Refrigerated preservation solution for stem cells

Abstract

Provided are a cryopreservation solution and a cryopreservation method suitable for cryopreservation of stem cells or embryos such as human or animal iPS cells in a non-frozen state. A preservation solution for refrigeration according to one embodiment of the present invention is obtained by modifying MEM-alpha (Minimum Essential Medium Eagle Medium Alpha) and a UW solution (UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln, UWsln; a liquid (HTM (-)) obtained by mixing BELZER UW (Registered Trademark) COLD STORAGE SOLUTION) or a modified UW liquid (a polymer component changed to PVA) at a ratio of about 1 / 1 to 1 / 2, or a refrigerated preservation liquid having a composition equivalent to that of the liquid, particularly having a potassium ion and sodium ion concentration, is used as a basis. In addition, it is particularly preferable to add 10 to 300 [mu] M of at least one first additive selected from the group consisting of deferoxamine (DFO: Deferoxamine), deferasirox (DFX: Deferasirox), deferiprone (DFP: Deferaprone), and ferristatin-1 (FST: Ferrostatin-1), 0.5 to 8 mM of a second additive that is water-soluble vitamin E (Trolox), vitamin E, or a water-soluble analogue / derivative thereof, and 10 to 300 [mu] M of sorbitol.

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, it relates to a refrigerated preservation solution for preservation at a temperature lower than normal temperature, such as 2 to 15 °C, 2 to 10 °C, 2 to 8 °C, or 2 to 6 °C. In addition, it particularly relates to a refrigerated preservation solution for floating or immersing pluripotent cells (such as human iPS cells) and other stem cells, or derivatives such as organoids obtained therefrom, and initial embryos. Or it relates to a cryopreservative for preventing or improving organ failure for organ transplantation, etc. Background Art

[0002] Simply, efficiently, and stably preserving and supplying stem cells such as iPS and ES cells is an essential and important technology in the basic research, clinical application research, or pharmaceutical research fields of regenerative medicine. Human iPS cells and ES cells need to be frozen for long-term preservation, but refrigerated preservation (non-frozen cryopreservation) is a very attractive technology as a short-term (e.g., 1 day to 1 week) to medium-term (e.g., 1 week to 1 month) preservation method. In addition, considering the industrialization of regenerative medicine technologies such as the preservation of cells induced to differentiate, organoids, and cells difficult to freeze, and their applications in pharmaceuticals, its advantages are great. Also, there are many advantages in terms of clinical utilization such as being able to be transplanted without going through processes such as thawing after preservation. However, it is difficult to cryopreserve 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 cryopreservation solution for organs, and new refrigerated preservation technologies need to be developed.

[0003] In the refrigerated preservation of organs for transplantation such as the liver and kidney, UW solution is widely used and has become a de facto gold standard (optimal benchmark). The UW solution developed in the 1980s has a relatively simple composition and contains lactobionate, raffinose, glutathion, hydroxy-ethyl starch, etc. in a solution with an intracellular-type salt composition of low Na ions and high K ions.

[0004] However, in the case of cryopreserving cells, etc., UW solution cannot sufficiently suppress cryopreservation stress. When solving this problem, the present inventors proposed such a hypothesis that the salt composition, which is intermediate between the intracellular type represented by UW solution and the extracellular type of high Na ion and low K ion with a normal culture medium, is important. In addition, compared with organ preservation, the addition of high-molecular substances such as proteins is considered important, and a preservation solution intermediate between UW solution and cell culture solution was developed.

[0005] There are few reports on the culture medium for cryopreserving cells. There are a few reports on preservation based on HTSFRS (Hypothermosol (registered trademark) FRS; BioLife Solutions) developed in recent years. There is a report that human skin fibroblasts and smooth muscle cells can be cryopreserved for 3 to 5 days with this HTS FRS, but there is no report on the cryopreservation of pluripotent cells such as human iPS cells.

[0006] In the attempt of various combinations, the present inventors tried using a liquid prepared by mixing MEM-alpha (Minimum Essential Medium Eagle (MEM) Alpha Modification) and the above UW solution at a ratio of about 1 / 1 to 1 / 2, or a preservation solution equivalent thereto (referred to as "HTM solution") (Patent Document 1). Here, water-soluble vitamin E (Trolox) was added. MEM-alpha is based on MEM medium and contains non-essential amino acids, sodium pyruvate, lipoic acid, biotin, and vitamin B12. Therefore, both the Na ion and low K ion concentrations are intermediate, and various vitamins, water-soluble vitamin E, various amino acids, and protective polymers are contained at appropriate concentrations. It is known that by using this improved cryopreservation solution, human iPS cells, etc. can be cryopreserved for several days or longer and the survival rate can be maintained to a certain extent.

[0007] Prior Art Documents

[0008] Patent Documents

[0009] Patent Document 1: WO2021 / 090869A (PCT / JP2020 / 041313)

[0010] Non-Patent Documents

[0011] Non-Patent Literature 1: COMPRE BELZER UWTM COLD STORAGE SOLUTION TO VIASPANhttps: / / bridgetolife.com / compare-belzer-uw-cold-storage-solution-to-viaspan / Summary of the Invention

[0012] Problems to be Solved by the Invention

[0013] However, in the case of the cold storage solution (“HTM solution”) of Patent Literature 1, when cells are stored in a state adhered to the culture vessel, almost no cell death is observed during 3 days of storage, showing high viability. However, the value remains at about 60% of the control during 6 days of cold storage. This time, by clarifying how the stress accumulated during cell storage causes apoptosis during re-culture after storage, an attempt was made to develop a higher-performance cold storage solution by screening new cryoprotectants.

[0014] <Exploration of Cryopreservation Protective Substances Targeting Ferroptosis>

[0015] Ferroptosis is one of the newly identified iron-dependent regulated cell deaths proposed in 2012 and was recognized as one of the programmed cell deaths by the Nomenclature Committee on Cell Death (NCCD) in 2018. The core role in cell death is due to the accumulation of lipid peroxides in the cell membrane depending on iron ions. Lipid peroxides are further converted into lipid free radicals, leading to cell death due to cell membrane rupture. As the main protective pathway to protect cells from ferroptosis, it is known that glutathione-dependent glutathione peroxidase 4 (GPX4) converts lipid peroxides into non-toxic lipid alcohols. Furthermore, it has been reported that a strong lipophilic antioxidant is generated by the enzymatic reduction of non-mitochondrial coenzyme Q10 by the oxidoreductase FSP1, thereby inhibiting the increase in lipid peroxides. In addition, Vitamin E is also known to inhibit lipid oxidation.

[0016] In the case of cold storage preservation, it was found that even if almost no cell death was observed shortly after preservation, apoptosis was induced during re-culture. As the preservation period was extended, the apoptosis induction rate increased. Therefore, it was considered that some kind of cell death stress accumulated during cold storage preservation and induced apoptosis during re-culture. When using an inhibitor that specifically inhibits possible cell death, it was found that the apoptosis inhibitor effectively inhibited apoptosis induction during re-culture. In summary, the following hypothesis was proposed: the stress that induces ferroptotic cell death during cold storage preservation induces apoptosis during re-culture. Based on this idea, it was considered that by blocking the pathway of ferroptosis occurrence or enhancing the ferroptosis protection pathway, apoptosis during cold storage preservation - re-culture could be inhibited. Agents considered to be related to the ferroptosis pathway (Table 1 described later) were selected to verify their cold storage protection effects.

[0017] Means for solving the problem

[0018] The present inventors replaced water-soluble vitamin E (Trolox) or a compound type similar thereto, which is considered to have excellent free radical scavenging effects, with "HTM solution", and used specific compound types such as iron ion chelating agents that are expected to have an anti-ferroptosis effect. Thus, with a smaller addition amount than in the case of water-soluble vitamin E, at least the same cold storage preservation effect as in the case of adding water-soluble vitamin E was obtained.

[0019] According to a preferred embodiment of the present invention, the following cryopreservation solutions (i) to (iii) or (i) to (v) can be used.

[0020] (i) A mixed solution prepared by mixing MEM-alpha and UW solution or modified UW solution (changing the polymer component to PVA) at a ratio of about 1 / 1 to 1 / 2, or a preservation solution equivalent thereto, is used as the basic cryopreservation solution. In particular, with respect to the concentrations of potassium ions and sodium ions and their mutual ratios, a preservation solution equivalent to this mixed solution is used.

[0021] (ii) As an additive having a free radical scavenging effect added to the basic cryopreservation solution, at least one selected from the following additive compound group is used as the first additive.

[0022] Additive compound group: Edaravone (EDV), Probucol (PBC), Deferoxamine (DFO), Deferasirox (DFX), Deferiprone (DFP), Ebselen (EBS), Idebenone (IDB), Necrostatin (NST), and Ferrostatin-1 (FST).

[0023] (iii) The concentration of each or the total concentration of the first additive is lower than 2 mM, which is generally required in the case of water-soluble vitamin E or its analogs. That is, it is 1 mM or less, 0.5 mM (500 μM) or less, 0.3 mM or less, 0.1 mM (100 μM) or less, 0.03 mM (30 μM) or less, 0.01 mM (1 μM) or less, or 0.03 μM or less. Additionally, it is 0.01 μM or more, 0.03 μM or more, 0.01 mM (10 μM) or more, or 0.03 mM (30 μM) or more.

[0024] (iv) It is preferable to use water-soluble vitamin E (Trolox) or a compound type similar thereto (especially vitamin E or its water-soluble analogs / derivatives) as the second additive. The concentration of the second additive can be 0.5 mM or more, 1 mM or more, 1.5 mM or more, or 2 mM or more, especially 10 mM or less, 8 mM or less, 7 mM or less, 5 mM or less, 4 mM or less, or 3 mM or less.

[0025] (v) It is particularly preferable to use sorbitol or its equivalent as the third additive. The concentration of the third additive can be 10 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, 50 mM or more, 60 mM or more, or 70 mM or more, especially 300 mM or less, 250 mM or less, 200 mM or less, 180 mM or less, 150 mM or less, or 120 mM or less.

[0026] In a particularly preferred embodiment, the basic cold storage solution is contained without dilution or diluted to 0.35 - 0.95 times, 0.4 - 0.9 times, or 0.5 - 0.8 times. Moreover, the content of sorbitol is within the range of the basic concentration multiplied by a specified magnification factor X2. The specified magnification factor X2 is 1.2 when the dilution rate X1 is 0.8 times, 1.5 when the dilution rate X1 is 0.75 times, 2.0 when the dilution rate X1 is 0.6 times, 3.0 when the dilution rate X1 is 0.5 times, and for other dilution rates, it can be determined by interpolation or extrapolation.

[0027] The basic concentration can be 20 - 100 mM, 30 - 80 mM, 40 - 60 mM, or 45 - 65 mM.

[0028] In a preferred embodiment, the basic cold storage solution can have substantially the same concentrations of potassium ions and sodium ions as those shown in Table 4 described later. Specifically, it is a liquid obtained by mixing the UW solution shown in Table 2 described later and the MEM alpha medium shown in Table 3 described later at a ratio of about 1 / 1 - 1 / 2, or a storage solution equivalent thereto with vitamin E omitted ("HTMX(-)").

[0029] In addition, according to the circumstances, sorbitol can be replaced or sugar alcohols can be used in place of sorbitol. As such sugar alcohols, sugar alcohols appropriately selected from maltitol, lactitol, erythritol, galactitol, mannitol, xylitol, etc. can be used.

[0030] In a preferred embodiment, the preferred concentration of each additive compound is as follows, for example.

[0031] Edaravone (EDV): 10 - 100 μM, or 3 - 300 μM

[0032] Probucol (PBC): 100 - 1000 μM, or 30 - 2000 μM

[0033] Deferoxamine (DFO): 10 - 100 μM, or 3 - 300 μM

[0034] Deferiprone (DFP): 100 - 1000 μM, or 50 - 2000 μM

[0035] Deferasirox (DFX): 10 - 100 μM, or 3 - 300 μM

[0036] Ebselen (EBS): 0.1 - 3 μM, or 0.03 - 10 μM

[0037] Idebenone (IDB): 0.3 - 3 μM, or 0.1 - 10 μM

[0038] Necrostatin (NST): 10 - 100 μM, or 3 - 300 μM

[0039] Ferrostatin-1 (FST): 0.3 - 3 μM, or 0.1 - 10 μM

[0040] MEM medium such as MEM-alpha (Eagle’s minimal essential medium) contains the following components (1) - (4).

[0041] (1) Amino acids (L-arginine, L-cysteine, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, L-threonine, L-tryptophan, L-tyrosine, L-valine),

[0042] (2) Salts (calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate),

[0043] (3) D-glucose, and

[0044] (4) Vitamins (folic acid, nicotinamide, riboflavin, B12, choline, inositol, pantothenic acid, pyridine sulfonic acid, thiamine).

[0045] On the other hand, UW fluid is an intracellular fluid composed of Ca 2+ It does not contain liquid, but is formulated with lactobionic acid or hydroxyethyl starch (HES) for regulating osmotic pressure and inhibiting cell swelling, adenosine as an anti-inflammatory, vascular relaxant and ATP precursor, glutathione and aromatic alcohol for anti-oxidation, and phosphate buffer as a buffer component.

[0046] A cold storage solution or a basic cold storage solution according to a preferred embodiment of the present invention has the following features a1 to a6 or a1 to a9.

[0047] a1. The potassium ion content may be 20-100mmoL / L, 20-90mmoL / L, 30-80mmoL / L or 30-100mmoL / L, and the sodium ion content may be 20-100mmoL / L, 20-90mmoL / L or 30-80mmoL / L.

[0048] a2. The amount of sodium ion species relative to potassium ion species based on the molar ratio of ions (Na + / K + The ratio of can be 0.5 to 1.5, 0.5 to 1.3, or 0.6 to 1.2.

[0049] a3. Contains adenine or a salt or derivative thereof 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.

[0050] a4. Regarding essential amino acids, all or all except one, two or three may be contained. The total content of essential amino acids may preferably be 50 to 250 mg / L, particularly preferably 50 to 200 mg / L or 80 to 150 mg / L.

[0051] a5. The total content of non-essential amino acids may preferably be 100 to 500 mg / L, particularly 50 to 200 mg / L or 80 to 150 mg / L.

[0052] The content of magnesium ion species (especially magnesium sulfate) can be 2 - 8 mmol / L, 2 - 5 mmol / L or 2 - 4 mmol / L, and the content of calcium can be 0.2 - 1.5 mmol / L, 0.2 - 1 mmol / L or 0.3 - 0.8 mmol / L.

[0053] a7. It contains glucose or other low - molecular polysaccharides (especially disaccharides such as maltose and monosaccharides such as fructose) at a concentration of 0.5 - 10 mmol / L.

[0054] a8. It contains the following vitamins. However, 1, 2, 3 or 4 of the following can be omitted.

[0055] Folic acid, nicotinamide, riboflavin, B12, choline, inositol, pantothenic acid, pyridoxal phosphate, and thiamine.

[0056] The preservation solution for refrigeration according to a preferred embodiment of the present invention contains: (i) lactobionic acid or lactobionate in terms of lactone conversion 30 - 100 mmol / L (or 10 - 40 g / L), (ii) crude saccharide hydrate 10 - 30 mmol / L (or 5 - 20 g / L), (iii) aromatic alcohol 0.3 - 1 mmol / L (or 0.05 - 0.1 g / L), (iv) glutathione (total glutathione) 1 - 3 mmol / L, (v) adenosine 2 - 10 mmol / L (or 0.3 - 0.9 g / L), and (vi) lipoic acid 0.05 - 1 μmol / L (or 0.03 - 0.2 mg / L), (vii) sodium pyruvate 0.1 - 1 mmol / L (or 10 - 100 mg / L), (viii) glucose 0.5 - 10 mmol / L (or 100 - 1000 mg / L), (ix) ascorbic acid 0.03 - 0.3 mmol / L, and vitamin E or its water - soluble analogues / derivatives 1 - 10 mM, (x) adenine or its salts or derivatives 0.1 - 4.2 mM, (xi) other vitamins or their water - soluble analogues / derivatives, (xii) total essential amino acids 50 - 200 mg / L, (xiii) total non - essential amino acids 100 - 500 mg / L, (xiv) potassium ion species 30 - 80 mmol / L, (xv) sodium ion species 20 - 90 mmol / L, and the physiological buffer solution has a pH set to 7 - 7.5.

[0057] The preservation solution for refrigerated storage preferably contains 20 to 50 g / L of (xv) hydroxyethyl starch. Additionally, it preferably contains (xvi) ribonucleosides at 4 to 40 mg / L and / or (xvii) deoxyribonucleosides at 4 to 40 mg / L. Further, it preferably contains potassium dihydrogen phosphate and sodium dihydrogenphosphate at 10 to 25 mmol / L.

[0058] Effects of the Invention

[0059] When cryopreserving pluripotent cells such as human iPS cells, it is possible to maintain a high survival rate maintenance effect and the proliferation ability after reculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a graph showing the cell viability after 24 hours of reculture in the case of cryopreserving at 4°C for 6 days using cryopreservation solutions respectively added with deferoxamine (DFO: Deferoxamine), deferasirox (DFX: Deferasirox), and deferiprone (DFP: Deferiprone) as iron ion chelators.

[0061] Figure 2 It is a graph showing the cell viability after 24 hours of reculture in the case of cryopreserving at 4°C for 6 days using cryopreservation solutions respectively added with edaravone (EDV: Edaravone) and probucol (PBC: Probucol) as lipophilic antioxidants.

[0062] Figure 3 It is a graph showing the cell viability after 24 hours of reculture in the case of cryopreserving at 4°C for 6 days using cryopreservation solutions respectively added with ebselen (EBS: Ebselen) as a GPX4 mimetic drug and idebenone (IDB: Idebenone) as a CoQ10 analogue.

[0063] Figure 4A It is a graph showing the death cell rate after 0 hours of reculture in the case of cryopreserving at 4°C for 6 days using the cryopreservation solutions of the comparative examples and reference examples.

[0064] Figure 4B It is a graph showing the apoptotic cell rate after 0 hours of reculture in the case of cryopreserving at 4°C for 6 days using the cryopreservation solutions of the comparative examples and reference examples.

[0065] Figure 4CIt is a graph showing the death cell rate after 8 hours of reculture when using the refrigerated preservation solutions of the comparative examples and reference examples and performing refrigerated preservation at 4°C for 6 days.

[0066] Figure 4D It is a graph showing the apoptotic cell rate after 8 hours of reculture when using the refrigerated preservation solutions of the comparative examples and reference examples and performing refrigerated preservation at 4°C for 6 days.

[0067] Figure 5A It is a graph showing the death cell rate after 24 hours of reculture when using the refrigerated preservation solutions of the examples, comparative examples, and reference examples and performing refrigerated preservation at 4°C for 6 days.

[0068] Figure 5B It is a graph showing the apoptotic cell rate after 24 hours of reculture when using the refrigerated preservation solutions of the examples, comparative examples, and reference examples and performing refrigerated preservation at 4°C for 6 days.

[0069] Figure 6 It is a schematic diagram of the pathway by which ferroptosis (iron-dependent cell death) leads to cell death.

[0070] Figure 7A It is a bar graph showing the viability when adding iron ion chelators (DFX, DFO, DFP) alone (single agent) or in combination with Trolox, performing refrigerated preservation for 6 days, and then performing 24 hours of recovery culture.

[0071] Figure 7B It is a bar graph showing the viability when adding iron ion chelators (DFX, DFO, DFP) alone (single agent) or in combination with Trolox, performing refrigerated preservation for 9 days, and then performing 24 hours of recovery culture.

[0072] Figure 8A It is a bar graph showing the cell death when adding iron ion chelators (DFX, DFO, DFP) alone (single agent) or in combination with Trolox, performing refrigerated preservation for 6 days, and then performing 24 hours of recovery culture.

[0073] Figure 8B It is a bar graph showing the cell death when adding iron ion chelators (DFX, DFO, DFP) alone (single agent) or in combination with Trolox, performing refrigerated preservation for 9 days, and then performing 24 hours of recovery culture.

[0074] Figure 9A It is a bar graph showing the viability when adding two iron ion chelators (DFX and EDV), performing refrigerated preservation for 8 days, and then performing 24 hours of recovery culture.

[0075] Figure 9B It is a bar graph showing cell death during 24-hour recovery culture after 8-day cold storage with the addition of two kinds of iron ion chelators (DFX and EDV).

[0076] Figure 10A It is a bar graph showing viability during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and EBS).

[0077] Figure 10B It is a bar graph showing cell death during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and EBS).

[0078] Figure 11A It is a bar graph showing viability during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and IDB).

[0079] Figure 11B It is a bar graph showing cell death during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and IDB).

[0080] Figure 12A It is a bar graph showing viability during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and DFO).

[0081] Figure 12B It is a bar graph showing cell death during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and DFO).

[0082] Figure 12C It is a bar graph showing viability during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and DFP).

[0083] Figure 12D It is a bar graph showing cell death during 24-hour recovery culture after 8-day cold storage with the addition of another two kinds of iron ion chelators (DFX and DFP).

[0084] Figure 13A It is a bar graph showing the change in viability after cold storage for 6 days based on the combination of Trolox and DFX.

[0085] Figure 13BIt is a bar graph showing the change in cell death after refrigerated storage for 6 days based on the combination of Trolox and DFX.

[0086] Figure 14 Summarize and show the structural formulas of 5 sugar alcohols and 3 sugars.

[0087] Figure 15 It is a bar graph showing the viability after refrigerated storage for 6 days and 24 - hour recovery culture when the sugar alcohols and sugars are added to the substance in which HTMX (“HTMX(-)”) is diluted to 4 / 5. Figure 14

[0088] Figure 16A It is a bar graph showing the viability after refrigerated storage for 6 days and 24 - hour recovery culture when, after diluting HTMX (“HTMX(-)”) to 4 / 5, 3 / 4, 3 / 5, and 1 / 2, sorbitol is added to each of them to 60 mM, 75 mM, 120 mM, and 150 mM, and Trolox, DFX, and both of them (Trolox + DFX) are added respectively.

[0089] Figure 16B It is a bar graph showing the cell death after refrigerated storage for 6 days and 24 - hour recovery culture under the same addition conditions as Figure 16A

[0090] Figure 17A It is showing the use of Figures 16A - 16B “HTMX_Trx2K” (HTMX(-)+Trolox 2 mM), “HTMX_T&D” (HTMX(-)+Trolox 2 mM+DFX 100 μm), and “4 / 5_S_T&D” (HTMX(-)×4 / 5+Sorbitol 60 μm+DFX 100 μm) in the experimental areas, and it is a bar graph showing the viability during refrigerated storage from 3 days to 6 days.

[0091] Figure 17B It is a bar graph showing the cell death during refrigerated storage from 3 days to 6 days in the same experimental areas as Figure 17A

[0092] Figure 18 It is a curve graph showing the investigation results of viability when Trolox or Trolox and DFX are added to HTMX (“HTMX(-)”) or the substance in which it is diluted to 1 / 2 and 150 mM of sorbitol is added (“HTMXSA” = HTMX(-)×1 / 2+Sorbitol 150 μm). Detailed implementation mode

[0093] The preservation solution for refrigeration used in the preferred embodiment of the present invention is a physiological buffer solution containing the following (i) to (vi), and the pH is set to 7 to 7.5.

[0094] (i) Lactobionic acid or lactobionate is 20 - 100 mmol / L, 30 - 100 mmol / L, 30 - 80 mmol / L or 30 - 60 mmol / L in terms of lactone, or 10 - 50 g / L, 10 - 40 g / L, 15 - 35 g / L or 20 - 30 g / L.

[0095] (ii) Raffinose hydrate is 10 - 40 mmol / L, 10 - 30 mmol / L or 10 - 20 mmol / L, or 5 - 20 g / L or 8 - 15 g / L.

[0096] (iii) Allopurinol is 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.

[0097] (iv) Glutathione (total glutathione) is 1 - 3 mmol / L or 1.5 - 2.5 mmol / L, or 0.3 - 1 g / L, 0.4 - 0.9 g / L or 0.4 - 0.8 g / L.

[0098] (v) Adenosine is 2 - 10 mmol / L, 2 - 8 mmol / L or 2 - 5 mmol / L, or 0.5 - 1.5 g / L or 0.5 - 1.3 g / L.

[0099] (vi) Alpha - lipoic acid is 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.

[0100] (vii) Sodium pyruvate is 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.

[0101] (viii) Glucose is 0.5 - 10 mmol / L, 1 - 5 mmol / L or 1 - 3 mmol / L, or 100 - 1000 mg / L or 200 - 500 mg / L.

[0102] (ix) The following antioxidant vitamins or their water - soluble analogs / derivatives

[0103] (Both can be in the form of salts. Depending on the situation, ix-1 in the following two can be omitted.)

[0104] ix-1. Ascorbic acid 0.03 - 0.3 mmol / L, 0.05 - 0.2 mmol / L or 0.05 - 0.15 mmol / L, or 5 - 20 mg / L or 10 - 15 mg / L.

[0105] ix-2. Vitamin E, water-soluble vitamin E (Trolox) or other water-soluble vitamin E analogs / derivatives 0.5 - 10 mM, 1 - 8 mM or 2 - 7 mM.

[0106] (x) Adenine or its salt or derivative 0.1 - 6 mM, 0.1 - 5 mM, 0.1 - 4.5 mM, 0.1 - 4.2 mM or 2 - 5 mM. Depending on the situation, 0.1 - 0.3 mM or 0.05 - 0.2 mM.

[0107] (xi) The above other vitamins or their water-soluble analogs / derivatives

[0108] (Both can be in the form of salts. Additionally, 1 - 5, 1 - 4 or 1 - 3 of the following 10 can be omitted.)

[0109] xi-1. Biotin 0.03 - 0.25 μmol / L, 0.05 - 0.2 μmol / L or 0.1 - 0.15 μmol / L, or 0.01 - 0.1 mg / L or 0.02 - 0.05 mg / L.

[0110] xi-2. Vitamin B12 0.03 - 3 μmol / L, 0.05 - 2 μmol / L or 0.1 - 0.5 μmol / L, or 0.1 - 0.8 mg / L or 0.2 - 0.6 mg / L.

[0111] xi-3. Folic acid 0.2 - 2 μmol / L, 0.3 - 1 μmol / L or 0.5 - 0.9 μmol / L, or 0.1 - 1 mg / L or 0.2 - 0.5 mg / L.

[0112] xi-4. Nicotinamide 0.5 - 8 μmol / L, 1 - 6 μmol / L or 1 - 4 μmol / L, or 0.03 - 0.2 mg / L or 0.05 - 0.1 mg / L.

[0113] 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.

[0114] xi - 6. Choline is 0.05 - 1 μmol / L, 0.1 - 0.5 μmol / L or 0.1 - 0.4 μmol / L, or 0.1 - 1 mg / L or 0.2 - 0.5 mg / L.

[0115] xi - 7. Inositol is 1 - 10 μmol / L, 1 - 8 μmol / L or 2 - 6 μmol / L, or 0.03 - 0.2 mg / L or 0.05 - 0.1 mg / L.

[0116] xi - 8. Pantothenic acid is 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.

[0117] xi - 9. Pyridoxal is 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.

[0118] xi - 10. Thiamine is 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.

[0119] (xii) The total of essential amino acids is 50 - 200 mg / L or 80 - 150 mg / L.

[0120] When the value obtained by dividing the content recorded in Table 3 by 3 (the value in "HTM - alpha" in the following examples) is set as A, the amount of each component can be appropriately set within the range of A / 3 - 3A (that is, within the range of 1 / 3 to 3 times the value in "HTM - alpha") or within the range of A / 2 - 2A. The same applies to the above - mentioned non - essential amino acids and the above vitamins.

[0121] xii - 1. Isoleucine

[0122] xii - 2. Leucine

[0123] xii - 3. Lysine

[0124] xii - 4. Methionine

[0125] xii - 5. Phenylalanine

[0126] xii - 6. Threonine

[0127] xii - 7. Tryptophan

[0128] xii - 8. Balin

[0129] xii - 9. Histidine

[0130] (xiii) The total amount of non-essential amino acids is 100 - 500 mg / L or 150 - 400 mg / L.

[0131] (Both can be in the form of salts. Additionally, 1 - 5, 1 - 4, or 1 - 3 of the following 10 types can be omitted.)

[0132] xiii-1. Glycine

[0133] xiii-2. Alanine

[0134] xiii-3. Arginine

[0135] xiii-4. Asparagine

[0136] xiii-5. Aspartic acid

[0137] xiii-6. Cysteine

[0138] xiii-7. Cystine

[0139] xiii-8. Glutamic acid

[0140] xiii-9. Glutamine

[0141] xiii-10. Proline

[0142] (xiv) The potassium ion species is 20 - 90 mmol / L, 30 - 80 mmol / L, 40 - 80 mmol / L, or 50 - 70 mmol / L. That is, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, or 50 mmol / L or more, and less than 90 mmol / L, less than 80 mmol / L, or less than 70 mmol / L.

[0143] (xv) The sodium ion species is 20 - 90 mmol / L, 30 - 80 mmol / L, 40 - 80 mmol / L, or 50 - 70 mmol / L. That is, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, or 50 mmol / L or more, and less than 90 mmol / L, less than 80 mmol / L, or less than 70 mmol / L.

[0144] This preservation solution for refrigeration preferably contains the following (xvi).

[0145] (xvi) Hydroxyethyl starch is 10 - 80 g / L, 20 - 50 g / L, or 20 - 40 g / L.

[0146] This preservation solution for refrigeration preferably contains at least one of the following (xvii) and (xviii).

[0147] (xvii) 2 to 4 ribonucleosides, with a total of 4 to 40 mg / L, 5 to 30 mg / L, or 10 to 15 mg / L.

[0148] (xviii) 2 to 4 deoxyribonucleosides, with a total of 4 to 40 mg / L, 5 to 30 mg / L, or 10 to 15 mg / L.

[0149] The preservation solution for refrigeration preferably contains the following (xix) to (xx).

[0150] (xix) Potassium dihydrogen phosphate or sodium dihydrogen phosphate, 10 to 30 mmol / L, 10 to 25 mmol / L, or 15 to 25 mmol / L.

[0151] (xx) Magnesium ion species (especially magnesium sulfate), 2 to 8 mmol / L, 2 to 5 mmol / L, or 2 to 4 mmol / L.

[0152] (xxi) 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.

[0153] In addition, in a preferred embodiment of the present invention, the sodium ion species, in terms of molar ratio (the ratio of Na / K ions), can be, for example, 0.7 to 1.3 times, 0.8 to 1.2 times, or 0.9 to 1.1 times that of the potassium ion species. In addition, in another preferred embodiment, the molar ratio of the sodium ion species can be, for example, 0.5 to 1.5 times, 0.5 to 1.3 times, or 0.6 to 1.2 times that of the potassium ion species.

[0154] As described above, the preservation solution for refrigeration used in the preferred embodiment of the present invention contains water-soluble vitamin E (Trolox) or other water-soluble vitamin analogs 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, 4 mM or more, or 5 mM or more, and 10 mM or less, 8 mM or less, or 6 mM or less.

[0155] As described above, the cold storage solution used in the preferred embodiment of the present invention contains adenine or its salt 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, at a concentration of 0.1 to 4.2 mM or 1 to 4.2 mM. In particular, it is considered that the addition of adenine shows a significant effect when cells adhere to each other to form a three-dimensional mass and an embryo or an organoid is preserved. In addition, when preserving an embryo or an organoid, even at a relatively low concentration, such as 0.01 to 0.2 mM (10 to 200 μM) or 0.05 to 0.3 mM (5 to 300 μM), an effect can be seen. On the other hand, it is considered that when culturing stem cells such as iPS cells in a state where an embryo or an organoid is not formed, it is particularly effective to add adenine at a concentration of 0.5 to 6 mM, particularly 1 to 5 mM or 2 to 4 mM.

[0156] The cold storage solution used in the preferred embodiment of the present invention also contains L-alanyl-L-glutamine or other dipeptide substitutes or their salts at a concentration of 1 to 6 mM, 2 to 5 mM, or 3 to 5 mM.

[0157] The cold storage solution used in a preferred embodiment of the present invention is obtained by mixing UW solution whose composition is shown in Table 2 and MEM alpha medium whose composition is shown in Table 3 at a volume ratio of UW solution to MEM alpha medium of 1 to 3 times, preferably 1.5 to 2.5 times, more preferably 1.8 to 2.2 times. In addition, the composition of Tables 1 and 2 and the content of each component obtained from this mixing ratio can be appropriately increased or decreased within a range of ±50%, ±40%, ±30%, or ±20%. In addition, in some components, particularly the components listed in Table 3, a part of non-essential amino acids (for example, 1 to 5 kinds), a part of nucleosides (for example, 1 to 5 kinds), a part of vitamins (for example, 1 to 5 kinds), a part of inorganic salts (for example, 1 to 3 kinds), and phenol red can be omitted.

[0158] According to a preferred embodiment of the present invention, the cold storage method is to disperse pluripotent cells into a single cell state in any of the above cold storage solutions so that the number of cells is 1×10 3 cells / mL to 1×10 9 cells / mL or 1×10 4 cells / mL to 1×10 8 cells / mL, and perform cold storage for, for example, 1 to 10 days or 1 to 7 days.

[0159] According to a preferred embodiment of the present invention, the cryopreservation method is carried out in a culture vessel containing any one of the above cryopreservation solutions, such that pluripotent cells are seeded in a manner of 1×10 3 cells / cm 2 ~1×10 9 cells / cm 2 or 1×10 4 cells / cm 2 ~1×10 8 cells / cm 2 . For example, they are cultured for 2 to 6 days and then cryopreserved for, for example, 1 to 10 days or 1 to 7 days.

[0160] According to a preferred embodiment of the present invention, the cryopreservation method can be carried out directly while maintaining the state of adhering cells to the culture medium, that is, in the state of normal culture. In particular, the state of adhering cells to the culture medium can be maintained throughout the preservation period including the awakening period.

[0161] According to a preferred embodiment of the present invention, when cryopreserving at 2 to 10°C for 2 to 10 days, 3 to 15 days, or 4 to 20 days, the awakening period is inserted at regular or irregular intervals. The awakening period can be set as follows.

[0162] · The length of each awakening period: 0.5 to 10 hours, especially 1 to 8 hours.

[0163] · The temperature of each awakening period: below 37°C and above 30°C, 32°C, 33°C, 34°C, or 35°C.

[0164] · The interval of the awakening period: 2 to 8 days, especially 2 to 5 days, for example, 3 to 4 days.

[0165] According to a preferred embodiment of the present invention, pluripotent cells or stem cells as the object of cryopreservation are, for example, universal cells called artificial pluripotent stem cells such as ES cells and iPS cells, or hematopoietic stem cells, neural stem cells, liver stem cells, skin stem cells, germ stem cells, etc. In addition, the pluripotent cells as the object of cryopreservation can be from humans or can be from mammals such as primates, mice, guinea pigs, etc.

[0166] In addition, according to another embodiment, the object of cryopreservation can be the embryo of a human or an animal, especially the embryo of domestic animals such as cattle, horses, pigs, goats, etc.

[0167] Examples

[0168] I. The first part (basis of priority)

[0169] 1. Preparation (maintenance culture) of cells for cryopreservation

[0170] The human iPS cell line (253G1) derived from healthy individuals without physical organ disorders obtained from the Kyoto University Institute for Integrated Cell-Material Sciences was maintained in feeder-free conditions. In this culture, the commercially available clinical research medium (for human ES / iPS cells) StemFit (registered trademark) AK02N (Ajinomoto) was used, and subculture was performed every 7 days at a density of 1.5E3 / cm 2 as follows.

[0171] At the time when confluence reached 70 - 80% on the 7th day of culture initiation, cells were detached enzymatically (using 0.5X TrypLE (registered trademark) Select), dispersed, and washed. Thereafter, they were suspended in fresh StemFit (registered trademark) AK02N medium containing 10 μM Y27362 (FUJIFILM Wako Pure Chemical Corporation), and as a further cell culture supplement, iMatrix-511 (MATRIXOME, Inc.) was added to 0.1 μg / cm 2 and seeded at a density of 1.5E3 / cm 2 (1.5 × 10 3 / cm 2 ). On the day after seeding, the medium was replaced with StemFit (registered trademark) AK02N medium without Y27362, and then the medium was replaced every 3 days for subculture or experiments on the 7th day.

[0172] In addition, in this application, unless otherwise specified, culture was performed in a 37°C incubator (5% CO2).

[0173] 2. Procedure for cryopreservation (cryopreservation of iPS clusters)

[0174] For the iPS cells obtained as described above, at the time when confluence reached 70 - 80% after seeding, cells were detached enzymatically (0.5x TrypLE (GIBCO)), dispersed, washed, and then suspended in fresh StemFit (registered trademark) AAK02N medium containing 10 μM Y27362 (FUJIFILM Wako), and iMatrix-511 (Matrixome) was added to 0.1 μg / cm 2 at a density slightly lower than that for maintenance culture (1.0E3 / cm 2)Sowing and culturing were carried out, and the medium was replaced with a medium without Y27362 on the next day. On the 5th day after sowing, when the number of cells in the cluster was 32 - 64, the medium was removed, replaced with a medium for cryopreservation, and then cryopreserved at 4°C in a sealed and static state for 6 days. After the end of the preservation period, the cryopreservation solution was aspirated and removed, a specified amount of other iPS culture medium (StemFit (registered trademark) AAK02) was added, and the cells were cultured at 37°C and 5% CO2 for 24 hours to measure viability and the number of cell deaths. As a control, organ preservation solution UW solution (UW) (Astellas Pharma) and commercially available cell preservation solution HTS - FRS (HTS) (Sigma - Aldrich) etc. were used.

[0175] 3. Cryopreservation solution

[0176] Prepare the following basic cryopreservation solution. For convenience, it is called "HTMX(-)". This is a cryopreservation solution with the same composition as "HTM" described in the patent literature 1, but replacing the UW solution in Table 2 below with the following modified UW solution composition and omitting water - soluble vitamin E (Trolox).

[0177] ·HTMX(-): According to the composition of the UW solution in Table 1 below, prepare a modified UW solution that omits hydroxyethyl starch and contains 90 mM mannitol and 0.25% polyvinyl alcohol, and use this modified UW solution by mixing it with MEM - alpha (12571 - MEM alpha, nucleosides, Thermo Fisher Scientific) at a volume ratio of 2 / 1. The polyvinyl alcohol (PVA) used here is #363146 from Merck, with a saponification degree of 99%, a weight - average molecular weight Mw of 85000 - 124000, and a rotational viscosity of 28 - 32 mPa / sec (28 - 32 cP, 4% in H2O (20°C) (lit.)) in a 4% aqueous solution at 20°C. In addition, GlutaMAX (registered trademark, GIBCO) was added at a 5 - fold concentration (x5; 10 mM).

[0178] ·"Trx2.0": 2.0 mM water - soluble vitamin E (Trolox) was added to the above - mentioned basic cryopreservation solution ("HTMX(-)"). Thus, it became a cryopreservation solution roughly the same as the cryopreservation solution in the patent literature 1 and was used as a reference example.

[0179] · Additive with free radical scavenging effect (cell death inhibitor): The additives shown in Table 1 below are mainly used. Specifically, each of the following agents, ZVAD-FMK (PEPTIDE Institute), Necrostatin (Chem Scene), Deferoxamine (Cayman Chemicals), Ferrostatin-1 (Cayman Chemicals), Edaravone (Sigma-Aldrich), Probucol (Tokyo Chemical Industry), Trolox (Cayman Chemicals), Deferasirox (Tokyo Chemical Industry), Deferiprone (Selleck Biotech), Ebselen (Tokyo Chemical Industry), Idebenone (Tokyo Chemical Industry), is added to the preservation solution or to the culture medium during subculture, and the survival rate and cell death rate are measured and evaluated.

[0180] [Table 1] Types of compounds expected to have an anti-ferroptosis effect

[0181]

[0182] In addition, the following two types of cryopreservation solutions, etc., are used as cryopreservation solutions for comparative examples.

[0183] · UW solution: BELZER UW (registered trademark) COLD STORAGE SOLUTION

[0184] · HTS FRS: HypoThermosol (registered trademark) FRS, BioLife Solutions

[0185] According to the above-mentioned non-patent literature 1, the composition of the UW solution (BELZER UW (registered trademark) COLD STORAGE SOLUTION) is as shown in the following table.

[0186] [Table 2] Composition of UW solution

[0187]

[0188] The MEM alpha medium (12571-MEM alpha, nucleosides, Thermo Fisher Scientific) used in "HTMX(-)" specifically has the following composition (https: / / www.thermofisher.com / jp / en / home / technical-resources / media-formulation.94.html).

[0189] [Table 3] Composition of MEM alpha medium

[0190]

[0191]

[0192]

[0193] GlutaMAX (registered trademark, GIBCO) commercially available as added to "HTMX(-)" is a 200 mM solution ("GlutaMAX TM I (100×)") in which L-alanyl-L-glutamine, a dipeptide substitute for L-glutamine, is dissolved in physiological saline (0.85N NaCl). It is diluted to 1 / 20 to 10 mM (5×) and added to "HTMX(-)" for use.

[0194] For the above-mentioned UW solution, HTMX(-), and HTS-FRS, the sodium ions, potassium ions, and their molar ratios are summarized in Table 4 below. In addition, the ion concentrations in StemFit (registered trademark) AK02N are the same as those in MEM-alpha. Referring to the experimental results described later, regarding Na + / K + molar ratio, it is considered that it should be about 1 or a value slightly less than 1.

[0195] [Table 4] Na + / K + molar ratio in commercially available media and media of the examples

[0196] UW solution MEM-alpha HTMX(-) HTS-FRS <![CDATA[Sodium + (mM)]]> 25.0 116.4 55.5 100.0 <![CDATA[K + (mM)]]> 125.0 5.4 85.1 42.5 Na / K 0.2：1 21.7：1 0.7：1 2.3：1

[0197] As described above, proteins, peptides, and growth factors are included in StemFit (registered trademark) AK02N but not in MEM-alpha. Therefore, they are not included in HTMX(-).

[0198] 4. Evaluation method

[0199] 1) Measurement of cell viability

[0200] In the measurement of cell viability, Cell Counting Kit-8 (Dojindo Laboratories) was used and the WST method was employed to measure the dehydrogenase activity in cells colorimetrically. The basic method followed the manufacturer's protocol. 10 μL of WST-8 was added to the medium after culturing and cryopreserving in a 96-well format and then culturing again for 24 hours. After culturing for 1 hour, the absorbance at 450 nm was measured using a microplate reader.

[0201] 2) Measurement of cell death rate

[0202] The number of dead cells was measured by determining the activity of lactate dehydrogenase (LDH) using the Cytotoxicity LDH Assay Kit - WST (Dojindo). According to the manufacturer's protocol, an equal volume of Assay Buffer was added to 50 μL of the cell supernatant collected after cold storage and after reculturing for 24 hours. After reacting at room temperature for 30 minutes, the absorbance at 490 nm was measured using a microplate reader, and the ratio of cell death was calculated as the ratio to the positive control (completely dead cells).

[0203] 3) Vital and dead cell staining and detection of apoptotic cells

[0204] To detect apoptotic cells, the activity of intracellular caspase was measured. The activity of intracellular caspase 3 / 7 was evaluated by staining using CellEvent TM Caspase - 3 / 7 Green Detection Reagent (ThermoFisher) according to the protocol. Dead cells were stained with PI (Propidium Iodide), and live cells were stained with Hoechst 33342 for separate staining. The fluorescence signals of each cell were quantified using the image analysis software ImageJ, and the ratios of live cells, apoptotic cells, and caspase - positive cells were calculated.

[0205] 5. Protective effect of cold storage of iron ion chelators

[0206] Figure 1 The effect of the added iron ion chelator on cell viability after reculturing for 24 hours when cold - stored for 6 days is shown. Cell viability was measured using the WST assay, and the substance obtained by adding Trolox to HTMX(-) to 2 mM was set as 1 and expressed as a relative value. Any iron ion chelator showed a concentration - dependent effect of improving viability and showed a higher protective effect compared to the Trolox - added group. Table 5 shows the optimal concentration, relative survival rate, and 95% survival limit of each iron ion chelator. Compared with 2 mM Trolox, Deferoxamine showed 1.3 - fold, and Deferasirox and Deriprone both showed more than 1.5 - fold higher viability. It was also found that the optimal concentration of all iron ion chelators was significantly lower than that of Trolox.

[0207] [Table 5]

[0208]

[0209] 6. Protective effect of cold storage of lipophilic antioxidants

[0210] Figure 2 The effect of lipophilic antioxidants added during 6-day cold storage on cell viability after 24-hour reculture is shown. Viability was measured using the WST assay, and the substance obtained by adding Trolox to 2 mM in HTMX(-) was set as 1 and expressed as a relative value. Any of the lipophilic antioxidants showed an effect of concentration-dependently increasing viability and showed a higher protective effect compared to the Trolox addition group. Table 6 shows the optimal concentration, relative survival rate, and 95% survival limit of each lipophilic antioxidant. Edaravone and Probucol both showed a viability about 1.2 times higher compared to 2 mM Trolox. In addition, the optimal concentration of any of the lipophilic antioxidants was significantly lower than that of Trolox.

[0211] [Table 6]

[0212]

[0213] 7. Protective effect of GPX4 mimetic drugs and CoQ10 analogs during cold storage

[0214] Figure 3 The effect of Ebselen, a GPX4 mimetic drug, and Idebenone, a CoQ10 analog, added during 6-day cold storage on cell viability after 24-hour reculture is shown. Viability was set as 1 and expressed as a relative value with the substance obtained by adding Trolox to 2 mM in HTMX(-). Any of these added drugs showed an effect of concentration-dependently increasing viability and showed a protective effect approximately the same as that of the Trolox addition group. Table 7 shows the optimal concentration, relative survival rate, and 95% survival limit of each added drug. Ebselen and Idebenone both showed a viability as high as that of Trolox. In addition, the optimal concentration of any of the added drugs was lower than that of Trolox.

[0215] [Table 7]

[0216]

[0217] 8. Elucidation of cell death occurring at the end of cell preservation and during reculture

[0218] Next, before the experiment related to Figures 1 - 3 the experiment to be prepared is described.

[0219] The ratios of cell death and apoptosis of cells cryopreserved for 6 days in the HTS-FRS, UW solution, HTMX(-), and Trx2.0 (HTMX(-) + Trolox 2 mM) regions and then recultured for 0 hours and 8 hours were measured. The results are as Figures 4A - 4D shown. In Figure 4A , the PI-positive cell rate shortly after 6 days of cryopreservation was expressed as the cell death rate. At this time, more than 90% of the cells died in the region preserved with HTS-FRS. In addition, 20% died out in the UW solution. On the other hand, almost no dead cells were found in the HTMX(-) and Trx2.0 regions.

[0220] In Figure 4B , the Caspase3 / 7-positive cell rate shortly after 6 days of cryopreservation was expressed as the cell rate causing apoptosis. The ratio of Caspase 3 / 7 positivity was slightly higher than the PI-positive cell rate in any addition region ( Figure 4A ). This indicates that although cell death was not induced, the presence of initial apoptotic cells was shown.

[0221] Figure 4C And Figure 4D show the cell death rate and apoptotic cell death rate after 6 days of cryopreservation - reculture for 8 hours. In the HTS-FRS region, it is known that all cells underwent cell death. In the UW region, it is known that more than 60% underwent cell death, all of which were apoptotic. In contrast, in the HTMX(-) region, the dead cells were less than 20%, and the apoptotic cells were about 30%. In the Trx2.0 region with Trolox added to HTMX(-), both the cell death rate and the apoptosis rate were suppressed relatively low at about 10%. Based on the above, it was clarified that without using the HTMX preservation solution, no cell death occurred shortly after cryopreservation, and as the reculture time progressed, cell death caused by apoptosis increased. The present inventors proposed a hypothesis that a certain cell stress accumulated during cryopreservation causes apoptosis during the reculture period after the end of cryopreservation.

[0222] 10. Presumption of stress generated during cryopreservation and during reculture

[0223] In comparison with Figures 4A - 4DAfter the relevant experiments, various cell death inhibitors were added to presume the cell death that occurred during and after cell preservation and the stress that caused cell death. Cell death after cryopreservation and re - culture for 24 hours was confirmed by the LDH test. As cell death inhibitors, apoptosis inhibitors (ZVAD: ZVAD - FMK) related to "regulated cell death" with a high possibility related to cryopreservation, necrostatin (NST), ferroptosis inhibitors (DFO: Deferoxamine, FST: Ferrostatin - 1) were added to the cryopreservation solution (HTMX) in the case of cryopreservation for 6 days, and added to the culture medium (StemFit) in the case of re - culture.

[0224] Figure 5A Shows the effect of cell death inhibitors during re - culture. The ratio of cell death when cryopreserved with HTMX(-) for 6 days and re - cultured for 1 day was approximately 0.5. When the apoptosis inhibitor (ZVAD) was added during re - culture, cell death could be inhibited to less than 10%. However, cell death was not inhibited by other cell death (ferroptosis, necroptosis) inhibitors. This indicates that, combined with the results of PI and Caspase3 / 7 activities after preservation, the cell death that occurs during cryopreservation and re - culture is mainly due to apoptosis.

[0225] Figure 5B Shows the effect of cell death inhibitors added during cryopreservation. When only ZVAD, an apoptosis inhibitor, was added during preservation, cell death after re - culture did not change compared to the control HTMX group. In addition, adding the necroptosis inhibitor (NST) did not inhibit cell death during re - culture. On the other hand, it was found that the two ferroptosis inhibitors (DFO, NST) inhibited cell death to approximately the same extent as when Trolox was added to HTMX(-). From the above, it can be seen that during cryopreservation, the stress that induces ferroptosis accumulates but does not cause cell death, and by inhibiting ferroptosis with ferroptosis inhibitors, apoptosis that occurs during re - culture can be inhibited.

[0226] 11. Rat kidney preservation

[0227] The kidneys of rats (Wistar strain, 380 g) were removed under anesthesia, and the preservation effects were compared and studied. As a comparative example, after washing with UW solution during kidney removal, the kidneys were cooled and preserved for 48 hours. In contrast, in the examples of the present invention, during kidney removal, the kidneys were washed with a preservation solution obtained by further adding the cryopreservation solution of the present invention to the UW solution, and were similarly cooled and preserved for 48 hours. Then, continuous perfusion was performed for 90 minutes using a kidney perfusion device, and the perfusion volume, urine volume, and creatinine clearance rate were compared. As a result, compared with the kidneys preserved with only UW solution used as a comparative control, in the cryopreservation solution system of the present invention, kidney failure was significantly alleviated.

[0228] <Conclusion>

[0229] There is no report on the cause of cell death during cryopreservation of cells. This time, it was clarified that cell death did not occur soon after cryopreservation, and apoptosis occurred during subsequent subculture. Furthermore, the apoptosis that occurred during subculture was caused by ferroptosis stress received during cryopreservation. We established a hypothesis that by suppressing the ferroptosis stress received during cryopreservation, apoptosis during subculture could be suppressed, and attempted to screen for new cryoprotective substances. As a result, by adding substances that inhibit various pathways of ferroptosis, the viability after cryopreservation was significantly improved. In particular, as iron ion chelators, Deferasirox and Deferiprone were able to increase the viability after 6 days of cryopreservation to more than 1.5 times. In addition, it was found that when any of the protectants was added at a low concentration of 1 / 10 to 1 / 100 compared with the current Trolox, there was an effect equal to or better than that of Trolox. This is an important factor conducive to expanding the adaptation range after cell cryopreservation.

[0230] In addition, when obtaining the basic cryopreservation solution "HTMX(-)" (a 2:1 mixture of UW solution and MEM-alpha solution), a modified MEM-alpha solution having the amino acid composition and vitamin composition shown in Tables 8 to 9 below can be used. By comparing with the composition of the MEM alpha medium in Table 2, it can be seen that this modified MEM-alpha solution omits some amino acids and vitamins.

[0231] [Table 8] Amino acid composition of the modified MEM-alpha solution

[0232]

[0233]

[0234] [Table 9] Vitamin composition of the modified MEM-alpha solution

[0235]

[0236]

[0237] Compared with cryopreservation, the cold storage technology of iPS cells limits the storage period, but it can maintain the culture state (adhesion) during storage, preserve non-freezable organoids undergoing differentiation induction, and for transportation, or provide on-demand cell supply in pharmaceutical research, etc. It is a technology with a very high application range. Therefore, this technology that enables the preservation of human iPS cells is very important.

[0238] Since the cell cryopreservation medium "HTM-alpha" of the embodiments of the present application, etc., has shown effects in the cold storage of mouse embryos, it is considered that it will also show effects in the cold storage of embryonic or non-embryonic stem cells of humans and various animals, and tissues containing such stem cells.

[0239] II. Second part (additional experiments)

[0240] After the basic application for the priority of this case, the results of additional experiments and investigations are as follows. In the following experiments, the cells used as objects, the order of cold storage, and the cold storage solution or its additives (cell death inhibitors), etc., are as described in the above "1." to "4.".

[0241] 12. Additive and synergistic effects based on the combined use of cryopreservation protective substances targeting ferroptosis

[0242] Figure 6 The outline of the pathway of cell death caused by ferroptosis (iron-dependent cell death) is shown. By applying various stresses to cells, the cell membrane phospholipid (PLH) "1" is oxidized by reactive oxygen species (ROS), etc., and accumulates as peroxidized phospholipid (POOH) "2". Inside the cell, this peroxidized phospholipid is usually reduced by glutathione peroxidase 4 (GPx4) with glutathione as the substrate and converted into non-toxic phosphatidyl alcohol "4".

[0243] By continuously stressing the cells, exceeding the capacity of this defense mechanism, the peroxidized phospholipid (PLH) in the "2" state generates more unstable phospholipid radicals (PLO·) "3" through hydroxyl radicals mainly generated by free divalent iron ions. The phospholipid radicals (PLO·) damage the cell membrane and cell death occurs. It is known that ferroptosis inhibitors inhibit the generation of ferroptosis by controlling (promoting or inhibiting) the specificity of each step ( Figure 6 each arrow in it). (Table 10)

[0244] [Table 10]

[0245]

[0246]

[0247] Considering that the main stress during cold storage is caused by ferroptosis, adding combinations of agents belonging to different groups may produce additive or synergistic effects. To verify the effects, Trolox 2 mM and the above-mentioned iron ion chelators (DFO, DFX, DFP; ferroptosis inhibitors) were added to HTMX (the "HTMX(-)" described in item "3." above) either as monomers (single agents) or in combinations, and after 6-day and 9-day cold storage, the viability and cell death were examined during 24-hour recovery culture ( Figures 7A - 7B and 8A to 8B). Figure 7A and Figure 7B The viabilities after 24-hour recovery culture following 6-day and 9-day cold storage are shown respectively.

[0248] Here, the vertical axis shows the relative viability with the viability of the non-preserved control set as 1. It was found that compared with the addition of single agents (left group; adding only one of Trolox and the ferroptosis inhibitor), the combined addition of two agents (right group; adding both Trolox and the ferroptosis inhibitor), especially the combined addition of Trolox + DFX and Trolox + DFO, increased the viability. In the case of 9-day cold storage, the difference between the left and right groups was large, and especially in the case of Trolox + DFX, an increase in viability was shown. Figure 8A and Figure 8B The cell death after 24-hour recovery culture following 6-day and 9-day cold storage is shown. The vertical axis is represented with the value of total cell death set as 1. In both 6-day and 9-day cold storage, the combined addition of two agents inhibited cell death lower than that of single agents.

[0249] [Table 11]

[0250]

[0251] Figure 9A And 9B shows the results of the combined addition of DFX and EDV. Figure 9AIt shows the viability of iPS cells 24 hours after resuming culture after storing them at 4°C for 8 days, with 0 - 100 μM of DFX added to HTMX (the "HTMX(-)" described in item "3." above) and 0 - 100 μM of EDV added. In the figure, the case where no substances such as Trolox or ferroptosis inhibitors are added to "HTMX(-)" is denoted as "HTMX(-)_0". Similar to the cases of Tables 5 - 7 described in items "5." - "8." above, the vertical axis sets the case where 2 mM of Trolox is added to HTMX as 1. In the DFX non - addition region (DFX 0 μM), as the added concentration of EDV increases, the viability increases, showing values approximately the same as those in the Trolox addition region.

[0252] As the added concentration of DFX increases, the overall viability improves. In the DFX 100 μM region, it reaches almost twice that of the case where only Trolox is added (the Trx addition region). When DFX is not added or at a low concentration of 10 - 50 μM, the combined use of EDV increases the viability, but in the DFX 100 μM addition region, no increase in viability due to the addition of EDV is found, and the combined use of EDV on the basis of DFX does not improve the viability after cold storage above that of the DFX single agent. Figure 9B It shows the ratio of cell death 24 hours after resuming culture after storing for 8 days. The vertical axis shows the relative value with the value of total cell death set as 1. Similar to the viability, in the region where DFX is 50 μM or more, even with the combined use of EDV, cell death cannot be particularly further inhibited.

[0253] Next, Figure 10A and Figure 10B It shows the effect of the combination of DFX and EBS. Similarly, at low DFX concentrations, due to the combined use of EBS, the viability slightly increases, but at DFX 50 μM or more, the addition of EBS instead slightly reduces the viability ( Figure 10A ). Also, regarding cell death, the same is true. No effect of the combined addition of EBS is observed, and instead, there is a result of a slight increase in cell death ( Figure 10B ).

[0254] Figure 11A and Figure 11B It shows the effect of the combined use of DFX and IDB. The addition of IDB, similar to the combined use of EBS, also slightly reduces the viability compared to the DFX single agent ( Figure 11A ). Regarding cell death, a tendency of a slight increase compared to the DFX monomer is also observed ( Figure 11B ).

[0255] Finally, Figure 12A and Figure 12BThe effects of the combination of DFX with DFO and DFP, which are also iron ion chelator groups, are shown. In the combination of DFX and DFO, an increase in viability was observed by adding DFO in the low concentration region of DFX, but this effect was not observed at DFX concentrations of 50 μM or higher ( Figure 12A ). Additionally, regarding cell death, the effect of adding DFO to inhibit cell death was also observed in the low concentration region of DFX, but there was no effect at high DFX concentrations ( Figure 12B ). In the combination of DFX and DFP, no effect of adding DFP was observed at any DFX concentration.

[0256] Based on the above results, it is clear that the combination of Trolox and DFX shows the highest cold storage preservation (additive effect).

[0257] Next, Figure 13A and Figure 13B show the changes in viability after cold storage for 6 days based on the combination of Trolox and DFX with the highest additive effect. When comparing at 80% viability, the 80% survival days with single-dose addition of Trolox were approximately 3 days, while in the single-dose addition region of DFX it was 5 days, and in the combined use region of Trx + DFX it was approximately 5 - 6 days, showing an extended survival effect ( Figure 13A ). Cell death showed that the combined use region of Trx + DFX was lower than that of other single-dose additions ( Figure 13B ). In summary, it was found that by combining inhibitors with different ferroptosis pathways, the cold storage ability of the HTMX preservation solution can be significantly improved.

[0258] 13. Additive effects of sugars and sugar alcohols

[0259] It is known that sugar alcohols such as glycerol, propylene glycol, and ethylene glycol penetrate into cells during cryopreservation of cells and inhibit freezing-induced failures by inhibiting ice crystal formation or stabilizing intracellular proteins. On the other hand, sugars such as sucrose that do not penetrate the cell membrane are considered to help protect the cell membrane from the outside. During cold storage, the cryo-stress inhibitory effects of such sugars and sugar alcohols were verified.

[0260] Five sugar alcohols (sorbitol, mannitol, xylitol, glycerol, propylene glycol) and three sugars (sucrose, trehalose, raffinose) ( Figure 14 ) were each added at 60 mM to 4 / 5 diluted HTMX (diluting the "HTMX(-)" described in item "3." above to 4 / 5 concentration). Then, the effects on viability after 6 days of cold storage and 24 hours of recovery culture were investigated. Figure 15The results are shown. The viability is expressed as a relative value with the viability of HTMX without the addition of a protective agent (Trolox, DFX) (the above-mentioned "HTMX(-)") being 1. As a result, only the section with the addition of sorbitol showed a viability higher than that of HTMX without addition, being a value close to that of HTMX+Trolox.

[0261] Next, in order to verify the effect of the addition concentration of highly effective sorbitol, sorbitol was added to 60 mM, 75 mM, 120 mM, and 150 mM, respectively, in substances obtained by diluting HTMX (the above-mentioned "HTMX(-)") to 4 / 5, 3 / 4, 3 / 5, and 1 / 2. In addition, Trolox, DFX, and both of them (Trolox+DFX) were added to these. The viability and cell death after refrigerated storage for 6 days and 24-hour recovery culture with such preservation solutions were investigated ( Figure 16A and 16B). The viability in the figure is expressed as a relative value with the viability of the substance with 2 mM Trolox added to HTMX ("HTMX_Trx2K") being 1 ( Figure 16A ). Figure 16A The compositions of the respective preservation solutions indicated by marks at the lower part of and 16B are as follows.

[0262] "HTMX_Trx2K": HTMX(-)+Trolox 2 mM

[0263] "HTMX_DFX100": HTMX(-)+DFX 100 μM

[0264] "HTMX_T&D": HTMX(-)+Trolox 2 mM+DFX 100 μM

[0265] "4 / 5_S_(-)": HTMX(-)×4 / 5+sorbitol 60 μM

[0266] "4 / 5_S_Trx2K": HTMX(-)×4 / 5+sorbitol 60 μM+Trolox 2 mM

[0267] "4 / 5_S_DFX100": HTMX(-)×4 / 5+sorbitol 60 μM+DFX 100 μM

[0268] "4 / 5_S_T&D": HTMX(-)×4 / 5+sorbitol 60 μM+Trolox 2 mM+DFX 100 μM

[0269] 「3 / 4_S_(-)」: HTMX(-) × 3 / 4 + Sorbitol 75μm

[0270] 「3 / 4_S_Trx2K」: HTMX(-) × 3 / 4 + Sorbitol 75μm + Trolox 2mM

[0271] 「3 / 4_S_DFX100」: HTMX(-) × 3 / 4 + Sorbitol 75μm + DFX 100μm

[0272] 「3 / 4_S_T&D」: HTMX(-) × 3 / 4 + Sorbitol 75μm + Trolox 2mM + DFX 100μm

[0273] 「3 / 5_S_(-)」: HTMX(-) × 3 / 5 + Sorbitol 120μm

[0274] 「3 / 5_S_Trx2K」: HTMX(-) × 3 / 5 + Sorbitol 120μm + Trolox 2mM

[0275] 「3 / 5_S_DFX100」: HTMX(-) × 3 / 5 + Sorbitol 120μm + DFX 100μm

[0276] 「3 / 5_S_T&D」: HTMX(-) × 3 / 5 + Sorbitol 120μm + Trolox 2mM + DFX 100μm

[0277] 「1 / 2_S_(-)」: HTMX(-) × 1 / 2 + Sorbitol 150μm

[0278] 「1 / 2_S_Trx2K」: HTMX(-) × 1 / 2 + Sorbitol 150μm + Trolox 2mM

[0279] 「1 / 2_S_DFX100」: HTMX(-) × 1 / 2 + Sorbitol 150μm + DFX 100μm

[0280] 「1 / 2_S_T&D」: HTMX(-) × 1 / 2 + Sorbitol 150μm + Trolox 2mM + DFX 100μm

[0281] From Figure 16AFrom the results, it can be seen that the viability increases with the increase in the addition amount of sorbitol. In addition, particularly in the DFX addition region ("4 / 5_S_DFX100", "3 / 5_S_DFX100", "1 / 2_S_DFX100") and the TRX+DFX addition region ("4 / 5_S_T&D", "3 / 5_S_T&D", "1 / 2_S_T&D"), high viability is shown. As Figure 16B shown, regarding cell death, a tendency to decrease with an increase in the sorbitol addition amount was also found, and the Trx+DFX addition region showed the lowest value.

[0282] Figure 17A And 17B shows the changes in viability and cell death during cold storage for 3 to 6 days in the experimental regions using Figures 16A - 16B "HTMX_Trx2K" (HTMX(-)+Trolox 2 mM), "HTMX_T&D" (HTMX(-)+Trolox 2 mM+DFX 100 μM), and "4 / 5_S_T&D" (HTMX(-)×4 / 5+Sorbitol 60 μM+DFX 100 μM) in Figure 17A ). Regarding the viability, about 80% viability was approximately 3 days in the substance with Torlox added to HTMX ("HTMX_Trx2K"). In contrast, the 80% survival days were 5 days in the HTMX+Trx+DFX combined use region ("HTMX_T&D"), and further, the 80% survival days were 6 days in the HTMX 4 / 5 dilution+Sorbitol 60 mM+Trx+DFX addition region ("4 / 5_S_T&D") ( Figure 17A ). Regarding Figure 17B the changes in cell death shown, compared with the single addition of HTMX+Trx ("HTMX_Trx2K"), both the "HTMX+Trx+DFX" ( Figure 17A and 17B "HTMX_T&D") addition region and the "HTMXSA" 4 / 5 dilution Sorbitol+Trx+DFX addition region ("HTMX_T&D") showed lower values. Based on these results, adding sorbitol to HTMX in combination with Trolox and DFX shows more inhibition of stress during cold storage and shows high viability.

[0283] Finally, to improve the effect of sorbitol addition, a new composition "HTMXSA" (HTMX(-)×1 / 2+Sorbitol 150 μM) containing 150 mM sorbitol was prepared to optimize the composition of HTMX. Figure 18The results of an investigation into the viability of HTMXA stored refrigerated for 7 to 14 days are shown. For "HTMX_Trx2K" with a single dose of Trx added to HTMX without Sorbitol, the 80% survival days were approximately 3 days. In contrast, in "HTMXSA+Trx" (HTMX(-)×1 / 2 + Sorbitol 150μm + Trolox 2mM) with Trolox added to the Sorbitol-containing preservation solution "HTMXSA", the survival days were approximately twice as long, at around 6 days (dashed line). In the Trx+DFX two-agent combined addition area "HTMXSA+Trx+DFX" (HTMX(-)×1 / 2 + Sorbitol 150μm + Trolox 2mM + DFX 100μm), the 80% survival days were approximately 11 days. That is, in the case of "HTMX+Trx+DFX" ("HTMX_T&D") without Sorbitol, it was approximately 5 days, while in the case of "HTMXSA+Trx+DFX" with 150mM of Sorbitol, it was approximately 11 days, showing a preservation period approximately twice as long.

[0284] The above results indicate that by adding Trolox (Trx) and Deverasirox (DFX), which are agents at different stages in ferroptosis and stress as controls, to the HTMX modified preservation solution (HTMXSA) with Soribitol added, unprecedented long-term refrigerated preservation can be achieved.

Claims

1. A refrigerated preservation solution for preserving human or animal stem cells, embryos, other cells or biological tissue in a non-frozen state, characterized in that, Comprising: A basic cryopreservation solution; At least one first additive selected from deferoxamine (DFO), deferasirox (DFX), deferiprone (DFP), ferrostatin-1 (FST), edaravone (EDV), probucol (PBC), deferoxamine (DFO), ebselen (EBS), and idebenone (IDB) at a concentration of 1 - 300 μM; And A second additive which is water-soluble vitamin E (Trolox), vitamin E or its water-soluble analog / derivative at a concentration of 0.5 - 8 mM; For the described basic cryopreservation solution, the content of potassium ion species is 30 to 100 mmol / L, the content of sodium ion species is 30 to 100 mmol / L, and the ion-based molar ratio (Na + / K + ) of the amount of sodium ion species relative to the amount of potassium ion species is 0.5 to 1.

5. Containing adenine or its salt or derivative at a concentration of 0.1 - 4.2 mM, Containing all or all except one, two, or three essential amino acids, with the total content of essential amino acids being 3 - 300 mg / L, The total content of non-essential amino acids being 30 - 300 mg / L, The basic cryopreservation solution is undiluted or diluted to 0.4 - 0.9 times.

2. The cryopreservation solution according to claim 1, which Further contains 10 - 300 μM of sorbitol.

3. The cryopreservation solution according to claim 1, wherein The basic cryopreservation solution is diluted at a dilution rate X1 of 0.4 - 0.9 times and contains, Further contains sorbitol, The content of sorbitol is within the range of 40 - 60 mM multiplied by a specified magnification factor X2, The specified magnification factor X2 is 1.2 when the dilution rate X1 is 0.8 times, 1.5 when the dilution rate X1 is 0.75 times, 2.0 when the dilution rate X1 is 0.6 times, 3.0 when the dilution rate X1 is 0.5 times, and for other dilution rates, it is determined by interpolation or extrapolation.

4. The cryopreservation solution according to any one of claims 1 to 3, characterized in that The cryopreservation solution contains: (i) lacturonic acid or its salt in terms of lactone conversion at 30 - 100 mmol / L, (ii) crude saccharide hydrate at 10 - 30 mmol / L, (iii) aromatic alcohol at 0.3 - 1 mmol / L, (iv) glutathione (total glutathione) at 1 - 3 mmol / L, (v) adenosine at 2 - 10 mmol / L, (vi) lipoic acid at 0.1 - 0.5 μmol / L, (vii) sodium pyruvate at 0.1 - 0.7 mmol / L, (viii) glucose at 1 - 5 mmol / L, (ix) vitamins other than vitamin E or their water-soluble analogs / derivatives (including folic acid, nicotinamide, riboflavin, B12, choline, inositol, pantothenic acid, pyridinecarboxylic acid phosphate, thiamine), (x) a total of 50 - 200 mg / L of essential amino acids, and (xi) a total of 100 - 500 mg / L of non-essential amino acids.

5. The cold storage preservation solution according to claim 1 or 2, wherein, The concentration of the additive compound should be within the following ranges for the compound type of the additive compound: Edaravone (EDV): 10 - 100 μM, Probucol (PBC): 100 - 1000 μM, Deferoxamine (DFO): 10 - 100 μM, Deferiprone (DFP): 100 - 1000 μM, Deferasirox (DFX): 10 - 100 μM, Ebselen (EBS): 0.1 - 3 μM, Idebenone (IDB): 0.3 - 3 μM, Necrostatin (NST): 10 - 100 μM, Ferrostatin-1 (FST): 0.3 - 3 μM.

6. The cold storage preservation solution according to any one of claims 1 to 3, characterized in that it contains 20 - 50 g / L hydroxyethyl starch, 5 - 50 g / L polyvinyl alcohol and / or 30 - 90 mmol / L mannitol.

7. A cold storage preservation method for preserving human or animal stem cells or embryos in a non-frozen state in the cold storage preservation solution according to any one of claims 1 to 3, characterized in that not only during the preservation at low temperature, but also during the re-culture after cold storage, the state of immersing the stem cells or embryos in the cold storage preservation solution is maintained.

8. The cold storage preservation agent according to any one of claims 1 to 3, wherein the biological tissue or organ includes skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta or pancreas.

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