Refrigerated storage solution for stem cells

Abstract

Provided are a cold storage solution that is suitable for the cold storage of human or animal stem cells such as iPS cells or embryos in a non-frozen state, and a cold storage method. In a cold storage solution according to one embodiment: the content of potassium ion species is 30-80 mmoL / L; the content of sodium ion species is 30-80 mmoL / L; the amount of sodium ion species represented by an ion molar ratio to potassium ion species (NA+ / K+ ratio) is 0.5-1.3; Trolox or an analog thereof is contained at a concentration of 1-8 mM; adenine or a salt or derivative thereof is contained at a concentration of 0.1-4.2 mM; all or all excluding one, two or three kinds of essential amino acids are contained; the total content of the essential amino acids is 50-200 mg; and the total content of non-essential amino acids is 50-200 mg / L.

Description

[Technical field]

[0001] The present invention relates to a preservation solution for preserving cells, tissues, organoids, organs, etc. in a non-frozen state. In particular, the present invention relates to a refrigerated preservation solution for preserving cells, tissues, organoids, organs, etc. at a temperature lower than room temperature, for example, at 2 to 15° C., 2 to 8° C., or 2 to 6° C. In particular, the present invention relates to a refrigerated preservation solution for preserving pluripotent stem cells such as human iPS cells and other stem cells, or derivatives such as organoids obtained therefrom, early embryos, etc., in a suspended or immersed state. [Background technology]

[0002] The simple, efficient and stable preservation and supply of stem cells such as iPS and ES cells is an essential technology in the fields of basic research, clinical application research and drug discovery research in regenerative medicine. Cryopreservation of human iPS cells and ES cells has many problems, such as a low survival rate after storage when using slow cryopreservation methods, and the cytotoxicity and differentiation-inducing properties of DMSO, a cryoresistant material used in cryopreservation. Non-freezing low-temperature preservation of cells is a very attractive technology as a short-term (e.g., 1 day to 2 weeks) to medium-term (e.g., half a month to 3 months) preservation method, and has great advantages when considering the industrialization of regenerative medicine technology and its application to drug discovery, such as preservation of differentiated cells and organoids. However, it is difficult to low-temperature preserve pluripotent stem cells, including human iPS cells, using UW solution (UW_sln; BELZER UW (registered trademark) COLD STORAGE SOLUTION), which is widely used as a low-temperature preservation solution for organs, and the development of a new refrigerated preservation technology is essential.

[0003] UW solution is widely used for the refrigerated storage of organs for transplantation, such as livers and kidneys, and is essentially the gold standard. UW solution, developed in the 1980s, has a relatively simple composition, consisting of intracellular low Na ion and high K ion salts, including lactobionate, raffinose, glutathione, and hydroxy-ethyl starch. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] PCT / JP2009 / 002941(JP5726525B) [Patent Document 2] JP6220108B (JP2012-217342A) [Patent Document 3] WO2016 / 076317A [Non-patent literature]

[0005] [Non-Patent Document 1] COMPARE BELZER UWTM COLD STORAGE SOLUTION TO VIASPAN https: / / bridgetolife.com / compare-belzer-uw-cold-storage-solution-to-viaspan / [Non-Patent Document 2] A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells, Scientific Reports 4 : 3594 doi: 10.1038 / srep03594 [Non-Patent Document 3] Cryopreservation and Hypothermal Storage of Hematopoietic Stem Cellshttps: / / etd.ohiolink.edu / !etd.send_file?accession=ucin1439307244&disposition=inline Summary of the Invention [Problem to be solved by the invention]

[0006] However, when storing cells in a refrigerator, UW solution cannot sufficiently suppress low-temperature storage stress. The inventors hypothesized that to solve this problem, it is important to have a salt composition intermediate between the intracellular type represented by UW solution and the high Na ion, low K ion extracellular type found in normal culture media. In addition, we believe that the addition of macromolecules such as proteins is more important than in organ storage, and so we developed a preservation solution intermediate between UW solution and cell culture solution.

[0007] There have been few reports on culture media for low-temperature storage of cells. There have been a few reports on storage using the recently developed HTS FRS (Hypothermosol (registered trademark) FRS; BioLife Solutions). It has been reported that this HTS FRS can store human skin fibroblasts and smooth muscle cells at low temperatures for 3 to 5 days, but there have been no reports on the refrigerated storage of pluripotent cells such as human iPS cells. [Means for solving the problem]

[0008] In the course of trying a wide variety of combinations, the present inventors attempted to use a liquid obtained by mixing MEM-alpha (Minimum Essential Medium Eagle (MEM) Alpha Modification) or StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) with the above-mentioned UW liquid or ES cell culture medium (modified based on DMEM:F12 medium described below; hereinafter referred to as "ESC medium") at a ratio of about 1 / 1 to 1 / 2. MEM-alpha is based on MEM medium and contains non-essential amino acids, sodium pyruvate, lipoic acid, biotin, and vitamin B12.

[0009] The Eagle's minimal essential medium (MEM) contains the following components (1) to (4). (1) Amino acids (L-arginine, L-cystine, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine), (2) Salts (calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate), (3) D-glucose, and (4) Vitamins (folic acid, nicotinamide (niacinamide), riboflavin, B12, choline, inositol, pantothenic acid, pyridoxal phosphate, thiamine).

[0010] On the other hand, UW solution has an intracellular fluid-type composition of Ca 2+ It is a liquid-free formulation that contains lactobionic acid and hydroxyethyl starch (HES) for osmotic pressure adjustment and cell swelling inhibition, adenosine for anti-inflammation, vasorelaxation, and as an ATP precursor, glutathione and allopurinol for antioxidant purposes, and phosphate buffer as a buffer component.

[0011] StemFit (registered trademark) AK02N is a feederless medium for human iPS cells, consisting of 400 mL of solution A, 100 mL of solution B, and 2 mL of solution C, which are mixed immediately before use, and the above-mentioned UW solution or ES cell culture solution was also mixed in at the time of mixing. Solution C is a solution of basic fibroblast growth factor (bFGF) dissolved in a physiological buffer solution.

[0012] In addition, compared to MEM-alpha, StemFit (registered trademark) AK02N can be said to have the same salt composition, the almost same essential amino acid composition, and a large part of the non-essential amino acid composition. As suggested by the experimental results described below, MEM-alpha and StemFit (registered trademark) AK02N could be used almost in the same way in the refrigerated preservation solution of the present invention. This is because these common parts, i.e., the main parts of the salt composition, essential amino acid composition, and non-essential amino acid composition, are considered to be important in exerting the effects of the present invention. On the other hand, the main difference between MEM-alpha and StemFit (registered trademark) AK02N is that both proteins and growth factors are contained only in StemFit (registered trademark) AK02N. It is presumed that these components are not essential and do not contribute much to the refrigerated preservation solution of the present invention. While StemFit (registered trademark) AK02N is a culture medium for iPS cells, MEM-alpha cannot culture iPS cells even with the addition of bFGF, a representative growth factor. Similarly, ES cell culture medium ("ESC medium") cannot culture iPS cells without feeder cells.

[0013] The DMEM:F12 medium that constitutes the base of the ES cell culture medium ("ESC medium") used herein is a mixture of Eagle's minimal essential medium (MEM) and Ham's F12 medium at a volume ratio of 1:1. Ham's F12 medium is a serum-free basal medium that contains putrescine, hypoxanthine, and thymidine.

[0014] A refrigerated storage solution according to a preferred embodiment of the present invention has the following characteristics a1 to a6 or a1 to a9. a1. The content of potassium ion species is 20~90mmoL / L or 30~80mmoL / L, and the content of sodium ion species is 20~90mmoL / L or 30~80mmoL / L. a2. The amount of sodium ion species is the molar ratio of ion-based to potassium ion species (Na + / K + The ratio of a3. Trolox or its analogues are contained at a concentration of 0.5-10 mM, 1-8 mM, or 2-7 mM. a4. Adenine or a salt or derivative thereof is contained at a concentration of 0.1 to 4.2 mM, 0.1 to 6 mM, 1 to 6 mM, 2 to 5 mM, or 3 to 5 mM. a5. All essential amino acids are contained, or all but one, two or three are contained, and the total content of essential amino acids is preferably 50 to 250 mg / L, particularly 50 to 200 mg / L or 80 to 150 mg / L. a6. The total content of non-essential amino acids is preferably 100 to 500 mg / L, particularly 50 to 200 mg / L or 80 to 150 mg / L. a7. The content of magnesium ion species (especially magnesium sulfate) is 2 to 8 mmol / L, 2 to 5 mmol / L, or 2 to 4 mmol / L, and the content of calcium is 0.2 to 1.5 mmol / L, 0.2 to 1 mmol / L, or 0.3 to 0.8 mmol / L. a8. Contains glucose or other low molecular weight polysaccharides (especially disaccharides such as maltose or monosaccharides such as fructose) at a concentration of 0.5 to 10 mmol / L. a9. Contains the following vitamins, provided that one, two, three or four of the following may be omitted. Folic acid, nicotinamide (niacinamide), riboflavin, B12, choline, inositol, pantothenic acid, pyridoxal phosphate, and thiamine.

[0015] A preferred embodiment of the refrigerated storage solution of the present invention contains: (i) lactobionic acid or a salt thereof at 30 to 100 mmol / L (or 10 to 40 g / L) calculated as lactone; (ii) raffinose hydrate at 10 to 30 mmol / L (or 5 to 20 g / L); (iii) allopurinol at 0.3 to 1 mmol / L (or 0.05 to 0.1 g / L); (iv) glutathione (total glutathione) at 1 to 3 mmol / L; and (v) adenosine at 2 to 10 mmol / L (or 0.3 to 0.9 g / L). (vi) 0.05 to 1 μmol / L (or 0.03 to 0.2 mg / L) of lipoic acid, (vii) 0.1 to 1 mmol / L (or 10 to 100 mg / L) of sodium pyruvate, (viii) 0.5 to 10 mmol / L (or 100 to 1000 mg / L) of glucose, (ix) 0.03 to 0.3 mmol / L of ascorbic acid, and 1 to 10 mM of vitamin E or a water-soluble analog or derivative thereof, (x) A physiological buffer solution containing 0.1 to 4.2 mM adenine or a salt or derivative thereof, (xi) other vitamins or water-soluble analogs or derivatives thereof, (xii) a total of 50 to 200 mg / L of essential amino acids, (xiii) a total of 100 to 500 mg / L of non-essential amino acids, (xiv) 30 to 80 mmol / L of potassium ion species, and (xv) 20 to 90 mmol / L of sodium ion species, and having a pH set at 7 to 7.5.

[0016] This refrigerated storage solution preferably contains (xv) hydroxyethyl starch at 20 to 50 g / L, (xvi) ribonucleoside at 4 to 40 mg / L and / or (xvii) deoxyribonucleoside at 4 to 40 mg / L, and (xviii) potassium dihydrogen phosphate or sodium dihydrogen phosphate at 10 to 25 mmol / L. Effect of the Invention

[0017] When pluripotent cells such as human iPS cells are stored in a refrigerator, it is possible to maintain a high viability and proliferation ability after re-culture. [Brief description of the drawings]

[0018] [Figure 1A] 7 is a graph showing the change in relative viability over the number of days of storage when the viability at the start of storage is set to 1.0, in the case of storage at 4° C. in a single cell state. In the experiments in FIGS. 1 to 7F, a refrigerated storage medium ("HTM1") was used, which was a 2 / 1 mixture of UW solution and clinical research medium StemFit (registered trademark) AK02N. [Figure 1B] FIG. 1B is a bar graph showing the results of determining the number of days for which 50% survival rate is maintained (ST50) from the graph in FIG. 1A. [Figure 2A] As in Fig. 1A, this is a graph showing the change in viability over the number of days of storage when the cells were stored in a single cell state at 4° C. However, as in Fig. 1A, the viability at the start of storage is set to 1.0. [Figure 2B] FIG. 2B is a bar graph showing the results of determining the number of days for which 80% survival rate is maintained (ST80) from the graph in FIG. 2A. [Figure 2C] 1 is a bar graph showing colony formation ability after 5 days of culture following 6 days of refrigerated storage. [Figure 3A] 1 is a bar graph showing the rate of apoptotic cell death immediately after 3 days of refrigerated storage. [Figure 3B] FIG. 3B is a bar graph showing the percentage of cell death due to necrosis immediately after 3 days of refrigerated storage, as in FIG. 3A. [Figure 4A] FIG. 3B is a bar graph showing the change in intracellular reactive oxygen species (ROS) concentration immediately after refrigerated storage for three days. [Figure 4B] 4A, this is a bar graph showing the change in intracellular caspase 3 / 7 activity, which is an index of apoptosis, immediately after refrigerated storage for three days. [Figure 5A]The chemical formulas of the radical scavengers used in the experiments are summarized below. [Figure 5B] FIG. 5B is a bar graph showing the number of days for which 80% viability is maintained (ST80) when various radical scavengers shown in FIG. 5A were added, similar to FIG. 2B. [Figure 6A] FIG. 2B is a graph similar to FIG. 2A, showing the change in viability depending on the number of days of storage when the concentration of the radical scavenger Trolox added is changed. [Figure 6B] FIG. 6B is a bar graph showing the results of determining the number of days for which 80% survival rate is maintained (ST80) from the graph in FIG. 6A. [Figure 6C] 1 is a bar graph showing the results of colony formation ability when cultured for 5 days after refrigerated storage for 6 days in media containing varying concentrations of Trolox. [Figure 6D] 1 is a bar graph showing the effect of addition of tocopherol and tocopherol acetate on relative viability after 6 days of storage and 1 day of recovery culture. [Figure 7A] Phase contrast images show colonies obtained after refrigerated storage for 3 days followed by re-cultivation for 4 days. [Figure 7B] The image in FIG. 7A has been binarized. [Figure 7C] Phase contrast images show colonies obtained after refrigeration for 6 days followed by refrigeration for 4 days. [Figure 7D] The image in FIG. 7B has been binarized. [Figure 7E] 1 is a bar graph summarizing the colony area ratios in media that were refrigerated for 3 and 6 days and then re-cultured for 4 days. [Figure 7F] This is a bar graph showing the increase in colony count when the cells were re-cultured for 4 days after refrigerated storage for 3 and 6 days, expressed as a ratio relative to the non-preserved control, which was cultured for 4 days without refrigeration, taken as 1. [Figure 8] This is a bar graph showing the results of FIG. 7F including the results without the addition of Trolox or the like, and with Trolox 5 mM and adenine at 1.4-8.2 mM. [Figure 9]This is a bar graph similar to that of Figures 7F and 8, showing the increase rate of colony counts when the refrigerated storage medium "HTM-alpha" (UW solution + MEM-alpha, 2:1), which is a 2:1 mixture of UW solution and MEM-alpha, was used as the refrigerated storage medium and then re-cultured for 4 days after 7 days of refrigeration. Here, 5 mM Trolox and 5 times concentration (x5; 10 mM) of GlutaMAX (registered trademark, GIBCO) were added to "HTM-alpha" and "HTM1". The results using HTS FRS, a commercially available refrigerated storage medium, are also shown. [Figure 10] This is a bar graph showing the rate at which mouse blastocyst-stage embryos (3.5 days after in vitro fertilization) escaped from the zona pellucida during a two-day culture period after being refrigerated for three days in the same medium as in Figure 9. [Figure 11] 11 is a bar graph similar to that of FIG. 10, showing the results of investigating the effect of the concentration of adenine hydrochloride added under the same conditions as in the experiment of FIG. [Figure 12A] FIG. 6B is a bar graph showing the protective effect when polyvinyl alcohol (PVA) was used instead of hydroxyethyl starch (HES) under similar conditions to the experiment of FIG. 6A. [Figure 12B] FIG. 12B is a bar graph similar to FIG. 12A, showing the effect of adding mannitol in addition to polyvinyl alcohol (PVA) under the same conditions as in the FIG. 12A experiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] A refrigerated storage solution according to a preferred embodiment of the present invention is a physiological buffer solution containing the following (i) to (vi), and has a pH set at 7 to 7.5.

[0020] (i) Lactobionic acid or lactobionate, calculated as lactone, at 20-100 mmol / L, 30-100 mmol / L, 30-80 mmol / L, or 30-60 mmol / L, or at 10-50 g / L, 10-40 g / L, 15-35 g / L, or 20-30 g / L. (ii) Raffinose hydrate 10-40mmol / L, 10-30mmol / L or 10-20mmol / L, or 5-20g / L or 8-15g / L. (iii) Allopurinol 0.3–2 mmol / L, 0.3–1 mmol / L, or 0.4–0.8 mmol / L, or 0.03–0.15 g / L, 0.05–0.1 g / L, or 0.06–0.08 g / L. (iv) Glutathione (total glutathione) 1-3mmol / L or 1.5-2.5mmol / L or 0.3-1g / L, 0.4-0.9g / L or 0.4-0.8g / L. (v) Adenosine 2-10mmol / L, 2-8mmol / L, or 2-5mmol / L, or 0.5-1.5g / L, or 0.5-1.3g / L.

[0021] (vi) Lipoic acid: 0.05–1 μmol / L, 0.1–0.5 μmol / L, or 0.2–0.4 μmol / L, or 0.03–0.2 mg / L, or 0.05–0.1 mg / L. (vii) Sodium pyruvate 0.1-1 mmol / L, 0.1-0.7 mmol / L, or 0.2-0.5 mmol / L, or 10-100 mg / L, or 20-50 mg / L. (viii) Glucose 0.5-10mmol / L, 1-5mmol / L, or 1-3mmol / L, or 100-1000mg / L, or 200-500mg / L.

[0022] (ix) The following antioxidant vitamins or their water-soluble analogs or derivatives: (Each of these may take the form of a salt. In some cases, ix-1 may be omitted from the following two types.) ix-1. Ascorbic acid 0.03~0.3mmoL / L, 0.05~0.2mmoL / L, or 0.05~0.15mmoL / L, or 5~20mg / L, or 10~15mg / L. ix-2. Vitamin E, Trolox or other water-soluble vitamin E analogues or derivatives 0.5-10 mM, 1-8 mM or 2-7 mM.

[0023] (x) adenine or a salt or derivative thereof 0.1-6 mM, 0.1-5 mM, 0.1-4.5 mM, 0.1-4.2 mM or 2-5 mM, optionally 0.1-0.3 mM or 0.05-0.2 mM.

[0024] (xi) Other vitamins listed above or their water-soluble analogues or derivatives (Each of these may take the form of a salt. In addition, of the following 10 types, 1 to 5 types, 1 to 4 types, or 1 to 3 types may be omitted.) xi-1. Biotin 0.03~0.25μmol / L, 0.05~0.2μmol / L, or 0.1~0.15μmol / L, or 0.01~0.1mg / L, or 0.02~0.05mg / L. xi-2. Vitamin B12 0.03~3μmoL / L, 0.05~2μmoL / L, or 0.1~0.5μmoL / L, or 0.1~0.8mg / L, or 0.2~0.6mg / L. xi-3. Folic acid 0.2~2μmoL / L, 0.3~1μmoL / L, or 0.5~0.9μmoL / L, or 0.1~1mg / L, or 0.2~0.5mg / L. xi-4. Niacinamide 0.5~8μmoL / L, 1~6μmoL / L, or 1~4μmoL / L, or 0.03~0.2mg / L, or 0.05~0.1mg / L. xi-5. Riboflavin 0.03~0.3 μmol / L, 0.04~0.2 μmol / L, or 0.05~0.15 μmol / L, or 0.03~0.2 mg / L, or 0.05~0.1 mg / L. xi-6. Choline 0.05~1μmoL / L, 0.1~0.5μmoL / L, or 0.1~0.4μmoL / L, or 0.1~1mg / L, or 0.2~0.5mg / L. xi-7. Inositol 1~10μmoL / L, 1~8μmoL / L, or 2~6μmoL / L, or 0.03~0.2mg / L, or 0.05~0.1mg / L. xi-8. Pantothenic acid 0.02~0.2 μmoL / L, 0.03~0.1 μmoL / L, or 0.04~0.08 μmoL / L, or 0.1~1 mg / L, or 0.2~0.5 mg / L. xi-9. Pyridoxal 0.5~3 μmoL / L, 0.1~2 μmoL / L, or 1~2 μmoL / L, or 0.1~1 mg / L, or 0.2~0.5 mg / L. xi-10. Thiamine 0.3~3 μmoL / L, 0.4~2 μmoL / L, or 0.5~1.5 μmoL / L, or 0.1~1 mg / L, or 0.2~0.5 mg / L.

[0025] (xii) Essential amino acids, total: 50-200 mg / L or 80-150 mg / L. The amount of each component can be appropriately set within the range of A / 3 to 3A (i.e., the range of one-third to three times the value in "HTM-alpha") or within the range of A / 2 to 2A, where A is the value obtained by dividing the content in Table 2 by 3 (the value in "HTM-alpha" in the examples below). The same applies to the non-essential amino acids described below and the vitamins mentioned above. xii-1. Isoleucine Leucine xii-3. Lysine Methionine 5. Phenylalanine 6. Threonine xii-7. Tryptophan xii-8. Valine XII-9. Histidine

[0026] (xiii) Non-essential amino acids, total: 100-500 mg / L or 150-400 mg / L. (Each of these may take the form of a salt. In addition, of the following 10 types, 1 to 5 types, 1 to 4 types, or 1 to 3 types may be omitted.) xiii-1. Glycine Alanine Arginine xiii-4. Asparagine xiii-5. Aspartic acid Cysteine 7. Cystine 8. Glutamic acid xiii-9. Glutamine xiii-10. Proline

[0027] (xiv) Potassium ion species: 20-90 mmoL / L, 30-80 mmoL / L, 40-80 mmoL / L, or 50-70 mmoL / L, i.e., 20 mmoL / L or more, 30 mmoL / L or more, 40 mmoL / L or more, or 50 mmoL / L or more, but less than 90 mmoL / L, less than 80 mmoL / L, or less than 70 mmoL / L.

[0028] (xv) Sodium ion species: 20 to 90 mmoL / L, 30 to 80 mmoL / L, 40 to 80 mmoL / L, or 50 to 70 mmoL / L, i.e., 20 mmoL / L or more, 30 mmoL / L or more, 40 mmoL / L or more, or 50 mmoL / L or more, but less than 90 mmoL / L, less than 80 mmoL / L, or less than 70 mmoL / L.

[0029] This refrigerated storage solution preferably contains at least any one of the following (xvi) to (xviii), and particularly preferably contains the following (xvi) to (xvii) and the following (xviii). (xvi) Hydroxyethyl starch, 10-80 g / L, 20-50 g / L or 20-40 g / L. (xvii) Polyvinyl alcohol, 3-100 g / L, 5-80 g / L, 5-50 g / L, 5-30 g / L, 5-25 g / L, or 5-20 g / L. (xviii) Mannitol 20-100mmol / L, 30-90mmol / L, or 20-90mmol / L.

[0030] This refrigerated storage solution preferably contains at least one of the following (xix) and (xx): (xix) 2 to 4 ribonucleosides, total 4 to 40 mg / L, 5 to 30 mg / L, or 10 to 15 mg / L. (xx) 2 to 4 deoxyribonucleosides total 4 to 40 mg / L, 5 to 30 mg / L or 10 to 15 mg / L.

[0031] This refrigerated storage solution preferably contains the following (xxi) to (xxiii): (xxi) Potassium dihydrogen phosphate or sodium dihydrogen phosphate, 10–30 mmol / L, 10–25 mmol / L, or 15–25 mmol / L. (xxii) Magnesium ion species (especially magnesium sulfate) from 2 to 8 mmol / L, from 2 to 5 mmol / L or from 2 to 4 mmol / L. (xxiii) Calcium ion species (especially magnesium chloride) 0.2 to 1.5 mmol / L, 0.2 to 1 mmol / L or 0.3 to 0.8 mmol / L.

[0032] In a preferred embodiment of the present invention, the sodium ion species may be, for example, 0.7 to 1.3 times, 0.8 to 1.2 times, or 0.9 to 1.1 times the potassium ion species in molar ratio (ratio of Na / K ions).In another preferred embodiment, the sodium ion species may be, for example, 0.5 to 1.5 times, 0.5 to 1.3 times, or 0.6 to 1.2 times the potassium ion species in molar ratio (ratio of Na / K ions).

[0033] A refrigerated storage solution according to a preferred embodiment of the present invention comprises Trolox or other water soluble vitamin analogues, as described above, at a concentration of at least 0.1 mM, at least 0.3 mM, at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, or at least 5 mM, but not more than 10 mM, not more than 8 mM, or not more than 6 mM.

[0034] As described above, the refrigerated preservation solution according to a preferred embodiment of the present invention contains adenine or a salt thereof at a concentration of 0.1 mM or more, 0.3 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 3 mM or more, and 8 mM or less, 6 mM or less, 5 mM or less, or 4 mM or less, for example, 0.1 to 4.2 mM or 1 to 4.2 mM. In particular, the addition of adenine is considered to be significantly effective in preserving embryos and organoids in which cells are adhered to each other to form a three-dimensional mass. In addition, the effect was observed even at a relatively low concentration, for example, 0.01 to 0.2 mM (10 to 200 μM) or 0.05 to 0.3 mM (5 to 300 μM), in preserving embryos and organoids (FIG. 11). On the other hand, when culturing stem cells such as iPS cells without forming embryos or organoids, it was thought that adding adenine at a concentration of, for example, 0.5 to 6 mM, particularly 1 to 5 mM or 2 to 4 mM, was particularly effective (Figures 7A to 8).

[0035] The refrigerated storage solution according to a preferred embodiment of the present invention further comprises L-alanyl-L-glutamine or other dipeptide substitute or a salt thereof at a concentration of 1-6 mM, 2-5 mM or 3-5 mM.

[0036] A refrigerated storage solution according to a preferred embodiment of the present invention is obtained by mixing UW solution, the composition of which is shown in Table 1, and MEM alpha medium, the composition of which is shown in Table 2, in a volume ratio such that the UW solution is 1 to 3 times, preferably 1.5 to 2.5 times, more preferably 1.8 to 2.2 times, the volume of the MEM alpha medium. The compositions in Tables 1 and 2 and the contents of each component obtained from the mixing ratios can be appropriately increased or decreased within a range of ±50%, ±40%, ±30%, or ±20%. Some components, particularly those listed in Table 2, can be omitted, including some of the non-essential amino acids (e.g., 1 to 5 types), some of the nucleosides (e.g., 1 to 5 types), some of the vitamins (e.g., 1 to 5 types), some of the inorganic salts (e.g., 1 to 3 types), and phenol red.

[0037] In a preferred embodiment of the present invention, the refrigerated preservation method comprises: incubating pluripotent cells in any one of the above-mentioned refrigerated preservation solutions at a temperature of 1×10 3 pieces / mL~1×10 9 / mL or 1 x 10 4 pieces / mL~1×10 8 The cells are dispersed in a single-cell state so as to have a concentration of 1 cell / mL, and then stored in a refrigerator for, for example, 1 to 10 days or 1 to 7 days.

[0038] In a preferred embodiment of the present invention, the refrigerated preservation method comprises culturing pluripotent cells at a concentration of 1×10 in a culture vessel containing any one of the above-mentioned refrigerated preservation solutions. 3 pieces / cm 2 ~1×10 9 pieces / cm 2 or 1×10 4 pieces / cm 2 ~1×10 8 pieces / cm 2 The cells are seeded so that the cells become 100% pure and 100% pure, and then cultured for, for example, 2 to 6 days, and then stored in a refrigerator for, for example, 1 to 10 days or 1 to 7 days.

[0039] According to a preferred embodiment of the present invention, the pluripotent cells or stem cells to be refrigerated are, for example, multipotent cells such as ES cells and induced pluripotent stem cells such as iPS cells, or hematopoietic stem cells, neural stem cells, hepatic stem cells, skin stem cells, germline stem cells, etc. Furthermore, the pluripotent cells to be refrigerated can be of human origin or of mammalian origin such as primates, mice, guinea pigs, etc.

[0040] According to another embodiment, the subject of cold storage may also be a human or animal embryo, in particular a livestock animal embryo, such as a cow, horse, pig or goat. EXAMPLES

[0041] 1. Preparing Cells for Cold Storage Experiments A human iPS cell line (253G1) derived from a healthy individual obtained from the Center for iPS Cell Research and Application, Kyoto University, was maintained and cultured under feeder-free conditions. StemFit (registered trademark) AK02N (Ajinomoto), a commercially available clinical research (for human ES / iPS cells) medium, was used for this culture, and when the cells reached 70-80% confluence on the 7th day after the start of culture, they were enzymatically detached (using 0.5X TrypLE (registered trademark) Select), dispersed, and washed. After this, they were suspended in new StemFit (registered trademark) AK02N medium containing 10 μM Y27362 (FUJIFILM Wako Pure Chemical Industries, Ltd.) as a ROCK inhibitor, and iMatrix-511 (MATRIXOME, Inc.) was added at 0.1 μg / cm as a further cell culture substrate. 2 Add it so that it becomes 1.3E3 / cm 2 (1.3×10 3 / cm 2 The day after seeding, the medium was replaced with StemFit (registered trademark) AK02N medium without Y27362, and thereafter, the medium was replaced every 3 days. On the 7th day, the cells were subcultured or used for experiments.

[0042] In the case of the experiment using HTM-alpha as the refrigerated storage medium described below (Figure 9), the same MEM-alpha medium used for HTM-alpha was used instead of StemFit (registered trademark) AK02N to prepare cells for the refrigerated storage experiment.

[0043] On the other hand, in order to attempt refrigerated storage of mouse embryos (Figures 10-11), in vitro fertilization was performed from female and male mice (both ICR), and the embryos were cultured for 3.5 days (84 hours). In this case, HTF (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the insemination medium, and KSOM (Arc Resources) was used for culture.

[0044] Unless otherwise specified in this application, the cells were cultured in a 37°C incubator (5% CO2).

[0045] 2. Refrigerated preservation solution Three types of refrigerated storage solutions were prepared, which for convenience are referred to as HTM1, HTM2, and HTM-alpha, respectively. HTM1: UW solution + the above clinical research medium StemFit® AK02N, mixing volume ratio: 2 / 1. HTM2: UW solution + human ES cell culture medium (ESC medium) below, mixed in a volume ratio of 2 / 1. HTM-alpha: UW solution + MEM-alpha (12571 - MEM alpha, nucleosides, Thermo Fisher Scientific), mixed volume ratio: 2 / 1. Trolox was added to a concentration of 5 mM, and GlutaMAX (registered trademark, GIBCO) was added to a concentration of 5 times (x5; 10 mM).

[0046] In addition, any one of the following three types of refrigerated preservation solutions was used as a refrigerated preservation solution in the comparative example. ·UW Solution (BELZER UW (registered trademark) COLD STORAGE SOLUTION) The above StemFit® AK02N ·HTS FRS (HypoThermosol® FRS, BioLife Solutions)

[0047] Human ES cell culture medium ("ESC medium") was prepared by adding the following to DMEM-F12 (Invitrogen 21331-020). The percentages and mM below indicate weight ratios or molar concentrations relative to the final human ES cell culture medium. Non-essential amino acids (NON-ESSENTIAL AMINO ACID (x100), invitrogen 11140) 1%, 200 mM L-glutamine (L-GLUTAMINE(x100), invitrogen 25030) 1%, StemSure® Serum Replacement (SSR) (FUJIFILM Wako Pure Chemical Industries, Ltd.) 20%, 2-Mercaptoethanol (x1000; concentration at time of addition) 0.1 mM, Penicillin and streptomycin.

[0048] According to the above-mentioned Non-Patent Document 1, the composition of UW solution (BELZER UW (registered trademark) COLD STORAGE SOLUTION) is as shown in the table below.

[0049] [Table 1] Composition of UW solution JPEG0007636797000001.jpg4982

[0050] The MEM alpha medium (12571 - MEM alpha, nucleosides, Thermo Fisher Scientific) used in "HTM-alpha" has the following specific composition (https: / / www.thermofisher.com / jp / en / home / technical-resources / media-formulation.94.html).

[0051] [Table 2] Composition of MEM alpha medium JPEG0007636797000002.jpg12482

[0052] GlutaMAX (registered trademark, GIBCO) added to "HTM-alpha" is a 200 mM solution of L-alanyl-L-glutamine, a dipeptide substitute for L-glutamine, dissolved in physiological saline (0.85N NaCl) (GlutaMAX TM This was diluted 1 / 20 to 10 mM (5x) and added to "HTM-alpha".

[0053] The sodium ions, potassium ions, and molar ratios thereof for the above UW solution, HTM1 to HTM2, HTM-alpha, and the commonly used culture solution Earls BSS (EBSS - Earle's Balanced Salt Solution; ThermoFisher Scientific) are summarized in Table 3 below. Note that the concentrations of these ions in StemFit (registered trademark) AK02N and MEM-alpha are the same as those in Earls BSS. Referring to the experimental results described later, Na + / K + It was thought that the molar ratio of should be around 1 or a little smaller than this.

[0054] [Table 3] Na in commercially available media and the media of the examples + / K + Molar ratio of JPEG0007636797000003.jpg2682

[0055] As mentioned above, proteins, peptides, and growth factors are contained in StemFit® AK02N but not in MEM-alpha, and therefore proteins, peptides, and growth factors are contained in HTM1 but not in HTM-alpha.

[0056] 3. Refrigerated storage in single cell state The human iPS cells obtained in "1." above were placed in the refrigerated storage solutions HTM1 and HTM2 in "2." above, and incubated at 1E6cells / mL (1 x 10 suspended cells). 6 The cells were suspended in a single cell state to a concentration of 0.1 cells / mL, placed in a plastic tube, and stored at 4° C. The viability was determined by counting the number of live cells after trypan blue staining.

[0057] The results of measuring the survival rate over time are shown in Figures 1A and 2A. The number of days at which 50% survival rate was maintained (ST50) was determined from the graph in Figure 1A and is shown in Figure 1B. The number of days at which 80% survival rate was maintained (ST80) was determined from the graph in Figure 2A and is shown in Figure 2B.

[0058] Statistical analysis in the present examples was performed as follows. The survival rate curves in Figures 1A and 2B were obtained by fitting a four-variable logistic curve using R version 3.5.1. One-way ANOVA, Bonferoni test, and Dunnetts test were calculated using Graphpad Prism 5.04.

[0059] As shown in FIG. 1A and FIG. 2A, the use of HTM1 (UW solution + StemFit (registered trademark), 2 / 1) and HTM2 (UW solution + ES cell culture medium (ESC medium), 2 / 1), which are refrigerated storage solutions according to the present invention, showed a higher viability maintenance effect than the use of UW solution. As shown in FIG. 1A and FIG. 1B, when the cells were preserved using HTM2, the viability maintenance period was extended to about twice that of the UW solution. When the cells were preserved using HTM1 and HTM2, the number of days for which 50% viability was maintained (ST50) was almost the same, and both values ​​were significantly higher than those of the UW solution. As shown in FIG. 2A and FIG. 2B, when the number of days for which 80% viability was maintained (Survival time 80: ST80) was compared, the number of days for which 80% viability was maintained was 4.6 days when HTM1 was used, which was 2.2 times higher than the 2.1 days when the UW solution was used.

[0060] Since the StemFit (registered trademark) medium used in HTM1 does not contain animal-derived components (Xenofree), the following experiments were conducted using HTM1. When ES cell culture medium (ESC medium) was used as is as a medium for refrigerated storage, the viability maintenance effect was significantly lower than that of UW medium. ES cell culture medium (ESC medium) was also used in the following experiments, but it is only shown in the graph and explanations and references will be omitted.

[0061] 4. Maintenance of colony formation ability The cell proliferation ability after refrigerated storage was verified as follows. First, the human iPS cells obtained in "1." above were placed in the refrigerated storage solution HTM1 in "2." above and refrigerated for 6 days as in "3." above. Next, they were suspended in StemFit (registered trademark) AK02N medium containing 10 μM Y27362, and iMatrix-511 was added at 0.1 μg / cm 2 Add it so that it becomes 1.3E3 / cm 2 The cells were seeded at a concentration of 1000 x 1000. On the fifth day of culture, the cells were stained with fixed alkaline phosphatase, and the number of colonies that had formed to a certain size or larger was counted using a colony counter. The number of colonies obtained after five days of culture without refrigeration was similarly counted and used as a control. The number of colonies obtained after five days of culture after refrigeration was then divided by the number of colonies obtained in the control to obtain the relative colony efficiency.

[0062] As shown in Figure 2C, when HTM1 was used, the value was significantly higher, 6.7 times higher than when UW solution was used. From the above, it was found that the novel cell cryopreservation solution HTM1 has a high effect of maintaining the viability of human iPS cells during cryopreservation and the ability to proliferate after reculture. In addition, no colony formation was observed when StemFit (registered trademark) AK02N was used.

[0063] 5. Detection and Measurement of Apoptotic Cells The human iPS cells obtained in "1." above were placed in the refrigerated storage solution HTM1 described in "2." above and stored for 3 days as described in "3." above, and the cell death that occurred immediately after this was classified using Annexin V / EthD-III. That is, to distinguish between apoptotic and necrotic cells, first, they were stained using the Apoptotic / Necrotic Cells Detection Kit (TAKARA) according to the protocol. Then, Annexin V-FITC positive cells (apoptosis), ethidium homodimer III positive cells (necrosis), and Hoechst33342 (Dojin) signals (total cells) were observed and counted under a fluorescent microscope to determine the cells.

[0064] The results are shown in the bar graphs of Figures 3A and 3B. As shown in Figure 3A, it was found that the use of HTM1 significantly suppressed the induction of apoptosis compared to the use of UW solution. This inhibitory effect on apoptosis immediately after storage is thought to be the reason why HTM1 has a higher proliferation ability (colony formation ability) than UW solution.

[0065] 6. Intracellular ROS measurement Although maintaining viability during cryopreservation is important, suppressing cell death during reculture after cryopreservation is even more important. To clarify the cause of cell death that occurs after cryopreservation and reculture, intracellular reactive oxygen species (ROS) were measured using aminophenyl fluorescein (APF). In detail, the measurement was performed as follows. First, aminophenyl fluorescein (APF) (Goryo Chemical) was added to the medium after refrigerated storage for 3 days in the same manner as in "5." above to a final concentration of 5 μM, and this was introduced for 30 minutes in a 37°C incubator (5% CO2, 5% O2). After this, the medium was replaced and counterstained by adding 10 μM Hoechst33342 (Dojin), and then the cells were observed under a fluorescent microscope and photographed. The fluorescent signal of each cell was quantified using the image analysis software ImageJ.

[0066] The results are shown in Figure 4A. It was found that the intracellular ROS concentration rose sharply within 90 min after the start of re-culture, and then tended to gradually decrease.

[0067] 7. Measurement of Intracellular Caspase Activity The activity of intracellular caspase 3 / 7, which is an indicator of apoptosis, was measured for the cells after refrigerated storage for three days in the same manner as in "5." above. TMCells were stained using Caspase-3 / 7 Green Detection Reagent (ThermoFisher) according to the protocol, and the fluorescent signal of each cell was quantified using the image analysis software ImageJ.

[0068] The results are shown in Figure 4B. Caspase-3 / 7 activity, an indicator of apoptosis, was found to gradually increase from 90 minutes after cryopreservation. This result indicates that apoptosis is induced by a transient increase in intracellular reactive oxygen species (ROS) at the start of cryopreservation and reculture.

[0069] 8. Testing the addition of antioxidants or radical scavengers Antioxidants or radical scavengers were added to HTM1 at various concentrations and subjected to a refrigerated storage test as described in "3." above. The antioxidants or radical scavengers added are as shown in FIG. 5A and are as follows: Trolox (Trx, Wako), EGCG (Teavigo (registered trademark), DSM), N-acetylcysteine ​​(NAC, Tokyo Chemical Industry), L-ascorbic acid (AA, WAKO), and Edaravone (3-Methyl-1-phenyl-2-pyrazoline-5-one, Edaravone: Sigma).

[0070] Figure 5B shows the results of the comparison of the number of days for which 80% viability was maintained, as in Figures 2A-2B. As shown in Figure 5B, the most effective treatment was the addition of Trolox (5 mM), a water-soluble vitamin E analogue, which extended the viability to 9.5 times that of UW solution and 4.4 times that of HTM1 alone, and maintained a viability of 80% or more for about 20 days (ST80: 20.11 days). Next to Trolox, Edaravone, known as a strong radical inhibitor, was highly effective, followed by EGCG and N-acetyl cysteine ​​(NAC). L-ascorbic acid (AA), which was expected to work in concert with Trolox, was not very effective. Based on the above results, subsequent experiments were carried out with the addition of Trolox, which is versatile and available at a low price.

[0071] 9. Trolox Addition Concentration The effect of Trolox, which had the highest viability-maintaining effect, was further examined by varying the concentration of Trolox from 0.1 mM to 0.5 mM. Figure 6A shows the time course of the viability, obtained in the same manner as in Figure 2A above, superimposed on Figure 2A. Figure 6B shows the results of the number of days at which 80% viability was maintained, as in Figures 2B and 5B above. As shown in Figure 6B, it was found that the number of days at which 80% viability was maintained increased almost with increasing concentration of Trolox. The number of days at which 80% viability was maintained was significantly higher with the addition of 0.5 mM or more, compared to unadded HTM1.

[0072] Next, to verify the proliferation ability after refrigerated storage and re-culture, cells that had been refrigerated for 6 days were seeded in the same manner as in "4." above, and the colony formation ability on the 5th day of culture was compared. As shown in Figure 6C, the colony formation ability evaluated in the same manner as in "4." above was also found to increase depending on the Trolox concentration. The colony formation ability of the most effective group, Trolox 5.0 mM, reached 2.3 times that of HTM1 alone and 41.2% of the unrefrigerated control. This value was almost the same as that of slow-frozen storage in a -80°C freezer according to the standard method. This indicates that the addition of 5 mM Trolox to HTM1 can maintain proliferation ability equivalent to or greater than that of frozen storage for up to 6 days.

[0073] 9A. Comparison with the addition of vitamin E and vitamin E derivatives Using HTM-alpha (UW solution + MEM-alpha, 2:1) described in "2." above, before adding Trolox and GlutaMAX (registered trademark, GIBCO), the effect of adding tocopherol (vitamin E) and tocopherol acetate (vitamin E derivative) during refrigerated storage was examined using the same procedure as in Figures 6A and 2A. Tocopherol and tocopherol acetate were added to 0.04 mM to 5.0 mM, the same as Trolox, and refrigerated storage was performed. At this time, a sample to which 2.0 mM Trolox, a water-soluble vitamin E analog, was added, was also tested at the same time. Figure 6D shows the relative survival rates after 6 days of storage and 1 day of recovery culture. The results showed that tocopherol (vitamin E) and tocopherol acetate were less effective during refrigerated storage compared to Trolox.

[0074] 10. Refrigerated storage in adhesive state (1) Human iPS cells obtained in the same manner as in "1." above were used. However, at the final stage of the culture, the concentration was slightly lower (1.0E3 / cm) than in the maintenance culture in "1." above. 2) and cultured. The day after seeding, the medium was replaced with StemFit (registered trademark) AK02N medium not containing Y27362, and then the medium was removed 4 days after seeding.

[0075] The medium was then replaced with HTM1, a medium for refrigerated storage, and stored stationary at 4°C for 3 or 6 days. That is, the cells were stored in a refrigerated state for 3 or 6 days while they were aggregated and adhered to each other and to the culture vessel. Trolox was added to this HTM1 medium for refrigerated storage. Adenine hydrochloride (Tokyo Kasei) was added to the medium at concentrations of 0 mM, 1.4 mM, and 4.1 mM, respectively. UW solution (BELZER UW (registered trademark) COLD STORAGE SOLUTION) was used as a comparative refrigerated storage medium.

[0076] After the cells were stored in a refrigerator, the refrigerated storage medium was removed, and the cells were gently washed with culture medium. Then, StemFit® AK02N medium was added, and the cells were cultured for 4 days. At the end of the culture, the cells were enzymatically detached (using 0.5X TrypLE® Select), dispersed, and the number of live cells was counted. The number of cells was then compared with the number at the start of the refrigerated storage. In addition, phase contrast images were taken at the end and start of the culture, and the images were processed with ImageJ to extract and digitize the colony area, and the colony area was measured to evaluate the proliferation ability.

[0077] The above experiment aimed to develop a method for refrigerated preservation of adherent iPS cells, and evaluated their proliferation ability by culturing for four days after refrigerated preservation for three or six days. The results of the experiment are shown in Figures 7A to 7F and Figure 8. Figures 7A to 7B show images of colonies obtained by re-culturing for four days after three days of refrigerated preservation, and Figures 7C to 7D show images of colonies obtained by re-culturing for four days after six days of refrigerated preservation. Here, Figures 7A and 7C are phase contrast images, and Figures 7B and 7D are the binarized versions of these, respectively.

[0078] The bar graph in Fig. 7E shows the colony area ratios in media recultured for 4 days after refrigerated storage for 3 and 6 days. The bar graphs in Fig. 7F and Fig. 8 show the increase rate of colony counts when recultured for 4 days after refrigerated storage for 3 and 6 days, expressed as a ratio of 1 for the non-preserved control, which was cultured for 4 days without refrigeration. The bar graph in Fig. 8 also shows the experimental results for 6 days of refrigerated storage using HTM1 as is (without addition of Trolox, etc.) and refrigerated media with Trolox added to HTM1 at 8.2 mM.

[0079] As shown in the upper left part of each of Figures 7A to 7D, when cells were refrigerated in UW solution, almost no proliferation was observed when they were recultured after refrigerated storage for 3 or 6 days. When cells were refrigerated for 3 days using refrigerated storage medium containing HTM1 and Trolox 5 mM, considerable proliferation was observed as shown in the upper right part of each of Figures 7A to 7B, and the cell proliferation rate when recultured after refrigerated storage for 3 days was almost the same as that of the non-preserved control as shown in the left part of Figure 7F. On the other hand, when cells were refrigerated for 6 days using refrigerated storage medium containing Trolox 5 mM in HTM1, some proliferation was observed as shown in the upper right part of each of Figures 7C to 7D, and the cell proliferation rate when recultured after refrigerated storage for 6 days was only about 10% of that of the non-preserved control as shown in the right part of Figure 7F.

[0080] On the other hand, as shown in the lower left and lower right parts of Figures 7A to 7D, the proliferation rate could be increased by adding adenine to HTM1 in addition to Trolox 5 mM. In particular, as shown in Figure 8, when cells in an adherent state were refrigerated for 6 days, it was found that the proliferation rate at the time of re-culture could be significantly increased by adding adenine at 2.8 mM and 4.2 mM in addition to Trolox 5 mM. It was found that the proliferation rate increased with increasing concentration up to 4.2 mM by the addition of adenine, but on the contrary, it decreased when 8.2 mM was added.

[0081] 11. Refrigerated storage in adhesive state (2) Using HTM-alpha (UW solution + MEM-alpha, 2:1) described in "2." above, refrigerated storage in an adherent state was evaluated by the same procedure as in "10." above. However, the cells were re-cultured after refrigerated storage for 7 days instead of 6 days, and then evaluated. Here, as described above, 5 mM Trolox and 5-fold concentration (x5; 10 mM) GlutaMAX (registered trademark, GIBCO) were added to HTM-alpha. In addition, HTM1 and HTS FRS were used at the same time under the same conditions for comparison. Note that, like HTM-alpha, 5 mM Trolox and 5-fold concentration (x5; 10 mM) GlutaMAX (registered trademark, GIBCO) were added to HTM1.

[0082] The results are shown in the graph in Figure 9. In the bar graph in Figure 9, as in Figures 7E and 8, the non-preserved control cultured for 4 days without refrigeration is shown as a ratio of 1. As shown in the graph in Figure 9, when HTM-alpha (middle) was used as the refrigerated preservation medium, a significantly higher proliferation rate was observed compared to when HTM1 was used. In addition, a much higher proliferation rate was observed compared to when HTS FRS, a commercially available refrigerated preservation solution, was used.

[0083] From these results, it is believed that the fibroblast growth factor (FGF) contained in Solution C of StemFit® AK02N is unnecessary or does not contribute much. In addition, among the components contained in StemFit® AK02N, those not contained in HTM-alpha are not considered essential. In particular, since HTM-alpha is a protein-free medium, it is believed that proteins are not essential or are better not to be present.

[0084] 12. Refrigerated Storage of Mouse Embryos As described above, blastocysts 3.5 days after in vitro fertilization were immediately used for refrigerated storage experiments. Specifically, the medium of these blastocysts was replaced with HTM-alpha, HTM1, and HTS FRS used in "11." above, and UW solution, then the tubes were placed in plastic tubes and stored at 4°C for 3 days (72 hours). Note that both HTM-alpha and HTM1 were supplemented with 5 mM Trolox and 5 times the concentration (x5; 10 mM) of GlutaMAX (registered trademark), as in "11." above.

[0085] The percentage of embryos that escaped the zona pellucida during the two-day culture period after further culture for two days is shown in Figure 10. Figure 10 also shows the results of a non-preserved control (F_Control) that was similarly preserved in a refrigerator and cultured.

[0086] As shown in FIG. 10, both HTM1 and HTM-alpha showed escape rates equivalent to that of the unpreserved control (F_Control), whereas both UW solution and HTS FRS showed significantly lower escape rates.

[0087] Next, the effect of adding adenine hydrochloride (Tokyo Kasei) was examined, as in Figures 7E to 7F. That is, under the same conditions as in the experiment in Figure 10, the rate of embryos that escaped from the zona pellucida was calculated when adenine hydrochloride (Tokyo Kasei) was added to each concentration. Figure 11 shows, from the left, the results of an unpreserved control (F_Control) and when adenine hydrochloride was added to each concentration of 1.4 mM (1400 μM), 140 μM, 14 μM, and 0 μM. As can be seen from Figure 11, in the case of low-temperature storage for 5 and 7 days, a relatively high rate of embryos that escaped from the zona pellucida was shown when the adenine concentration was 14 μM to 140 μM (0.014 to 0.14 mM).

[0088] 13.Effect of adding polyvinyl alcohol (PVA) As shown in Table 1 above, UW solution contains 5.0% hydroxyethyl starch (HES), so HTM-alpha (UW solution + MEM-alpha, 2:1) described in "2." above contains 3.3% hydroxyethyl starch (HES). Here, the effect of adding polyvinyl alcohol (PVA) instead of hydroxyethyl starch (HES) was examined. Here, a refrigerated storage solution was used that was the same as HTM-alpha (UW solution + MEM-alpha, 2:1) except for hydroxyethyl starch (HES) from the composition of UW solution in Table 1 above, and contained polyvinyl alcohol (PVA) in the range of 0.25% to 2.5% (by weight). Then, cell viability was evaluated in the same manner as in Figure 6D above. That is, the protective effect of polyvinyl alcohol (PVA) was examined by the same procedure as in Figure 6A and Figure 2A. At the same time, a test was also conducted on a sample containing 5% (by weight) hydroxyethyl starch (HES). Figure 12A shows the relative survival rates after 6 days of storage and 1 day of recovery culture. These results show that polyvinyl alcohol (PVA) exhibits a protective effect equivalent to that of 5% hydroxyethyl starch (HES) in all concentration ranges. Polyvinyl alcohol (PVA) is cheaper than hydroxyethyl starch (HES) and is also considered to be safer for biological tissues.

[0089] 14. Effect of adding mannitol Next, in the same manner as in "13." above, mannitol was added together with polyvinyl alcohol (PVA) to examine their synergistic effect. That is, polyvinyl alcohol (PVA) was added in the range of 0.5% to 5% to HTM-alpha (UW solution + MEM-alpha, 2:1) without hydroxyethyl starch (HES), and mannitol, which is thought to have a cell membrane-impermeable osmotic pressure adjusting effect, was added at 0, 30, 60, and 90 mM. In addition, the protective effect was examined by the same procedure as in Figure 6A and Figure 2A. Figure 12B shows the relative survival rate after 6 days of storage and 1 day of recovery culture, as in Figure 12A. The results in Figure 12B show that the addition of mannitol in the range of 30 mM to 90 mM together with 0.5% polyvinyl alcohol (PVA) increased cell viability after refrigerated storage compared to the addition of hydroxyethyl starch (HES) at a concentration of 5%.

[0090] 15. Summary of experimental results When using the cell cryopreservation medium HTM1 of the present embodiment, which is a 2 / 1 mixture of UW solution and the commercially available clinical research medium StemFit (registered trademark) AK02N, it became possible to extend the viability maintenance time of human iPS cells in cryopreservation at 4°C by about two times compared to UW solution, which is widely used as a refrigerated tissue preservation solution, as shown in Figures 1B and 2B. In addition, as shown in Figure 6C, the cell proliferation ability after refrigerated storage for six days and reculture was increased to about seven times that of UW solution.

[0091] In addition, it is believed that the addition of antioxidants and radical scavengers to HTM1 makes it possible to suppress the rapid rise in ROS concentration that occurs at the start of reculture after refrigerated storage, and with the addition of Trolox 5mM, the period during which 80% viability was maintained was 20 days or more, approximately four times longer than that of HTM1 without Trolox, as shown in Figure 6A and 6B. In addition, as shown in Figure 6C, it was confirmed that the colony formation rate after low-temperature storage and reculture was approximately twice that of the control group.

[0092] On the other hand, when cells were stored at low temperatures while remaining in an attached state, as shown in Figs. 7A to 7F and Fig. 8, it was revealed that the addition of Adenine in addition to Trolox increased the subsequent proliferation rate.

[0093] On the other hand, when a particularly preferred embodiment of the cell cryopreservation medium "HTM-alpha" (UW solution + MEM-alpha, 2 / 1), obtained by mixing UW solution and MEM alpha, was used, it was considered to be more effective in maintaining cell proliferation ability than HTM1 (UW solution + StemFit (registered trademark) AK02, 2 / 1), as shown in Figure 9.

[0094] Although the storage period of iPS cell refrigeration is limited compared to freezing, it has a wide range of applications, including preservation in a cultured state (adherent), preservation and transportation of differentiated organoids that cannot be frozen, and on-demand cell supply in drug discovery research. Therefore, this technology, which has made it possible to preserve human iPS cells, is extremely important.

[0095] Since the low-temperature cell preservation medium "HTM-alpha" of the present embodiment was effective for the refrigerated preservation of mouse embryos, it is believed that it will also be effective for the refrigerated preservation of embryonic and non-embryonic stem cells and tissues containing these from humans and various animals.

Claims

1. The potassium ion content is 30-80mmol / L, the sodium ion content is 30-80mmol / L, and the amount of sodium ion is determined by the ion-based molar ratio to potassium ion (Na + / K + The ratio of Trolox at a concentration of 0.5 to 8 mM, adenine or a salt thereof at a concentration of 0.1 to 4.2 mM; Contains all essential amino acids or all but one, two or three essential amino acids, and the total content of essential amino acids is 50-200 mg / L; A refrigerated storage solution for storing human or animal stem cells or embryos in an unfrozen state, characterized in that the total content of non-essential amino acids is 50 to 200 mg / L.

2. (i) 30 to 100 mmol / L of lactobionic acid or a salt thereof, calculated as lactone; (ii) 10 to 30 mmol / L of raffinose hydrate; (iii) 0.3 to 1 mmol / L of allopurinol; (iv) 1 to 3 mmol / L of glutathione (total glutathione); (v) 2 to 10 mmol / L of adenosine; (vi) 0.1 to 0.5 μmol / L of lipoic acid; (vii) 0.1 to 0.7 mmol / L of sodium pyruvate; (viii) 1 to 5 mmol / L of glucose; (ix) 0.03 to 0.3 mmol / L of ascorbic acid and 0.5 to 8 mM of vitamin E; (x) 0.1 to 4.2 mM of adenine or a salt thereof; (xi) A refrigerated storage solution for storing human or animal stem cells or embryos in an unfrozen state, characterized in that it is a physiological buffer solution containing other vitamins (including folic acid, nicotinamide (niacinamide), riboflavin, B12, choline, inositol, pantothenic acid, pyridoxal phosphate, and thiamine), (xii) a total of 50 to 200 mg / L of essential amino acids, (xiii) a total of 100 to 500 mg / L of non-essential amino acids, (xiv) 30 to 80 mmol / L of potassium ions, and (xv) 20 to 90 mmol / L of sodium ions.

3. 3. A refrigerated storage solution according to claim 1 or 2, characterized in that it contains 20 to 50 g / L of hydroxyethyl starch.

4. 4. The refrigerated preservation solution according to claim 1, which contains L-alanyl-L-glutamine or a salt thereof at a concentration of 2 to 5 mM.

5. A refrigerated storage method comprising suspending or immersing human or animal iPS cells or other artificial stem cells, other stem cells, organoids obtained therefrom, or embryos in the refrigerated storage solution according to any one of claims 1 to 4, and storing the cells at 2 to 8°C for 1 to 10 days.

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