JP2021000027A - Medical cell cryopreservation solution - Google Patents

Abstract

To provide animal cell cryopreservation solutions that cause decreased cell viability when animal cells are cryopreserved and a small amount of cell death when animal cells are immersed and further that can be directly administered to humans after thawed.SOLUTION: The present invention relates to: an animal cell cryopreservation solution containing at least one selected from the group consisting of taurine, glycine, and derivatives thereof, a cryoprotectant other than dimethyl sulfoxide, a water-soluble polysaccharide and an oligosaccharide; an animal cell dispersion in which animal cells are present in a dispersed state in such an animal cell cryopreservation solution; and a method for cryopreserving animal cells comprising the steps of suspending animal cells in such an animal cell cryopreservation solution, and freezing it.SELECTED DRAWING: None

Description

The present invention relates to an animal cell cryopreservation solution that can be directly administered to humans after thawing.

Cryopreservation of cells has become widespread as an indispensable technique in cell biology. In recent years, the technique of cell cryopreservation has been applied to the preservation of various established cells and living (including human) cells, and the preservation of cells collected for medical purposes.

In recent years, research on cell therapy such as stem cell therapy and cancer immunotherapy using cells collected from patients or other donors for treatment has been actively conducted. In addition, attempts have been made to administer various cells such as cardiomyocytes and chondrocytes to patients, and future development is drawing attention.

Stem cell therapy is a medical treatment that aims to restore the function of cells and tissues damaged by various diseases by utilizing the self-renewal ability and pluripotency of stem cells and various factors secreted by stem cells. .. Bone marrow transplantation is performed as a treatment method for patients with intractable blood diseases such as leukemia and aplastic anemia, and hematopoietic stem cells engraft in the patient's body, and hematopoietic ability can be maintained for almost a lifetime. Recently, many researchers have identified stem cells in various tissues such as central nerves, peripheral nerves, bone marrow, heart, small intestine, and blood vessels, aiming for clinical application using stem cells other than hematopoietic stem cells, and traumatic diseases and tissues. Tissue stem cell transplantation treatment for degenerative diseases is beginning to be practiced.

Among the stem cells, there are stem cells whose differentiation direction is determined and mesenchymal stem cells having the ability to differentiate into various cells such as fat, bone marrow, and cartilage. In stem cell therapy as well, treatment in which mesenchymal stem cells are administered is actively performed. Here, the mesenchymal stem cell refers to a cell in which CD73 / CD90 / CD105, which is a cell membrane antigen, is positive and CD14 / CD34 / CD45 / CD19 / HLA-DR is negative, and is in vivo. It can be collected from various tissues of. The tissue to be collected is not limited, but various tissues such as bone marrow, fat, umbilical cord, umbilical cord blood, dental pulp, synovium, uterus, placenta, peripheral blood, and skin can be exemplified.

On the other hand, cancer immuno-cell therapy is cell medicine in which immune cells that have the function of attacking cancer are taken out of the body, strengthened, and then returned to the body. Dendritic cell vaccine therapy, Alpha Beta T Therapy using immune cells such as cell therapy (αβT cell therapy), gamma delta T cell therapy (γδT cell therapy), CTL therapy, and natural killer cell therapy (NK cell therapy) is practiced.

When a stem cell for transplantation or a T cell for transplantation is stored for a long period of time, it is generally cryopreserved, and a cell cryopreservation solution containing dimethyl sulfoxide (DMSO) as a cryoprotectant is usually used. DMSO used in cryopreservation solutions has a high cell cryoprotective ability, and is a very useful substance in an environment where cells can be frozen immediately after suspension. On the other hand, DMSO is also well known as a cytotoxic substance, and cells die when immersed in a solution containing DMSO at room temperature. Furthermore, DMSO has been reported to be toxic to treated patients (Non-Patent Documents 1 and 2). For example, in bone marrow transplantation, patients transplanted with cells cryopreserved in DMSO cause side effects, and in some cases, serious complications develop. Direct side effects and delayed-onset side effects during infusion have been observed in a dose-dependent manner in DMSO. After thawing the cryopreserved cell preparation in a cryopreservation solution containing DMSO, it is administered systemically as it is. Then, side effects and complications may occur.

In addition, administration via direct injection into tissues (for example, intracranial administration, intramuscular administration, or intracardiac administration) locally increases the DMSO concentration, and thus there is concern about local toxicity. From these problems, when cells are preserved using a cell cryopreservation solution using DMSO, it is common to remove the cell cryopreservation solution and replace it with the administration solution before administration. The method of removing DMSO components from cryopreserved cells in this way reduces DMSO-related complications and side effects (Non-Patent Document 3). However, there are problems that the number of cells recovered by the centrifugation process decreases, that the replacement process of the cell cryopreservation solution takes time and effort, and that equipment such as CPC is required for replacement. (Non-Patent Document 4).

As the development of stem cells and T cells as cell preparations progresses, issues during mass production as products have become apparent. The process for producing a cell preparation is roughly divided into (1) cell culture step, (2) cell recovery, washing step, (3) cell dispensing, and cryopreservation step. In the case of mass production of cell medicines, after the recovery and washing steps of (2), the cells are suspended in a cryopreservation solution, and until they are frozen through the dispensing and freezing steps of (3). It will be immersed in a non-frozen cell cryopreservation solution at room temperature, but the immersion time before freezing will increase as the production scale expands. DMSO is known to exhibit high cytotoxicity under conditions above room temperature, and cells are cryopreserved at room temperature for a long time in the process of dispersing cells in mass production in a cell cryopreservation solution and freezing it. Conditions exposed to the fluid can lead to excessive cell death and impair the quality of cell medicines.

Due to the risk problem during administration of DMSO, by adding polysaccharides such as trehalose and dextran to the suspension of mesenchymal stem cells, the cells are kept in a solution state for several hours to several days for replacement. Development of an administration solution that can be administered as it is without washing is underway (Patent Document 1). However, these administration solutions can only be stored under refrigerated conditions and can only be stored for a few days at the longest, so they are only used for transportation from other medical institutions and are long-term. It is not intended for preservation. Since cell production is expected to shift to mass production with the spread of cell medicines, long-term storage is an essential technique, and refrigerated storage is insufficient.

When cryopreserving cells, it is necessary that the cell cryopreservation solution does not freeze extracellularly, or that the ice crystals formed even when frozen are sufficiently small and do not damage the cells. As a substance having properties similar to the cell cryoprotective ability of DMSO, a cell cryopreservation solution using propylene glycol or glycerol has been developed. However, these substances are inferior in the cell cryoprotective ability of DMSO, and no substance that can completely replace DMSO has been found.

Trehalose, which is a kind of oligosaccharide, has high biocompatibility and is known to function as an antifreeze in insects, plants, yeasts and the like (Non-Patent Document 5). It is also known to have an action of stabilizing proteins and phospholipids, which are cell membrane components (Non-Patent Document 6). In addition, other oligosaccharides are also considered to have a similar protective effect. Based on these facts, although studies have been conducted as an effective substance in cell cryopreservation solutions, oligosaccharides themselves have weak cell membrane permeability and a weak function of suppressing the formation of ice crystals that occur inside cells. The saccharide alone does not show a sufficient cell cryoprotective effect (Non-Patent Document 7).

In addition, attempts have been made to combine various substances with cell cryoprotectants in order to supplement the cell cryoprotective effect in the form of containing no DMSO. For example, it has been reported that the cell viability can be improved by combining propylene glycol and a saccharide (Patent Document 2). However, the survival rate after cryopreservation of cells is not sufficient, and the adverse effects on cells due to immersion at room temperature in the manufacturing process and the use process cannot be suppressed. Although various studies have been conducted on cell cryopreservation solutions, no cell cryopreservation solution having both properties of cell preservation in a solution state and cell cryopreservation in a frozen state has been found yet. , Development of such a cell cryopreservation solution is required.

In addition, a method of cryopreserving cells in a form that does not contain DMSO by containing a polymer in which the amino group of ε-poly-L-lysine is carboxylated has also been reported (Patent Document 3). It cannot be administered directly into the body and needs to be washed.

International Publication 2014/208053 Specification U.S. Application Publication 2016/0369227 International Publication 2009/169183 specification

Allison Hubel et al., Transfusion. 2001 May; 41 (5): 579-80 Sauer-Heilborn A et al., Transfusion. 2004 Jun; 44 (6): 907-16 R Syme et al., Biology of Blood and Marrow Transplantation Volume 10, Issue 2, February 2004, Pages 135-141 Calmels B et al., Bone Marrow Transplant. 2003 May; 31 (9): 823-8 Ryo Shirakashi, Production Research Vol. 55, No. 2 (2003), 150-152 Hiroshi Takahashi, Membrane 24 (4), (1999), 207-214 D.M.C. Sharp et al., Cryobiology 67 (2013), 305-311, Volume 10, Issue 2, February 2004, 135-141

An object of the present invention is that the decrease in cell viability when animal cells are cryopreserved is suppressed, the cell death when the animal cells are immersed is also suppressed, and the cells are directly administered to humans after thawing. The purpose is to provide an animal cell cryopreservation solution that can be produced.

Furthermore, another object of the present invention is to provide an animal cell cryopreservation solution having good initial adhesion of animal cells after thawing and a good cell proliferation rate.

The present invention comprises cryopreservation of animal cells containing (at least one selected from the group consisting of taurine, glycine, and derivatives thereof), cryoprotectants other than dimethyl sulfoxide, water-soluble polysaccharides, and oligosaccharides. It is a liquid.

Further, the present invention is an animal cell cryopreservation solution containing taurine and having an improved cell proliferation rate after thawing.

Further, the present invention is an animal cell dispersion in which animal cells are dispersed in these animal cell cryopreservation solutions.

Furthermore, the present invention is a method for cryopreserving animal cells, which comprises a step of suspending animal cells in the above-mentioned animal cell cryopreservation solution and a step of freezing the suspension.

According to the animal cell cryopreservation solution of the present invention, the decrease in cell viability when the animal cells are cryopreserved using the solution is suppressed, and the cell death when the animal cells are immersed therein is also suppressed, and it is used. It is possible to directly administer the animal cell suspension after thawing the cryopreserved product of animal cells to humans.

Further, according to the animal cell cryopreservation solution of the present invention containing taurine, the proliferation rate of the animal cells after thawing the cryopreserved product of the animal cells using the taurine is good, and particularly for adherent animal cells. If so, the initial adhesion to the incubator is also good.

The figure which compared the number of cells after cryopreserving and thawing animal cells using the animal cell cryopreservation solution of Example 1 and Comparative Example 4 in a plate culture for 1-3 days. The figure which compared the cell growth rate after cryopreserving and thawing animal cells using the animal cell cryopreservation solution of Example 1 and Comparative Example 4 in a plate culture for 1-3 days.

1. 1. Animal cell cryopreservation solution The present invention comprises freezing animal cells containing at least one selected from the group consisting of taurine, glycine, and derivatives thereof, a cryoprotectant other than DMSO, a water-soluble polysaccharide, and an oligosaccharide. It is a preservation solution. Hereinafter, the "animal cell cryopreservation solution" is also abbreviated as "cryopreservation solution".

The "derivative of taurine" used in the present invention refers to N-substituted taurine, hypotaurine, thiotaurine, or salts thereof. Examples of such N-substituted taurine include N- (2-acetamide) -2-aminoethanesulfonic acid, N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, and N-cyclohexyl-2-aminoethane. Sulfonic acid, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid, 2-morpholinoethanesulfonic acid, piperazin-1,4-bis (2-ethanesulfonic acid), N-tris ( Hydroxymethyl) methyl-2-aminoethanesulfonic acid, and salts thereof. Among these, as the taurine derivative, hypotaurine and / or thiotaurine is preferable in that the decrease in survival rate is efficiently suppressed.

The "glycine derivative" used in the present invention refers to an ester of glycine, an N-substituted glycine, an ester of N-substituted glycine, a salt thereof, or a salt of glycine. Examples of such glycine derivatives include sarcosine (N-methylglycine), N-ethylglycine, N-propylglycine, N, N-diethylglycine, N, N-dimethylglycine, N-amidinoglycine, and N-amidino-N. -Methylglycine, glycylglycine, phenaceturic acid, glycine methyl ester hydrochloride, glycine ethyl ester hydrochloride, glycine n-butyl ester hydrochloride, glycine t-butyl ester hydrochloride, glycine n-propyl ester hydrochloride, glycine n- Examples thereof include pentyl ester hydrochloride and glycine benzyl ester hydrochloride.

Among taurine, glycine, and derivatives thereof used in the present invention, taurine and glycine are most preferable because they have a high effect of suppressing a decrease in survival rate.

The concentration of at least one selected from the group consisting of taurine, glycine, and derivatives thereof used in the present invention is preferably 0.5 to 2.0 (mass / volume)%, particularly 1.0 to 1.0 to 2.0. It is preferably 1.5 (mass / volume)%.

The "freeze protective agent excluding dimethyl sulfoxide" used in the present invention refers to a substance other than DMSO that suppresses the formation of ice crystals when the solution is frozen, and among them, propylene glycol and / or glycerol are preferable, and propylene glycol is particularly preferable. preferable.

When the cryoprotectant other than dimethyl sulfoxide used in the present invention is propylene glycol and / or glycerol, the concentration thereof is preferably 5 to 20 (volume / volume)%, particularly 8 to 10 (volume / volume). It is preferably%.

The "water-soluble polysaccharide" used in the present invention refers to a water-soluble substance in which a large number of monosaccharide molecules are polymerized by glycosidic bonds, and among them, at least selected from the group consisting of dextran, hydroxyethyl starch, and derivatives thereof. One can be used.

The dextran and dextran moiety in its derivative used in the present invention, there is provided a polysaccharide _{(C 6 H 10 O 5)} n composed of D- glucose is not particularly limited as long as the main chain [alpha] 1 → 6 bond ..

The dextran derivative used in the present invention includes dextran sulfate, carboxylated dextran, diethylaminoethyl (DEAE) -dextran and the like.

The molecular weight of dextran or a derivative thereof used in the present invention is not particularly limited, but the weight average molecular weight (Mw) is preferably 40,000 to 200,000.

Further, among the dextran and the dextran derivative, dextran is preferable, and for example, dextran 40 (Mw = 40,000) and dextran 70 (Mw = 70,000) are preferably used.

The hydroxyethyl starch moiety in the hydroxyethyl starch and its derivatives used in the present invention consists of amylose in which α-D-glucose is linearly bound (α-1,4 bond) and a branch (α-1,6 bond). ) Is a mixture with amylopectin, which is a substance in which the glucose units C2, C3, and C6 are hydroxyethylated. The most common hydroxyethyl starch, hetastarch, is derived from cornstarch and has a molecular weight of about 450,000 Da. Pentastarch is also an analog of hetastarch with a molecular weight of about 264,000 Da.

The molecular weight of hydroxyethyl starch and its derivatives used in the present invention is not particularly limited, but the weight average molecular weight (Mw) is preferably 130,000 to 450,000.

Dextran is preferable as the water-soluble polysaccharide used in the present invention.

The concentration of at least one selected from the group consisting of dextran, hydrosiethyl starch, and derivatives thereof used in the present invention is preferably 5 to 20 (mass / volume)%, particularly 10 to 15 (mass / volume). / Volume)% is preferable.

The "oligosaccharide" used in the present invention refers to an oligomer of a saccharide in which several monosaccharides are bound by glycoside bonds, among which trehalose, sorbitol, mannitol, galactose, fructose, raffinose, sucrose, and them (that is, trehalose, Salts of sorbitol, mannitol, galactose, fructose, raffinose, sucrose) can be mentioned.

Here, as the "salt" of (trehalose, sorbitol, mannitol, galactose, fructose, raffinose, and sucrose), for example, hydrochloride, hydrobromide, hydroiodide, as long as chemically possible, Phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate , Citrate, fumarate, maleate, malate and other acid addition salts, sodium salt, potassium salt, calcium salt and other metal salts, ammonium salt, alkylammonium salt and the like.

Among these oligosaccharides used in the present invention, trehalose is particularly preferable.

The concentration of the oligosaccharide used in the present invention is preferably 0.05 to 0.3 mol / L, particularly preferably 0.1 to 0.2 mol / L.

Further, the present invention is a cryopreservation solution containing taurine and having an improved cell proliferation rate after thawing. The cryopreservation solution of the present invention can be obtained by adding taurine to a known cryopreservation solution, for example, a cryopreservation solution containing DMSO, but the cryopreservation solution before the addition of taurine has a function as a cryopreservation solution. There are no particular restrictions. Then, by adding taurine, the proliferation rate of animal cells when they are thawed after cryopreservation is improved. Further, in the case of adhesive animal cells, the addition of taurine improves the initial adhesion of the thawed animal cells to the incubator. Here, the preferred concentration of taurine is 0.5 to 2.0 (mass / volume)%.

The cryopreservation solution of the present invention preferably contains one or more isotonic solutions selected from the group consisting of physiological saline, phosphate buffer, Ringer's acetate solution, and Ringer's bicarbonate solution as the main component, and particularly bicarbonate. It preferably contains Ringer's solution.

Further, it is preferable that the cryopreservation solution of the present invention is safe for humans and can be directly administered to humans. In order to be able to be directly administered to humans, it must be sterile and, for example, the endotoxin content measured in accordance with the Pharmacopoeia must be less than or equal to the specified amount.

A suitable combination of each component of the cryopreservation solution of the present invention is a combination of those listed as preferable for each component. Further, a suitable combination of the concentration ranges of each component is also a combination of the concentration ranges preferred for each component.

2. 2. Animal cell dispersions The present invention also includes 1. This is an animal cell dispersion in which animal cells are dispersed in the cryopreservation solution of the present invention described in the above.

The type of animal cells in the animal cell dispersion of the present invention is not particularly limited, and animal cells suitable for the intended use may be selected, and mammalian cells such as primates can be exemplified. Of these, human cells are preferable, and in particular, one or more cells selected from the group consisting of human mesenchymal stem cells, human immune cells, human nerve cells, human hematopoietic stem cells, and human cardiomyocytes are preferable.

The density of animal cells in the animal cell dispersion of the present invention is also not particularly limited as long as the cell viability is not significantly reduced due to the high density, but for example, 1 × 10 ³ to 1 × 10 ⁸ cells / mL. It is preferably 1 × 10 ^{5 to} 5 × 10 ⁷ cells / mL, and more preferably 1 × 10 ⁶ to 2 × 10 ⁷ cells / mL.

The animal cell dispersion of the present invention includes an animal cell suspension at a stage where animal cells are suspended in the cryopreservation solution of the present invention, and an animal cell suspension obtained by freezing the animal cell suspension. Also included are animal cell suspensions obtained by thawing the fluid and such frozen dispersions of animal cells.

The animal cell dispersion of the present invention is free from harmful substances to humans to the extent that it can be directly administered to humans without performing a dilution operation or a washing operation of the animal cells contained therein. preferable. For example, the content of animal cell aggregates and dead cell fragments is sufficiently low. In particular, it is preferable to satisfy the standard as a cell pharmaceutical composition.

3. 3. Method for cryopreserving animal cells The present invention further comprises 1. A method for cryopreserving animal cells, which comprises a step of suspending the suspension in the cryopreservation solution of the present invention described in the above and a step of freezing the suspension.

The method for cryopreserving animal cells of the present invention includes a method in which the animal cell suspension is in the state of a frozen animal cell dispersion, and the frozen animal cell dispersion is thawed and used again as an animal cell suspension. It also includes a method that includes a step of performing.

In the method for cryopreserving animal cells of the present invention, the operation of suspending the animal cells in the cryopreservation solution of the present invention, the operation of freezing the obtained animal cell suspension, and the operation of thawing the frozen animal cell dispersion are performed. , Suspension conditions, freezing conditions, and thawing conditions similar to those in the prior art using cryopreservation solutions containing DMSO can be adopted. However, those skilled in the art can easily use it for cell density, suspension temperature, and suspension operation according to the characteristics and conditions of individual cells to be targeted or the composition of the cryopreservation solution by trial as appropriate. You will be able to optimize equipment selection, suspension operation strength, cooling temperature programming for freezing, and heating conditions for thawing.

Whether it is a frozen animal cell dispersion obtained by the method for cryopreserving animal cells of the present invention or an animal cell suspension obtained by thawing it, they satisfy the requirements for pharmaceuticals. Is preferable. In that case, the method for cryopreserving animal cells of the present invention is also understood as a method for producing a cytopharmaceutical composition.

Further, in the cell suspension step using the cryopreservation solution of the present invention, the decrease in cell viability is small and excessive cell death does not occur as the period during which the animal cells are maintained in the cryopreservation state in the cryopreservation solution. The period is preferred, for example 2 hours, 6 hours, 12 hours, 1 day, 2 days, or 3 days. If the storage period of animal cells is too long, it may adversely affect the survival of cells. Therefore, from the viewpoint of avoiding adverse effects on the survival rate of cells, it is preferably 12 hours or less, but some animal cells It may be longer than that as long as it survives.

The storage temperature when the animal cells are stored in a solution state in the cryopreservation solution of the present invention may be a temperature at which the cell suspension does not freeze and the cells can survive, and is usually 0 to 37 ° C. It is preferably in the range of 0 to 25 ° C, particularly preferably 0 to 20 ° C.

The cryopreservation solution of the present invention includes stabilizers (eg, human serum albumin, polyethylene glycol), buffers (eg, phosphate buffer, sodium acetate buffer), chelating agents (eg, EDTA, EGTA, citric acid, salicylate). , Vitamins (eg, vitamin A or its derivatives, vitamin B2 or its derivatives, vitamin B6 or its derivatives, vitamin B12 or its derivatives, pantothenic acid or its derivatives, α-tocopherol, β-tocopherol, γ-tocopherol, vitamin E acetate , Vitamin D, biotin, pantothenic acid, pantetin, pyroquinolinquinone, etc.), antioxidants (mannitol, ascorbic acid, trolox, etc.), Rho inhibitors, etc. may be supplemented as needed. ..

Hereinafter, embodiments of the present invention will be described with reference to examples, but these do not limit the scope of the present invention.

The materials and equipment used in the Examples and / or Comparative Examples are as follows.
(I) Propylene glycol; Wako Pure Chemical Industries, Ltd.
(Ii) Dextran 40; Wako Pure Chemical Industries, Ltd.
(Iii) Trehalose dihydrate: Wako Pure Chemical Industries, Ltd.
(Iv) Distilled water: Otsuka distilled water (v) DMEM, High Glucose: Life Technologies
(Vi) Inactivated neonatal bovine serum: Life Technologies
(Vii) Penicillin-Spretomycin Solution (× 100): Wako Pure Chemical Industries, Ltd.
(Viii) Bicarbon (ringel bicarbonate solution); AY Pharma Co., Ltd.
(Ix) Phosphate buffered saline (PBS) pH7.4; Wako Pure Chemical Industries, Ltd.
(X) 0.25% Trypsin-EDTA: Wako Pure Chemical Industries, Ltd.
(Xi) 0.4 w / v% Trypan Blue Solution: Wako Pure Chemical Industries, Ltd.
(Xii) Taurine; Wako Pure Chemical Industries, Ltd.
(Xiii) Dimethyl sulfoxide: Wako Pure Chemical Industries, Ltd.
(Xiv) Glycine; Wako Pure Chemical Industries, Ltd.
(Xv) Mannitol; Wako Pure Chemical Industries, Ltd.
(Xvi) Sorbitol; Wako Pure Chemical Industries, Ltd.
(Xvii) Galactose; Wako Pure Chemical Industries, Ltd.
(Xviii) Raffinose pentahydrate; Wako Pure Chemical Industries, Ltd.
(Xix) Sucrose; Wako Pure Chemical Industries, Ltd.
(Xx) Fructose; Wako Pure Chemical Industries, Ltd.
(Xxi) Solacet F (Ringel acetate solution); Terumo Corporation
(Xxii) Glycerol; Wako Pure Chemical Industries, Ltd.
(Xxiii) Phosphate buffer; Wako Pure Chemical Industries, Ltd.
(Xxiv) Saline; Otsuka Pharmaceutical Factory Co., Ltd.
(Xxv) Bovine serum albumin; Wako Pure Chemical Industries, Ltd.
(Xxvi) Oil Red O; Wako Pure Chemical Industries, Ltd.
(Xxvii) Isopropyl alcohol; Wako Pure Chemical Industries, Ltd.
(Xxviii) 28% aqueous ammonium hydroxide solution; Alfa Aesar
(Xxix) Alizarin Red S; Wako Pure Chemical Industries, Ltd.
(Xxx) ALP staining kit; Muto Chemical Co., Ltd.
(Xxxi) Ethanol; Wako Pure Chemical Industries, Ltd.
(Xxxii) Acetone; Wako Pure Chemical Industries, Ltd.
(Xxxxiii) Mesenchymal stem cell adipocyte differentiation medium 2; PromoCell Co., Ltd.
(Xxxiv) Mesenchymal stem cell osteoblast differentiation medium 2; PromoCell Co., Ltd.
(Xxxv) BD Stemflow Human MSC Analysis Kit; Becton Dickinson Co., Ltd.
(Xxxvi) BV421 Mouse Anti-Human CD14; Becton Dickinson Co., Ltd.
(Xxxvii) Acetic acid; Fujifilm Wako Pure Chemical Industries, Ltd.

For the tests, human bone marrow-derived mesenchymal stem cells (hBM-MSC; Human Born marrow-derived Mesenchymal Stem Cell) and human adipose-derived mesenchymal stem cells (hADSC; Human Adipose-Derived Stem Cell) shown in Table 1 below were used. Was used.

The cell viability was calculated from the following formula.
Cell viability (%) = number of living cells / (number of living cells + number of dead cells) x 100 (%)
Further, in the following examples, "v / v%" represents (volume / volume)%, and "w / v%" represents (mass / volume)%. Here, the unit of volume is mL, and the unit of mass is g.

・ Culture of animal cells
hBM-MSC was cultured by the following method. That is, 10% of deactivated newborn fetal bovine serum and 1% of penicillin-spretomycin solution were added to Dulbecco's modified Eagle's medium (DMEM) to prepare a serum-containing medium. Hereinafter, the term "medium" without comment refers to this medium. The hBM-MSC and serum-containing medium were placed in a 225 cm ² culture flask and subcultured in a CO ₂ incubator at 37 ° C. The serum-containing medium was replaced every 3 days.

1. 1. Cell viability in various components [Example 1]
(1) Preparation of cryopreservation solution Add and dissolve 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine to the bicarbonate ringer solution, and add 0.22 μm. It was sterilized by filtration through a filter to prepare a cryopreservation solution. The composition of the cryopreservation solution is shown in Table 2.

(2) Cell cryopreservation and survival rate calculation (non-immersion method)
HBM-MSCs cultured in a CO ₂ incubator were stripped with 0.25% trypsin-EDTA and cell pellet was collected by centrifugation (500xg, 5 minutes, room temperature). The cells were suspended in the cryopreservation solution prepared in (1) to a concentration of 1 × 10 ⁶ cells / mL, and the cell suspension was dispensed into a serum tube to a concentration of 1 mL / tube. Each tube was placed in a bicell freezing container (manufactured by Nippon Freezer), placed in a -80 ° C deep freezer, frozen overnight, and then cryopreserved in liquid nitrogen for 1 week.

Cell tubes cryopreserved in liquid nitrogen for 1 week were lysed at 37 ° C for 1 minute. The cells were collected while lysing the sherbet-like cryopreservation solution in a prepared medium of 10 mL. After preparing a cell suspension by adding 1 mL of medium to the cell pellet collected by centrifugation (500 xg, 5 minutes, room temperature), the number of live cells and dead cells in the cell suspension was determined by trypan blue staining. I counted. Table 3 shows the results of calculating the cell viability by the above formula.

(3) Cryopreservation of cells and calculation of survival rate (pre-immersion method)
HBM-MSCs cultured in a CO ₂ incubator were stripped with 0.25% trypsin-EDTA and cell pellet was collected by centrifugation. The cells were suspended in the cryopreservation solution prepared in (1) to a concentration of 1 × 10 ⁶ cells / mL, and the cell suspension was dispensed into a serum tube to a concentration of 1 mL / tube. After holding for 2 hours under room temperature (25 ° C) conditions, each tube is placed in a Bisel freezing container (manufactured by Nippon Freezer), frozen overnight in a -80 ° C deep freezer, and then frozen in liquid nitrogen for 1 week. saved.

Cell tubes cryopreserved in liquid nitrogen for 1 week were lysed at 37 ° C for 1 minute. The cells were collected while lysing the sherbet-like cryopreservation solution in a prepared medium of 10 mL. After preparing a cell suspension by adding 1 mL of medium to the cell pellet collected by centrifugation (500 xg, 5 minutes, room temperature), the number of live cells and dead cells in the cell suspension was determined by trypan blue staining. I counted. Table 3 shows the results of calculating the cell viability by the above formula.

(4) Cryopreservation of cells and calculation of survival rate (post-immersion method)
HBM-MSCs cultured in a CO ₂ incubator were stripped with 0.25% trypsin-EDTA and cell pellet was collected by centrifugation. The cells were suspended in the cryopreservation solution prepared in (1) to a concentration of 1 × 10 ⁶ cells / mL, and the cell suspension was dispensed into a serum tube to a concentration of 1 mL / tube. Each tube was placed in a -80 ° C deep freezer, frozen overnight, and then cryopreserved in liquid nitrogen for 1 week.

Cell tubes cryopreserved in liquid nitrogen for 1 week were completely lysed at 37 ° C for 3 minutes. The lysed cell tube was held at room temperature (25 ° C.) for 1 hour, and then suspended in a prepared medium of 10 mL to collect the cells. After preparing a cell suspension by adding 1 mL of medium to the cell pellet collected by centrifugation (500 xg, 5 minutes, room temperature), the number of live cells and dead cells in the cell suspension was determined by trypan blue staining. I counted. Table 3 shows the results of calculating the cell viability by the above formula.

[Example 2]
The cryopreservation solution was prepared in the same manner as in Example 1 except that 1.0 w / v% glycine was used instead of 1.0 w / v% taurine. The composition of the cryopreservation solution is shown in Table 2, and the results of cell viability are shown in Table 3.

[Comparative Example 1]
The cryopreservation solution was prepared in the same manner as in Example 1 (3) except that 10 v / v% propylene glycol was added and dissolved in the bicarbonate ringer solution. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Comparative Example 2]
As a cryopreservation solution, 10 v / v% propylene glycol and 10 w / v% dextran were added to and dissolved in a bicarbonate ringer solution, and the same procedure as in Example 1 (3) was carried out. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Comparative Example 3]
As a cryopreservation solution, 10 v / v% propylene glycol and 0.1 M trehalose were added to and dissolved in a bicarbonate ringer solution, and the same procedure as in Example 1 (3) was carried out. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Comparative Example 4]
As a cryopreservation solution, 10 v / v% propylene glycol, 10 w / v% dextran, and 0.1 M trehalose were added to and dissolved in a bicarbonate ringer solution in the same manner as in Example 1. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 3]
Example 1 (3) except that 5 v / v% propylene glycol, 10 w / v% dextran, 0.1 M trehalose, and 0.5 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 4]
Example 1 (3) except that 5 v / v% propylene glycol, 10 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 5]
Example 1 (3) except that 5 v / v% propylene glycol, 10 w / v% dextran, 0.1 M trehalose, and 2.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Comparative Example 5]
As a cryopreservation solution, 10 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added to and dissolved in a bicarbonate ringer solution in the same manner as in Example 1 (3). The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 6]
Example 1 (3) except that 5 v / v% propylene glycol, 20 w / v% dextran, 0.2 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 7]
Example 1 (3) except that 10 v / v% propylene glycol, 20 w / v% dextran, 0.2 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 8]
Example 1 (3) except that 20 v / v% propylene glycol, 20 w / v% dextran, 0.2 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Comparative Example 6]
As a cryopreservation solution, 10 v / v% propylene glycol, 0.1 M trehalose, and 1.0 w / v% taurine were added to and dissolved in a bicarbonate ring gel solution, and the same procedure as in Example 1 (3) was carried out. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 9]
Example 1 (3) except that 10 v / v% propylene glycol, 5 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 10]
Example 1 (3) except that 10 v / v% propylene glycol, 20 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Comparative Example 7]
As a cryopreservation solution, 10 v / v% propylene glycol, 10 w / v% dextran, and 1.0 w / v% taurine were added to and dissolved in a bicarbonate ringer solution in the same manner as in Example 1 (3). did. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 11]
Example 1 (3) except that 10 v / v% propylene glycol, 20 w / v% dextran, 0.05 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 12]
Example 1 (3) except that 10 v / v% propylene glycol, 20 w / v% dextran, 0.2 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. The composition of the cryopreservation solution is shown in Table 2, and the survival rate after freezing and thawing is shown in Table 3.

[Example 13]
Example 1 (3) except that 10 v / v% glycerol, 10 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. And-in the same way. Table 3 shows the survival rate after freezing and thawing.

[Example 14]
Example 1 except that 10 v / v% propylene glycol, 5 w / v% hydroxyethyl starch, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. It was carried out in the same manner as in (3). Table 3 shows the survival rate after freezing and thawing.

[Example 15]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M sorbitol, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. Table 3 shows the survival rate after freezing and thawing.

[Example 16]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M sucrose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. Table 3 shows the survival rate after freezing and thawing.

[Example 17]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M fructose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. Table 3 shows the survival rate after freezing and thawing.

[Example 18]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M galactose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. Table 3 shows the survival rate after freezing and thawing.

[Example 19]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M mannitol, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. Table 3 shows the survival rate after freezing and thawing.

[Example 20]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M raffinose, and 1.0 w / v% taurine were added and dissolved in a bicarbonate ring gel solution as a cryopreservation solution. ) Was carried out in the same manner. Table 3 shows the survival rate after freezing and thawing.

[Example 21]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in physiological saline as a cryopreservation solution. ) Was carried out in the same manner. Table 3 shows the survival rate after freezing and thawing.

[Example 22]
Example 1 (Except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in a phosphate buffer solution as a cryopreservation solution. It was carried out in the same manner as in 3). Table 3 shows the survival rate after freezing and thawing.

[Example 23]
Example 1 (3) except that 10 v / v% propylene glycol, 10 w / v% dextran, 0.1 M trehalose, and 1.0 w / v% taurine were added and dissolved in a ring acetate solution as a cryopreservation solution. It was carried out in the same manner as. Table 3 shows the survival rate after freezing and thawing.

[Comparative Example 8]
The same procedure as in Example 1 was carried out except that a cryopreservation solution in which 10% DMSO and 5% BSA were added to a bicarbonate ringer solution was used. Table 3 shows the survival rate after freezing and thawing.

The composition of the cryopreservation solution used in the test is shown in Table 2.

The above composition was dissolved in a bicarbonate ringer solution.
Each solution was sterilized by filtration through a 0.22 μm filter to obtain a cryopreservation solution.
Table 3 shows the results of calculating the cell viability by the above formula.

[Results of examination of preservation solution composition]
Table 3 shows the viability of hBM-MSC stored with different cryopreservation solutions (n = 4). According to Table 3, the addition of taurine or glycine improved cell viability during pre- and post-immersion. High cell viability of 85% or higher was maintained when 5-20 v / v% propylene glycol or glycerol was used as the cryoprotectant. High cell viability of 80% or higher was maintained when 5-20 w / v% dextran or hydroxyethyl starch was used as the polysaccharide. When 0.05 to 0.2 M of trehalose, sorbitol, sucrose, fructose, galactose, mannitol, or raffinose was used as oligosaccharides, a high cell viability of 80% or more was maintained. When a bicarbonate ring gel, a physiological saline solution, a phosphate buffer solution, or a ring acetate solution was used as the isotonic solution, a high cell viability of 80% or more was maintained.

2. 2. Confirmation test of differentiation into adipocytes in frozen cells (1) Induction of differentiation into adipocytes Freezing by the method of Example 1 (3) using the cryopreservation solutions of Examples 1, 2 and Comparative Example 8. hBM-MSC was seeded in 6 well plates at 2.0 × 10 ⁵ cells / well and cultured in DMEM for 3 days. The medium was then replaced with DMEM (negative control) or adipocyte differentiation-inducing medium. The medium was changed every 2 days, and the cell morphology was observed over time until the 14th day.
(2) Preparation of adipocyte staining solution (Oil Red O staining solution)
50 mg of Oil Red O was added to 10 mL of isopropyl alcohol and dissolved overnight in a hot water bath at 60 ° C. 4 mL of milliQ water was added to 6 mL of the above solution to prepare a staining solution.
(3) After confirming the staining of adipocytes (1), the culture supernatant was removed and immersed in 60% isopropyl alcohol for 1 minute. After removing isopropyl alcohol, Oil Red O stain was added and stained at 37 ° C for 15 minutes. After removing the stain, it was immersed in isopropyl alcohol for 2 minutes. After removing the isopropyl alcohol, it was washed 3 times with MilliQ water. It was confirmed that the cells stored in any of the cryopreservation solutions maintained undifferentiation in the DMEM culture and differentiated into adipocytes in the adipocyte differentiation-inducing medium.
(4) Results To the adipocytes of hBM-MSC cryopreserved in the cryopreservation solutions of Examples 1 and 2 as in the case of cryopreservation in the cryopreservation solution of Comparative Example 8 which is a general cryopreservation solution composition. When the differentiation induction was performed, a stained image showing differentiation into adipocytes was observed in the cells induced to differentiate, and no stained image was observed in the cells cultured in DMEM. Therefore, the cryopreservation solution of the present invention was used. It was confirmed that the cryopreservation used did not affect the differentiation of hBM-MSC.

3. 3. Test for confirming differentiation of frozen cells into osteoblasts (1) Induction of differentiation into osteoblasts hBM-MSC frozen by the method of Example 1 (3) using the cryopreservation solutions of Examples 1 and 2. Was seeded on a 6-well plate so as to have 2.0 × 10 ⁵ cells / well, and cultured in DMEM for 3 days. The medium was then replaced with DMEM (negative control) or osteoblast differentiation induction medium. The medium was changed every two days, and the cell morphology was observed over time until the 21st day.
(2) Preparation of Alizarin Red S Staining Solution 1 g of Alizarin Red S was dissolved in 100 mL of milliQ water. 0.1 mL of 28% aqueous ammonium hydroxide solution was mixed with 100 mL of milliQ water. A diluted aqueous solution of ammonium hydroxide was added to the alizarin red S solution to adjust the pH to 6.4.
(3) Confirmation of osteoblast staining (Alizarin Red S staining)
After (1), the culture supernatant was removed and washed twice with PBS. After removing PBS, 100% ethanol was added to immobilize the cells. Ethanol was removed and washed twice with MilliQ water. Immersed in 1.3% Alizarin Red S stain for 2 minutes. It was washed 3 times with MilliQ water and dried.
According to such Arizarin Red S staining, it can be confirmed that the cells after storage in the cryopreservation solution of the present invention are differentiated into osteoblasts by the osteoblast differentiation-inducing medium.
(4) Preparation of alkaline phosphatase stain
Staining was performed using an ALP staining kit (Muto Kagaku). The stain was prepared according to the instructions for use.
(5) Confirmation of osteoblast staining (alkaline phosphatase staining)
After (1), the culture supernatant was removed and washed twice with PBS. After removing PBS, acetone was added at 4 ° C. and cells were immobilized for 5 minutes. Acetone was removed and washed twice with MilliQ water. It was immersed in an alkaline phosphatase stain and stained at 37 ° C. for 2 hours. Washed 3 times with MilliQ water. It was confirmed that the cells stored in any of the cryopreservation solutions maintained undifferentiation in DMEM culture and differentiated into osteoblasts in the osteoblast differentiation-inducing medium.
(6) Results Osteoblasts of hBM-MSC cryopreserved in the cryopreservation solutions of Examples 1 and 2 in the same manner as in the case of cryopreservation in Comparative Example 8 which has a general cryopreservation solution composition. When the differentiation induction was performed, a stained image showing differentiation into osteoblasts was observed in the cells induced to differentiate, and no stained image was observed in the cells cultured in DMEM. Therefore, the cryopreservation of the present invention was performed. It was confirmed that cryopreservation using a solution did not affect the differentiation of hBM-MSC.

4. Undifferentiated maintenance evaluation test in frozen cells (1) Confirmation of undifferentiated maintenance of stem cells Using the cryopreservation solution of Example 1, hBM-MSC frozen by the method of Example 1 (3) was applied at 37 ° C for 3 minutes. The cells were completely dissolved, diluted with 10 mL of medium, and then the cells were collected by centrifugation. The collected cells were stained with BD Stemflow Human MSC Analysis Kit and BV421 Mouse Anti-Human CD14, and the surface antigens of the cells were confirmed by flow cytometry.
(2) Results In both Example 1 and Comparative Example 8, the mesenchymal stem cell positive markers CD90, CD105, and CD73 were 90% or more positive, and the negative markers CD14, CD11b, CD34, CD45, Since CD19 and HLA-DR were 5% or less, it was confirmed that the phenotype as mesenchymal stem cells could be maintained.

5. Cell Adhesion and Proliferation Test (1) Measurement of the number of viable cells after cryopreservation and thawing Using the cryopreservation solutions of Example 1 and Comparative Example 4, cells were cryopreserved according to the method of Example 1 (3). After measuring the number of thawed cells by the trypan blue staining method, the cells were seeded on a 6-well plate so that the number of cells in one well was 1 x 10 ⁵ . The seeded cells were cultured in a 37 ° C. incubator, exfoliated with 0.25% trypsin-EDTA on days 1, 2, and 3 from the culture, and the cell pellet was collected by centrifugation (500xg, 5 minutes, room temperature). After preparing a cell suspension by adding 1 mL of medium to the collected cell pellet, the number of live cells and the number of dead cells in the cell suspension are counted by trypan blue staining, and the cell adhesion and proliferation rate are evaluated. did.
(2) Results The number of cells and the cell proliferation rate in the plate culture after cryopreservation and thawing of cells using the cryopreservation solution of Example 1 and Comparative Example 4 are shown in FIGS. 1 and 2, respectively.
Since the cells that had undergone cryopreservation and thawing using the cryopreservation solution of Example 1 had a larger number of cells on the day after culture than those using the cryopreservation solution of Comparative Example 4, the initial adhesion of the cells was good. It can be seen that it is. In addition, since the cell proliferation rate was also higher when the cryopreservation solution of Example 1 was used, the cells could be cryopreserved in a state where the cell proliferation after thawing was maintained. You can see that.

6. Examination of Mesenchymal Stem Cells of Different Origins (1) Cell Cryopreservation and Survival Rate Calculation Using cryopreservation solutions of Examples 1, 2 and Comparative Example 8, adipose-derived mesenchymal stem cells The cells were cryopreserved and the cell viability after thawing was calculated. The method of cryopreservation was in accordance with the method of Example 1 (3).
(2) Results The cell viability was 83.2 ± 8.4% when the cryopreservation solution of Comparative Example 8, which is a general cell cryopreservation solution, was used, whereas the cryopreservation solution of Example 1 was used. The cell viability was 93.9 ± 2.7%, and the cell viability was 90.0 ± 4.5% when the cryopreservation solution of Example 2 was used. Therefore, it was confirmed that when the cryopreserved solutions of Examples 1 and 2 were used, a high cell viability was obtained even when targeting mesenchymal mesenchymal stem cells.

7. Intramuscular administration of cryopreservation solution Local irritation test (1) Intramuscular local irritation test In order to examine local muscle irritation, the cell cryopreservation solution prepared in Example 1 was used as a male Kbl: JW rabbit (Kitayama Labes Co., Ltd.). )) (6 animals / group) was administered once in the vastus lateralis muscle at 1 mL / site, and local muscle irritation was examined 2 and 7 days after administration. A 0.425 volume% acetic acid solution was administered as a positive control substance, and a physiological saline solution was administered as a negative control substance in the same manner as the test substance. As pathological examinations, gross examination and histopathological examination were performed. In the physical examination, changes on the cut surface (hyperemia, bleeding, swelling, white change and brown change) were judged and evaluated based on the macroscopic examination criteria. In the histopathological examination, bleeding, edema, cell infiltration, degeneration, necrosis, and fibrosis were evaluated based on the histopathological examination criteria. Table 4 shows the criteria for macroscopic examination, and Table 5 shows the criteria for histopathological examination.

(2) Results During the observation period, no death was observed in all cases, and no abnormalities were observed in the general condition or body weight transition. In addition, neither macroscopic examination nor histopathological examination showed a significant difference compared with physiological saline.

Since the cryopreservation solution of the present invention can suppress cell death in a frozen or refrigerated state of animal cells, it can be used, for example, in the manufacturing industry of cell pharmaceutical preparations.

Claims (22)

An animal cell cryopreservation solution containing at least one selected from the group consisting of taurine, glycine, and derivatives thereof, a cryoprotectant excluding dimethyl sulfoxide, a water-soluble polysaccharide, and an oligosaccharide. The animal cell cryopreservation solution according to claim 1, wherein the concentration of at least one selected from the group consisting of taurine, glycine, and derivatives thereof is 0.5 to 2.0 (mass / volume)%. The animal cell cryopreservation solution according to claim 1 or 2, wherein the cryoprotectant is propylene glycol and / or glycerol. The animal cell cryopreservation solution according to claim 3, wherein the concentration of the cryoprotectant is 5 to 20 (volume / volume)%. The animal cell cryopreservation solution according to any one of claims 1 to 4, wherein the water-soluble polysaccharide is at least one selected from the group consisting of dextran, hydroxyethyl starch, and derivatives thereof. The animal cell cryopreservation solution according to claim 5, wherein the concentration of the water-soluble polysaccharide is 5 to 20 (mass / volume)%. The animal cell cryopreservation solution according to any one of claims 1 to 6, wherein the oligosaccharide is at least one selected from the group consisting of trehalose, sorbitol, mannitol, galactose, fructose, raffinose, sucrose, and salts thereof. .. The animal cell cryopreservation solution according to claim 7, wherein the concentration of oligosaccharides is 0.05 to 0.3 mol / L. An animal cell cryopreservation solution containing taurine and having an improved cell proliferation rate after thawing. The animal cell cryopreservation solution according to claim 9, wherein the concentration of taurine is 0.5 to 2.0 (mass / volume)%. The animal cell cryopreservation solution according to any one of claims 1 to 10, which contains an isotonic solution as a main component. The animal cell cryopreservation solution according to claim 11, wherein the isotonic solution is one or more selected from the group consisting of physiological saline, phosphate buffer, Ringer's acetate solution, and Ringer's bicarbonate solution. The animal cell cryopreservation solution according to any one of claims 1 to 12, wherein the safety to humans is such that it can be directly administered to humans. An animal cell dispersion in which animal cells are dispersed in the animal cell cryopreservation solution according to any one of claims 1 to 13. The animal cell dispersion according to claim 14, wherein the animal cell is one or more selected from the group consisting of human mesenchymal stem cells, human immune cells, human nerve cells, human cardiomyocytes, and human hematopoietic stem cells. The animal cell dispersion according to claim 14 or 15, wherein the density of animal cells is 1 × 10 ⁶ to 2 × 10 ⁷ cells / mL. The animal cell dispersion according to any one of claims 14 to 16, which is in a frozen state. An animal cell dispersion obtained by thawing the animal cell dispersion according to claim 17. The animal cell dispersion according to any one of claims 14, 16 and 18, which can be directly administered to humans without performing the dilution operation or the washing operation of the animal cells contained therein. The animal cell dispersion according to any one of claims 14 to 19, which is a cell pharmaceutical composition. A method for cryopreserving an animal cell, comprising the step of suspending the animal cell in the animal cell cryopreservation solution according to any one of claims 1 to 13 and the step of freezing the suspension. A method for producing a cell pharmaceutical composition, comprising a step of suspending an animal cell in the animal cell cryopreservation solution according to any one of claims 1 to 13 and a step of freezing the suspension.

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