー（ＣＤ３）；ＣＤ８＋Ｔ細胞及び細胞傷害性Ｔ細胞の細胞表面マーカー（ＣＤ８）；ＣＤ４＋Ｔ細胞及びヘルパーＴ細胞の細胞表面マーカー（ＣＤ４）；又は、γδＴ細胞の細胞表面マーカー（ＣＤ３９）；に対する抗体を用いた蛍光活性化セルソーター（ＦＡＣＳ）や、蛍光物質やビオチン、アビジン等の標識物質で標識した上記細胞表面マーカーに対する抗体と、かかる標識物質に対する抗体とＭＡＣＳビーズ（磁性ビーズ）とのコンジュゲート抗体とを用いた自動磁気細胞分離装置（ａｕｔｏＭＡＣＳ）により、これら血球系細胞を単離することができる。上記蛍光物質としては、アロフィコシアニン（ＡＰＣ）、フィコエリトリン（ＰＥ）、ＦＩＴＣ（fluorescein isothiocyanate）、Ａｌｅｘａ Ｆｌｕｏｒ ４８８、Ａｌｅｘａ Ｆｌｕｏｒ ６４７、Ａｌｅｘａ Ｆｌｕｏｒ ７００、ＰＥ－Ｔｅｘａｓ Ｒｅｄ、ＰＥ－Ｃｙ５、ＰＥ－Ｃｙ７等を挙げることができる。なお、Ｔ細胞には、ＣＡＲ－Ｔ細胞等の遺伝子改変したＴ細胞も含まれる。
In the present specification, the “blood cell lineage cell” means a cell derived from blood, and the blood cell lineage cell includes a blood cell (specifically, a cell in which differentiation is induced from a blood cell hematopoietic stem cell to a specific blood cell. ) And hematopoietic stem cells are included. Here, the hematopoietic stem cells, even those collected from the bone marrow, are embryonic stem cells (ES cells), EG cells (Embryonic germ cells), and induced pluripotent stem cells (iPS cells). ) Etc. may be obtained by inducing differentiation from pluripotent stem cells. Examples of the blood cells include erythrocytes; lymphocytes (eg, CD8 + T cells, CD4 + T cells, alpha beta [αβ] T cells, gamma delta [γδ] T cells, cytotoxic T cells [cytotoxic T lymphocytes; CTL]. ], T cells such as helper T cells; natural killer (NK) cells; B cells); leukocytes (eg, macrophages, neutrophils, eosinophils); platelets; etc. It can be exemplified. These cells can be isolated by known common methods. For example, from hemolytically treated peripheral or umbilical cord blood samples, leukocyte cell surface markers (CD45); T cell cell surface markers (CD3); CD8 + T cells and cytotoxic T cell cell surface markers (CD8); CD4 + T cells. And helper T cell cell surface marker (CD4); or γδT cell cell surface marker (CD39); labeled with a fluorescence activated cell sorter (FACS) using an antibody, or a fluorescent substance or a labeling substance such as biotin or avidin. These blood cell lineage cells can be isolated by an automatic magnetic cell separator (autoMACS) using an antibody against the above-mentioned cell surface marker, an antibody against such a labeling substance, and a conjugated antibody with MACS beads (magnetic beads). can. Examples of the fluorescent substance 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. The T cells also include genetically modified T cells such as CAR-T cells.
本明細書において、血球系細胞は、通常哺乳動物由来のものであり、ここで哺乳動物としては、マウス、ラット、ハムスター、モルモット等のげっ歯類、ウサギ等のウサギ目、ブタ、ウシ、ヤギ、ウマ、ヒツジ等の有蹄目、イヌ、ネコ等のネコ目、ヒト、サル、アカゲザル、カニクイザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を例示することができ、中でも、マウス、ブタ、ヒトを好適に例示することができる。
In the present specification, the blood cell lineage cells are usually derived from mammals, and the mammals include rodents such as mice, rats, hamsters and guinea pigs, lagomorphs such as rabbits, pigs, cows and goats. , Lagomorphs such as horses and sheep, cats such as dogs and cats, humans, monkeys, red-tailed monkeys, crab monkeys, marmosets, orangutans, chimpanzees and other primates, among others, mice, pigs, humans Can be preferably exemplified.
本明細書において、ｐＨが「４．５～７．２」には、例えば、
４．５～７．１；４．５～７．０；４．５～６．９；４．５～６．８；４．５～６．７；４．５～６．６；４．５～６．５；４．５～６．４；４．５～６．３；４．５～６．２；４．５～６．１；４．５～６．０；４．５～５．９；４．５～５．８；４．５～５．７；４．５～５．６；４．５～５．５；４．５～５．４；４．５～５．３；４．５～５．２；４．５～５．１；４．５～５．０；４．５～４．９；４．５～４．８；４．５～４．７；４．６～７．２；４．７～７．２；４．８～７．２；４．９～７．２；５．０～７．２；５．１～７．２；５．２～７．２；５．３～７．２；５．４～７．２；５．５～７．２；５．６～７．２；５．７～７．２；５．８～７．２；５．９～７．２；６．０～７．２；６．１～７．２；６．２～７．２；６．３～７．２；６．４～７．２；６．５～７．２；６．６～７．２；６．７～７．２；６．８～７．２；６．９～７．２；７．０～７．２；４．６～７．１；４．６～７．０；４．６～６．９；４．６～６．８；４．６～６．７；４．６～６．６；４．６～６．５；４．６～６．４；４．６～６．３；４．６～６．２；４．６～６．１；４．６～６．０；４．６～５．９；４．６～５．８；４．６～５．７；４．６～５．６；４．６～５．５；４．６～５．４；４．６～５．３；４．６～５．２；４．６～５．１；４．６～５．０；４．６～４．９；４．６～４．８；４．７～７．１；４．７～７．０；４．７～６．９；４．７～６．８；４．７～６．７；４．７～６．６；４．７～６．５；４．７～６．４；４．７～６．３；４．７～６．２；４．７～６．１；４．７～６．０；４．７～５．９；４．７～５．８；４．７～５．７；４．７～５．６；４．７～５．５；４．７～５．４；４．７～５．３；４．７～５．２；４．７～５．１；４．７～５．０；４．７～４．９；４．８～７．１；４．８～７．０；４．８～６．９；４．８～６．８；４．８～６．７；４．８～６．６；４．８～６．５；４．８～６．４；４．８～６．３；４．８～６．２；４．８～６．１；４．８～６．０；４．８～５．９；４．８～５．８；４．８～５．７；４．８～５．６；４．８～５．５；４．８～５．４；４．８～５．３；４．８～５．２；４．８～５．１；４．８～５．０；４．９～７．１；４．９～７．０；４．９～６．９；４．９～６．８；４．９～６．７；４．９～６．６；４．９～６．５；４．９～６．４；４．９～６．３；４．９～６．２；４．９～６．１；４．９～６．０；４．９～５．９；４．９～５．８；４．９～５．７；４．９～５．６；４．９～５．５；４．９～５．４；４．９～５．３；４．９～５．２；４．９～５．１；５．０～７．１；５．０～７．０；５．０～６．９；５．０～６．８；５．０～６．７；５．０～６．６；５．０～６．５；５．０～６．４；５．０～６．３；５．０～６．２；５．０～６．１；５．０～６．０；５．０～５．９；５．０～５．８；５．０～５．７；５．０～５．６；５．０～５．５；５．０～５．４；５．０～５．３；５．０～５．２；５．１～７．１；５．１～７．０；５．１～６．９；５．１～６．８；５．１～６．７；５．１～６．６；５．１～６．５；５．１～６．４；５．１～６．３；５．１～６．２；５．１～６．１；５．１～６．０；５．１～５．９；５．１～５．８；５．１～５．７；５．１～５．６；５．１～５．５；５．１～５．４；５．１～５．３；５．２～７．１；５．２～７．０；５．２～６．９；５．２～６．８；５．２～６．７；５．２～６．６；５．２～６．５；５．２～６．４；５．２～６．３；５．２～６．２；５．２～６．１；５．２～６．０；５．２～５．９；５．２～５．８；５．２～５．７；５．２～５．６；５．２～５．５；５．２～５．４；５．３～７．１；５．３～７．０；５．３～６．９；５．３～６．８；５．３～６．７；５．３～６．６；５．３～６．５；５．３～６．４；５．３～６．３；５．３～６．２；５．３～６．１；５．３～６．０；５．３～５．９；５．３～５．８；５．３～５．７；５．３～５．６；５．３～５．５；５．４～７．１；５．４～７．０；５．４～６．９；５．４～６．８；５．４～６．７；５．４～６．６；５．４～６．５；５．４～６．４；５．４～６．３；５．４～６．２；５．４～６．１；５．４～６．０；５．４～５．９；５．４～５．８；５．４～５．７；５．４～５．６；５．５～７．１；５．５～７．０；５．５～６．９；５．５～６．８；５．５～６．７；５．５～６．６；５．５～６．５；５．５～６．４；５．５～６．３；５．５～６．２；５．５～６．１；５．５～６．０；５．５～５．９；５．５～５．８；５．５～５．７；５．６～７．１；５．６～７．０；５．６～６．９；５．６～６．８；５．６～６．７；５．６～６．６；５．６～６．５；５．６～６．４；５．６～６．３；５．６～６．２；５．６～６．１；５．６～６．０；５．６～５．９；５．６～５．８；５．７～７．１；５．７～７．０；５．７～６．９；５．７～６．８；５．７～６．７；５．７～６．６；５．７～６．５；５．７～６．４；５．７～６．３；５．７～６．２；５．７～６．１；５．７～６．０；５．７～５．９；５．８～７．１；５．８～７．０；５．８～６．９；５．８～６．８；５．８～６．７；５．８～６．６；５．８～６．５；５．８～６．４；５．８～６．３；５．８～６．２；５．８～６．１；５．８～６．０；５．９～７．１；５．９～７．０；５．９～６．９；５．９～６．８；５．９～６．７；５．９～６．６；５．９～６．５；５．９～６．４；５．９～６．３；５．９～６．２；５．９～６．１；６．０～７．１；６．０～７．０；６．０～６．９；６．０～６．８；６．０～６．７；６．０～６．６；６．０～６．５；６．０～６．４；６．０～６．３；６．０～６．２；６．１～７．１；６．１～７．０；６．１～６．９；６．１～６．８；６．１～６．７；６．１～６．６；６．１～６．５；６．１～６．４；６．１～６．３；６．２～７．１；６．２～７．０；６．２～６．９；６．２～６．８；６．２～６．７；６．２～６．６；６．２～６．５；６．２～６．４；６．３～７．１；６．３～７．０；６．３～６．９；６．３～６．８；６．３～６．７；６．３～６．６；６．３～６．５；６．４～７．１；６．４～７．０；６．４～６．９；６．４～６．８；６．４～６．７；６．４～６．６；６．５～７．１；６．５～７．０；６．５～６．９；６．５～６．８；６．５～６．７；６．６～７．１；６．６～７．０；６．６～６．９；６．６～６．８；６．７～７．１；６．７～７．０；６．７～６．９；６．８～７．１；６．８～７．０；６．９～７．１；等が含まれる。 In the present specification, when the pH is "4.5 to 7.2", for example,
4.5-7.1; 4.5-7.0; 4.5-6.9; 4.5-6.8; 4.5-6.7; 4.5-6.6; 4. 5 to 6.5; 4.5 to 6.4; 4.5 to 6.3; 4.5 to 6.2; 4.5 to 6.1; 4.5 to 6.0; 4.5 to 5.9; 4.5 to 5.8; 4.5 to 5.7; 4.5 to 5.6; 4.5 to 5.5; 4.5 to 5.4; 4.5 to 5. 3; 4.5-5.2; 4.5-5.1; 4.5-5.0; 4.5-4.9; 4.5-4.8; 4.5-4.7; 4.6-7.2; 4.7-7.2; 4.8-7.2; 4.9-7.2; 5.0-7.2; 5.1-7.2; 5. 2-7.2; 5.3-7.2; 5.4-7.2; 5.5-7.2; 5.6-7.2; 5.7-7.2; 5.8- 7.2; 5.9 to 7.2; 6.0 to 7.2; 6.1 to 7.2; 6.2 to 7.2; 6.3 to 7.2; 6.4 to 7. 2; 6.5-7.2; 6.6-7.2; 6.7-7.2; 6.8-7.2; 6.9-7.2; 7.0-7.2; 4.6-7.1; 4.6-7.0; 4.6-6.9; 4.6-6.8; 4.6-6.7; 4.6-6.6; 4. 6-6.5; 4.6-6.4; 4.6-6.3; 4.6-6.2; 4.6-6.1; 4.6-6.0; 4.6- 5.9; 4.6 to 5.8; 4.6 to 5.7; 4.6 to 5.6; 4.6 to 5.5; 4.6 to 5.4; 4.6 to 5. 3; 4.6-5.2; 4.6-5.1; 4.6-5.0; 4.6-4.9; 4.6-4.8; 4.7-7.1; 4.7-7.0; 4.7-6.9; 4.7-6.8; 4.7-6.7; 4.7-6.6; 4.7-6.5; 4. 7-6.4; 4.7-6.3; 4.7-6.2; 4.7-6.1; 4.7-6.0; 4.7-5.9; 4.7- 5.8; 4.7-5.7; 4.7-5.6; 4.7-5.5; 4.7-5.4; 4.7-5.3; 4.7-5.5. 2; 4.7-5.1; 4.7-5.0; 4.7-4.9; 4.8-7.1; 4.8-7.0; 4.8-6.9; 4.8 to 6.8; 4.8 to 6.7; 4.8 to 6.6; 4.8 to 6.5; 4.8 to 6.4; 4.8 to 6.3; 4. 8 to 6.2; 4.8 to 6.1; 4.8 to 6.0; 4.8 to 5.9; 4.8 to 5.8; 4.8 to 5.7; 4.8 to 5.6; 4.8-5.5; 4.8-5.4; 4.8-5.3; 4.8-5.2; 4.8-5.1; 4.8-5.5. 0; 4.9 to 7.1; 4.9 to 7.0; 4.9 to 6.9; 4.9 to 6.8; 4.9 to 6.7; 4.9 to 6.6; 4.9-6.5; 4.9-6.4; 4.9-6.3; 4.9-6.2; 4.9-6.1; 4.9-6.0; 4. 9-5.9; 4.9-5.8; 4.9-5.7; 4.9-5.6; 4.9-5.5; 4.9-5.4; 4.9- 5.3; 4.9-5.2; 4.9-5.1; 5.0-7.1; 5.0-7.0; 5.0-6.9; 5.0-6. 8; 5.0-6.7; 5.0-6.6; 5.0-6.5; 5.0-6.4; 5.0-6.3; 5.0-6.2; 5.0-6.1; 5.0-6.0; 5.0-5.9; 5.0-5.8; 5.0-5.7; 5.0-5.6; 5. 0-5.5; 5.0-5.4; 5.0-5.3; 5.0-5.2; 5.1-7.1; 5.1-7.0; 5.1- 6.9; 5.1 to 6.8; 5.1 to 6.7; 5.1 to 6.6; 5.1 to 6.5; 5.1 to 6.4; 5.1 to 6. 3; 5.1 to 6.2; 5.1 to 6.1; 5.1 to 6.0; 5.1 to 5.9; 5.1 to 5.8; 5.1 to 5.7; 5.1 to 5.6; 5.1 to 5.5; 5.1 to 5.4; 5.1 to 5.3; 5.2 to 7.1; 5.2 to 7.0; 5. 2-6.9; 5.2-6.8; 5.2-6.7; 5.2-6.6; 5.2-6.5; 5.2-6.4; 5.2- 6.3; 5.2 to 6.2; 5.2 to 6.1; 5.2 to 6.0; 5.2 to 5.9; 5.2 to 5.8; 5.2 to 5. 7; 5.2 to 5.6; 5.2 to 5.5; 5.2 to 5.4; 5.3 to 7.1; 5.3 to 7.0; 5.3 to 6.9; 5.3-6.8; 5.3-6.7; 5.3-6.6; 5.3-6.5; 5.3-6.4; 5.3-6.3; 5. 3-6.2; 5.3-6.1; 5.3-6.0; 5.3-5.9; 5.3-5.8; 5.3-5.7; 5.3- 5.6; 5.3-5.5; 5.4-7.1; 5.4-7.0; 5.4-6.9; 5.4-6.8; 5.4-6. 7; 5.4 to 6.6; 5.4 to 6.5; 5.4 to 6.4; 5.4 to 6.3; 5.4 to 6.2; 5.4 to 6.1; 5.4 to 6.0; 5.4 to 5.9; 5.4 to 5.8; 5.4 to 5.7; 5.4 to 5.6; 5.5 to 7.1; 5. 5 to 7.0; 5.5 to 6.9; 5.5 to 6.8; 5.5 to 6.7; 5.5 to 6.6; 5.5 to 6.5; 5.5 to 6.4; 5.5 to 6.3; 5.5 to 6.2; 5.5 to 6.1; 5.5 to 6.0; 5.5 to 5.9; 5.5 to 5.5. 8; 5.5 to 5.7; 5.6 to 7.1; 5.6 to 7.0; 5.6 to 6.9; 5.6 to 6.8; 5.6 to 6.7; 5.6 to 6.6; 5.6 to 6.5; 5.6 to 6.4; 5.6 to 6.3; 5.6 to 6.2; 5.6 to 6.1; 5. 6-6.0; 5.6-5.9; 5.6-5.8; 5.7-7.1; 5.7-7.0; 5.7-6.9; 5.7- 6.8; 5.7 to 6.7; 5.7 to 6.6; 5.7 to 6.5; 5.7 to 6.4; 5.7 to 6.3; 5.7 to 6.6. 2; 5.7 to 6.1; 5.7 to 6.0; 5.7 to 5.9; 5.8 to 7.1; 5.8 to 7.0; 5.8 to 6.9; 5.8-6.8; 5.8-6.7; 5.8-6.6; 5.8-6.5; 5.8-6.4; 5.8-6.3; 5. 8 to 6.2; 5.8 to 6.1; 5.8 to 6.0; 5.9 to 7.1; 5.9 to 7.0; 5.9 to 6.9; 5.9 to 6.8; 5.9 to 6.7; 5.9 to 6.6; 5.9 to 6.5; 5.9 to 6.4; 5.9 to 6.3; 5.9 to 6. 2; 5.9 to 6.1; 6.0 to 7.1; 6.0 to 7.0; 6.0 to 6.9; 6.0 to 6.8; 6.0 to 6.7; 6.0 to 6.6; 6.0 to 6.5; 6.0 to 6.4; 6.0 to 6.3; 6.0 to 6.2; 6.1 to 7.1; 6. 1 to 7.0; 6.1 to 6.9; 6.1 to 6.8; 6.1 to 6.7; 6.1 to 6.6; 6.1 to 6.5; 6.1 to 6.4; 6.1 to 6.3; 6.2 to 7.1; 6.2 to 7.0; 6.2 to 6.9; 6.2 to 6.8; 6.2 to 6. 7; 6.2 to 6.6; 6.2 to 6.5; 6.2 to 6.4; 6.3 to 7.1; 6.3 to 7.0; 6.3 to 6.9; 6.3-6.8; 6.3-6.7; 6.3-6.6; 6.3-6.5; 6.4-7.1; 6.4-7.0; 6. 4 to 6.9; 6.4 to 6.8; 6.4 to 6.7; 6.4 to 6.6; 6.5 to 7.1; 6.5 to 7.0; 6.5 to 6.9; 6.5 to 6.8; 6.5 to 6.7; 6.6 to 7.1; 6.6 to 7.0; 6.6 to 6.9; 6.6 to 6.6. 8; 6.7 to 7.1; 6.7 to 7.0; 6.7 to 6.9; 6.8 to 7.1; 6.8 to 7.0; 6.9 to 7.1; Etc. are included.
４．５～７．１；４．５～７．０；４．５～６．９；４．５～６．８；４．５～６．７；４．５～６．６；４．５～６．５；４．５～６．４；４．５～６．３；４．５～６．２；４．５～６．１；４．５～６．０；４．５～５．９；４．５～５．８；４．５～５．７；４．５～５．６；４．５～５．５；４．５～５．４；４．５～５．３；４．５～５．２；４．５～５．１；４．５～５．０；４．５～４．９；４．５～４．８；４．５～４．７；４．６～７．２；４．７～７．２；４．８～７．２；４．９～７．２；５．０～７．２；５．１～７．２；５．２～７．２；５．３～７．２；５．４～７．２；５．５～７．２；５．６～７．２；５．７～７．２；５．８～７．２；５．９～７．２；６．０～７．２；６．１～７．２；６．２～７．２；６．３～７．２；６．４～７．２；６．５～７．２；６．６～７．２；６．７～７．２；６．８～７．２；６．９～７．２；７．０～７．２；４．６～７．１；４．６～７．０；４．６～６．９；４．６～６．８；４．６～６．７；４．６～６．６；４．６～６．５；４．６～６．４；４．６～６．３；４．６～６．２；４．６～６．１；４．６～６．０；４．６～５．９；４．６～５．８；４．６～５．７；４．６～５．６；４．６～５．５；４．６～５．４；４．６～５．３；４．６～５．２；４．６～５．１；４．６～５．０；４．６～４．９；４．６～４．８；４．７～７．１；４．７～７．０；４．７～６．９；４．７～６．８；４．７～６．７；４．７～６．６；４．７～６．５；４．７～６．４；４．７～６．３；４．７～６．２；４．７～６．１；４．７～６．０；４．７～５．９；４．７～５．８；４．７～５．７；４．７～５．６；４．７～５．５；４．７～５．４；４．７～５．３；４．７～５．２；４．７～５．１；４．７～５．０；４．７～４．９；４．８～７．１；４．８～７．０；４．８～６．９；４．８～６．８；４．８～６．７；４．８～６．６；４．８～６．５；４．８～６．４；４．８～６．３；４．８～６．２；４．８～６．１；４．８～６．０；４．８～５．９；４．８～５．８；４．８～５．７；４．８～５．６；４．８～５．５；４．８～５．４；４．８～５．３；４．８～５．２；４．８～５．１；４．８～５．０；４．９～７．１；４．９～７．０；４．９～６．９；４．９～６．８；４．９～６．７；４．９～６．６；４．９～６．５；４．９～６．４；４．９～６．３；４．９～６．２；４．９～６．１；４．９～６．０；４．９～５．９；４．９～５．８；４．９～５．７；４．９～５．６；４．９～５．５；４．９～５．４；４．９～５．３；４．９～５．２；４．９～５．１；５．０～７．１；５．０～７．０；５．０～６．９；５．０～６．８；５．０～６．７；５．０～６．６；５．０～６．５；５．０～６．４；５．０～６．３；５．０～６．２；５．０～６．１；５．０～６．０；５．０～５．９；５．０～５．８；５．０～５．７；５．０～５．６；５．０～５．５；５．０～５．４；５．０～５．３；５．０～５．２；５．１～７．１；５．１～７．０；５．１～６．９；５．１～６．８；５．１～６．７；５．１～６．６；５．１～６．５；５．１～６．４；５．１～６．３；５．１～６．２；５．１～６．１；５．１～６．０；５．１～５．９；５．１～５．８；５．１～５．７；５．１～５．６；５．１～５．５；５．１～５．４；５．１～５．３；５．２～７．１；５．２～７．０；５．２～６．９；５．２～６．８；５．２～６．７；５．２～６．６；５．２～６．５；５．２～６．４；５．２～６．３；５．２～６．２；５．２～６．１；５．２～６．０；５．２～５．９；５．２～５．８；５．２～５．７；５．２～５．６；５．２～５．５；５．２～５．４；５．３～７．１；５．３～７．０；５．３～６．９；５．３～６．８；５．３～６．７；５．３～６．６；５．３～６．５；５．３～６．４；５．３～６．３；５．３～６．２；５．３～６．１；５．３～６．０；５．３～５．９；５．３～５．８；５．３～５．７；５．３～５．６；５．３～５．５；５．４～７．１；５．４～７．０；５．４～６．９；５．４～６．８；５．４～６．７；５．４～６．６；５．４～６．５；５．４～６．４；５．４～６．３；５．４～６．２；５．４～６．１；５．４～６．０；５．４～５．９；５．４～５．８；５．４～５．７；５．４～５．６；５．５～７．１；５．５～７．０；５．５～６．９；５．５～６．８；５．５～６．７；５．５～６．６；５．５～６．５；５．５～６．４；５．５～６．３；５．５～６．２；５．５～６．１；５．５～６．０；５．５～５．９；５．５～５．８；５．５～５．７；５．６～７．１；５．６～７．０；５．６～６．９；５．６～６．８；５．６～６．７；５．６～６．６；５．６～６．５；５．６～６．４；５．６～６．３；５．６～６．２；５．６～６．１；５．６～６．０；５．６～５．９；５．６～５．８；５．７～７．１；５．７～７．０；５．７～６．９；５．７～６．８；５．７～６．７；５．７～６．６；５．７～６．５；５．７～６．４；５．７～６．３；５．７～６．２；５．７～６．１；５．７～６．０；５．７～５．９；５．８～７．１；５．８～７．０；５．８～６．９；５．８～６．８；５．８～６．７；５．８～６．６；５．８～６．５；５．８～６．４；５．８～６．３；５．８～６．２；５．８～６．１；５．８～６．０；５．９～７．１；５．９～７．０；５．９～６．９；５．９～６．８；５．９～６．７；５．９～６．６；５．９～６．５；５．９～６．４；５．９～６．３；５．９～６．２；５．９～６．１；６．０～７．１；６．０～７．０；６．０～６．９；６．０～６．８；６．０～６．７；６．０～６．６；６．０～６．５；６．０～６．４；６．０～６．３；６．０～６．２；６．１～７．１；６．１～７．０；６．１～６．９；６．１～６．８；６．１～６．７；６．１～６．６；６．１～６．５；６．１～６．４；６．１～６．３；６．２～７．１；６．２～７．０；６．２～６．９；６．２～６．８；６．２～６．７；６．２～６．６；６．２～６．５；６．２～６．４；６．３～７．１；６．３～７．０；６．３～６．９；６．３～６．８；６．３～６．７；６．３～６．６；６．３～６．５；６．４～７．１；６．４～７．０；６．４～６．９；６．４～６．８；６．４～６．７；６．４～６．６；６．５～７．１；６．５～７．０；６．５～６．９；６．５～６．８；６．５～６．７；６．６～７．１；６．６～７．０；６．６～６．９；６．６～６．８；６．７～７．１；６．７～７．０；６．７～６．９；６．８～７．１；６．８～７．０；６．９～７．１；等が含まれる。 In the present specification, when the pH is "4.5 to 7.2", for example,
4.5-7.1; 4.5-7.0; 4.5-6.9; 4.5-6.8; 4.5-6.7; 4.5-6.6; 4. 5 to 6.5; 4.5 to 6.4; 4.5 to 6.3; 4.5 to 6.2; 4.5 to 6.1; 4.5 to 6.0; 4.5 to 5.9; 4.5 to 5.8; 4.5 to 5.7; 4.5 to 5.6; 4.5 to 5.5; 4.5 to 5.4; 4.5 to 5. 3; 4.5-5.2; 4.5-5.1; 4.5-5.0; 4.5-4.9; 4.5-4.8; 4.5-4.7; 4.6-7.2; 4.7-7.2; 4.8-7.2; 4.9-7.2; 5.0-7.2; 5.1-7.2; 5. 2-7.2; 5.3-7.2; 5.4-7.2; 5.5-7.2; 5.6-7.2; 5.7-7.2; 5.8- 7.2; 5.9 to 7.2; 6.0 to 7.2; 6.1 to 7.2; 6.2 to 7.2; 6.3 to 7.2; 6.4 to 7. 2; 6.5-7.2; 6.6-7.2; 6.7-7.2; 6.8-7.2; 6.9-7.2; 7.0-7.2; 4.6-7.1; 4.6-7.0; 4.6-6.9; 4.6-6.8; 4.6-6.7; 4.6-6.6; 4. 6-6.5; 4.6-6.4; 4.6-6.3; 4.6-6.2; 4.6-6.1; 4.6-6.0; 4.6- 5.9; 4.6 to 5.8; 4.6 to 5.7; 4.6 to 5.6; 4.6 to 5.5; 4.6 to 5.4; 4.6 to 5. 3; 4.6-5.2; 4.6-5.1; 4.6-5.0; 4.6-4.9; 4.6-4.8; 4.7-7.1; 4.7-7.0; 4.7-6.9; 4.7-6.8; 4.7-6.7; 4.7-6.6; 4.7-6.5; 4. 7-6.4; 4.7-6.3; 4.7-6.2; 4.7-6.1; 4.7-6.0; 4.7-5.9; 4.7- 5.8; 4.7-5.7; 4.7-5.6; 4.7-5.5; 4.7-5.4; 4.7-5.3; 4.7-5.5. 2; 4.7-5.1; 4.7-5.0; 4.7-4.9; 4.8-7.1; 4.8-7.0; 4.8-6.9; 4.8 to 6.8; 4.8 to 6.7; 4.8 to 6.6; 4.8 to 6.5; 4.8 to 6.4; 4.8 to 6.3; 4. 8 to 6.2; 4.8 to 6.1; 4.8 to 6.0; 4.8 to 5.9; 4.8 to 5.8; 4.8 to 5.7; 4.8 to 5.6; 4.8-5.5; 4.8-5.4; 4.8-5.3; 4.8-5.2; 4.8-5.1; 4.8-5.5. 0; 4.9 to 7.1; 4.9 to 7.0; 4.9 to 6.9; 4.9 to 6.8; 4.9 to 6.7; 4.9 to 6.6; 4.9-6.5; 4.9-6.4; 4.9-6.3; 4.9-6.2; 4.9-6.1; 4.9-6.0; 4. 9-5.9; 4.9-5.8; 4.9-5.7; 4.9-5.6; 4.9-5.5; 4.9-5.4; 4.9- 5.3; 4.9-5.2; 4.9-5.1; 5.0-7.1; 5.0-7.0; 5.0-6.9; 5.0-6. 8; 5.0-6.7; 5.0-6.6; 5.0-6.5; 5.0-6.4; 5.0-6.3; 5.0-6.2; 5.0-6.1; 5.0-6.0; 5.0-5.9; 5.0-5.8; 5.0-5.7; 5.0-5.6; 5. 0-5.5; 5.0-5.4; 5.0-5.3; 5.0-5.2; 5.1-7.1; 5.1-7.0; 5.1- 6.9; 5.1 to 6.8; 5.1 to 6.7; 5.1 to 6.6; 5.1 to 6.5; 5.1 to 6.4; 5.1 to 6. 3; 5.1 to 6.2; 5.1 to 6.1; 5.1 to 6.0; 5.1 to 5.9; 5.1 to 5.8; 5.1 to 5.7; 5.1 to 5.6; 5.1 to 5.5; 5.1 to 5.4; 5.1 to 5.3; 5.2 to 7.1; 5.2 to 7.0; 5. 2-6.9; 5.2-6.8; 5.2-6.7; 5.2-6.6; 5.2-6.5; 5.2-6.4; 5.2- 6.3; 5.2 to 6.2; 5.2 to 6.1; 5.2 to 6.0; 5.2 to 5.9; 5.2 to 5.8; 5.2 to 5. 7; 5.2 to 5.6; 5.2 to 5.5; 5.2 to 5.4; 5.3 to 7.1; 5.3 to 7.0; 5.3 to 6.9; 5.3-6.8; 5.3-6.7; 5.3-6.6; 5.3-6.5; 5.3-6.4; 5.3-6.3; 5. 3-6.2; 5.3-6.1; 5.3-6.0; 5.3-5.9; 5.3-5.8; 5.3-5.7; 5.3- 5.6; 5.3-5.5; 5.4-7.1; 5.4-7.0; 5.4-6.9; 5.4-6.8; 5.4-6. 7; 5.4 to 6.6; 5.4 to 6.5; 5.4 to 6.4; 5.4 to 6.3; 5.4 to 6.2; 5.4 to 6.1; 5.4 to 6.0; 5.4 to 5.9; 5.4 to 5.8; 5.4 to 5.7; 5.4 to 5.6; 5.5 to 7.1; 5. 5 to 7.0; 5.5 to 6.9; 5.5 to 6.8; 5.5 to 6.7; 5.5 to 6.6; 5.5 to 6.5; 5.5 to 6.4; 5.5 to 6.3; 5.5 to 6.2; 5.5 to 6.1; 5.5 to 6.0; 5.5 to 5.9; 5.5 to 5.5. 8; 5.5 to 5.7; 5.6 to 7.1; 5.6 to 7.0; 5.6 to 6.9; 5.6 to 6.8; 5.6 to 6.7; 5.6 to 6.6; 5.6 to 6.5; 5.6 to 6.4; 5.6 to 6.3; 5.6 to 6.2; 5.6 to 6.1; 5. 6-6.0; 5.6-5.9; 5.6-5.8; 5.7-7.1; 5.7-7.0; 5.7-6.9; 5.7- 6.8; 5.7 to 6.7; 5.7 to 6.6; 5.7 to 6.5; 5.7 to 6.4; 5.7 to 6.3; 5.7 to 6.6. 2; 5.7 to 6.1; 5.7 to 6.0; 5.7 to 5.9; 5.8 to 7.1; 5.8 to 7.0; 5.8 to 6.9; 5.8-6.8; 5.8-6.7; 5.8-6.6; 5.8-6.5; 5.8-6.4; 5.8-6.3; 5. 8 to 6.2; 5.8 to 6.1; 5.8 to 6.0; 5.9 to 7.1; 5.9 to 7.0; 5.9 to 6.9; 5.9 to 6.8; 5.9 to 6.7; 5.9 to 6.6; 5.9 to 6.5; 5.9 to 6.4; 5.9 to 6.3; 5.9 to 6. 2; 5.9 to 6.1; 6.0 to 7.1; 6.0 to 7.0; 6.0 to 6.9; 6.0 to 6.8; 6.0 to 6.7; 6.0 to 6.6; 6.0 to 6.5; 6.0 to 6.4; 6.0 to 6.3; 6.0 to 6.2; 6.1 to 7.1; 6. 1 to 7.0; 6.1 to 6.9; 6.1 to 6.8; 6.1 to 6.7; 6.1 to 6.6; 6.1 to 6.5; 6.1 to 6.4; 6.1 to 6.3; 6.2 to 7.1; 6.2 to 7.0; 6.2 to 6.9; 6.2 to 6.8; 6.2 to 6. 7; 6.2 to 6.6; 6.2 to 6.5; 6.2 to 6.4; 6.3 to 7.1; 6.3 to 7.0; 6.3 to 6.9; 6.3-6.8; 6.3-6.7; 6.3-6.6; 6.3-6.5; 6.4-7.1; 6.4-7.0; 6. 4 to 6.9; 6.4 to 6.8; 6.4 to 6.7; 6.4 to 6.6; 6.5 to 7.1; 6.5 to 7.0; 6.5 to 6.9; 6.5 to 6.8; 6.5 to 6.7; 6.6 to 7.1; 6.6 to 7.0; 6.6 to 6.9; 6.6 to 6.6. 8; 6.7 to 7.1; 6.7 to 7.0; 6.7 to 6.9; 6.8 to 7.1; 6.8 to 7.0; 6.9 to 7.1; Etc. are included.
本明細書において、「４．５～７．２のｐＨ」は、通常、室温（具体的には、２０～２８℃の範囲内、好ましくは２４～２６℃、より好ましくは２５℃）条件下で測定した値である。したがって、室温以外の温度（例えば、冷温［０～１０℃等］）条件下で、ｐＨが４．５～７．２の範囲外のトレハロース類含有液であっても、室温条件下にしたときにｐＨが４．５～７．２となるトレハロース類含有液であって、「血球系細胞を保存するため」に用いるものは、本件血球系細胞保存用液に包含される。また、本明細書において、「４．５～７．２のｐＨ」には、室温条件下で測定したｐＨの値を、小数点以下第２位で四捨五入した場合に、４．５～７．２となるｐＨ、具体的には、４．４５以上でかつ、７．２５未満のｐＨも含まれる。
In the present specification, "pH of 4.5 to 7.2" is usually under room temperature (specifically, in the range of 20 to 28 ° C., preferably 24-26 ° C., more preferably 25 ° C.). It is a value measured in. Therefore, even if the pH is a trehalose-containing solution outside the range of 4.5 to 7.2 under a temperature other than room temperature (for example, cold temperature [0 to 10 ° C.]), when the trehalose-containing solution is subjected to room temperature conditions. A trehalose-containing solution having a pH of 4.5 to 7.2 and used for "preserving blood cell lineage cells" is included in the blood cell lineage cell preservation solution. Further, in the present specification, "pH of 4.5 to 7.2" is 4.5 to 7.2 when the value of pH measured under room temperature conditions is rounded to the second decimal place. The pH to be, specifically, the pH of 4.45 or more and less than 7.25 is also included.
本件血球系細胞保存用液や本件トレハロース類含有液は、血球系細胞の保存が可能な液（例えば、等張液、低張液、高張液）であり、等張液が好ましい。本明細書において「等張液」とは、体液や細胞液の浸透圧とほぼ同じ浸透圧を有する液を意味し、具体的には、２５０～３８０ｍＯｓｍ／Ｌの範囲内の浸透圧を有する液を意味する。また、本明細書において「低張液」とは、体液や細胞液の浸透圧よりも低い浸透圧を有する液を意味し、具体的には、２５０ｍＯｓｍ／Ｌ未満の浸透圧を有する液を意味する。かかる低張液としては、細胞が破裂しない程度の低張液（具体的には、１００～２５０ｍＯｓｍ／Ｌ未満の範囲内の浸透圧を有する液）が好ましい。また、本明細書において「高張液」とは、体液や細胞液の浸透圧よりも高い浸透圧を有する液を意味し、具体的には、浸透圧が３８０ｍＯｓｍ／Ｌ超（好ましくは３８０ｍＯｓｍ／Ｌ超～１０００ｍＯｓｍ／Ｌの範囲内）を意味する。
The blood cell cell preservation solution and the trehalose-containing solution are solutions capable of preserving blood cell cells (for example, isotonic solution, hypotonic solution, hypertonic solution), and isotonic solution is preferable. As used herein, the term "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. Further, in the present specification, 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. do. As such 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. Further, in the present specification, 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).
上記等張液としては、体液や細胞液の浸透圧とほぼ同じになるようにナトリウムイオン、カリウムイオン、カルシウムイオン等によって塩濃度や糖濃度等を調整した等張液であれば特に制限されず、具体的には生理食塩水や、緩衝効果のある生理食塩水（例えば、ＰＢＳ、トリス緩衝生理食塩水［Tris Buffered Saline；ＴＢＳ］、ＨＥＰＥＳ緩衝生理食塩水）、リンゲル液、乳酸リンゲル液、酢酸リンゲル液、重炭酸リンゲル液、５％グルコース水溶液、動物細胞培養用基礎培地（例えば、ＤＭＥＭ、ＥＭＥＭ、ＲＰＭＩ－１６４０、α－ＭＥＭ、Ｆ－１２、Ｆ－１０、Ｍ－１９９）、等張剤（例えば、ブドウ糖、Ｄ－ソルビトール、Ｄ－マンニトール、ラクトース、塩化ナトリウム）等を挙げることができ、これらの中でも乳酸リンゲル液が好ましい。等張液は、市販のものであっても、自ら調製したものであってもよい。市販のものとしては、大塚生食注（大塚製薬工場社製）（生理食塩液）、リンゲル液「オーツカ」（大塚製薬工場社製）（リンゲル液）、ラクテック（登録商標）注（大塚製薬工場社製）（乳酸リンゲル液）、ヴィーン（登録商標）Ｆ輸液（扶桑薬品工業社製）（酢酸リンゲル液）、大塚糖液５％（大塚製薬工場社製）（５％グルコース水溶液）、ビカネイト（登録商標）輸液（大塚製薬工場社製）（重炭酸リンゲル液）を挙げることができる。
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 be substantially the same as the osmotic pressure of body fluid or cell fluid. , Specifically, physiological saline, physiological saline having 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, α-MEM, F-12, F-10, M-199), isotonic (eg, glucose) , D-sorbitol, D-mannitol, lactose, sodium chloride) and the like, and among these, lactated Ringer's solution is preferable. 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). (Lactated Ringer's solution), Vein (registered trademark) F infusion (manufactured by Fuso Pharmaceutical Co., Ltd.) (Ringel acetate solution), Otsuka sugar solution 5% (manufactured by Otsuka Pharmaceutical Factory) (5% glucose aqueous solution), Vicanate (registered trademark) infusion (registered trademark) Otsuka Pharmaceutical Factory Co., Ltd.) (Ringel bicarbonate solution) can be mentioned.
上記トレハロース類におけるトレハロースとしては、２つのα-グルコースが１，１－グリコシド結合した二糖類であるα，α－トレハロースの他に、α-グルコースとβ－グルコースとが１，１－グリコシド結合した二糖類であるα，β－トレハロースや、２つのβ－グルコースが１，１－グリコシド結合した二糖類であるβ，β－トレハロースを挙げることができるが、これらの中でもα，α－トレハロースが好ましい。これらトレハロースは、化学合成、微生物による生産、酵素による生産等のいずれの公知の方法によっても製造することができるが、市販品を用いることもできる。例えば、α，α－トレハロース二水和物（富士フイルム和光純薬社製）、α，α－トレハロース二水和物（和光純薬社製）、α，α－トレハロース二水和物（林原社製）などの市販品を挙げることができる。
As trehalose in the above trehalose, in addition to α and α-trehalose, which are disaccharides in which two α-glucoses are 1,1-glycosidated, α-glucose and β-glucose are 1,1-glycosidated. Examples thereof include α and β-trehalose, which are disaccharides, and β, β-trehalose, which is a disaccharide in which two β-glucoses are 1,1-glycosidated, and among these, α and α-trehalose are preferable. .. These 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. For example, α, α-trehalose dihydrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), α, α-trehalose dihydrate (manufactured by Wako Pure Chemical Industries, Ltd.), α, α-trehalose dihydrate (manufactured by Hayashibara Co., Ltd.) (Manufactured) and other commercially available products can be mentioned.
上記トレハロース類におけるトレハロース誘導体としては、二糖類のトレハロースに１又は複数の糖単位が結合したグリコシルトレハロース類であれば特に制限されず、グリコシルトレハロース類には、グルコシルトレハロース、マルトシルトレハロース、マルトトリオシルトレハロースなどが含まれる。
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.
上記トレハロース類におけるトレハロースやその誘導体の塩としては、例えば塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、リン酸塩、硝酸塩、硫酸塩、酢酸塩、プロピオン酸塩、トルエンスルホン酸塩、コハク酸塩、シュウ酸塩、乳酸塩、酒石酸塩、グリコール酸塩、メタンスルホン酸塩、酪酸塩、吉草酸塩、クエン酸塩、フマル酸塩、マレイン酸塩、リンゴ酸塩等の酸付加塩や、ナトリウム塩、カリウム塩、カルシウム塩等の金属塩や、アンモニウム塩、アルキルアンモニウム塩などを挙げることができる。なお、これらの塩は使用時において溶液として用いられ、その作用は、トレハロースの場合と同効なものが好ましい。これらの塩類は、水和物又は溶媒和物を形成していてもよく、またいずれかを単独で又は２種以上を適宜組み合わせて用いることができる。
Examples of 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. In addition, 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.
本件血球系細胞保存用液や本件トレハロース類含有液中のトレハロース類の濃度としては、トレハロース類による細胞生存率低下の抑制効果、細胞凝集の抑制効果、及び／又は、保存容器への細胞接着の抑制効果が発揮される濃度であればよく、例えば、トレハロース換算で０．１（ｗ／ｖ）％以上、好ましくは０．３（ｗ／ｖ）％以上、より好ましくは０．６（ｗ／ｖ）％以上、さらに好ましくは１．０（ｗ／ｖ）％以上、最も好ましくは２．０（ｗ／ｖ）％以上である。また、細胞への悪影響を回避する観点から、例えば、トレハロース換算で４０（ｗ／ｖ）％以下、好ましくは２０（ｗ／ｖ）％以下、より好ましくは１５（ｗ／ｖ）％以下、さらに好ましくは１０（ｗ／ｖ）％以下、最も好ましくは６．０（ｗ／ｖ）％以下である。したがって、本件血球系細胞保存用液や本件トレハロース類含有液中のトレハロース類の濃度としては、例えば、トレハロース換算で０．１～４０（ｗ／ｖ）％の範囲内、好ましくは０．３～２０（ｗ／ｖ）％、より好ましくは０．６～１５（ｗ／ｖ）％、さらに好ましくは１．０～１０（ｗ／ｖ）％、最も好ましくは２．０～６．０（ｗ／ｖ）％である。
The concentration of trehalose in the blood cell cell preservation solution and the trehalose-containing solution is as follows: the effect of suppressing the decrease in cell viability by the trehalose, the effect of suppressing cell aggregation, and / or the effect of cell adhesion to the storage container. The concentration may be any concentration at which the inhibitory effect is exhibited, for example, 0.1 (w / v)% or more, preferably 0.3 (w / v)% or more, more preferably 0.6 (w / v) in terms of trehalose. v)% or more, more preferably 1.0 (w / v)% or more, and most preferably 2.0 (w / v)% or more. Further, from the viewpoint of avoiding adverse effects on cells, for example, 40 (w / v)% or less, preferably 20 (w / v)% or less, more preferably 15 (w / v)% or less in terms of trehalose, and further. It is preferably 10 (w / v)% or less, and most preferably 6.0 (w / v)% or less. Therefore, the concentration of trehaloses in the blood cell preservation solution and the trehalose-containing solution is, for example, in the range of 0.1 to 40 (w / v)% in terms of trehalose, preferably 0.3 to 0.3. 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)%.
本件血球系細胞保存用液は、血球系細胞を含む本件トレハロース類含有液が液体の状態で存在する温度条件下で、血球系細胞を任意の期間保存するために用いるものである。本件血球系細胞保存用液は、血球系細胞を液体中で保存したときに生じる細胞生存率低下、細胞凝集、及び／又は、保存容器への細胞接着を効果的に抑制する作用を有し、かつ、当該液を哺乳動物に投与したときに、哺乳動物の生態に悪影響を及ぼす可能性の低い液である。このため、本件血球系細胞保存用液は、さらに、「血球系細胞の生存率低下を抑制するため」という用途；「血球系細胞の細胞凝集を抑制するため」という用途；「血球系細胞の保存溶液への接着を抑制するための」という用途；及び／又は「血球系細胞を移植するため」という用途；に特定されたものが好ましい。
The Blood Cell Cell Preservation Solution is used to store blood cells for an arbitrary period under temperature conditions in which the trehalose-containing solution containing blood cell cells exists in a liquid state. The Blood Cell Cell Preservation Solution has the effect of effectively suppressing cell viability reduction, cell aggregation, and / or cell adhesion to the storage container, which occurs when blood cell cells are stored in the liquid. Moreover, when the solution is administered to a mammal, it is unlikely to adversely affect the ecology of the mammal. Therefore, the Blood Cell Cell Preservation Solution is further used for "to suppress a decrease in the viability of blood cells"; "to suppress cell aggregation of blood cells"; "for suppressing blood cell cell aggregation". Those specified for "inhibiting adhesion to storage solutions"; and / or "for transplanting blood cell lineage cells"; are preferred.
本件血球系細胞保存用液や本件トレハロース類含有液は、トレハロース類を、単独で含む液であってもよいし、トレハロース類から選択される２種類以上を含む液や、トレハロース類以外に、さらに任意成分を含む液であってもよい。
The blood cell cell preservation solution and the trehalose-containing solution may be a solution containing trehalose alone, a solution containing two or more types selected from trehalose, and other than trehalose. It may be a liquid containing an arbitrary component.
本明細書において「任意成分」としては、例えば、等張剤（例えば、グルコース、ソルビトール、マンニトール、ラクトース、塩化ナトリウム）、キレート剤（例えば、ＥＤＴＡ、ＥＧＴＡ、クエン酸、サリチレート）、溶解補助剤、保存剤、酸化防止剤、アミノ酸（例えば、プロリン、グルタミン）、ポリマー（例えば、ポリエーテル）、リン脂質（例えば、リゾホスファチジン酸［ＬＰＡ；Lysophosphatidic acid］）、上述のｐＨ調整剤を挙げることができる。本明細書において「任意成分」とは、含んでもよいし含まなくてもよい成分のことを意味する。
また、本発明には、トレハロース類を含む、本件血球系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。 In the present specification, the “arbitrary component” includes, for example, isotonic agents (for example, glucose, sorbitol, mannitol, lactose, sodium chloride), chelating agents (for example, EDTA, EGTA, citric acid, salicylate), lysis aids, and the like. Preservatives, antioxidants, amino acids (eg, proline, glutamine), polymers (eg, polyether), phospholipids (eg, lysophosphatidic acid [LPA]), pH regulators mentioned above can be mentioned. .. As used herein, the term "arbitrary component" means a component that may or may not be contained.
In addition, the present invention includes a powder preparation for preparing the Blood Cell Cell Preservation Solution, which contains trehalose. The powder formulation may contain the above-mentioned optional ingredients.
また、本発明には、トレハロース類を含む、本件血球系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。 In the present specification, the “arbitrary component” includes, for example, isotonic agents (for example, glucose, sorbitol, mannitol, lactose, sodium chloride), chelating agents (for example, EDTA, EGTA, citric acid, salicylate), lysis aids, and the like. Preservatives, antioxidants, amino acids (eg, proline, glutamine), polymers (eg, polyether), phospholipids (eg, lysophosphatidic acid [LPA]), pH regulators mentioned above can be mentioned. .. As used herein, the term "arbitrary component" means a component that may or may not be contained.
In addition, the present invention includes a powder preparation for preparing the Blood Cell Cell Preservation Solution, which contains trehalose. The powder formulation may contain the above-mentioned optional ingredients.
本件血球系細胞保存用液や本件トレハロース類含有液としては、トレハロース類以外に、血球系細胞の生存率低下や凝集の抑制作用を有する成分（例えば、アカルボース、スタキオース、デキストラン、ヒドロキシエチルスターチ［Hydroxyethyl starch；ＨＥＳ］等の多糖類若しくはその誘導体又はこれらの塩；グルコース等の単糖類若しくはその誘導体又はこれらの塩）を含まないものであってもよい。
In addition to trehalose, the blood cell cell preservation solution and the trehalose-containing solution include components having an action of reducing the viability of blood cell lines and suppressing aggregation (for example, acarbose, stachyose, dextran, hydroxyethyl starch [Hydroxyethyl]. It may not contain polysaccharides such as starch; HES] or derivatives thereof or salts thereof; monosaccharides such as glucose or derivatives thereof or salts thereof).
本件血球系細胞保存用液としては、血球系細胞を含む本件血球系細胞保存用液をそのまま移植に用いる場合、血球系細胞移植に適した液が好ましく、かかる血球系細胞移植に適した液は、血球系細胞移植に適さない物質、例えば、生体由来の成分（例えば、血清又は血清由来成分［例えば、アルブミン］）；や、血球系細胞を凍結保存又は凍結乾燥保存したときの血球系細胞の生存率低下を抑制する作用を有する成分、例えば、ジメチルスルホキシド［Dimethyl sulfoxide；ＤＭＳＯ］、グリセリン、エチレングリコール、トリメチレングリコール、ジメチルアセトアミド、ポリエチレングリコール［ＰＥＧ］、ポリビニルピロリドン、血清又は血清由来成分（例えば、アルブミン）等の凍結保護剤又は凍結乾燥保護剤；を含まないことが好ましい。
As the blood cell lineage preservation solution, when the blood cell lineage cell preservation solution containing blood cell lineage cells is used as it is for transplantation, a solution suitable for blood cell lineage cell transplantation is preferable, and a solution suitable for such blood cell lineage cell transplantation is suitable. , Substances unsuitable for blood cell transplantation, such as biological components (eg, serum or serum-derived components [eg, albumin]); Ingredients that suppress the decline in viability, such as dimethylsulfoxide [Dimethylsulfoxide; DMSO], glycerin, ethylene glycol, trimethylene glycol, dimethylacetamide, polyethylene glycol [PEG], polyvinylpyrrolidone, serum or serum-derived components (eg, , Albumin) and other cryoprotectants or cryoprotectants; preferably not contained.
本発明において、血球系細胞を保存する期間としては、血球系細胞を含む本件トレハロース類含有液を液体の状態で保存したときに、当該液中の血球系細胞の細胞生存率低下、細胞凝集、及び／又は、保存容器への細胞接着を抑制し、生細胞の割合を高める、細胞凝集塊数を減少できる、及び／又は、保存容器からの細胞の回収率を高める期間が好ましく、例えば、６時間以上、１２時間以上、１日（２４時間）以上、１．５日（３６時間）以上、２日（４８時間）以上、３日（７２時間）以上、４日（９６時間）以上、７日（１６８時間）以上であり、また、血球系細胞の保存期間が長すぎると細胞の生存に悪影響を及ぼす可能性があるため、細胞の生存率への悪影響を回避する観点から、例えば、２１日以下、１６日以下、１４日以下、１０日以下、７日以下、４日以下である。したがって、上記保存期間としては、例えば、６時間～２１日；１２時間～２１日；１～２１日；１．５～２１日；２～２１日；３～２１日；４～２１日；７～２１日；６時間～１６日；６時間～１４日；６時間～１０日；６時間～７日；６時間～４日；１２時間～１６日；１２時間～１４日；１２時間～１０日；１２時間～７日；１２時間～４日；１～１６日；１～１４日；１～１０日；１～７日；１～４日；１．５～１６日；１．５～１４日；１．５～１０日；１．５～７日；１．５～４日；２～１６日；２～１４日；２～１０日；２～７日；２～４日；３～１６日；３～１４日；３～１０日；３～７日；３～４日；４～１６日；４～１４日；４～１０日；４～７日；７～１６日；７～１４日；７～１０日；等を挙げることができる。本件トレハロース類含有液中に保存した血球系細胞について、細胞死が抑制されたことは、トリパンブルー（Trypan Blue）染色法、ＴＵＮＥＬ法、Ｎｅｘｉｎ法、ＦＬＩＣＡ法などの細胞死を検出できる公知の方法を用いて確認することができる。また、本件トレハロース類含有液中に保存した血球系細胞について、細胞凝集が抑制されたことは、位相差顕微鏡による細胞観察により確認することができる。
In the present invention, the period for storing blood cell lines is such that when the Trehalose-containing solution containing blood cells is stored in a liquid state, the cell viability of the blood cells in the solution decreases, cell aggregation, and the like. And / or a period in which cell adhesion to the storage vessel can be suppressed, the proportion of viable cells can be increased, the number of cell aggregates can be reduced, and / or the recovery rate of cells from the storage container can be increased, 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 If it is more than a day (168 hours) and the storage period of blood cell lineage cells is too long, it may adversely affect the survival of the cells. Therefore, from the viewpoint of avoiding an adverse effect on the viability of the cells, for example, 21 Days or less, 16 days or less, 14 days or less, 10 days or less, 7 days or less, 4 days or less. Therefore, 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 to 10 days; 1.5 to 7 days; 1.5 to 4 days; 2 to 16 days; 2 to 14 days; 2 to 10 days; 2 to 7 days; 2 to 4 days; 3 -16 days; 3-14 days; 3-10 days; 3-7 days; 3-4 days; 4-16 days; 4-14 days; 4-10 days; 4-7 days; 7-16 days; 7 ~ 14 days; 7 ~ 10 days; etc. can be mentioned. The suppression of cell death in blood cell lines stored in the Trehalose-containing solution is a known method capable of detecting cell death such as trypan blue staining method, TUNEL method, Nexin method, and FLICA method. Can be confirmed using. In addition, it can be confirmed by cell observation with a phase-contrast microscope that cell aggregation was suppressed in the blood cell lineage cells stored in the trehalose-containing solution of the present case.
本発明において、「血球系細胞を含む本件トレハロース類含有液が液体の状態で存在する温度」としては、血球系細胞を含む本件トレハロース類含有液が、凍結せずに液体の状態で存在し、かつ当該液中の血球系細胞が生育可能な温度であればよく、通常０～４０℃の範囲内、好ましくは０～３０℃（室温）の範囲内である。さらに、本発明において、血球系細胞を保存するために好適な温度は、８～４０℃、好ましくは１０～４０℃、より好ましくは１２～３８℃、最も好ましくは１５～３７℃である。
In the present invention, the "temperature at which the trehalose-containing solution containing blood cell-based cells exists in a liquid state" is defined as the temperature at which the trehalose-containing solution containing blood cell-based cells exists in a liquid state without freezing. Moreover, the temperature may be such that the blood cell cells in the liquid can grow, and it is usually in the range of 0 to 40 ° C, preferably in the range of 0 to 30 ° C (room temperature). Further, in the present invention, the temperature suitable for preserving blood cell lineage cells is 8 to 40 ° C., preferably 10 to 40 ° C., more preferably 12 to 38 ° C., and most preferably 15 to 37 ° C.
本発明において、血球系細胞の保存容器としては、ガラス製の容器、プラスチック製の容器等が挙げられるが、細胞投与用のバッグとして用いられる、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、架橋エチレン・酢酸ビニル共重合体、エチレン・α―オレフィン共重合体、及びこれら樹脂のブレンド樹脂からなる群より選ばれる樹脂製の容器が好ましい。また、保存容器にはカニューレ等の細胞投与用バッグに付随するデバイスも含まれる。
In the present invention, examples of the storage container for blood cell lineage cells include a glass container, a plastic container, and the like. Polyethylene, polypropylene, polyvinyl chloride, crosslinked ethylene / vinyl acetate, which are used as bags for cell administration. A resin container selected from the group consisting of a copolymer, an ethylene / α-olefin copolymer, and a blended resin of these resins is preferable. The storage container also includes a device attached to a cell administration bag such as a cannula.
本発明において、血球系細胞（集団）は、生体内から分離されたものであっても、インビトロで継代培養されたものであってもよいが、単離又は精製されていることが好ましい。本明細書中、「単離又は精製」とは、目的とする成分以外の成分を除去する操作が施されていることを意味する。単離又は精製された血球系細胞の純度（全細胞数に対する血球系細胞細胞の割合）は、通常３０％以上、好ましくは５０％以上、より好ましくは７０％以上、さらに好ましくは９０％以上（例えば１００％）である。
In the present invention, the blood cell lineage cells (population) may be those isolated from the living body or those subcultured in vitro, but are preferably isolated or purified. In the present specification, "isolation or purification" means that an operation for removing a component other than the target component has been performed. The purity of the isolated or purified blood cell line (ratio of blood cell line cell to the total number of cells) is usually 30% or more, preferably 50% or more, more preferably 70% or more, still more preferably 90% or more (). For example, 100%).
本件血球系細胞保存用液や本件トレハロース類含有液中に保存する血球系細胞（集団）は、単一細胞（シングルセル）の状態であってもよい。本明細書において、「単一細胞の状態」とは、他の細胞と寄り集まって塊を形成していないこと（即ち、凝集していない状態）を意味する。単一細胞の状態の血球系細胞は、インビトロで培養した血球系細胞をトリプシン／ＥＤＴＡ等で酵素処理することにより調製することができる。血球系細胞中に含まれる単一細胞の状態の血球系細胞の割合は、例えば７０％以上、好ましくは９０％以上、より好ましくは９５％以上、さらに好ましくは９９％以上（例えば１００％）である。単一細胞の状態の細胞の割合は、血球系細胞をＰＢＳ中に分散させ、これを顕微鏡下で観察し、無作為に選択された複数個（例えば、１０００個）の細胞について凝集の有無を調べることにより決定することができる。
The blood cell lineage cells (population) to be stored in the blood cell lineage cell preservation solution or the trehalose-containing solution may be in a single cell state. As used herein, the term "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). Blood cell lineage cells in a single cell state can be prepared by enzymatically treating blood cell lineage cells cultured in vitro with trypsin / EDTA or the like. The proportion of single-celled blood cells contained in blood 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%). be. The percentage of cells in the single cell state is that blood cell lineage cells are dispersed in PBS and observed under a microscope to determine the presence or absence of aggregation in a plurality of randomly selected cells (eg, 1000 cells). It can be determined by examining.
本件血球系細胞保存用液や本件トレハロース類含有液中に保存する血球系細胞（集団）は、浮遊していてもよい。本明細書において、「浮遊」とは、血球系細胞が、保存用液を収容した容器の内壁に接触することなく、液中に保持されていることをいう。
The blood cell line cells (population) to be stored in the blood cell line cell preservation solution or the trehalose-containing solution may be suspended. As used herein, the term "floating" means that blood cell lineage cells are retained in the liquid without contacting the inner wall of the container containing the storage liquid.
本件血球系細胞保存用液や本件トレハロース類含有液中に保存した血球系細胞が、凝集又は沈殿している場合、移植前にピペッティングやタッピング等の当該技術分野における周知の方法により血球系細胞を懸濁することが好ましい。
When the blood cell cells stored in the blood cell cell preservation solution or the trehalose-containing solution are aggregated or precipitated, the blood cell cells are collected by a well-known method in the art such as pipetting or tapping before transplantation. Is preferably suspended.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。
Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.
（１）本発明の血球系細胞保存液の調製及び評価
１－１．材料及び方法
［血球系細胞］
血球系細胞として、以下の表１に記載のヒト末梢血由来ＣＤ８＋Ｔ細胞（以下、単に「Ｔ細胞」という）を用いた。 (1) Preparation and Evaluation of Blood Cell Cell Preservation Solution of the Present Invention 1-1. Materials and methods [Blood cell lineage cells]
As the blood cell lineage cells, CD8 + T cells derived from human peripheral blood (hereinafter, simply referred to as “T cells”) shown in Table 1 below were used.
１－１．材料及び方法
［血球系細胞］
血球系細胞として、以下の表１に記載のヒト末梢血由来ＣＤ８＋Ｔ細胞（以下、単に「Ｔ細胞」という）を用いた。 (1) Preparation and Evaluation of Blood Cell Cell Preservation Solution of the Present Invention 1-1. Materials and methods [Blood cell lineage cells]
As the blood cell lineage cells, CD8 + T cells derived from human peripheral blood (hereinafter, simply referred to as “T cells”) shown in Table 1 below were used.
［被験液の調製］
ｐＨが６．５７４の乳酸リンゲル液であるラクテック注（大塚製薬工場社製、表２参照）（以下、「ＬＲ」ともいう）に、α，α－トレハロース二水和物（富士フイルム和光純薬社製）を、トレハロースの終濃度が３（ｗ／ｖ）％となるように添加し、ＬＲＴ－５（表３参照）を得た。ＬＲＴ－５に、０．１ｍｏｌ／Ｌの塩酸を、ｐＨがそれぞれ４．４６８、５．０２１、５．９９７、及び６．４８２となるように添加し、ＬＲＴ－１～４（表３参照）を得た。また、ＬＲＴ－５に、０．５ｍｏｌ／Ｌの水酸化ナトリウム溶液を、ｐＨがそれぞれ７．２０７、７．５１５、及び８．０３３となるように添加し、ＬＲＴ－６～８（表３参照）を得た。なお、各被験液におけるｐＨは、室温（２０．６～２５．８℃）で測定した際の値である。 [Preparation of test solution]
Lactec injection (manufactured by Otsuka Pharmaceutical Factory, see Table 2) (hereinafter also referred to as "LR"), which is a lactated Ringer's solution with a pH of 6.574, and α, α-trehalose dihydrate (Fujifilm Wako Pure Chemical Industries, Ltd.) Was added so that the final concentration of trehalose was 3 (w / v)%, and LRT-5 (see Table 3) was obtained. 0.1 mol / L hydrochloric acid was added to LRT-5 so that the pH was 4.468, 5.021, 5.997, and 6.482, respectively, and LRT-1 to 4 (see Table 3). Got Further, 0.5 mol / L sodium hydroxide solution was added to LRT-5 so that the pH was 7.207, 7.515, and 8.033, respectively, and LRT-6 to 8 (see Table 3). ) Was obtained. The pH of each test solution is a value measured at room temperature (20.6 to 25.8 ° C.).
ｐＨが６．５７４の乳酸リンゲル液であるラクテック注（大塚製薬工場社製、表２参照）（以下、「ＬＲ」ともいう）に、α，α－トレハロース二水和物（富士フイルム和光純薬社製）を、トレハロースの終濃度が３（ｗ／ｖ）％となるように添加し、ＬＲＴ－５（表３参照）を得た。ＬＲＴ－５に、０．１ｍｏｌ／Ｌの塩酸を、ｐＨがそれぞれ４．４６８、５．０２１、５．９９７、及び６．４８２となるように添加し、ＬＲＴ－１～４（表３参照）を得た。また、ＬＲＴ－５に、０．５ｍｏｌ／Ｌの水酸化ナトリウム溶液を、ｐＨがそれぞれ７．２０７、７．５１５、及び８．０３３となるように添加し、ＬＲＴ－６～８（表３参照）を得た。なお、各被験液におけるｐＨは、室温（２０．６～２５．８℃）で測定した際の値である。 [Preparation of test solution]
Lactec injection (manufactured by Otsuka Pharmaceutical Factory, see Table 2) (hereinafter also referred to as "LR"), which is a lactated Ringer's solution with a pH of 6.574, and α, α-trehalose dihydrate (Fujifilm Wako Pure Chemical Industries, Ltd.) Was added so that the final concentration of trehalose was 3 (w / v)%, and LRT-5 (see Table 3) was obtained. 0.1 mol / L hydrochloric acid was added to LRT-5 so that the pH was 4.468, 5.021, 5.997, and 6.482, respectively, and LRT-1 to 4 (see Table 3). Got Further, 0.5 mol / L sodium hydroxide solution was added to LRT-5 so that the pH was 7.207, 7.515, and 8.033, respectively, and LRT-6 to 8 (see Table 3). ) Was obtained. The pH of each test solution is a value measured at room temperature (20.6 to 25.8 ° C.).
［血球系細胞の培養］
１×１０６個のＴ細胞を、TLY CULTURE（登録商標）キット２５（GCリンフォテック社製）、すなわち、インターロイキン（ＩＬ）－２、固相化抗ヒトＣＤ３抗体、及び１０％ＦＣＳを含むＲＰＭＩ－１６４０培養液の存在下の培養用フラスコに添加し、当該製品の添付文書に記載の方法に従い、３７℃、５％ＣＯ２インキュベーター内で７日間培養した。 [Culture of blood cell lineage cells]
1 × 10 6 T cells with TLY CULTURE® Kit 25 (GC Lymphotech), ie interleukin (IL) -2, immobilized anti-human CD3 antibody, and 10% FCS. It was added to the culture flask in the presence of the containing RPMI-1640 culture solution and cultured in a 5% CO 2 incubator at 37 ° C. for 7 days according to the method described in the attached document of the product.
１×１０６個のＴ細胞を、TLY CULTURE（登録商標）キット２５（GCリンフォテック社製）、すなわち、インターロイキン（ＩＬ）－２、固相化抗ヒトＣＤ３抗体、及び１０％ＦＣＳを含むＲＰＭＩ－１６４０培養液の存在下の培養用フラスコに添加し、当該製品の添付文書に記載の方法に従い、３７℃、５％ＣＯ２インキュベーター内で７日間培養した。 [Culture of blood cell lineage cells]
1 × 10 6 T cells with TLY CULTURE® Kit 25 (GC Lymphotech), ie interleukin (IL) -2, immobilized anti-human CD3 antibody, and 10% FCS. It was added to the culture flask in the presence of the containing RPMI-1640 culture solution and cultured in a 5% CO 2 incubator at 37 ° C. for 7 days according to the method described in the attached document of the product.
［被験液の評価］
血球系細胞の被験液中での保存と、その後の細胞生存率及び細胞凝集塊数の測定については、以下の〔１〕～〔７〕の手順に従って行った。
〔１〕上記［血球系細胞の培養］の項目に記載の方法に従って培養したＴ細胞を含む培養用フラスコに、培養液と等量のＬＲを加え、５０ｍＬのコニカルチューブに移した後、細胞塊を懸濁した。
〔２〕細胞懸濁液の一部（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、保存前の細胞生存率を算出した。
〔３〕残りの細胞懸濁液は、Ｔ細胞が５×１０５個となるように、低接着性のコニカルチューブであるステムフル（住友ベークライト社製）に移し、遠心処理（３００×ｇ、１０分間、室温）後、上清を除去し、表３に示す９種類の被験液（実施例１～６及び比較例１～３）１ｍＬを加え、細胞ペレットを懸濁し、２５℃にて２４時間保存した。
〔４〕保存後、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（１ｍＬのピペットを用いて、５００μＬの液量でのピペッティングを５回）した。
〔５〕撹拌直後の１０μＬの液を、ワンセルカウンターに分取し、ワンセルカウンターの細胞計数部の９カ所のエリアの細胞凝集塊の数を計測した。なお、３つ以上の細胞が接着した状態のものを細胞凝集塊として判断した。
〔６〕再度、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（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを２回）した。
〔７〕撹拌直後の２０μＬの液を、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（％）」を用いて算出した。 [Evaluation of test solution]
The storage of blood cell lines in the test solution and the subsequent measurement of the cell viability and the number of cell aggregates were carried out according to the following procedures [1] to [7].
[1] Add an equal amount of LR to the culture solution to a culture flask containing T cells cultured according to the method described in the above item [Culture of blood cell lineage cells], transfer to a 50 mL conical tube, and then cell mass. Was suspended.
[2] A part (20 μL) of the cell suspension was divided into 1.5 mL microtubes to which 20 μL of trypan blue staining solution (manufactured by Gibco) was previously added. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
[3] The remaining cell suspension, so that T cells is of 5 × 10 5 cells were transferred to a low adhesion conical tube Sutemufuru (manufactured by Sumitomo Bakelite), centrifuged (300 × g, 10 After (minutes, room temperature), the supernatant is removed, 1 mL of 9 types of test solutions (Examples 1 to 6 and Comparative Examples 1 to 3) shown in Table 3 is added, cell pellets are suspended, and the cells are suspended at 25 ° C. for 24 hours. saved.
[4] After storage, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, pipette with a liquid volume of 500 μL). Petting was done 5 times).
[5] 10 μL of the solution immediately after stirring was separated into a one-cell counter, and the number of cell aggregates in nine areas of the cell counting section of the one-cell counter was measured. In addition, those in which three or more cells were adhered were judged as cell aggregates.
[6] Again, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, pipetting with a liquid volume of 250 μL). Twice).
[7] 20 μL of the solution immediately after stirring was collected in a 1.5 mL microtube, mixed with 20 μL of trypan blue staining solution (manufactured by Gibco), and then separated into a one-cell counter, and then separated into an optical microscope (ECLIPSE TS100, Nikon). The cell viability in each storage period was calculated by measuring the total number of cells and the number of trypan blue-positive cells (dead cells) using (manufactured by). The cell viability is calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". Calculated.
血球系細胞の被験液中での保存と、その後の細胞生存率及び細胞凝集塊数の測定については、以下の〔１〕～〔７〕の手順に従って行った。
〔１〕上記［血球系細胞の培養］の項目に記載の方法に従って培養したＴ細胞を含む培養用フラスコに、培養液と等量のＬＲを加え、５０ｍＬのコニカルチューブに移した後、細胞塊を懸濁した。
〔２〕細胞懸濁液の一部（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、保存前の細胞生存率を算出した。
〔３〕残りの細胞懸濁液は、Ｔ細胞が５×１０５個となるように、低接着性のコニカルチューブであるステムフル（住友ベークライト社製）に移し、遠心処理（３００×ｇ、１０分間、室温）後、上清を除去し、表３に示す９種類の被験液（実施例１～６及び比較例１～３）１ｍＬを加え、細胞ペレットを懸濁し、２５℃にて２４時間保存した。
〔４〕保存後、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（１ｍＬのピペットを用いて、５００μＬの液量でのピペッティングを５回）した。
〔５〕撹拌直後の１０μＬの液を、ワンセルカウンターに分取し、ワンセルカウンターの細胞計数部の９カ所のエリアの細胞凝集塊の数を計測した。なお、３つ以上の細胞が接着した状態のものを細胞凝集塊として判断した。
〔６〕再度、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（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを２回）した。
〔７〕撹拌直後の２０μＬの液を、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（％）」を用いて算出した。 [Evaluation of test solution]
The storage of blood cell lines in the test solution and the subsequent measurement of the cell viability and the number of cell aggregates were carried out according to the following procedures [1] to [7].
[1] Add an equal amount of LR to the culture solution to a culture flask containing T cells cultured according to the method described in the above item [Culture of blood cell lineage cells], transfer to a 50 mL conical tube, and then cell mass. Was suspended.
[2] A part (20 μL) of the cell suspension was divided into 1.5 mL microtubes to which 20 μL of trypan blue staining solution (manufactured by Gibco) was previously added. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
[3] The remaining cell suspension, so that T cells is of 5 × 10 5 cells were transferred to a low adhesion conical tube Sutemufuru (manufactured by Sumitomo Bakelite), centrifuged (300 × g, 10 After (minutes, room temperature), the supernatant is removed, 1 mL of 9 types of test solutions (Examples 1 to 6 and Comparative Examples 1 to 3) shown in Table 3 is added, cell pellets are suspended, and the cells are suspended at 25 ° C. for 24 hours. saved.
[4] After storage, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, pipette with a liquid volume of 500 μL). Petting was done 5 times).
[5] 10 μL of the solution immediately after stirring was separated into a one-cell counter, and the number of cell aggregates in nine areas of the cell counting section of the one-cell counter was measured. In addition, those in which three or more cells were adhered were judged as cell aggregates.
[6] Again, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, pipetting with a liquid volume of 250 μL). Twice).
[7] 20 μL of the solution immediately after stirring was collected in a 1.5 mL microtube, mixed with 20 μL of trypan blue staining solution (manufactured by Gibco), and then separated into a one-cell counter, and then separated into an optical microscope (ECLIPSE TS100, Nikon). The cell viability in each storage period was calculated by measuring the total number of cells and the number of trypan blue-positive cells (dead cells) using (manufactured by). The cell viability is calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". Calculated.
１－２．結果
細胞保存後の生じる細胞凝集塊を測定したところ、Ｔ細胞を、ＬＲＴ－７（比較例２）やＬＲＴ－８（比較例３）中に保存すると、細胞凝集塊が認められたのに対して、Ｔ細胞を、ＬＲＴ－１～６（実施例１～６）中に保存した場合や、ＬＲ中に保存した場合は、細胞凝集塊はほとんど又は全く認められなかった（図１Ａ及び表４参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる細胞凝集塊を効果的に抑制できることを示している。 1-2. Results When the cell aggregates generated after cell preservation were measured, when T cells were preserved in LRT-7 (Comparative Example 2) or LRT-8 (Comparative Example 3), cell aggregates were observed. When T cells were stored in LRT-1 to 6 (Examples 1 to 6) or stored in LR, almost or no cell aggregates were observed (FIGS. 1A and 4). reference).
This result shows that when blood cell line cells (particularly T cells) are stored in a solution containing trehalose and having a pH in the range of about 4.5 to about 7.2, trehalose is contained, but the pH is outside the above range. It is shown that the cell aggregates generated when stored can be effectively suppressed as compared with the case where the cells are stored in the liquid of.
細胞保存後の生じる細胞凝集塊を測定したところ、Ｔ細胞を、ＬＲＴ－７（比較例２）やＬＲＴ－８（比較例３）中に保存すると、細胞凝集塊が認められたのに対して、Ｔ細胞を、ＬＲＴ－１～６（実施例１～６）中に保存した場合や、ＬＲ中に保存した場合は、細胞凝集塊はほとんど又は全く認められなかった（図１Ａ及び表４参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる細胞凝集塊を効果的に抑制できることを示している。 1-2. Results When the cell aggregates generated after cell preservation were measured, when T cells were preserved in LRT-7 (Comparative Example 2) or LRT-8 (Comparative Example 3), cell aggregates were observed. When T cells were stored in LRT-1 to 6 (Examples 1 to 6) or stored in LR, almost or no cell aggregates were observed (FIGS. 1A and 4). reference).
This result shows that when blood cell line cells (particularly T cells) are stored in a solution containing trehalose and having a pH in the range of about 4.5 to about 7.2, trehalose is contained, but the pH is outside the above range. It is shown that the cell aggregates generated when stored can be effectively suppressed as compared with the case where the cells are stored in the liquid of.
また、Ｔ細胞を、ＬＲＴ－１～６（実施例１～６）中に保存すると、ＬＲ（比較例１）中に保存した場合と比べ、いずれの液についても細胞生存率が上昇した。特に、Ｔ細胞をＬＲＴ－３～５（実施例３～５）中に保存した場合にその効果が高かった（図１Ｂ及び表５参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、ｐＨが上記範囲内であるが、トレハロース不含の液中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 In addition, when T cells were stored in LRT-1 to 6 (Examples 1 to 6), the cell viability was increased in all the solutions as compared with the case where they were stored in LR (Comparative Example 1). In particular, the effect was high when T cells were stored in LRT-3 to 5 (Examples 3 to 5) (see FIGS. 1B and 5).
This result shows that when blood cell line cells (particularly T cells) are stored in a solution containing trehalose and having a pH in the range of about 4.5 to about 7.2, the pH is within the above range, but trehalose. It is shown that cell death that occurs when stored can be effectively suppressed as compared with the case where it is stored in a non-containing liquid.
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、ｐＨが上記範囲内であるが、トレハロース不含の液中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 In addition, when T cells were stored in LRT-1 to 6 (Examples 1 to 6), the cell viability was increased in all the solutions as compared with the case where they were stored in LR (Comparative Example 1). In particular, the effect was high when T cells were stored in LRT-3 to 5 (Examples 3 to 5) (see FIGS. 1B and 5).
This result shows that when blood cell line cells (particularly T cells) are stored in a solution containing trehalose and having a pH in the range of about 4.5 to about 7.2, the pH is within the above range, but trehalose. It is shown that cell death that occurs when stored can be effectively suppressed as compared with the case where it is stored in a non-containing liquid.
（２）トレハロース含有細胞保存液で血球系細胞を保存したときの温度と細胞凝集の関係２－１．材料及び方法
［血球系細胞］
上記（１）で使用したＴ細胞を用いた。 (2) Relationship between temperature and cell aggregation when blood cell lineage cells are stored in a trehalose-containing cell preservation solution 2-1. Materials and methods [Blood cell lineage cells]
The T cells used in (1) above were used.
［血球系細胞］
上記（１）で使用したＴ細胞を用いた。 (2) Relationship between temperature and cell aggregation when blood cell lineage cells are stored in a trehalose-containing cell preservation solution 2-1. Materials and methods [Blood cell lineage cells]
The T cells used in (1) above were used.
［被験液の調製］
３（ｗ／ｖ）％のトレハロースを含む細胞洗浄保存液であるセルストアＷ（大塚製薬工場社製、表６参照）（以下、「Ｗ」ともいう）を室温（２５℃）又は冷蔵（５℃）でインキュベートし、Ｗ（２５℃）（以下「比較例４」ともいう。）及びＷ（５℃）（以下「比較例５」ともいう。）（表７参照）を得た。なお、被験液におけるｐＨは、室温（２３．５℃）で測定時が７．３３１、氷冷（４．３℃）で測定時が７．２３１である。 [Preparation of test solution]
Cell store W (manufactured by Otsuka Pharmaceutical Factory, see Table 6) (hereinafter, also referred to as “W”), which is a cell washing preservation solution containing 3 (w / v)% trehalose, is stored at room temperature (25 ° C.) or refrigerated (5). Incubation at (° C.) gave W (25 ° C.) (hereinafter, also referred to as "Comparative Example 4") and W (5 ° C.) (hereinafter, also referred to as "Comparative Example 5") (see Table 7). The pH of the test solution is 7.331 when measured at room temperature (23.5 ° C.) and 7.231 when measured at ice-cooled (4.3 ° C.).
３（ｗ／ｖ）％のトレハロースを含む細胞洗浄保存液であるセルストアＷ（大塚製薬工場社製、表６参照）（以下、「Ｗ」ともいう）を室温（２５℃）又は冷蔵（５℃）でインキュベートし、Ｗ（２５℃）（以下「比較例４」ともいう。）及びＷ（５℃）（以下「比較例５」ともいう。）（表７参照）を得た。なお、被験液におけるｐＨは、室温（２３．５℃）で測定時が７．３３１、氷冷（４．３℃）で測定時が７．２３１である。 [Preparation of test solution]
Cell store W (manufactured by Otsuka Pharmaceutical Factory, see Table 6) (hereinafter, also referred to as “W”), which is a cell washing preservation solution containing 3 (w / v)% trehalose, is stored at room temperature (25 ° C.) or refrigerated (5). Incubation at (° C.) gave W (25 ° C.) (hereinafter, also referred to as "Comparative Example 4") and W (5 ° C.) (hereinafter, also referred to as "Comparative Example 5") (see Table 7). The pH of the test solution is 7.331 when measured at room temperature (23.5 ° C.) and 7.231 when measured at ice-cooled (4.3 ° C.).
［血球系細胞の培養］
上記（１）に記載の方法に従ってＴ細胞を培養した。 [Culture of blood cell lineage cells]
T cells were cultured according to the method described in (1) above.
上記（１）に記載の方法に従ってＴ細胞を培養した。 [Culture of blood cell lineage cells]
T cells were cultured according to the method described in (1) above.
［被験液の評価］
上記（１）に記載の方法に従い、比較例４及び５の被験液に細胞ペレットを懸濁し、２４時間２５℃又は５℃で保存し、細胞凝集塊形成を評価した。 [Evaluation of test solution]
According to the method described in (1) above, cell pellets were suspended in the test solutions of Comparative Examples 4 and 5 and stored at 25 ° C. or 5 ° C. for 24 hours to evaluate cell agglutination formation.
上記（１）に記載の方法に従い、比較例４及び５の被験液に細胞ペレットを懸濁し、２４時間２５℃又は５℃で保存し、細胞凝集塊形成を評価した。 [Evaluation of test solution]
According to the method described in (1) above, cell pellets were suspended in the test solutions of Comparative Examples 4 and 5 and stored at 25 ° C. or 5 ° C. for 24 hours to evaluate cell agglutination formation.
２－２．結果
細胞保存後の生じる細胞凝集塊を測定したところ、Ｔ細胞を、２５℃で保存すると、細胞凝集塊が認められたのに対して（比較例４）、Ｔ細胞を、５℃で保存した場合は（比較例５）、細胞凝集塊は全く認められなかった（図２及び表８参照）。
この結果は、血球系細胞（特にＴ細胞）を冷蔵（１０℃前後）よりも高い温度で保存すると、細胞凝集塊が形成される傾向にあることを示している。 2-2. Results When the cell aggregates generated after cell storage were measured, cell aggregates were observed when the T cells were stored at 25 ° C (Comparative Example 4), whereas the T cells were stored at 5 ° C. In the case (Comparative Example 5), no cell agglutination was observed (see FIGS. 2 and 8).
This result indicates that when blood cell lineage cells (particularly T cells) are stored at a temperature higher than refrigeration (around 10 ° C.), cell aggregates tend to be formed.
細胞保存後の生じる細胞凝集塊を測定したところ、Ｔ細胞を、２５℃で保存すると、細胞凝集塊が認められたのに対して（比較例４）、Ｔ細胞を、５℃で保存した場合は（比較例５）、細胞凝集塊は全く認められなかった（図２及び表８参照）。
この結果は、血球系細胞（特にＴ細胞）を冷蔵（１０℃前後）よりも高い温度で保存すると、細胞凝集塊が形成される傾向にあることを示している。 2-2. Results When the cell aggregates generated after cell storage were measured, cell aggregates were observed when the T cells were stored at 25 ° C (Comparative Example 4), whereas the T cells were stored at 5 ° C. In the case (Comparative Example 5), no cell agglutination was observed (see FIGS. 2 and 8).
This result indicates that when blood cell lineage cells (particularly T cells) are stored at a temperature higher than refrigeration (around 10 ° C.), cell aggregates tend to be formed.
（３）本発明の血球系細胞保存液の評価
３－１．材料及び方法
［血球系細胞］
上記（１）で使用したＴ細胞を用いた。 (3) Evaluation of Blood Cell Cell Preservation Solution of the Present Invention 3-1. Materials and methods [Blood cell lineage cells]
The T cells used in (1) above were used.
３－１．材料及び方法
［血球系細胞］
上記（１）で使用したＴ細胞を用いた。 (3) Evaluation of Blood Cell Cell Preservation Solution of the Present Invention 3-1. Materials and methods [Blood cell lineage cells]
The T cells used in (1) above were used.
［被験液の調製］
生理食塩液である大塚生食注（大塚製薬工場社製）に、アルブミナー（登録商標）２５％静注１２．５ｇ／５０ｍＬ（ＣＳＬベーリング社製）を、アルブミンの終濃度が２．５（ｗ／ｖ）％となるように添加し、２．５％アルブミン含有生理食塩液（２．５％ＡＬＢＳ）（表９参照）を調製した。ＬＲＴ－３とＬＲＴ－７（表９参照）は、上記（１）で調製した被験液を用いた。 [Preparation of test solution]
Albuminer (registered trademark) 25% intravenous injection 12.5 g / 50 mL (manufactured by CSL Behring) was added to Otsuka saline injection (manufactured by Otsuka Pharmaceutical Factory), which is a physiological saline solution, and the final concentration of albumin was 2.5 (w / w /). V)% was added to prepare a 2.5% albumin-containing physiological saline solution (2.5% ALBS) (see Table 9). For LRT-3 and LRT-7 (see Table 9), the test solution prepared in (1) above was used.
生理食塩液である大塚生食注（大塚製薬工場社製）に、アルブミナー（登録商標）２５％静注１２．５ｇ／５０ｍＬ（ＣＳＬベーリング社製）を、アルブミンの終濃度が２．５（ｗ／ｖ）％となるように添加し、２．５％アルブミン含有生理食塩液（２．５％ＡＬＢＳ）（表９参照）を調製した。ＬＲＴ－３とＬＲＴ－７（表９参照）は、上記（１）で調製した被験液を用いた。 [Preparation of test solution]
Albuminer (registered trademark) 25% intravenous injection 12.5 g / 50 mL (manufactured by CSL Behring) was added to Otsuka saline injection (manufactured by Otsuka Pharmaceutical Factory), which is a physiological saline solution, and the final concentration of albumin was 2.5 (w / w /). V)% was added to prepare a 2.5% albumin-containing physiological saline solution (2.5% ALBS) (see Table 9). For LRT-3 and LRT-7 (see Table 9), the test solution prepared in (1) above was used.
［血球系細胞の培養］
上記（１）に記載の方法に従ってＴ細胞を培養した。 [Culture of blood cell lineage cells]
T cells were cultured according to the method described in (1) above.
上記（１）に記載の方法に従ってＴ細胞を培養した。 [Culture of blood cell lineage cells]
T cells were cultured according to the method described in (1) above.
［被験液の評価］
血球系細胞の被験液中での保存と、その後の細胞回収率及び細胞生存率の測定については、以下の〔１〕～〔７〕の手順に従って行った。
〔１〕上記［血球系細胞の培養］の項目に記載の方法に従って培養したＴ細胞を含む培養用フラスコに、培養液と等量のＬＲを加え、５０ｍＬのコニカルチューブに移した後、細胞塊を懸濁した。
〔２〕細胞懸濁液の一部（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、保存前の細胞生存率を算出した。
〔３〕残りの細胞懸濁液は、Ｔ細胞が４．５×１０５個となるように、Ｆａｌｃｏｎ（登録商標）１５ｍＬ高透明度ポリプロピレンコニカルチューブ（コーニング社製）に移し、遠心処理（３００×ｇ、１０分間、室温）後、上清を除去し、表９に示す３種類の被験液（実施例３並びに比較例６及び比較例２）０．９ｍＬを加え、細胞ペレットを懸濁し、２５℃にて保存した。
〔４〕保存６時間後及び２４時間後に、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（１ｍＬのピペットを用いて、４５０μＬの液量でのピペッティングを５回）して、手順〔５〕の細胞回収率及び細胞生存率の測定に供した。
〔５〕撹拌直後の２０μＬの液を、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回収率及び細胞生存率を算出した。なお、細胞回収率は、式「（全細胞数／保存前の全細胞数）×１００＝細胞回収率（％）」を用いて算出した。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（％）」を用いて算出した。 [Evaluation of test solution]
The storage of blood cells in the test solution and the subsequent measurement of the cell recovery rate and the cell viability were performed according to the following procedures [1] to [7].
[1] Add an equal amount of LR to the culture solution to a culture flask containing T cells cultured according to the method described in the above item [Culture of blood cell lineage cells], transfer to a 50 mL conical tube, and then cell mass. Was suspended.
[2] A part (20 μL) of the cell suspension was divided into 1.5 mL microtubes to which 20 μL of trypan blue staining solution (manufactured by Gibco) was previously added. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
[3] The remaining cell suspension, so that T cells becomes 4.5 × 10 5 cells were transferred to Falcon (TM) 15 mL high transparency polypropylene conical tube (Corning), centrifuged (300 × g, 10 minutes, room temperature), the supernatant is removed, 0.9 mL of the three test solutions shown in Table 9 (Example 3, Comparative Example 6 and Comparative Example 2) is added, the cell pellet is suspended, and 25 Stored at ° C.
[4] After 6 hours and 24 hours of storage, insert the tip of the tip visually from the bottom of the tube containing the cells to a position of about 5 mm, and gently stir (450 μL using a 1 mL pipette). Pipetting with the liquid volume of (5 times) was performed to measure the cell recovery rate and cell viability in step [5].
[5] 20 μL of the solution immediately after stirring is collected in a 1.5 mL microtube, mixed with 20 μL of trypan blue staining solution (manufactured by Gibco), and then divided into a one-cell counter, and then separated into an optical microscope (ECLIPSE TS100, Nikon). By measuring the total number of cells and the number of trypan blue-positive cells (dead cells) using (manufactured by), the cell recovery rate and cell viability rate during each storage period were calculated. The cell recovery rate was calculated using the formula "(total number of cells / total number of cells before storage) x 100 = cell recovery rate (%)". The cell viability was calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". ..
血球系細胞の被験液中での保存と、その後の細胞回収率及び細胞生存率の測定については、以下の〔１〕～〔７〕の手順に従って行った。
〔１〕上記［血球系細胞の培養］の項目に記載の方法に従って培養したＴ細胞を含む培養用フラスコに、培養液と等量のＬＲを加え、５０ｍＬのコニカルチューブに移した後、細胞塊を懸濁した。
〔２〕細胞懸濁液の一部（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、保存前の細胞生存率を算出した。
〔３〕残りの細胞懸濁液は、Ｔ細胞が４．５×１０５個となるように、Ｆａｌｃｏｎ（登録商標）１５ｍＬ高透明度ポリプロピレンコニカルチューブ（コーニング社製）に移し、遠心処理（３００×ｇ、１０分間、室温）後、上清を除去し、表９に示す３種類の被験液（実施例３並びに比較例６及び比較例２）０．９ｍＬを加え、細胞ペレットを懸濁し、２５℃にて保存した。
〔４〕保存６時間後及び２４時間後に、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（１ｍＬのピペットを用いて、４５０μＬの液量でのピペッティングを５回）して、手順〔５〕の細胞回収率及び細胞生存率の測定に供した。
〔５〕撹拌直後の２０μＬの液を、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回収率及び細胞生存率を算出した。なお、細胞回収率は、式「（全細胞数／保存前の全細胞数）×１００＝細胞回収率（％）」を用いて算出した。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（％）」を用いて算出した。 [Evaluation of test solution]
The storage of blood cells in the test solution and the subsequent measurement of the cell recovery rate and the cell viability were performed according to the following procedures [1] to [7].
[1] Add an equal amount of LR to the culture solution to a culture flask containing T cells cultured according to the method described in the above item [Culture of blood cell lineage cells], transfer to a 50 mL conical tube, and then cell mass. Was suspended.
[2] A part (20 μL) of the cell suspension was divided into 1.5 mL microtubes to which 20 μL of trypan blue staining solution (manufactured by Gibco) was previously added. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
[3] The remaining cell suspension, so that T cells becomes 4.5 × 10 5 cells were transferred to Falcon (TM) 15 mL high transparency polypropylene conical tube (Corning), centrifuged (300 × g, 10 minutes, room temperature), the supernatant is removed, 0.9 mL of the three test solutions shown in Table 9 (Example 3, Comparative Example 6 and Comparative Example 2) is added, the cell pellet is suspended, and 25 Stored at ° C.
[4] After 6 hours and 24 hours of storage, insert the tip of the tip visually from the bottom of the tube containing the cells to a position of about 5 mm, and gently stir (450 μL using a 1 mL pipette). Pipetting with the liquid volume of (5 times) was performed to measure the cell recovery rate and cell viability in step [5].
[5] 20 μL of the solution immediately after stirring is collected in a 1.5 mL microtube, mixed with 20 μL of trypan blue staining solution (manufactured by Gibco), and then divided into a one-cell counter, and then separated into an optical microscope (ECLIPSE TS100, Nikon). By measuring the total number of cells and the number of trypan blue-positive cells (dead cells) using (manufactured by), the cell recovery rate and cell viability rate during each storage period were calculated. The cell recovery rate was calculated using the formula "(total number of cells / total number of cells before storage) x 100 = cell recovery rate (%)". The cell viability was calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". ..
３－２．結果
細胞保存後の細胞回収率を算出した結果、２．５％ＡＬＢＳ（比較例６）やＬＲＴ－３（実施例３）中にＴ細胞を保存すると、いずれの保存時間においてもほぼ全ての細胞が回収できているのに対して、ＬＲＴ－７（比較例２）中に保存した場合には、いずれの保存時間においても１０％程度の細胞が回収されていないことが示された（表１０、図３Ａ及び図４Ａ参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる保存容器への接着を効果的に抑制できることを示している。 3-2. Results As a result of calculating the cell recovery rate after cell storage, when T cells were stored in 2.5% ALBS (Comparative Example 6) or LRT-3 (Example 3), almost all cells were stored at any storage time. However, when stored in LRT-7 (Comparative Example 2), it was shown that about 10% of the cells were not recovered at any storage time (Table 10). , See FIGS. 3A and 4A).
This result shows that when blood cell line cells (particularly T cells) are stored in a liquid containing trehalose and having a pH in the range of about 4.5 to about 7.2, trehalose is contained, but the pH is outside the above range. It is shown that the adhesion to the storage container that occurs when the cells are stored can be effectively suppressed as compared with the case where the cells are stored in the liquid.
細胞保存後の細胞回収率を算出した結果、２．５％ＡＬＢＳ（比較例６）やＬＲＴ－３（実施例３）中にＴ細胞を保存すると、いずれの保存時間においてもほぼ全ての細胞が回収できているのに対して、ＬＲＴ－７（比較例２）中に保存した場合には、いずれの保存時間においても１０％程度の細胞が回収されていないことが示された（表１０、図３Ａ及び図４Ａ参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる保存容器への接着を効果的に抑制できることを示している。 3-2. Results As a result of calculating the cell recovery rate after cell storage, when T cells were stored in 2.5% ALBS (Comparative Example 6) or LRT-3 (Example 3), almost all cells were stored at any storage time. However, when stored in LRT-7 (Comparative Example 2), it was shown that about 10% of the cells were not recovered at any storage time (Table 10). , See FIGS. 3A and 4A).
This result shows that when blood cell line cells (particularly T cells) are stored in a liquid containing trehalose and having a pH in the range of about 4.5 to about 7.2, trehalose is contained, but the pH is outside the above range. It is shown that the adhesion to the storage container that occurs when the cells are stored can be effectively suppressed as compared with the case where the cells are stored in the liquid.
また、細胞保存後の細胞生存率を算出した結果、トレハロースを含む液であるＬＲＴ－３及びＬＲＴ－７に保存すると（実施例３及び比較例２）、トレハロースを含まない液である２．５％ＡＬＢＳ中に保存した場合（比較例６）と比べ、いずれの保存時間においても細胞生存率が上昇し、特に、Ｔ細胞をＬＲＴ－３（実施例３）中に保存した場合にその効果が高いことが示された（表１１、図３Ｂ及び図４Ｂ参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、ｐＨが上記範囲内であるが、トレハロース不含の液中に保存した場合や、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 Further, as a result of calculating the cell viability after cell preservation, when stored in LRT-3 and LRT-7 which are solutions containing trehalose (Example 3 and Comparative Example 2), it is a solution containing no trehalose 2.5. Compared with the case of storage in% ALBS (Comparative Example 6), the cell viability increased at any storage time, and the effect was particularly effective when T cells were stored in LRT-3 (Example 3). It was shown to be high (see Table 11, FIGS. 3B and 4B).
This result shows that when blood cell line cells (particularly T cells) are stored in a liquid containing trehalose and having a pH in the range of about 4.5 to about 7.2, the pH is within the above range, but trehalose. It is shown that cell death that occurs when stored can be effectively suppressed as compared with the case where it is stored in a liquid containing no trehalose or when it is stored in a liquid containing trehalose but having a pH outside the above range.
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、ｐＨが上記範囲内であるが、トレハロース不含の液中に保存した場合や、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 Further, as a result of calculating the cell viability after cell preservation, when stored in LRT-3 and LRT-7 which are solutions containing trehalose (Example 3 and Comparative Example 2), it is a solution containing no trehalose 2.5. Compared with the case of storage in% ALBS (Comparative Example 6), the cell viability increased at any storage time, and the effect was particularly effective when T cells were stored in LRT-3 (Example 3). It was shown to be high (see Table 11, FIGS. 3B and 4B).
This result shows that when blood cell line cells (particularly T cells) are stored in a liquid containing trehalose and having a pH in the range of about 4.5 to about 7.2, the pH is within the above range, but trehalose. It is shown that cell death that occurs when stored can be effectively suppressed as compared with the case where it is stored in a liquid containing no trehalose or when it is stored in a liquid containing trehalose but having a pH outside the above range.
（４）本発明の血球系細胞保存液の評価
４－１．材料及び方法
［血球系細胞］
上記（１）で使用したＴ細胞を用いた。 (4) Evaluation of Blood Cell Cell Preservation Solution of the Present Invention 4-1. Materials and methods [Blood cell lineage cells]
The T cells used in (1) above were used.
４－１．材料及び方法
［血球系細胞］
上記（１）で使用したＴ細胞を用いた。 (4) Evaluation of Blood Cell Cell Preservation Solution of the Present Invention 4-1. Materials and methods [Blood cell lineage cells]
The T cells used in (1) above were used.
［被験液の調製］
２．５％ＡＬＢＳ（表１２参照）は、上記（３）のとおり調製した。ＬＲに、α，α－トレハロース二水和物（富士フイルム和光純薬社製）を、トレハロースの終濃度が３（ｗ／ｖ）％となるように添加し、ｐＨが６．５３８のＬＲＴ－９を得た（表１２参照）。ＬＲＴ－９に、０．５ｍｏｌ／Ｌの水酸化ナトリウム溶液を、ｐＨが７．９６８となるように添加し、ＬＲＴ－１０を得た（表１２参照）。 [Preparation of test solution]
2.5% ALBS (see Table 12) was prepared as described in (3) above. Α, α-Trehalose dihydrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to LR so that the final concentration of trehalose was 3 (w / v)%, and LRT- with a pH of 6.538 was added. 9 was obtained (see Table 12). A 0.5 mol / L sodium hydroxide solution was added to LRT-9 so as to have a pH of 7.968 to obtain LRT-10 (see Table 12).
２．５％ＡＬＢＳ（表１２参照）は、上記（３）のとおり調製した。ＬＲに、α，α－トレハロース二水和物（富士フイルム和光純薬社製）を、トレハロースの終濃度が３（ｗ／ｖ）％となるように添加し、ｐＨが６．５３８のＬＲＴ－９を得た（表１２参照）。ＬＲＴ－９に、０．５ｍｏｌ／Ｌの水酸化ナトリウム溶液を、ｐＨが７．９６８となるように添加し、ＬＲＴ－１０を得た（表１２参照）。 [Preparation of test solution]
2.5% ALBS (see Table 12) was prepared as described in (3) above. Α, α-Trehalose dihydrate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to LR so that the final concentration of trehalose was 3 (w / v)%, and LRT- with a pH of 6.538 was added. 9 was obtained (see Table 12). A 0.5 mol / L sodium hydroxide solution was added to LRT-9 so as to have a pH of 7.968 to obtain LRT-10 (see Table 12).
［血球系細胞の培養］
上記（１）に記載の方法に従ってＴ細胞を培養した。 [Culture of blood cell lineage cells]
T cells were cultured according to the method described in (1) above.
上記（１）に記載の方法に従ってＴ細胞を培養した。 [Culture of blood cell lineage cells]
T cells were cultured according to the method described in (1) above.
［被験液の評価］
血球系細胞の被験液中での保存と、その後の生細胞回収率及び細胞回収率の測定については、以下の〔１〕～〔６〕の手順に従って行った。
〔１〕上記［血球系細胞の培養］の項目に記載の方法に従って培養したＴ細胞を含む培養用フラスコに、培養液と等量のＬＲを加え、５０ｍＬのコニカルチューブに移した後、細胞塊を懸濁した。
〔２〕細胞懸濁液の一部（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、保存前の細胞生存率を算出した。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（％）」を用いて算出した。
〔３〕残りの細胞懸濁液は、Ｔ細胞が５×１０５個となるように、Ｆａｌｃｏｎ（登録商標）１５ｍＬ高透明度ポリプロピレンコニカルチューブ（コーニング社製）に移し、遠心処理（３００×ｇ、１０分間、室温）後、上清を除去し、表１２に示す３種類の被験液（実施例７並びに比較例７及び比較例８）１ｍＬを加え、細胞ペレットを懸濁し、５℃にて保存した。
〔４〕保存２４時間後に、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（１ｍＬのピペットを用いて、５００μＬの液量でのピペッティングを５回）して、手順〔５〕の細胞回収率及び手順〔６〕の生細胞回収率の測定に供した。
〔５〕撹拌直後の１０μＬの液を、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）を用いて全細胞数を計測することにより、各保存期間における細胞回収率を算出した。なお、細胞回収率は、式「（全細胞数／保存前の全細胞数）×１００＝細胞回収率（％）」を用いて算出した。
〔６〕再度、緩やかに攪拌（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを２回）し、撹拌直後の２０μＬの液を、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生細胞回収率を算出した。なお、生細胞回収率は、式「（［全細胞数－死細胞数］／保存前の生細胞数）×１００＝生細胞回収率（％）」を用いて算出した。 [Evaluation of test solution]
The storage of blood cells in the test solution and the subsequent measurement of the viable cell recovery rate and the cell recovery rate were carried out according to the following procedures [1] to [6].
[1] Add an equal amount of LR to the culture solution to a culture flask containing T cells cultured according to the method described in the above item [Culture of blood cell lineage cells], transfer to a 50 mL conical tube, and then cell mass. Was suspended.
[2] A part (20 μL) of the cell suspension was divided into 1.5 mL microtubes to which 20 μL of trypan blue staining solution (manufactured by Gibco) was previously added. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated. The cell viability was calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". ..
[3] The remaining cell suspension, so that T cells is of 5 × 10 5 cells were transferred to Falcon (TM) 15 mL high transparency polypropylene conical tube (Corning), centrifuged (300 × g, After 10 minutes at room temperature), the supernatant is removed, 1 mL of the three test solutions shown in Table 12 (Example 7, Comparative Example 7 and Comparative Example 8) is added, the cell pellet is suspended, and the cells are stored at 5 ° C. did.
[4] After 24 hours of storage, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, with a liquid volume of 500 μL). Pipetting was performed 5 times) to measure the cell recovery rate of the procedure [5] and the viable cell recovery rate of the procedure [6].
[5] The cell recovery rate in each storage period is calculated by dividing 10 μL of the solution immediately after stirring into a one-cell counter and measuring the total number of cells using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). did. The cell recovery rate was calculated using the formula "(total number of cells / total number of cells before storage) x 100 = cell recovery rate (%)".
[6] Again, gently stir (using a 1 mL pipette, pipetting with a liquid volume of 250 μL twice), collect 20 μL of the liquid immediately after stirring in a 1.5 mL microtube, and stain with trypan blue. After mixing with 20 μL of the solution (Gibco), the cells were separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) were measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon). , The viable cell recovery rate in each storage period was calculated. The viable cell recovery rate was calculated using the formula "([total number of cells-number of dead cells] / number of viable cells before storage) x 100 = viable cell recovery rate (%)".
血球系細胞の被験液中での保存と、その後の生細胞回収率及び細胞回収率の測定については、以下の〔１〕～〔６〕の手順に従って行った。
〔１〕上記［血球系細胞の培養］の項目に記載の方法に従って培養したＴ細胞を含む培養用フラスコに、培養液と等量のＬＲを加え、５０ｍＬのコニカルチューブに移した後、細胞塊を懸濁した。
〔２〕細胞懸濁液の一部（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、保存前の細胞生存率を算出した。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（％）」を用いて算出した。
〔３〕残りの細胞懸濁液は、Ｔ細胞が５×１０５個となるように、Ｆａｌｃｏｎ（登録商標）１５ｍＬ高透明度ポリプロピレンコニカルチューブ（コーニング社製）に移し、遠心処理（３００×ｇ、１０分間、室温）後、上清を除去し、表１２に示す３種類の被験液（実施例７並びに比較例７及び比較例８）１ｍＬを加え、細胞ペレットを懸濁し、５℃にて保存した。
〔４〕保存２４時間後に、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（１ｍＬのピペットを用いて、５００μＬの液量でのピペッティングを５回）して、手順〔５〕の細胞回収率及び手順〔６〕の生細胞回収率の測定に供した。
〔５〕撹拌直後の１０μＬの液を、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）を用いて全細胞数を計測することにより、各保存期間における細胞回収率を算出した。なお、細胞回収率は、式「（全細胞数／保存前の全細胞数）×１００＝細胞回収率（％）」を用いて算出した。
〔６〕再度、緩やかに攪拌（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを２回）し、撹拌直後の２０μＬの液を、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生細胞回収率を算出した。なお、生細胞回収率は、式「（［全細胞数－死細胞数］／保存前の生細胞数）×１００＝生細胞回収率（％）」を用いて算出した。 [Evaluation of test solution]
The storage of blood cells in the test solution and the subsequent measurement of the viable cell recovery rate and the cell recovery rate were carried out according to the following procedures [1] to [6].
[1] Add an equal amount of LR to the culture solution to a culture flask containing T cells cultured according to the method described in the above item [Culture of blood cell lineage cells], transfer to a 50 mL conical tube, and then cell mass. Was suspended.
[2] A part (20 μL) of the cell suspension was divided into 1.5 mL microtubes to which 20 μL of trypan blue staining solution (manufactured by Gibco) was previously added. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated. The cell viability was calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". ..
[3] The remaining cell suspension, so that T cells is of 5 × 10 5 cells were transferred to Falcon (TM) 15 mL high transparency polypropylene conical tube (Corning), centrifuged (300 × g, After 10 minutes at room temperature), the supernatant is removed, 1 mL of the three test solutions shown in Table 12 (Example 7, Comparative Example 7 and Comparative Example 8) is added, the cell pellet is suspended, and the cells are stored at 5 ° C. did.
[4] After 24 hours of storage, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, with a liquid volume of 500 μL). Pipetting was performed 5 times) to measure the cell recovery rate of the procedure [5] and the viable cell recovery rate of the procedure [6].
[5] The cell recovery rate in each storage period is calculated by dividing 10 μL of the solution immediately after stirring into a one-cell counter and measuring the total number of cells using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). did. The cell recovery rate was calculated using the formula "(total number of cells / total number of cells before storage) x 100 = cell recovery rate (%)".
[6] Again, gently stir (using a 1 mL pipette, pipetting with a liquid volume of 250 μL twice), collect 20 μL of the liquid immediately after stirring in a 1.5 mL microtube, and stain with trypan blue. After mixing with 20 μL of the solution (Gibco), the cells were separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) were measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon). , The viable cell recovery rate in each storage period was calculated. The viable cell recovery rate was calculated using the formula "([total number of cells-number of dead cells] / number of viable cells before storage) x 100 = viable cell recovery rate (%)".
４－２．結果
細胞保存後の細胞回収率を算出した結果、２．５％ＡＬＢＳ（比較例７）やＬＲＴ－９（実施例７）中にＴ細胞を保存すると、いずれの保存時間においてもほぼ全ての細胞が回収できているのに対して、ＬＲＴ－１０（比較例８）中に保存した場合には、１０％程度の細胞が回収されていないことが示された（表１３、図５Ａ参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる保存容器への接着を効果的に抑制できることを示している。 4-2. Results As a result of calculating the cell recovery rate after cell storage, when T cells were stored in 2.5% ALBS (Comparative Example 7) or LRT-9 (Example 7), almost all cells were stored at any storage time. However, when stored in LRT-10 (Comparative Example 8), it was shown that about 10% of the cells were not recovered (see Table 13, FIG. 5A).
This result shows that when blood cell line cells (particularly T cells) are stored in a liquid containing trehalose and having a pH in the range of about 4.5 to about 7.2, trehalose is contained, but the pH is outside the above range. It is shown that the adhesion to the storage container that occurs when the cells are stored can be effectively suppressed as compared with the case where the cells are stored in the liquid.
細胞保存後の細胞回収率を算出した結果、２．５％ＡＬＢＳ（比較例７）やＬＲＴ－９（実施例７）中にＴ細胞を保存すると、いずれの保存時間においてもほぼ全ての細胞が回収できているのに対して、ＬＲＴ－１０（比較例８）中に保存した場合には、１０％程度の細胞が回収されていないことが示された（表１３、図５Ａ参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる保存容器への接着を効果的に抑制できることを示している。 4-2. Results As a result of calculating the cell recovery rate after cell storage, when T cells were stored in 2.5% ALBS (Comparative Example 7) or LRT-9 (Example 7), almost all cells were stored at any storage time. However, when stored in LRT-10 (Comparative Example 8), it was shown that about 10% of the cells were not recovered (see Table 13, FIG. 5A).
This result shows that when blood cell line cells (particularly T cells) are stored in a liquid containing trehalose and having a pH in the range of about 4.5 to about 7.2, trehalose is contained, but the pH is outside the above range. It is shown that the adhesion to the storage container that occurs when the cells are stored can be effectively suppressed as compared with the case where the cells are stored in the liquid.
また、細胞保存後の生細胞回収率を算出した結果、トレハロースを含む液であるＬＲＴ－９（実施例７）及びＬＲＴ－１０（比較例８）に保存すると、トレハロースを含まない液である２．５％ＡＬＢＳ（比較例７）中に保存した場合と比べ、細胞生存率が上昇し、特に、Ｔ細胞をＬＲＴ－９（実施例７）中に保存した場合にその効果が高いことが示された（表１３、図５Ｂ参照）。
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロース不含の液中に保存した場合や、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 Further, as a result of calculating the viable cell recovery rate after cell preservation, when stored in LRT-9 (Example 7) and LRT-10 (Comparative Example 8), which are solutions containing trehalose, the solutions do not contain trehalose2. It was shown that the cell viability was increased as compared with the case of storing in 5.5% ALBS (Comparative Example 7), and the effect was particularly high when the T cells were stored in LRT-9 (Example 7). (See Table 13, FIG. 5B).
This result shows that when blood cell line cells (particularly T cells) are stored in a solution containing trehalose and having a pH in the range of about 4.5 to about 7.2, they are stored in a solution containing no trehalose. It is shown that cell death caused by storage can be effectively suppressed as compared with the case where trehalose is contained but the pH is outside the above range.
この結果は、血球系細胞（特にＴ細胞）を、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存すると、トレハロース不含の液中に保存した場合や、トレハロースを含むが、ｐＨが上記範囲外の液中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 Further, as a result of calculating the viable cell recovery rate after cell preservation, when stored in LRT-9 (Example 7) and LRT-10 (Comparative Example 8), which are solutions containing trehalose, the solutions do not contain trehalose2. It was shown that the cell viability was increased as compared with the case of storing in 5.5% ALBS (Comparative Example 7), and the effect was particularly high when the T cells were stored in LRT-9 (Example 7). (See Table 13, FIG. 5B).
This result shows that when blood cell line cells (particularly T cells) are stored in a solution containing trehalose and having a pH in the range of about 4.5 to about 7.2, they are stored in a solution containing no trehalose. It is shown that cell death caused by storage can be effectively suppressed as compared with the case where trehalose is contained but the pH is outside the above range.
（５）非血球系細胞をトレハロース含有細胞保存液中で保存したときの細胞凝集レベル及び細胞生存率の評価
５－１．材料及び方法
［非血球系細胞］
非血球系細胞として、以下の表１４に記載のヒト骨髄由来間葉系幹細胞（ｈＢＭ－ＭＳＣ）を用いた。 (5) Evaluation of cell agglutination level and cell viability when non-hematological cells are stored in a trehalose-containing cell preservation solution 5-1. Materials and methods [Non-blood cell lineage cells]
As non-blood cell lineage cells, human bone marrow-derived mesenchymal stem cells (hBM-MSC) shown in Table 14 below were used.
５－１．材料及び方法
［非血球系細胞］
非血球系細胞として、以下の表１４に記載のヒト骨髄由来間葉系幹細胞（ｈＢＭ－ＭＳＣ）を用いた。 (5) Evaluation of cell agglutination level and cell viability when non-hematological cells are stored in a trehalose-containing cell preservation solution 5-1. Materials and methods [Non-blood cell lineage cells]
As non-blood cell lineage cells, human bone marrow-derived mesenchymal stem cells (hBM-MSC) shown in Table 14 below were used.
［被験液の調製］
表２のＬＲ（以下、便宜上「ＬＲ－６．５」という）（表１５参照）に、０．５ｍｏｌ／Ｌの水酸化ナトリウム溶液を、ｐＨが７．１６４となるように添加し、ＬＲ－７．２を得た（表１５参照）。表６のＷ（以下、便宜上「Ｗ－７．２」という）（表１５参照）に、０．１ｍｏｌ／Ｌの塩酸を、ｐＨが、６．５２６となるように添加し、Ｗ－６．５を得た（表１５参照）。なお、各被験液におけるｐＨは、室温（２１．６～２１．７℃）で測定した際の値である。 [Preparation of test solution]
To the LR in Table 2 (hereinafter referred to as “LR-6.5” for convenience) (see Table 15), a 0.5 mol / L sodium hydroxide solution was added so that the pH became 7.164, and LR- 7.2 was obtained (see Table 15). 0.1 mol / L hydrochloric acid was added to W in Table 6 (hereinafter referred to as “W-7.2” for convenience) (see Table 15) so that the pH became 6.526, and W-6. 5 was obtained (see Table 15). The pH of each test solution is a value measured at room temperature (21.6 to 21.7 ° C.).
表２のＬＲ（以下、便宜上「ＬＲ－６．５」という）（表１５参照）に、０．５ｍｏｌ／Ｌの水酸化ナトリウム溶液を、ｐＨが７．１６４となるように添加し、ＬＲ－７．２を得た（表１５参照）。表６のＷ（以下、便宜上「Ｗ－７．２」という）（表１５参照）に、０．１ｍｏｌ／Ｌの塩酸を、ｐＨが、６．５２６となるように添加し、Ｗ－６．５を得た（表１５参照）。なお、各被験液におけるｐＨは、室温（２１．６～２１．７℃）で測定した際の値である。 [Preparation of test solution]
To the LR in Table 2 (hereinafter referred to as “LR-6.5” for convenience) (see Table 15), a 0.5 mol / L sodium hydroxide solution was added so that the pH became 7.164, and LR- 7.2 was obtained (see Table 15). 0.1 mol / L hydrochloric acid was added to W in Table 6 (hereinafter referred to as “W-7.2” for convenience) (see Table 15) so that the pH became 6.526, and W-6. 5 was obtained (see Table 15). The pH of each test solution is a value measured at room temperature (21.6 to 21.7 ° C.).
［ｈＢＭ－ＭＳＣを含む乳酸リンゲル液の調製］
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、室温で約４分間静置した。
〔４〕ｈＢＭー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ー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、１２ｍＬ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。 [Preparation of lactated ringer solution containing hBM-MSC]
A lactated ringer solution containing hBM-MSC was prepared according to the following procedures [1] to [8].
[1] 4 × 10 5 hBM-MSCs were added to a 75 cm 2 flask, and the temperature was 37 ° C., 5% in the presence of a medium kit for human mesenchymal stem cells (manufactured by Lonza) (hereinafter referred to as “MSC medium”). 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.
[2] The MSC medium was removed, and hBM-MSC was rinsed with 10 mL of PBS (−).
[3] PBS (-) was removed, 4 mL of trypsin-EDTA (CC-3232, manufactured by Lonza) was added, and the mixture was allowed to stand at room temperature for about 4 minutes.
[4] The hBM-MSC was slowly shaken while observing under a microscope until about 90% of the hBM-MSC was exfoliated.
[5] 5 mL of MSC medium was added to stop the trypsin reaction, and hBM-MSC was collected by pipetting and transferred to a 50 mL centrifuge tube.
[6] Centrifugation was carried out at room temperature for 5 minutes at 600 × g.
[7] The supernatant (MSC medium) was removed, 12 mL of lactated Ringer's solution was added, and the precipitate (hBM-MSC) was suspended.
[8] measures the number of cells was adjusted with lactated Ringer's solution so that the 5 × 10 5 cells / mL, to prepare a lactic acid Ringer's solution containing hBM-MSC.
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、室温で約４分間静置した。
〔４〕ｈＢＭー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ー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、１２ｍＬ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。 [Preparation of lactated ringer solution containing hBM-MSC]
A lactated ringer solution containing hBM-MSC was prepared according to the following procedures [1] to [8].
[1] 4 × 10 5 hBM-MSCs were added to a 75 cm 2 flask, and the temperature was 37 ° C., 5% in the presence of a medium kit for human mesenchymal stem cells (manufactured by Lonza) (hereinafter referred to as “MSC medium”). 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.
[2] The MSC medium was removed, and hBM-MSC was rinsed with 10 mL of PBS (−).
[3] PBS (-) was removed, 4 mL of trypsin-EDTA (CC-3232, manufactured by Lonza) was added, and the mixture was allowed to stand at room temperature for about 4 minutes.
[4] The hBM-MSC was slowly shaken while observing under a microscope until about 90% of the hBM-MSC was exfoliated.
[5] 5 mL of MSC medium was added to stop the trypsin reaction, and hBM-MSC was collected by pipetting and transferred to a 50 mL centrifuge tube.
[6] Centrifugation was carried out at room temperature for 5 minutes at 600 × g.
[7] The supernatant (MSC medium) was removed, 12 mL of lactated Ringer's solution was added, and the precipitate (hBM-MSC) was suspended.
[8] measures the number of cells was adjusted with lactated Ringer's solution so that the 5 × 10 5 cells / mL, to prepare a lactic acid Ringer's solution containing hBM-MSC.
［被験液の評価］
ｈＢＭ－ＭＳＣの被験液中での保存と、その後の細胞生存率及び細胞凝集塊数の測定については、以下の〔１〕～〔６〕の手順に従って行った。
〔１〕上記［ｈＢＭ－ＭＳＣを含む乳酸リンゲル液の調製］の項目に記載の方法に従って培養し、調製したｈＢＭーＭＳＣを含む乳酸リンゲル液の一部（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、保存前の細胞生存率を算出した。
〔２〕残りの細胞懸濁液は、ｈＢＭ－ＭＳＣが２．５×１０５個となるように、低接着性のコニカルチューブであるステムフル（住友ベークライト社製）に０．５ｍＬずつ分注し、遠心処理（６００×ｇ、５分間、室温）後、上清を除去し、表１５に示す４種類の被験液（ＬＲ－６．５、Ｗ－６．５、ＬＲ－７．２、及びＷ－７．２）を０．５ｍＬ加え、細胞ペレットを懸濁し、２５℃にて２４時間保存した。
〔３〕２４時間保存後、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（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを１０回）した。
〔４〕撹拌直後の１０μＬの液を、ワンセルカウンターに分取し、ワンセルカウンターの細胞計数部の９カ所のエリアの細胞凝集塊の数を計測した。なお、３つ以上の細胞が接着した状態のものを細胞凝集塊として判断した。
〔５〕再度、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（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを２回）した。
〔６〕撹拌直後の２０μＬの液を、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、細胞生存率を算出した。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（％）」を用いて算出した。 [Evaluation of test solution]
The storage of hBM-MSC in the test solution and the subsequent measurement of the cell viability and the number of cell aggregates were carried out according to the following procedures [1] to [6].
[1] A part (20 μL) of the lactated Ringer's solution containing hBM-MSC prepared by culturing according to the method described in the above item [Preparation of lactated Ringer's solution containing hBM-MSC] is previously subjected to trypan blue staining solution (Gibco). (Manufactured by) 20 μL was added to a 1.5 mL microtube. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
[2] The remaining cell suspension, as hBM-MSC becomes 2.5 × 10 5 cells, was dispensed into a low adhesion conical tube Sutemufuru (manufactured by Sumitomo Bakelite) by 0.5mL min After centrifugation (600 × g, 5 minutes, room temperature), the supernatant was removed, and the four test solutions (LR-6.5, W-6.5, LR-7.2, and LR-7.2) shown in Table 15 and 0.5 mL of W-7.2) was added, the cell pellet was suspended, and the cells were stored at 25 ° C. for 24 hours.
[3] After storage for 24 hours, insert the tip of the tip visually from the bottom of the tube containing the cells to a position of about 5 mm, and gently stir (using a 1 mL pipette, with a liquid volume of 250 μL). Pipetting was performed 10 times).
[4] 10 μL of the solution immediately after stirring was separated into a one-cell counter, and the number of cell aggregates in nine areas of the cell counting section of the one-cell counter was measured. In addition, those in which three or more cells were adhered were judged as cell aggregates.
[5] Again, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, pipetting with a liquid volume of 250 μL). Twice).
[6] 20 μL of the solution immediately after stirring is collected in a 1.5 mL microtube, mixed with 20 μL of trypan blue staining solution (manufactured by Gibco), and then divided into a one-cell counter, and then separated into an optical microscope (ECLIPSE TS100, Nikon). The cell viability was calculated by measuring the total number of cells and the number of trypan blue positive cells (dead cells). The cell viability is calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". Calculated.
ｈＢＭ－ＭＳＣの被験液中での保存と、その後の細胞生存率及び細胞凝集塊数の測定については、以下の〔１〕～〔６〕の手順に従って行った。
〔１〕上記［ｈＢＭ－ＭＳＣを含む乳酸リンゲル液の調製］の項目に記載の方法に従って培養し、調製したｈＢＭーＭＳＣを含む乳酸リンゲル液の一部（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、保存前の細胞生存率を算出した。
〔２〕残りの細胞懸濁液は、ｈＢＭ－ＭＳＣが２．５×１０５個となるように、低接着性のコニカルチューブであるステムフル（住友ベークライト社製）に０．５ｍＬずつ分注し、遠心処理（６００×ｇ、５分間、室温）後、上清を除去し、表１５に示す４種類の被験液（ＬＲ－６．５、Ｗ－６．５、ＬＲ－７．２、及びＷ－７．２）を０．５ｍＬ加え、細胞ペレットを懸濁し、２５℃にて２４時間保存した。
〔３〕２４時間保存後、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（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを１０回）した。
〔４〕撹拌直後の１０μＬの液を、ワンセルカウンターに分取し、ワンセルカウンターの細胞計数部の９カ所のエリアの細胞凝集塊の数を計測した。なお、３つ以上の細胞が接着した状態のものを細胞凝集塊として判断した。
〔５〕再度、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（１ｍＬのピペットを用いて、２５０μＬの液量でのピペッティングを２回）した。
〔６〕撹拌直後の２０μＬの液を、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、細胞生存率を算出した。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（％）」を用いて算出した。 [Evaluation of test solution]
The storage of hBM-MSC in the test solution and the subsequent measurement of the cell viability and the number of cell aggregates were carried out according to the following procedures [1] to [6].
[1] A part (20 μL) of the lactated Ringer's solution containing hBM-MSC prepared by culturing according to the method described in the above item [Preparation of lactated Ringer's solution containing hBM-MSC] is previously subjected to trypan blue staining solution (Gibco). (Manufactured by) 20 μL was added to a 1.5 mL microtube. The cell suspension mixed with the trypan blue stain solution is separated into a one-cell counter, and the total number of cells and the number of trypan blue positive cells (dead cells) are measured using an optical microscope (ECLIPSE TS100, manufactured by Nikon Corporation). Therefore, the cell viability before storage was calculated.
[2] The remaining cell suspension, as hBM-MSC becomes 2.5 × 10 5 cells, was dispensed into a low adhesion conical tube Sutemufuru (manufactured by Sumitomo Bakelite) by 0.5mL min After centrifugation (600 × g, 5 minutes, room temperature), the supernatant was removed, and the four test solutions (LR-6.5, W-6.5, LR-7.2, and LR-7.2) shown in Table 15 and 0.5 mL of W-7.2) was added, the cell pellet was suspended, and the cells were stored at 25 ° C. for 24 hours.
[3] After storage for 24 hours, insert the tip of the tip visually from the bottom of the tube containing the cells to a position of about 5 mm, and gently stir (using a 1 mL pipette, with a liquid volume of 250 μL). Pipetting was performed 10 times).
[4] 10 μL of the solution immediately after stirring was separated into a one-cell counter, and the number of cell aggregates in nine areas of the cell counting section of the one-cell counter was measured. In addition, those in which three or more cells were adhered were judged as cell aggregates.
[5] Again, insert the tip of the tip visually from the bottom of the tube containing the cell-containing solution to a position of about 5 mm, and gently stir (using a 1 mL pipette, pipetting with a liquid volume of 250 μL). Twice).
[6] 20 μL of the solution immediately after stirring is collected in a 1.5 mL microtube, mixed with 20 μL of trypan blue staining solution (manufactured by Gibco), and then divided into a one-cell counter, and then separated into an optical microscope (ECLIPSE TS100, Nikon). The cell viability was calculated by measuring the total number of cells and the number of trypan blue positive cells (dead cells). The cell viability is calculated using the formula "(total number of living cells / total number of cells) x 100 = ([total number of cells-number of dead cells] / total number of cells) x 100 = cell viability (%)". Calculated.
５－２．結果
細胞保存後の生じる細胞凝集塊を測定したところ、ｈＢＭ－ＭＳＣを、Ｗ－６．５やＷ－７．２中に保存すると、細胞凝集塊が認められたのに対して、ｈＢＭ－ＭＳＣを、ＬＲ－６．５やＬＲ－７．２に保存した場合は、細胞凝集塊はほとんど認められなかった（表１６、図６Ａ参照）。
この結果は、ｈＢＭ－ＭＳＣにおいては、トレハロースを含み、かつ、ｐＨが約６．５～約７．２の範囲の液中に保存しても、保存中に生じる細胞凝集塊を抑制できないことを示している。 5-2. Results When the cell aggregates generated after cell storage were measured, when hBM-MSCs were stored in W-6.5 or W-7.2, cell aggregates were observed, whereas hBM-MSCs were observed. When stored in LR-6.5 or LR-7.2, almost no cell aggregates were observed (see Table 16, FIG. 6A).
This result shows that hBM-MSC cannot suppress cell aggregates generated during storage even if it contains trehalose and is stored in a liquid having a pH in the range of about 6.5 to about 7.2. Shown.
細胞保存後の生じる細胞凝集塊を測定したところ、ｈＢＭ－ＭＳＣを、Ｗ－６．５やＷ－７．２中に保存すると、細胞凝集塊が認められたのに対して、ｈＢＭ－ＭＳＣを、ＬＲ－６．５やＬＲ－７．２に保存した場合は、細胞凝集塊はほとんど認められなかった（表１６、図６Ａ参照）。
この結果は、ｈＢＭ－ＭＳＣにおいては、トレハロースを含み、かつ、ｐＨが約６．５～約７．２の範囲の液中に保存しても、保存中に生じる細胞凝集塊を抑制できないことを示している。 5-2. Results When the cell aggregates generated after cell storage were measured, when hBM-MSCs were stored in W-6.5 or W-7.2, cell aggregates were observed, whereas hBM-MSCs were observed. When stored in LR-6.5 or LR-7.2, almost no cell aggregates were observed (see Table 16, FIG. 6A).
This result shows that hBM-MSC cannot suppress cell aggregates generated during storage even if it contains trehalose and is stored in a liquid having a pH in the range of about 6.5 to about 7.2. Shown.
また、ｈＢＭ－ＭＳＣを、Ｗ－６．５やＷ－７．２中に保存すると、ＬＲ－６．５やＬＲ－７．２に保存した場合と比べ、いずれの液についても細胞生存率が上昇した（表１６、図６Ｂ参照）。
この結果は、ｈＢＭ－ＭＳＣを、トレハロースを含み、かつ、ｐＨが約６．５～約７．２の範囲の液中に保存すると、同じｐＨのＬＲ中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 In addition, when hBM-MSC was stored in W-6.5 or W-7.2, the cell viability of each solution was higher than that in the case of storing in LR-6.5 or LR-7.2. It increased (see Table 16, FIG. 6B).
This result shows that when hBM-MSC is stored in a liquid containing trehalose and having a pH in the range of about 6.5 to about 7.2, it is compared with the case where it is stored in LR having the same pH. It is shown that the cell death that occurs in the disease can be effectively suppressed.
この結果は、ｈＢＭ－ＭＳＣを、トレハロースを含み、かつ、ｐＨが約６．５～約７．２の範囲の液中に保存すると、同じｐＨのＬＲ中に保存した場合と比べ、保存したときに生じる細胞死を効果的に抑制できることを示している。 In addition, when hBM-MSC was stored in W-6.5 or W-7.2, the cell viability of each solution was higher than that in the case of storing in LR-6.5 or LR-7.2. It increased (see Table 16, FIG. 6B).
This result shows that when hBM-MSC is stored in a liquid containing trehalose and having a pH in the range of about 6.5 to about 7.2, it is compared with the case where it is stored in LR having the same pH. It is shown that the cell death that occurs in the disease can be effectively suppressed.
これらの結果は、ｈＢＭ－ＭＳＣにおいては、トレハロースを含み、かつ、ｐＨが約６．５～約７．２の範囲の液中に保存すると細胞死は抑制できるが、保存中に生じる細胞凝集塊を抑制できないことを示しており、トレハロースを含み、かつ、ｐＨが約４．５～約７．２の範囲の液中に保存した際の細胞凝集塊の抑制効果は、血球系細胞（特にＴ細胞）に特異的であることを示している。
These results show that in hBM-MSC, cell death can be suppressed when stored in a liquid containing trehalose and having a pH in the range of about 6.5 to about 7.2, but cell aggregates generated during storage can be suppressed. The effect of suppressing cell agglutination when stored in a liquid containing trehalose and having a pH in the range of about 4.5 to about 7.2 is that blood cell cells (particularly T) cannot be suppressed. It shows that it is specific to cells).
本発明によると、良質な血球系細胞による細胞移植を実施することができ、がん等の疾患に対する治療効果の向上が期待できる。また、血球系細胞の凝集物が、カニューレ中に詰まったり、肺静脈等の細い血管中に塞栓を形成してしまうリスクを低減することができる。さらに、血球系細胞を含む本件血球系細胞保存用液を、新しい移植用液に置換することなく、そのまま生体内へ投与することができる。また生体由来成分を含まず細胞保存容器や投与バッグへの細胞接着を抑制し、正確な細胞数を生体内に投与できる。このため、本発明は血球系細胞を利用した移植医療分野に資するものである。
According to the present invention, cell transplantation using high-quality blood cell lineage cells can be performed, and improvement in therapeutic effect for diseases such as cancer can be expected. In addition, it is possible to reduce the risk that aggregates of blood cell lineage cells may become clogged in the cannula or form an embolus in a small blood vessel such as a pulmonary vein. Furthermore, the blood cell cell preservation solution containing blood cell cells can be directly administered into the living body without being replaced with a new transplantation solution. In addition, it does not contain biological components, suppresses cell adhesion to cell storage containers and administration bags, and can administer an accurate number of cells in vivo. Therefore, the present invention contributes to the field of transplantation medicine using blood cell lineage cells.
Claims (15)
- トレハロース若しくはその誘導体又はこれらの塩を含み、かつ、ｐＨが４．５～７．２である血球系細胞保存用液。 A blood cell cell preservation solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
- 等張液である、請求項１に記載の液。 The liquid according to claim 1, which is an isotonic liquid.
- 等張液が、乳酸リンゲル液である、請求項２に記載の液。 The liquid according to claim 2, wherein the isotonic liquid is a lactated Ringer liquid.
- 血球系細胞を０～４０℃で保存するための、請求項１～３のいずれか１項に記載の液。 The solution according to any one of claims 1 to 3, for storing blood cell lineage cells at 0 to 40 ° C.
- 血球系細胞の生存率低下を抑制するために用いられる、請求項１～４のいずれか１項に記載の液。 The solution according to any one of claims 1 to 4, which is used for suppressing a decrease in the survival rate of blood cell lineage cells.
- 血球系細胞の細胞凝集を抑制するために用いられる、請求項１～５のいずれか１項に記載の液。 The solution according to any one of claims 1 to 5, which is used for suppressing cell aggregation of blood cell lineage cells.
- 血球系細胞の保存容器への接着を抑制するために用いられる、請求項１～６のいずれか１項に記載の液。 The solution according to any one of claims 1 to 6, which is used for suppressing adhesion of blood cell lineage cells to a storage container.
- 血球系細胞の移植に用いられる、請求項１～７のいずれか１項に記載の液。 The solution according to any one of claims 1 to 7, which is used for transplantation of blood cell lineage cells.
- 血球系細胞がＴ細胞である、請求項１～８のいずれか１項に記載の液。 The solution according to any one of claims 1 to 8, wherein the blood cell lineage cell is a T cell.
- トレハロース若しくはその誘導体又はこれらの塩を含む、請求項１～９のいずれか１項に記載の液を調製するための粉末製剤。 A powder preparation for preparing the liquid according to any one of claims 1 to 9, which comprises trehalose or a derivative thereof or a salt thereof.
- トレハロース若しくはその誘導体又はこれらの塩を含み、かつ、ｐＨが４．５～７．２である液中で、血球系細胞を保存する工程を含む、血球系細胞の保存方法。 A method for preserving blood cells, which comprises a step of preserving blood cells in a solution containing trehalose or a derivative thereof or a salt thereof and having a pH of 4.5 to 7.2.
- 液が、等張液である、請求項１１に記載の保存方法。 The storage method according to claim 11, wherein the liquid is an isotonic liquid.
- 等張液が、乳酸リンゲル液である、請求項１２に記載の保存方法。 The storage method according to claim 12, wherein the isotonic solution is a lactated ringer solution.
- 血球系細胞を０～４０℃で保存する、請求項１１～１３のいずれか１項に記載の保存方法。 The storage method according to any one of claims 11 to 13, wherein the blood cell line cells are stored at 0 to 40 ° C.
- 血球系細胞がＴ細胞である、請求項１１～１４のいずれか１項に記載の保存方法。 The storage method according to any one of claims 11 to 14, wherein the blood cell lineage cells are T cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006230396A (en) * | 2004-10-12 | 2006-09-07 | Nipro Corp | Cell preservative liquid |
| WO2012063870A1 (en) * | 2010-11-09 | 2012-05-18 | 株式会社大塚製薬工場 | Stem cell suspension |
| WO2014208053A1 (en) * | 2013-06-28 | 2014-12-31 | 株式会社大塚製薬工場 | Trehalose and dextran-containing solution for transplanting mammalian cells |
| WO2020218461A1 (en) * | 2019-04-26 | 2020-10-29 | 株式会社大塚製薬工場 | Trehalose-containing liquid for mammalian cell preservation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006230396A (en) * | 2004-10-12 | 2006-09-07 | Nipro Corp | Cell preservative liquid |
| WO2012063870A1 (en) * | 2010-11-09 | 2012-05-18 | 株式会社大塚製薬工場 | Stem cell suspension |
| WO2014208053A1 (en) * | 2013-06-28 | 2014-12-31 | 株式会社大塚製薬工場 | Trehalose and dextran-containing solution for transplanting mammalian cells |
| WO2020218461A1 (en) * | 2019-04-26 | 2020-10-29 | 株式会社大塚製薬工場 | Trehalose-containing liquid for mammalian cell preservation |
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