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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/56—Physical preservation processes for animal cells or human cells
  • C12N5/562—Temperature processes, e.g. following predefined temperature changes over time
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  • 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
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
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  • 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
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
• • 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
  • C12N5/52—Chemical aspects of preservation of animal cells or human cells
  • C12N5/522—Preservation media
  • C12N5/525—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators

Definitions

• the present invention provides a mammalian cell cryopreservation solution (hereinafter referred to as “the present cryopreservation solution”) containing 2.5 to 8.75 (v/v)% propylene glycol (hereinafter sometimes referred to as "PG"). ), a method for cryopreserving mammalian cells (hereinafter sometimes referred to as “the cryopreservation method”), etc., which includes the step (a) of cryopreserving the cryopreservation solution containing mammalian cells. Regarding.
• Cryopreservation of cells has been widely used as an essential technique in cell biology research.
• cell cryopreservation technology has been used not only for the preservation of various cell lines in cell banks around the world, but also for the preservation of seeds in the livestock industry, the cryopreservation of sperm, eggs, or fertilized eggs for increasing livestock production, It is applied to cryopreservation of germ cells in reproductive medicine.
• Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), have unlimited proliferation ability and multipotency into various tissue cells. It is a cell. Human pluripotent stem cells are expected to be applied to regenerative medicine by utilizing their properties, and in order to realize this, high-quality cell freezing technology, that is, high cell survival rate and undifferentiated state after thawing, is required. It is essential to establish a cell freezing technology that guarantees this.
• Cell cryopreservation methods are generally divided into slow freezing methods and quick freezing methods (vitrification methods).
• the slow freezing method uses a cryopreservation solution containing glycerin, dimethyl sulfoxide (hereinafter sometimes referred to as "DMSO"), keratin hydrolyzate, hydrolyzed gelatin, serum, serum albumin, etc. as a cryoprotectant (Patent Documents 1 to 5). ) in which cells are suspended and gradually frozen by lowering the temperature to about 1°C per minute.
• Slow cooling replaces intracellular water molecules with the cryoprotectant and dehydrates the cells, suppressing the growth of ice crystals inside and around the cells, causing damage to cell membranes and intracellular structures, and denaturation and denaturation of proteins.
• Non-Patent Document 1 Prevents cutting (Non-Patent Document 1).
• general cells can be cryopreserved using Styrofoam boxes or commercially available cell freezing boxes, without the need to use a programmed freezer to tightly control the temperature.
• slow freezing can be achieved by using Since such a method is simple, it is also called a simple slow freezing method, and is widely used in laboratories, cell banks, and the like.
• the vitrification method is a method of freezing cells into a glass-like state by rapid cooling in order to suppress the formation of ice crystals inside and outside cells due to freezing.
• the vitrification method was first reported in 1937 and took a long time to develop until it was put to practical use, but in 1985 it was developed using highly concentrated DMSO, acetamide, PG, and polyethylene glycol (hereinafter referred to as "PEG").
• PEG polyethylene glycol
• a method using a vitrification preservation solution containing a cryoprotectant was developed. The development of this method has made it possible to cryopreserve early mouse embryos, as well as cow and pig embryos, which was difficult to do using slow freezing methods.
• the vitrification method is used in many institutions including embryo banks.
• Non-Patent Document 3 when human ES cells and iPS cells are suspended in cryopreservation solution (STEM-CELLBANKER [manufactured by Nippon Zenyaku Kogyo Co., Ltd.]) and cryopreserved using a simple slow freezing method, the cell survival rate after thawing increases. It has also been reported (Non-Patent Document 3).
• a cell cryopreservation solution contains sugars such as trehalose and PG, but does not contain DMSO, thickeners, or natural animal-derived components (Patent Document 6). Furthermore, an animal cell cryopreservation solution 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 has also been proposed. (Patent Document 7).
• An object of the present invention is to provide a cell cryopreservation solution that can effectively suppress cell death caused by freezing and thawing of mammalian cells, and that can effectively increase the proliferation ability of mammalian cells after freezing and thawing.
• An object of the present invention is to provide a method for cryopreserving mammalian cells using a cell cryopreservation solution.
• the present invention is as follows.
• Cell cryopreservation solution [3] The mammalian cell according to [1] or [2] above, further comprising a polymer compound selected from dextran, a derivative thereof, or a salt thereof; and hydroxyethyl starch, a derivative thereof, or a salt thereof; Cryopreservation solution.
• the mammalian cell cryopreservation solution according to [8] above, wherein the mammalian cells are mesenchymal stem cells, T cells, or hematopoietic stem cells.
• a method for cryopreserving mammalian cells comprising the step (a) of cryopreserving the mammalian cell cryopreservation solution according to any one of [8] to [10] above. [12] The method according to [11] above, further comprising a step (b) of freezing and thawing the mammalian cells and culturing them for 5 days or more after step (a).
• a cell cryopreservation solution containing a concentration of PG other than 2.5-8.75% Compared to the case where mammalian cells are cryopreserved in a cell cryopreservation solution containing 2.5 to 5% PG, cell death caused by freezing and thawing of mammalian cells can be effectively suppressed.
• cryopreserving cells mammalian cells are cryopreserved in a cell cryopreservation solution containing a concentration of PG other than 2.5-5%, or in a cell cryopreservation solution containing 2.5-5% DMSO.
• Figure 1B shows four types of cell cryopreservation solutions containing 1.25-10% PG (LR containing 1.25% PG, 2.7% trehalose, and 4.5% dextran [in the figure "1.25% PG + Tre + D”]; LR containing 2.5% PG, 2.7% trehalose, and 4.5% dextran ["2.5% PG + Tre + D” in the figure]; 5% PG, LR containing 2.7% trehalose and 4.5% dextran [“5% PG+Tre+D” in the figure]; and LR containing 10% PG, 2.7% trehalose, and 4.5% dextran Diagram showing the results of cryopreserving hAD-MSCs in [“10%PG+Tre+D” in the figure] for 31 days and analyzing the cell proliferation ability of hAD-MSCs after thawing (
• FIG. 2 is a diagram showing the results of a comparison of cell cryopreservation solutions containing the same concentration of DMSO and PG based on the results of FIG. 1.
• "**” and “***” in the figure indicate that there is a statistically significant difference (p ⁇ 0.01 and p ⁇ 0.001), respectively, between samples cultured for the same number of days.
• Trehalose and dextran-containing cell cryopreservation solution containing 4% PG or 4% DMSO were cryopreserved for 23 days in LR containing 4% DMSO, 2.88% trehalose, and 4.8% dextran [4% DMSO+Tre+D] in the figure, and after thawing.
• FIG. 1 shows that Trehalose and dextran-containing cell cryopreservation solution containing 4% PG or 4% DMSO (LR containing 4% PG, 2.88% trehalose, and 4.8% dextran ["4% PG+Tre+D" in the figure] hAD-MSCs were cryopreserved for 23 days in LR containing 4% DMSO, 2.88% trehalose, and 4.8% dextran [4% DMSO+Tre+D] in the figure, and after thawing.
• FIG. 6C shows the results in Ringer's acetate solution containing 5% PG ("PG/AR" in the figure) or Ringer's acetate solution containing 5% PG and 4.75% dextran ("PG+D/AR" in the figure).
• FIG. 6D shows the results in lactated Ringer's solution containing 5% PG ("PG/LR” in the figure) or lactated Ringer's solution containing 5% PG and 4.75% dextran (“PG+D/LR” in the figure).
• Cell cryopreservation solutions containing 1.25 to 10% PG and dextran that is, 8 types of cell cryopreservation solutions (LR containing 1.25% PG and 4.94% dextran ["1.25%” in the figure]) ; LR containing 2.5% PG and 4.88% dextran ["2.5%” in the figure]; LR containing 3.75% PG and 4.81% dextran ["3.75%” in the figure] ; LR containing 5% PG and 4.75% dextran ["5%” in the figure]; LR containing 6.25% PG and 4.69% dextran ["6.25%” in the figure]; 7.5 LR containing %PG and 4.63% dextran ["7.5%” in the figure]; LR containing 8.75%
• FIG. "*" in the figure indicates that there is a statistically significant difference (p ⁇ 0.05) by Dunnett's test, and " ⁇ " and “ ⁇ ” in the figure indicate that there is a statistically significant difference (p ⁇ 0.05) by Dunnett's test. This indicates that there is a scientifically significant difference (p ⁇ 0.05 and p ⁇ 0.01).
• FOG. 11A cell survival rate
• FIG. 11B viable cell recovery rate
• FIG. 11C annexin V positivity rate
• “*" and "**” in the figure indicate statistically significant differences (p ⁇ 0.05 and p ⁇ 0.01), respectively, according to Dunnett's test
• " ⁇ " in the figure indicates that there is a statistically significant difference (p ⁇ 0.05 and p ⁇ 0.01), respectively.
• “*”, “**”, and “***” in the figure indicate that there is a statistically significant difference from “PG+Med” (p ⁇ 0.05, p ⁇ 0.01, and p ⁇ 0.001), and " ⁇ ", " ⁇ ", and “ ⁇ ” in the figure indicate statistically significant differences in the two-group comparison of "PG+Tre” and "PG", respectively. (p ⁇ 0.05, p ⁇ 0.01, and p ⁇ 0.001).
• FIG. 17B is a diagram showing the results of analyzing the annexin V positivity rate (FIG.
• the mammalian cell cryopreservation solution of the present invention is a solution containing 2.5 to 8.75 (v/v)% propylene glycol (i.e., This cryopreservation solution).
• the present cryopreservation solution with a propylene glycol concentration of 2.5 to 5.0 (v/v)% is a cell cryopreservation solution containing propylene glycol at a concentration other than 2.5 to 5.0 (v/v)%.
• cryopreservation solution which has a propylene glycol concentration of 2.5 to 5.0 (v/v)%, was specified for use "to increase the proliferation ability of mammalian cells after freezing and thawing.” It may be something.
• the present cryopreservation solution (a solution containing 2.5 to 8.75 (v/v)% propylene glycol) can be used in cell cryopreservation solutions containing DMSO and 2.5 to 8.75 (v/v)% propylene glycol.
• cell viability can be maintained at a high value when stored at room temperature (1°C to 30°C) after thawing. . Therefore, the present cryopreservation solution may be used specifically for "increasing the cell survival rate of mammalian cells after freezing and thawing.”
• the method of cryopreserving mammalian cells of the present invention includes the step (a) of cryopreserving the cryopreservation solution containing the mammalian cells (in other words, cryopreserving the mammalian cells in the cryopreservation solution). ) (i.e., the present cryopreservation method), there are no particular limitations on the method, and in step (a), mammalian cells may be frozen using a slow freezing method and then stored, or may be stored rapidly. It may be frozen using a freezing method (vitrification method) and then stored.
• Such slow freezing methods include, for example, transferring the subject cryopreservation solution containing mammalian cells to a cell storage tube or vial and placing it in a low-temperature freezer or ultra-low temperature freezer (usually within the range of -20°C to -150°C). Examples include a method of freezing and then storing in liquid nitrogen (usually within the range of -150°C to -196°C).
• a quick freezing method for example, after suspending mammalian cells in the present cryopreservation solution, if necessary, transfer them into a straw, and rapidly freeze them in liquid nitrogen (usually within the range of -150°C to -196°C). Examples include freezing and preserving methods.
• the concentration of propylene glycol may be within the range of 2.5 to 8.75 (v/v)%, for example, 2.5-7.5%, 2.5-6.25%, 2.5-5.0%, 2.5-4.9%, 2.5-4.8%, 2.5-4 .6%, 2.5-4.4%, 2.5-4.2%, 2.5-4.0%, 2.5-3.8%, 2.5-3.6%, 2 .5-3.4%, 2.5-3.2%, 2.5-3.0%, 2.5-2.8%, 2.5-2.6%, 2.6-8. 75%, 2.8-8.75%, 3.0-8.75%, 3.2-8.75%, 3.4-8.75%, 3.6-8.75%, 3.
• the cryopreservation solution of the present invention preferably further contains a polymer compound.
• a polymer compound means a compound having a weight average molecular weight (Mw) of 1 ⁇ 10 4 or more.
• Such polymer compounds include, for example, dextran or its derivatives or salts thereof (hereinafter sometimes referred to as "dextrans”); hydroxyethyl starch (also referred to as hydroxyethyl starch) or its derivatives or salts thereof (hereinafter referred to as "dextrans”);Albumin; carboxymethyl cellulose or its salt; xanthan gum or its salt; gelatin; amylopectin or its salt; Preferred examples include polymer compounds selected from dextrans and hydroxyethyl starches, since their effects have been demonstrated in this example, which will be described later.
• the cryopreservation solution may or may not contain polymeric compounds other than dextran and hydroxyethyl starch.
• Mw weight average molecular weight of the dextran
• These dextran can be produced by any known method such as chemical synthesis, microbial production, enzymatic production, etc., but commercially available products can also be used.
• Dextran 40 manufactured by Tokyo Kasei Kogyo Co., Ltd.
• Dextran 70 manufactured by Tokyo Kasei Kogyo Co., Ltd.
• Examples of the dextran derivatives among the above dextrans include carboxylated dextran, diethylaminoethyl (DEAE)-dextran, and the like.
• Hydroxyethyl starch in the above hydroxyethyl starches is composed of amylose in which ⁇ -D-glucose is linearly linked ( ⁇ -1,4 linkage) and amylopectin having branches ( ⁇ -1,6 linkage).
• the mixture is not particularly limited as long as one or more of C2, C3, and C6 of the glucose units are hydroxyethylated (amylopectin derivative), and the Mw of hydroxyethyl starch is, for example, A range of 5 ⁇ 10 4 to 5 ⁇ 10 6 (eg, 7 ⁇ 10 4 , 2 ⁇ 10 5 ) can be mentioned.
• the degree of substitution (the number of hydroxyethyl groups per glucose unit) of the hydroxyethyl starch is not particularly limited, and is, for example, within the range of 0.4 to 0.8 (for example, 0.50 to 0.55). can be mentioned.
• These hydroxyethyl starches 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, commercially available products such as HES (manufactured by Fresenius Kabi Austria GmbH) can be used.
• hydroxyethyl starch derivatives in the above hydroxyethyl starches include DEAE-hydroxyethyl starch.
• the concentration of the polymer compound in the cryopreservation solution is not particularly limited as long as the combined effect with PG is recognized, and the lower limit of the concentration of the polymer compound is, for example, 1.0 (w/v)%, 1.2 (w/v)%, 1.4 (w/v)%, 1.6 (w/v)%, 1.8 (w/v)%, 2.0 (w/v)%, 2.2 (w/v)%, 2.4 (w/v)%, 2.6 (w/v)%, 2.8 (w/v)%, 3.0 (w/v)%, 3.2 (w/v)%, 3.4 (w/v)%, 3.6 (w/v)%, 3.8 (w/v)%, 4.0 (w/v)%, Examples include 4.2 (w/v)%, 4.4 (w/v)%, 4.6 (w/v)%, and 4.8 (w/v)%.
• the upper limit of the concentration of the polymer compound in the cryopreservation solution is, for example, 20 (w/v)%, 16 (w/v)%, 13 (w/v)%, 10 (w/v). %, 9.8 (w/v)%, 9.6 (w/v)%, etc.
• the concentration of the polymer compound is, for example, 1.0-20%, 1.2-20%, 1.4-20%, 1.6-20%, 1.8-20%, 2.0-20%, 2.2-20%, 2 .4-20%, 2.6-20%, 2.8-20%, 3.0-20%, 3.2-20%, 3.4-20%, 3.6-20%, 3.
• the range of the weight ratio of propylene glycol and the polymer compound in the cryopreservation solution further containing the polymer compound is, for example, 1:0.1 to 1:10, 1:0.1 to 1:8, 1:0. .1 ⁇ 1:6, 1:0.1 ⁇ 1:4, 1:0.2 ⁇ 1:10, 1:0.2 ⁇ 1:8, 1:0.2 ⁇ 1:6, 1:0 .2 ⁇ 1:4, 1:0.3 ⁇ 1:10, 1:0.3 ⁇ 1:8, 1:0.3 ⁇ 1:6, 1:0.3 ⁇ 1:4, 1:0 .4 ⁇ 1:10, 1:0.4 ⁇ 1:8, 1:0.4 ⁇ 1:6, 1:0.4 ⁇ 1:4, 1:0.5 ⁇ 1:10, 1:0 .5 to 1:8, 1:0.5 to 1:6, 1:0.5 to 1:4, etc., and when the polymer compound is dextran, 1:0.1 to 1:
• the ratio is preferably 4, more preferably 1:0.3 to 1:4, even more preferably 1:0.5 to 1:4.
• the present cryopreservation solution preferably further contains trehalose, a derivative thereof, or a salt thereof (hereinafter sometimes referred to as "trehalose”).
• trehalose includes ⁇ , ⁇ -trehalose, which is a disaccharide in which two ⁇ -glucoses are linked together in a 1,1-glycosidic bond, and trehalose, which is a disaccharide in which two ⁇ -glucoses are linked together in a 1,1-glycoside bond.
• Examples include ⁇ , ⁇ -trehalose, which is a saccharide, and ⁇ , ⁇ -trehalose, which is a disaccharide in which two ⁇ -glucoses are linked with a 1,1-glycoside bond, and among these, ⁇ , ⁇ -trehalose is preferred.
• These trehaloses can be produced by any known method such as chemical synthesis, microbial production, enzymatic production, etc., but commercially available products can also be used. For example, commercially available products such as trehalose dihydrate (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) can be mentioned.
• the trehalose derivatives in the trehaloses mentioned above are not particularly limited as long as they are glycosyltrehaloses in which one or more sugar units are bonded to the disaccharide trehalose. Contains trehalose, etc.
• the concentration of trehalose in the cryopreservation solution is not particularly limited as long as the combined effect with PG is recognized, and the lower limit of the concentration of trehalose is, for example, 0.7 (w/v)%, 0. 8 (w/v)%, 0.9 (w/v)%, 1.0 (w/v)%, 1.1 (w/v)%, 1.2 (w/v)%, 1. Examples include 3 (w/v)%, 1.4 (w/v)%, and the like.
• the upper limit of the concentration of trehalose in this cryopreservation solution is, for example, 40 (w/v)%, 35 (w/v)%, 30 (w/v)%, 25 (w/v)%. , 20 (w/v)%, 15 (w/v)%, 12 (w/v)%, 6.0 (w/v)%, and the like.
• the concentration of the above-mentioned trehaloses is, for example, 0.7-40%, 0.8-40%, 0.9-40%, 1.0-40%, 1.1-40%, 1.2-40%, 1.3-40%, 1 .4-40%, 0.7-35%, 0.8-35%, 0.9-35%, 1.0-35%, 1.1-35%, 1.2-35%, 1.
• the range of the weight ratio of propylene glycol and trehalose in the cryopreservation solution further containing trehalose is, for example, 1:0.08 to 1:16, 1:0.08 to 1:14, 1:0.08.
• the cryopreservation solution in question is a solution (e.g., isotonic solution, hypotonic solution, hypertonic solution) that contains 2.5 to 8.75 (v/v)% propylene glycol and is capable of cryopreservation of mammalian cells.
• An isotonic solution containing 2.5-8.75 (v/v)% propylene glycol is preferred.
• isotonic fluid means a fluid that has approximately the same osmotic pressure as that of body fluids or cell fluids, and specifically, a fluid that has an osmotic pressure within the range of 250 to 380 mOsm/L. means.
• hypotonic solution refers to a solution that has an osmotic pressure lower than that of body fluids or cell fluids, and specifically refers to a solution that has an osmotic pressure of less than 250 mOsm/L. do.
• a hypotonic solution is preferably a hypotonic solution that does not cause cell rupture (specifically, a solution having an osmotic pressure within a range of 100 to less than 250 mOsm/L).
• hypothermic solution means a solution having an osmotic pressure higher than that of body fluids or cell fluids, and specifically, the osmotic pressure is more than 380 mOsm/L (preferably 380 mOsm/L). (within the range of more than 1000 mOsm/L).
• the above-mentioned isotonic solution is not particularly limited as long as it is an isotonic solution whose salt concentration and sugar concentration are adjusted using sodium ions, potassium ions, calcium ions, etc. so that the osmotic pressure is almost the same as that of body fluids and cell fluids.
• physiological saline physiological saline with a buffering effect (e.g., PBS, Tris Buffered Saline; TBS, HEPES buffered saline), Ringer's solution, lactated Ringer's solution, acetate Ringer's solution, Ringer's bicarbonate solution, 5% glucose aqueous solution, basal medium for animal cell culture (e.g.
• DMEM fetal calf serum
• EMEM fetal calf serum
• F-12, F-10 M-199
• isotonic agent e.g. glucose , D-sorbitol, D-mannitol, lactose, sodium chloride
• lactated Ringer's solution physiological saline, Ringer's solution
• a suitable example is an isotonic solution selected from Ringer's acetate solution.
• Isotonic solutions may be commercially available or self-prepared.
• Otsuka raw food injection manufactured by Otsuka Pharmaceutical Factory Co., Ltd.
• physiological saline solution Ringer's solution "Otsuka” (manufactured by Otsuka Pharmaceutical Factory Co., Ltd.) (Ringer's solution)
• Lactech registered trademark
• Injection manufactured by Otsuka Pharmaceutical Factory Co., Ltd.
• Veen registered trademark
• F infusion manufactured by Fuso Pharmaceutical Industries, Ltd.
• Otsuka sugar solution 5% manufactured by Otsuka Pharmaceutical Factory
• 5% glucose aqueous solution 5% glucose aqueous solution
• Bikanate registered trademark
• the cryopreservation solution may contain cryoprotective components other than PG (for example, DMSO; glycerol; ethylene glycol; PEG; sericin; isomalto-oligosaccharide; serum derived from human, bovine, etc.); Those containing no cryoprotective components other than PG are preferred because they alone can effectively enhance the proliferation ability of mammalian cells after freezing and thawing.
• cryoprotective components other than PG for example, DMSO; glycerol; ethylene glycol; PEG; sericin; isomalto-oligosaccharide; serum derived from human, bovine, etc.
• the cryopreservation solution may contain oligosaccharides other than trehalose (for example, disaccharides such as sucrose, lactose, and maltose; trisaccharides such as maltotriose, raffinose, and melezitose; tetrasaccharides such as acarbose and stachyose), and monosaccharides.
• oligosaccharides other than trehalose for example, disaccharides such as sucrose, lactose, and maltose; trisaccharides such as maltotriose, raffinose, and melezitose; tetrasaccharides such as acarbose and stachyose
• monosaccharides for example, glucose, fructose, galactose
• glucose, fructose, galactose may or may not be included.
• the cryopreservation solution of the present invention usually does not contain a substance selected from taurine or a derivative thereof, or a salt thereof; and glycine or a derivative thereof or a salt thereof.
• taurine derivatives include, for example, N-substituted taurine (for example, N-(2-acetamido)-2-aminoethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, -Cyclohexyl-2-aminoethanesulfonic acid, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, 2-morpholinoethanesulfonic acid, piperazine-1,4-bis(2-ethanesulfonic acid) (acid), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, etc.), hypotaurine, thiotaurine, etc.
• Examples of the above glycine derivatives include sarcosine (N-methylglycine), N-ethylglycine, N-propylglycine, N,N-diethylglycine, N,N-dimethylglycine, N-amidinoglycine, N-amidino-N - Methylglycine, glycylglycine, phenacetulic acid, glycine methyl ester, glycine ethyl ester, glycine n-butyl ester, glycine t-butyl ester, glycine n-propyl ester, glycine n-pentyl ester, glycine benzyl ester, etc.
• taurine, taurine derivatives, glycine, and "salts" of glycine derivatives include hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, acetate, propionate, Toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate
• acid addition salts such as, metal salts such as sodium salts, potassium salts, calcium salts, ammonium salts, alkylammonium salts, and the like.
• salts include, for example, hydrochloride, hydrobromide, etc.
• hydroiodide phosphate, nitrate, sulfate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate , acid addition salts such as butyrate, valerate, citrate, fumarate, maleate, malate, metal salts such as sodium salt, potassium salt, calcium salt, ammonium salt, alkylammonium salt. etc. can be mentioned.
• salts are used as a solution, and their effects are the same as when using dextran, dextran derivatives, hydroxyethyl starch, hydroxyethyl starch derivatives, trehalose, trehalose derivatives, carboxymethylcellulose, xanthan gum, and amylopectin. Preferably something that is effective.
• the "salts" of (dextran, dextran derivatives, hydroxyethyl starch, hydroxyethyl starch derivatives, trehalose, trehalose derivatives, carboxymethylcellulose, xanthan gum, and amylopectin) may form hydrates or solvates, and Either one can be used alone or two or more kinds can be used in an appropriate combination.
• Optional ingredients in the cryopreservation solution include, for example, vitamins (e.g., choline chloride, pantothenic acid, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, ascorbic acid, biotin, inositol), chelating agents (e.g., EDTA , EGTA, citric acid, salicylate), antibiotics (eg, penicillin, streptomycin), proteins, antioxidants (eg, polyphenols, quercetin), and the like.
• vitamins e.g., choline chloride, pantothenic acid, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, ascorbic acid, biotin, inositol
• chelating agents e.g., EDTA , EGTA, citric acid, salicylate
• antibiotics
• mammalian cells include, for example, stem cells (for example, stem cells administered via blood vessels for regenerative medicine, etc.); pancreatic islet cells (for example, pancreatic islet cells administered intravenously to type I diabetic patients); Lymphoid cells such as T cells, natural killer (NK) cells, and B cells; Antigen presenting cells such as monocytes, macrophages, and dendritic cells; Granules such as neutrophils, eosinophils, basophils, and mast cells.
• T cells can be suitably exemplified because their effects have been demonstrated in the present example described below.
• T cells can include alpha beta T cells, gamma delta T cells, CD8 positive T cells, CD4 positive T cells, tumor infiltrating T cells, memory T cells, naive T cells, and NKT cells.
• T cells can be collected from mammalian bone marrow, peripheral blood, umbilical cord blood, etc. by known general methods.
• mammals include rodents such as mice, rats, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, horses, and sheep, dogs, cats, etc.
• lagomorphs such as rabbits
• ungulates such as pigs, cows, goats, horses, and sheep, dogs, cats, etc.
• primates such as cats, humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees, among which mice, pigs, and humans are preferred.
• stem cell means an immature cell that has self-renewal ability and differentiation/proliferation ability.
• Stem cells include subpopulations such as pluripotent stem cells (pluripotent stem ce11), multipotent stem cells (multipotent stem ce11), and unipotent stem cells (unipotent stem ce11), depending on their differentiation ability.
• pluripotent stem cell refers to a cell that cannot become an individual by itself, but has the ability to differentiate into all tissues and cells that make up a living body.
• Multipotent stem cells refer to cells that have the ability to differentiate into multiple types of tissues and cells, although not all types.
• Unipotent stem cells refer to cells that have the ability to differentiate into specific tissues or cells.
• pluripotent stem cells examples include embryonic stem cells (ES cells), EG cells, iPS cells, and the like.
• ES cells can be produced by culturing the inner cell mass on feeder cells or in a medium containing LIF. Methods for producing ES cells are described, for example, in WO96/22362, WO02/101057, US5,843,780, US6,200,806, US6,280,718, and the like.
• EG cells can be produced by culturing primordial germ cells in a medium containing mSCF, LIF, and bFGF (Ce11, 70:841-847, 1992).
• iPS cells are produced by introducing reprogramming factors such as Oct3/4, Sox2, and Klf4 (and further c-Myc or n-Myc as necessary) into somatic cells (e.g., fibroblasts, skin cells, etc.) (Ce11,126:p.663-676,2006;Nature,448:p.313-317,2007;Nat Biotechno1,26;p,101-106,2008;Cel1 131:p.861-872, 2007;Science,318:p.1917-1920,2007;Ce11 Stem Cells 1:p.55-70,2007;Nat Biotechnol,25:p.1177-1181,2007;Nature,448:p.318-324, 2007;Cell Stem Cells 2:p.10-12,2008;Nature451:p.141-146,2008;Science,318:p.1917-1920,2007).
• somatic cells e.g., fibroblasts,
• stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (Nature, 385, 810 (1997); Science, 280, 1256 (1998); Nature Biotechnology, 17, 456 (1999); Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Nat1. Acad. Sci. USA, 96, 14984 (1999)), Rideout III et al. 2000)).
• Multipotent stem cells include mesenchymal stem cells that can differentiate into cells such as adipocytes, osteocytes, and chondrocytes; hematopoietic stem cells that can differentiate into blood cells such as white blood cells, red blood cells, and platelets (e.g., CD34, CD110 , CD111, CD112, and CD117 (preferably CD34-positive hematopoietic stem cells); nervous system cells capable of differentiating into cells such as neurons, astrocytes, and oligodendrocytes.
• mesenchymal stem cells that can differentiate into cells such as adipocytes, osteocytes, and chondrocytes
• hematopoietic stem cells that can differentiate into blood cells such as white blood cells, red blood cells, and platelets (e.g., CD34, CD110 , CD111, CD112, and CD117 (preferably CD34-positive hematopoietic stem cells)
• nervous system cells capable of differentiating into cells such as neurons,
• so mesenchymal stem cells and hematopoietic stem cells can be preferably exemplified.
• Multipotent stem cells can be isolated from a living body by a method known per se.
• mesenchymal stem cells can be collected from mammalian bone marrow, adipose tissue, peripheral blood, umbilical cord blood, etc. by known general methods.
• human mesenchymal stem cells can be isolated by culturing and passage of hematopoietic stem cells etc.
• hematopoietic stem cells can be collected from mammalian bone marrow, peripheral blood, umbilical cord blood, etc. by known general methods. Multipotent stem cells can also be obtained by culturing the above-mentioned pluripotent stem cells under appropriate induction conditions.
• the mesenchymal stem cells are preferably human adipose-derived mesenchymal stem cells.
• the hematopoietic stem cells are preferably human-derived CD34-positive hematopoietic stem cells.
• the present cryopreservation solution may be in a form that does not contain mammalian cells or in a form that contains mammalian cells, and when implementing the present cryopreservation method, it may be in a form that contains mammalian cells (i.e., a form that contains mammalian cells).
• the present cryopreservation solution containing animal cells is preferred.
• the cryopreservation solution containing mammalian cells can be prepared by any method.
• the mammalian cell-containing solution is centrifuged to separate the supernatant (liquid) and precipitate (mammalian cells); 2) A method in which the cryopreservation solution is prepared by removing the supernatant (liquid) and adding the subject cryopreservation solution to the precipitate (mammalian cells), or 2) when mixed with the mammalian cell-containing solution, A solution that has been adjusted so that the final concentration of the components contained in the solution is the desired concentration (for example, 2.5 to 8.75 (v/v)% in the case of PG) is mixed with a solution containing mammalian cells.
• a method for preparing the same can be mentioned.
• the present cryopreservation solution containing umbilical cord blood is preferred.
• the present cryopreservation solution containing umbilical cord blood can be obtained by mixing umbilical cord blood (i.e., a solution containing mammalian cells such as hematopoietic stem cells) and a solution containing PG in an arbitrary ratio (for example, according to method 2) above. 10:1 to 1:10 [for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.]).
• adherent cells An example of mammalian cells to be preserved in the present cryopreservation solution is adherent cells.
• adherent cells refer to anchorage-dependent cells that can survive, proliferate, and produce substances by adhering to the scaffold.
• adherent stem cells include pluripotent stem cells, mesenchymal stem cells, neural stem cells, bone marrow stem cells, and germline stem cells.
• Adherent stem cells are preferably mesenchymal stem cells.
• the mammalian cells (population) to be preserved in this cryopreservation solution may be isolated from the living body or subcultured in vitro, but they may be isolated or purified. It is preferable.
• isolated or purification means that an operation is performed to remove components other than the target component.
• the purity of the isolated or purified mammalian cells is usually 30% or more, preferably 50% or more, more preferably 70% or more, More preferably, it is 90% or more (for example, 100%).
• the mammalian cells (population) to be stored in the present cryopreservation solution are preferably in the state of a single cell (single cell).
• single cell state means that the cells do not gather together with other cells to form a mass (ie, a non-aggregated state).
• Mammalian cells in a single cell state can be obtained by treating mammalian cells cultured in vitro with enzymes such as trypsin/EDTA, and then suspending the cells by methods well known in the art such as pipetting and tapping. It can be prepared.
• the percentage of single-cell mammalian cells contained in mammalian cells is usually 70% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more (for example, 100%). be.
• mammalian cells are dispersed in PBS, observed under a microscope, and a plurality of randomly selected cells (for example, 1000 cells) are examined for the presence or absence of aggregation. It can be determined by
• the present cryopreservation method preferably includes a step (b) of freezing and thawing the mammalian cells and culturing them after the step (a).
• Examples of the culture period include 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, but 5 days or more preferable.
• the method of freezing and thawing the mammalian cells is not particularly limited, and the container containing the cryopreservation solution containing frozen mammalian cells (cell storage tube, cell storage vial, straw) etc.) may be naturally thawed at room temperature (for example, within the range of 10 to 28 °C), but from the viewpoint of preventing refreezing, soak it in warm water (for example, water within the range of 30 to 40 °C), Rapid thawing is preferred.
• step (b) above the mammalian cells after freezing and thawing are cultured in an appropriate container (culture plate, culture dish, culture flask, etc.) in the presence of an appropriate culture medium.
• culture media include animal cell culture media (DMEM, EMEM, IMDM, RPMI1640, ⁇ MEM, F-12, F-10, M-199, AIM-V, etc.), MyeloCult H5100 medium (manufactured by STEMCELL Technologies, ST-05150), and the like.
• serum-free culture solution for example, an appropriate amount of a serum substitute such as the above-mentioned commercially available B27 supplement (-insulin), N2 supplement, B27 supplement, Knockout Serum Replacement, etc.
• a serum substitute such as the above-mentioned commercially available B27 supplement (-insulin), N2 supplement, B27 supplement, Knockout Serum Replacement, etc.
• examples include the above-mentioned culture solution for animal cell culture, in which 1% to 30%) have been added.
• the culture temperature of the mammalian cells after freezing and thawing is usually within the range of about 30 to 40°C, preferably 37°C.
• the CO 2 concentration during culture is usually within the range of about 1 to 10%, preferably about 5%.
• the humidity during culturing is usually within the range of about 70 to 100%, preferably within the range of about 95 to 100%.
• the O 2 concentration during culture may be normal oxygen concentration (18-22% O 2 ) or low oxygen concentration (0-10% O 2 ).
• LR containing 10 (v/v)% PG was prepared by mixing Lactech Injection (manufactured by Otsuka Pharmaceutical Factory) and the above PG at a ratio of 9:1.
• Lactech Injection manufactured by Otsuka Pharmaceutical Factory
• 5(v/v)% PG-containing LR, 2.5(v/v)% PG-containing LR, and 1.25(v/v)% PG-containing LR are v)% PG-containing LR'' was prepared by diluting it with Lactech Injection in a 2-fold common ratio.
• LR containing 10 (v/v)% PG and 2.7 (w/v)% trehalose was prepared using 0.5 mL of the above PG and 0.5 mL of Cellstore (registered trademark) W (Otsuka Pharmaceutical Factory). Co., Ltd.) (i.e., lactated Ringer's solution containing 3 (w/v)% trehalose).
• AR containing 5 (v/v)% PG and 4.75 (w/v)% dextran was prepared by first dissolving 500 mg of the above Dextran 40 in 10 mL of Veen F infusion, )% dextran-containing AR was prepared by mixing 0.5 mL of the above PG and 9.5 mL of 5 (w/v) % dextran-containing AR.
• LR containing 5 (v/v)% PG and 4.75 (w/v)% dextran was prepared by first dissolving 500 mg of the above Dextran 40 in 10 mL of Lactech Injection, % dextran-containing LR was prepared by mixing 0.5 mL of the above PG and 9.5 mL of 5 (w/v) % dextran-containing LR.
• 7.5 (v/v)% PG and 4.63 (w/v)% dextran-containing LR is the above-mentioned “5 (v/v)% PG and 4.75 (w/v)% and the above-mentioned "LR containing 10 (v/v)% PG and 4.5 (w/v)% dextran" at a ratio of 1:1. 3) LR containing 3.75 (v/v)% PG and 4.81 (w/v)% dextran was )% dextran-containing LR" and the above-mentioned "5 (v/v)% PG and 4.75 (w/v)% dextran-containing LR" in a 1:1 ratio.
• the 6.25 (v/v)% PG and 4.69 (w/v)% dextran-containing LR is the same as the above “5 (v/v)% PG and 4.75 (w/v)% and the above-mentioned "LR containing 7.5 (v/v)% PG and 4.63 (w/v)% dextran" at a ratio of 1:1. 5) LR containing 8.75 (v/v)% PG and 4.56 (w/v)% dextran was )% dextran-containing LR" and the above-mentioned "10 (v/v)% PG and 4.5 (w/v)% dextran-containing LR" in a 1:1 ratio.
• the LR containing 4 (v/v)% PG and 4.8 (w/v)% dextran is the same as the above-mentioned “10 (v/v)% PG and 4.5 (w/v)% dextran.” It was prepared by mixing the above-mentioned "LR containing 5 (w/v)% dextran” and the above "LR containing 5 (w/v)% dextran” at a ratio of 4:6.
• 4 (v/v)% PG and 4.8 (w/v)% HES-containing LR is the above-mentioned “10 (v/v)% PG and 4.5 (w/v)% HES It was prepared by mixing the above-mentioned "LR containing 5 (w/v)% HES” and the above "LR containing 5 (w/v)% HES” at a ratio of 4:6. 7) LR containing 10 (v/v)% PG and 2.7 (w/v)% trehalose was prepared in 5) of [Preparation of cell cryopreservation solution used in Study 3] above. there was.
• 4 (v/v)% PG and 2.88 (w/v)% trehalose-containing LR is the above-mentioned “10 (v/v)% PG and 2.7 (w/v)% trehalose It was prepared by mixing "Containing LR" and Cellstore W at a ratio of 4:6. 9) LR containing 10 (v/v)% PG, 2.7 (w/v)% trehalose, and 4.5 (w/v)% dextran was prepared by first adding 500 mg of the above Dextran 40 to 10 mL.
• Lactated Ringer's solution (LR) containing 2.5 (v/v)% PG was prepared by mixing Lactec Injection and PG in a ratio of 39:1.
• LR containing 2.5 (v/v)% PG and 2.925 (w/v)% trehalose was prepared by mixing Cellstore W and the above PG at a ratio of 39:1.
• LR containing 2.5 (v/v)% PG, 2.925 (w/v)% trehalose, and 4.875 (w/v)% dextran contains Cellstore S and the above PG at 39 It was prepared by mixing at a ratio of :1.
• LR containing 4 (v/v)% PG and 1.44 (w/v)% fructose is prepared by first adding 150 mg of D(-)-fructose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to 10 mL of After preparing LR containing 1.5 (w/v)% fructose by dissolving it in Lactec Injection, mix 0.4 mL of the above PG and 9.6 mL LR containing 1.5 (w/v) % fructose.
• LR containing 4 (v/v)% PG and 2.88% sucrose was prepared by first dissolving 300 mg of sucrose (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) in 10 mL of Lactech Injection, and then preparing the LR containing 3% sucrose. After preparing the LR, it was prepared by mixing 0.4 mL of the above PG and 9.6 mL of LR containing 3% sucrose. 6) For LR containing 4 (v/v)% PG and 2.88 (w/v)% lactose, first add 316 mg of lactose monohydrate (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) to 10 mL of Lactech Injection. After preparing 3 (w/v)% lactose-containing LR by dissolving it in the following, 0.4 mL of the above PG and 9.6 mL of 3 (w/v)% lactose-containing LR were mixed.
• LR containing 2 (v/v)% PG, 2.7 (w/v)% trehalose, and 4.5 (w/v)% dextran; 3 (v/v)% PG, 2.7 LR containing (w/v)% trehalose, and 4.5 (w/v)% dextran; 4 (v/v)% PG, 2.7 (w/v)% trehalose, and 4.5% dextran; LR containing (w/v)% dextran; LR containing 5 (v/v)% PG, 2.7 (w/v)% trehalose, and 4.5 (w/v)% dextran; and LR containing 7.5 (v/v)% PG, 2.7 (w/v)% trehalose, and 4.5 (w/v)% dextran; first, Cellstore S and the above PG were mixed.
• hAD-MSCs Human adipose-derived mesenchymal stem cells (manufactured by Lonza Walkersville) were cultured according to Lonza Walkersville's human adipocyte-derived stem cell culture procedure manual. Specifically, hAD-MSCs were seeded in a 75 cm 2 flask with 15 mL of a medium prepared from a medium kit (PT-4505, ADSC BulletKit, manufactured by Lonza Walkersville) (hereinafter referred to as "MSC medium”), and incubated at 37°C. , and cultured under a 5% CO 2 incubator. Medium exchange was performed every 3 or 4 days.
• hAD-MSCs were prepared according to the following procedures [1] to [11].
• a personal incubator PIC-100, manufactured by As One Corporation
• PBS product number: 14190-144, manufactured by GIBCO
• trypsin/EDTA product code: CC-5012, manufactured by Lonza Walkersville
• trypsin/EDTA product code: CC-5012, manufactured by Lonza Walkersville
• hCD8-positive T cells 1.5 x 106 human CD8-positive T cells (hCD8-positive T cells) (manufactured by STEMCELL Technologies) were placed in a flask containing culture medium for T lymphocyte culture (product name: TLY CULTURE Kit 25, TLY-FK25, GC). (manufactured by Lymphotec) and culture was started in a 37° C., 5% CO 2 incubator. On the third day after culturing, the cells were transferred to 8 or 10 25 cm 2 flasks. Thereafter, the flasks on the 9th day of culture were used in the method described in the following section [Preparation of hCD8-positive T cells (Studies 11 and 12)].
• hCD8-positive T cells were prepared according to the following procedures [1] to [4]. [1] hCD8-positive T cells were transferred from the flask to 50 mL Proteosave (MS-52550, manufactured by Sumitomo Bakelite). [2] Cells were collected by centrifugation at 300 ⁇ g for 10 minutes at room temperature. [3] The supernatant was aspirated and suspended in Lactech Injection.
• hCD8-positive T cells or hCD4-positive T cells were transferred to three 75 cm 2 flasks, respectively.
• one flask each was used in the method described in the following section [Preparation of hCD8-positive T cells and hCD4-positive T cells (Studies 13 and 14)].
• the cells were collected from the remaining flask and divided into three 75 cm 2 flasks, and each flask was filled with T cell medium (product name: X-VIVO15 serum-free lymphocytes). 10 mL of culture medium (04-418Q, manufactured by Lonza Walkersville) was added. Thereafter, three flasks on the 11th day after culture were used in the method described in the following section [Preparation of hCD8-positive T cells and hCD4-positive T cells (Studies 13 and 14)].
• hCD8-positive T cells were prepared according to the following procedures [1] to [5]. [1] hCD8-positive T cells or hCD4-positive T cells were transferred from the flask to 50 mL Proteosave (MS-52550, manufactured by Sumitomo Bakelite). [2] Cells were collected by centrifugation at 300 ⁇ g for 10 minutes at room temperature. [3] The supernatant was aspirated and suspended in Cell Store W.
• Hematopoietic progenitor cell proliferation culture solution XF (manufactured by Takara Bio) containing 2 ⁇ 10 4 cells/mL of cord blood-derived human CD34-positive hematopoietic stem cells (hCD34-positive hematopoietic stem cells) (manufactured by Takara Bio) was added to a 75 cm 2 The seeds were seeded in a suspension culture flask (product name: Super Quality Suspension Cell Culture Flask 75 cm 2 Filter Caps, MS-2325RS, manufactured by Sumitomo Bakelite), and culture was started in a 37° C., 5% CO 2 incubator.
• a suspension culture flask product name: Super Quality Suspension Cell Culture Flask 75 cm 2 Filter Caps, MS-2325RS, manufactured by Sumitomo Bakelite
• hematopoietic progenitor cell proliferation culture medium XF was added and cultured for an additional 3 days.
• the culture solution was transferred to 50 mL Proteosave (MS-52550, manufactured by Sumitomo Bakelite) and centrifuged at 240 ⁇ g for 10 minutes at room temperature to collect cells.
• the supernatant was aspirated, fresh hematopoietic progenitor cell proliferation culture medium XF was added, the cells were suspended, and then seeded in a new 75 cm 2 suspension culture flask.
• the above operations 1) and 2) were repeated every 2 to 3 days, and the flask on the 12th day was used in the method described in the following section [Preparation of hCD34-positive hematopoietic stem cells (Study 15)].
• hCD34-positive hematopoietic stem cells were prepared according to the following procedures [1] to [4]. [1] The hCD34-positive hematopoietic stem cells were transferred from the flask to 50 mL Proteosave (MS-52550, manufactured by Sumitomo Bakelite). [2] Cells were collected by centrifugation at 240 ⁇ g for 10 minutes at room temperature. [3] The supernatant was aspirated and suspended in Lactech Injection.
• Four types of mammalian cells hAD-MSCs, hCD8-positive T cells, hCD4-positive T cells, and hCD34-positive hematopoietic stem cells
• hAD-MSCs hCD8-positive T cells
• hCD4-positive T cells hCD4-positive T cells
• hCD34-positive hematopoietic stem cells were cryopreserved according to the following procedures [1] to [4].
• Various mammalian cells suspended in Cell Store W or Lactech Injection were transferred to 15 mL Stemful (product number MS-90150, size 15 mL, Sumitomo Bakelite Co., Ltd.).
• [Cell sample preparation after freezing and thawing] Evaluate the survival rate of four types of mammalian cells (hAD-MSC, hCD8-positive T cells, hCD4-positive T cells, and hCD34-positive hematopoietic stem cells) after freezing and thawing according to the following steps [1] to [6].
• a sample was prepared for this purpose.
• a constant temperature bath water bath was set at 37°C and heated.
• Vials containing frozen cell cryopreservation solutions containing various mammalian cells were taken out of the liquid nitrogen tank and transferred to a container containing dry ice.
• hCD34-positive hematopoietic stem cell suspension 800 ⁇ L was collected. In addition, in order to carry out Studies 11 and 12, the fractionated cell suspension was stored at room temperature with shaking (100 rpm, 25 mm).
• Annexin V staining solution 400 ⁇ L was added, transferred to a round-bottomed standard tube through a cell strainer, and the Annexin V positivity rate was immediately evaluated using flow cytometry (Gallios, manufactured by Beckman Coulter). Annexin V positive rate was analyzed and calculated using Kaluza Analysis Software (Ver. 1.5a, manufactured by Beckman Coulter).
• FBS Stain Buffer
• hAD-MSCs cryopreserved in a cell cryopreservation solution containing 1.25-10% DMSO showed no difference in cell proliferation efficiency due to differences in DMSO concentration. ( Figure 1A and Table 1).
• hAD-MSCs cryopreserved in a cell cryopreservation solution containing 2.5-5% PG were significantly more efficient than hAD-MSCs cryopreserved in a cell cryopreservation solution containing 10% PG. It was shown that the cells proliferated well (FIG. 1B and Table 1).
• hAD-MSCs cryopreserved in a cell cryopreservation solution containing 2.5-5% PG were compared to hAD-MSCs cryopreserved in a cell cryopreservation solution containing 2.5-5% DMSO. , was shown to grow significantly more efficiently (FIGS. 2B and 2C).
• hAD-MSCs cryopreserved in cell cryopreservation solution containing trehalose and dextran containing 4% DMSO were Compared to hAD-MSCs that were not preserved, the proliferation level was shown to be significantly reduced from the 3rd day of culture onwards (FIG. 3 and Table 3).
• hAD-MSCs cryopreserved in a cell cryopreservation solution containing 2.5 to 5% PG did not contain 10% PG. It was shown that hAD-MSCs proliferated more efficiently compared to hAD-MSCs cryopreserved in a cell cryopreservation solution containing the following (Fig. 4 and Table 4). In particular, when three types of base solutions (LR containing dextran; LR containing trehalose; and LR containing trehalose and dextran) were used, the cell proliferation ability of hAD-MSCs was significantly higher ( Figures 4B to D and Table 4). .
• the cell survival rate of hAD-MSC when using a cell cryopreservation solution containing 5% PG and 4.75% dextran is as follows:
• the cell survival rate of hAD-MSCs was higher than that obtained when a solution was used, and was particularly significantly higher when Ringer's solution or Ringer's acetate solution was used as the base solution (FIG. 5A and Table 5).
• the survival rate of hAD-MSCs had a similar tendency, and was significantly higher especially when Ringer's acetate solution was used as the base solution (FIG. 5B and Table 5).
• the annexin V positive rate of hAD-MSCs was as follows: The cell survival rate was lower than that when a preservation solution was used (FIG. 5C and Table 5).
• Cell cryopreservation solution (LR containing 2.5% PG and 4.88% dextran; LR containing 3.75% PG and 4.81% dextran; LR containing 5% PG and 4.75% dextran; 6.25% hAD-MSCs cryopreserved in cell cryopreservation solution containing 10% PG (LR containing PG and 4.69% dextran; and LR containing 7.5% PG and 4.63% dextran) hAD-MSCs were shown to proliferate more efficiently than hAD-MSCs cryopreserved in 4.5% dextran-containing LR), especially when the concentration of PG in the cell cryopreservation solution was 2.5-5%. It was shown that the cells proliferated ( Figure 8 and Table 8).
• hAD-MSCs were cryopreserved in a cell cryopreservation solution containing 4% PG and 1.2-9.6% dextran
• hAD-MSCs were cryopreserved in a cell cryopreservation solution containing 4% PG without dextran.
• the annexin V positivity rate of hAD-MSCs immediately after freezing and thawing was lower than when the cells were cryopreserved, especially when the concentration of dextran in the cell cryopreservation solution was 4.8 to 9.6% ( Figures 9A and 9B, Table 9).
• hAD-MSCs were frozen in cell cryopreservation solution containing PG and HES (LR containing 4% PG and 4.8% HES; and LR containing 10% PG and 4.5% HES).
• hAD-MSCs are cryopreserved in cell cryopreservation solution containing PG and dextran (LR with 4% PG and 4.8% dextran; and LR with 10% PG and 4.5% dextran).
• hAD-MSCs immediately after freezing and thawing were The cell survival rate and viable cell recovery rate were high, and the annexin V positive rate was low ( Figure 10 and Table 10).
• the PG concentration in the cell cryopreservation solution containing PG and HES was 4%, the cell survival rate and viable cell recovery rate of hAD-MSCs immediately after freezing and thawing were lower than when the PG concentration was 10%.
• the annexin V positive rate was low (FIG. 10 and Table 10).
• LR containing 2.5% PG, 2.925% trehalose, and 4.875% dextran When cryopreserved, the cell viability and viable cell recovery rate of hAD-MSCs immediately after thawing were higher than when hAD-MSCs were cryopreserved in 2.5% PG-containing LR.
• These values were significantly higher when using LR containing PG, 2.925% trehalose, and 4.875% dextran (FIGS. 12A and 1B, Table 12).
• LR containing 2.5% PG, 2.925% trehalose, and 4.875% dextran Upon storage, the annexin V positivity rate of hAD-MSCs immediately after thawing was significantly lower than when hAD-MSCs were cryopreserved in 2.5% PG-containing LR (FIG. 12C, Table 12).
• LR containing 72% trehalose; LR containing 4% PG and 1.44% trehalose; LR containing 4% PG and 2.88% trehalose; LR containing 4% PG and 5.76% trehalose; and hAD-MSCs were cryopreserved in LR containing 4% PG and 11.52% trehalose, and the cell viability, viable cell recovery rate, and annexin V positivity rate of hAD-MSCs were analyzed immediately after thawing.
• cell cryopreservation solution containing 4% PG and 0.72-11.52% trehalose (LR containing 4% PG and 0.72% trehalose; containing 4% PG and 1.44% trehalose) LR; LR containing 4% PG and 2.88% trehalose; LR containing 4% PG and 5.76% trehalose;
• MSCs are cryopreserved, cell survival of hAD-MSCs immediately after freezing and thawing is improved compared to when hAD-MSCs are cryopreserved in 4% PG-containing cell cryopreservation solution (LR containing 4% PG) without trehalose.
• hAD-MSCs were cryopreserved in a cell cryopreservation solution containing 4% PG and 0.72-11.52% trehalose
• hAD-MSCs were cryopreserved in a cell cryopreservation solution containing 4% PG without trehalose.
• the annexin V positivity rate of hAD-MSCs immediately after freezing and thawing was lower than that when the hAD-MSCs were cryopreserved ( Figure 14C, Table 14).
• hCD8-positive T cells were frozen in three types of cells. Cryopreserve in storage solution (serum-containing culture medium containing 4% PG; trehalose-containing LR containing 4% PG; and LR containing 4% PG) immediately after thawing, 1 hour after thawing, 3 hours after thawing, Then, the cell survival rate and viable cell recovery rate of hCD8-positive T cells for 6 hours were analyzed.
• hCD8-positive T cells were cryopreserved in serum-containing culture medium containing 4% PG or trehalose-containing LR containing 4% PG, hCD8-positive T cells were cryopreserved in LR containing 4% PG.
• the cell survival rate and viable cell recovery rate of hCD8-positive T cells immediately after or after thawing were higher than in the case of thawing (FIG. 15 and Table 15).
• the hCD8-positive T cells were cryopreserved in LR containing PG, 2.7% trehalose, and 4.5% dextran, and the cell viability and viable cell recovery rate of hCD8-positive T cells were analyzed immediately after thawing and 3 hours after thawing.
• hCD4-positive T cells are cryopreserved in the above four types of cell cryopreservation solutions containing 3 to 7.5% PG
• hCD4-positive T cells are Compared to when cells were cryopreserved, the annexin V positivity rate of hCD4-positive T cells immediately after freezing and thawing was significantly lower, especially when the concentration of PG in the cell cryopreservation solution was 4 to 5% ( Figures 18C and 18C). Table 18)
• cell death caused by freezing and thawing of mammalian cells can be effectively suppressed, and the proliferation ability of mammalian cells after freezing and thawing can be effectively increased, so transplantation therapy in regenerative medicine etc. It is useful in the field and cancer treatment field.

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