DMSO-FREE CRYOPRESERVATION SOLUTION AND PREPARATION METHOD AND USE THEREOF

Abstract

A cryopreservation solution contains 0.01-50.0 g of bionic ice control materials, 5.0-30 mL of polyols, 1-30 g of water-soluble sugar, and 0-30 mL of serum, and a buffer in every 100 mL of the cryopreservation solution. It does not contain DMSO. When being used for the cryopreservation of mouse oocytes and embryos, the solution may achieve the same or an even higher cell and tissue survival rate and functional expression stability as or than a commercial cryopreservation solution (containing 15% DMSO), and has high preservation efficiency. The cryopreservation solution without DMSO or serum reduces parasitic biological contaminants in the commercial cryopreservation solution containing serum currently used in clinical practice.

Claims

1. A DMSO-free cryopreservation solution, comprising, per 100 mL, 0.01-50 g of a biomimetic ice growth inhibition material, 5.0-45 mL of a polyalcohol, a water-soluble saccharide at 0.1-1.0 mol L.sup.−1, 0-30 mL of serum and the balance of a buffer, wherein the biomimetic ice growth inhibition material is selected from a PVA and/or an amino acid biomimetic ice growth inhibition material.

2. The cryopreservation solution according to claim 1, wherein the amino acid biomimetic ice growth inhibition material is selected from one or two or more of a polyamino acid with a degree of polymerization ≥2, an amino acid and a peptide compound; preferably, the peptide compound is a polypeptide, a glycopeptide derivative or a compound of formula (I): ##STR00006## wherein R is selected from substituted or unsubstituted alkyl, and the substituent may be selected from —OH, —NH.sub.2, —COOH, —CONH.sub.2 and the like; for example, R is substituted or unsubstituted C.sub.1-6 alkyl, and preferably R is —CH.sub.3, —CH.sub.2CH.sub.3 or —CH.sub.2CH.sub.2COOH; n is an integer greater than or equal to 1 and less than or equal to 1000.

3. The cryopreservation solution according to claim 1, wherein the ice growth inhibition material is a PVA, and the content of the PVA is 0.1-6.0 g; preferably, the polyalcohol may be C2-5 polyalcohol, such as any one of ethylene glycol, propylene glycol and glycerol; preferably, the water-soluble saccharide may be at least one of a non-reducing disaccharide, a water-soluble polysaccharide and a glycoside, for example, selected from sucrose, water-soluble cellulose (e.g., hydroxypropyl methylcellulose), trehalose and polysucrose; preferably, the buffer may be at least one of DPBS, hepes-buffered HTF buffer and other cell buffers; according to the present invention, the serum can be selected from human serum albumin or a substitute thereof, such as sodium dodecyl sulfate, for a human-derived cryopreservation object, and can be fetal bovine serum or bovine serum albumin for a non-human-derived cryopreservation object.

4. The cryopreservation solution according to claim 1, wherein the biomimetic ice growth inhibition material is a polyamino acid or an amino acid, and the content of the ice growth inhibition material is 0.01-50 g; preferably, the ice growth inhibition material is a combination of a PVA and an amino acid and/or a polyamino acid, for example consists of 0.1-5.0 g of the PVA, 8.0-35 g of the amino acid and/or 1.0-9.0 g of the polyamino acid.

5. The cryopreservation solution according to claim 1, wherein the PVA is selected from one of or a combination of two or more of an isotactic PVA, a syndiotactic PVA and an atactic PVA; for example, the PVA has a syndiotacticity of 15%-60%, preferably 50%-60%; preferably, the PVA is selected from a PVA having a molecular weight of 10-500 kDa or higher; preferably, the PVA is selected from a PVA having a degree of hydrolysis of greater than 80%; preferably, the polyamino acid is a homopolymer (with a degree of polymerization ≥2) of at least one selected from lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine and the like; preferably, the polypeptide is selected from a peptide consisting of 2 to 8 different amino acids, such as a dipeptide, a tripeptide or a tetrapeptide, for example selected from one or two or more of L-Thr-L-Arg (TR), L-Thr-L-Pro (TP), L-Arg-L-Thr (RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), and L-Ala-L-Ala-L-Thr (AAT); preferably, the glycopeptide derivative is synthesized by a saccharide and an amino acid, for example is a molecule consisting of gluconolactone (GDL) and an ice-philic amino acid through chemical bonding, and for example is at least one of GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Val and GDL-L-Ser.

6. The cryopreservation solution according to claim 1, wherein the content of the polyalcohol content is 6.0-28 mL; preferably, the content of serum is 0; preferably, the content of the water-soluble saccharide is 0.1-1.0 mol L.sup.−1; preferably, the pH of the cryopreservation solution is 6.5-7.6.

7. A preparation method of the cryopreservation solution according to claim 1, comprising: dissolving an ice growth inhibition material in a buffer, cooling to room temperature before adjusting the pH, dissolving other components in the rest buffer, and mixing after cooling; wherein preferably, the preparation method comprises: (1) dissolving a PVA in a portion of a buffer and cooling to room temperature before adjusting the pH to give a solution 1; (2) optionally, dissolving a polyamino acid or an amino acid in a portion of the buffer and cooling to room temperature before adjusting the pH to form a solution 2; (3) dissolving a water-soluble saccharide in a portion of the buffer and adding other components except serum after the water-soluble saccharide is completely dissolved to prepare a solution 3; and (4) mixing the solution 1, optionally the solution 2 and the solution 3 after they are cooled to room temperature, and adjusting the pH and making up to a predetermined volume with the buffer to give the cryopreservation solution; optionally, when the cryopreservation solution comprises serum, the serum is added when the cryopreservation solution is used.

8. A DMSO-free freezing equilibration solution comprising, per 100 mL, 0-5.0 g of a PVA, 5.0-45 mL of a polyalcohol, 0-30 mL of serum and the balance of a buffer; wherein preferably, the content of the PVA is 0.1-4.0 g.

9. The freezing equilibration solution according to claim 8, wherein the freezing equilibration solution comprises, per 100 mL, 7.5-15 mL of the polyalcohol, 10-20 mL of the serum and the balance of the buffer; preferably, the freezing equilibration solution comprises, per 100 mL, 0.5-3.5 g of the PVA, 7.5-15 mL of the polyalcohol and the balance of the buffer.

10. A DMSO-free cryopreservation reagent comprising the cryopreservation solution according to claim 1, wherein the cryopreservation solution and the freezing equilibration solution are independently present; preferably, the content of serum is 0, and the freezing equilibration solution comprises, per 100 mL, 0.5-2.5 g of a PVA, 7.5-15 mL of a polyalcohol and the balance of a buffer.

11. Use of the cryopreservation solution according to claim 1 and the freezing equilibration solution thereof in cryopreservation of biological tissues.

12. The use according to claim 11, wherein the biological tissue is selected from at least one of an oocyte, an embryo, a stem cell, an organ and tissue.

13. A DMSO-free cryopreservation reagent comprising the cryopreservation solution according to claim 8, wherein the cryopreservation solution and the freezing equilibration solution are independently present; preferably, the content of serum is 0, and the freezing equilibration solution comprises, per 100 mL, 0.5-2.5 g of a PVA, 7.5-15 mL of a polyalcohol and the balance of a buffer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0134] FIG. 1 is a picture of a stained slice of a fresh (unfrozen) ovarian organ of a 3-day newborn mouse;

[0135] FIG. 2 is a picture of a stained slice of the cryopreserved intact ovarian organ of Comparative Embodiment 7 after thawing;

[0136] FIG. 3 is a picture of a stained slice of the cryopreserved intact ovarian organ of Application Embodiment 14 after thawing;

[0137] FIG. 4 is a picture of a stained slice of the cryopreserved intact ovarian organ of Application Embodiment 15 after thawing;

[0138] FIG. 5 is a picture of a stained slice of the cryopreserved ovarian organ of Embodiment 16 after thawing;

[0139] FIG. 6 is a picture of a stained slice of fresh (unfrozen) ovarian tissue of a sexually mature mouse;

[0140] FIG. 7 is a picture of a stained slice of the cryopreserved ovarian tissue of Comparative Embodiment 8 after thawing;

[0141] FIG. 8 is a picture of a stained slice of the cryopreserved ovarian tissue of Application Embodiment 17 after thawing;

[0142] FIG. 9 is a picture of a stained slice of the cryopreserved ovarian tissue of Application Embodiment 18 after thawing; and

[0143] FIG. 10 is a picture of a stained slice of the cryopreserved ovarian tissue of Application Embodiment 19 after thawing.

DETAILED DESCRIPTION

[0144] The preparation method of the present invention will be further illustrated in detail with reference to the following specific examples. It should be understood that the following examples are merely exemplary illustration and explanation of the present invention, and should not be construed as limiting the protection scope of the present invention. All techniques implemented based on the aforementioned contents of the present invention are encompassed within the protection scope of the present invention.

[0145] Unless otherwise stated, the experimental methods used in the following examples are conventional methods. Unless otherwise stated, the reagents, materials, and the like used in the following examples are commercially available.

[0146] The PVA used in the examples of the present invention has a syndiotacticity of 50%-55%, a molecular weight of 13-23 kDa and a degree of hydrolysis of 98%.

[0147] In the examples of the present invention, poly-L-proline used in the freezing solution has a degree of polymerization of 8 or 15 and a molecular weight of 795 or 1475, and poly-L-arginine used has a degree of polymerization of 8 and a molecular weight of 1267. Poly-L-proline in a thawing solution has a degree of polymerization of 8 and a molecular weight of 795.

[0148] The survival rate in the examples of the present invention is the average survival rate of 3-12 repeated experiments.

Example 1. Cryopreservation of Mouse Oocytes and Embryos

[0149] 1. Preparation of cryopreservation solutions: the cryopreservation solutions were prepared according to the following formulations

[0150] A cryopreservation solution A comprises, per 100 mL, the following components:

TABLE-US-00001 Substances Content Poly-L-proline (g) 1.5 PVA (g) 2.0 Ethylene glycol (mL) 10 Sucrose (mol .Math. L.sup.−1) 0.5 DPBS (mL) Balance

[0151] 2.0 g of a PVA was dissolved in 25 mL of DPBS in a water bath at 80° C. by heating magnetic stirring, and the pH was adjusted to 7.0 to give a solution 1; 1.5 g of poly-L-proline was ultrasonically dissolved in another 20 mL, of DPBS, and the pH was adjusted to 7.0 to give a solution 2; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L:.sup.−1) was ultrasonically dissolved in 25 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 3; after returning to room temperature, the solution 1, the solution 2 and the solution 3 were well mixed, the pH was adjusted to 7.0, and the volume was made up to a total volume of 100 mL using DPBS to give the cryopreservation solution A for later use.

[0152] A cryopreservation solution B comprises, per 100 mL, the following components:

TABLE-US-00002 Substances Content L-Arg (g) 8.0 L-Thr (g) 4.0 PVA (g) 2.0 Ethylene glycol (mL) 10 Sucrose (mol .Math. L.sup.−1) 0.5 Fetal bovine serum (mL) 20 DPBS (mL) Balance

[0153] Steps of preparing the cryopreservation solution B: 2.0 g of a PVA was dissolved in 20 mL of DPBS in a water bath at 80° C. by heating and magnetic stirring, and the pH was adjusted to 7.1 to give a solution 1; 8.0 g of L-Arg and 4.0 g of L-Thr were dissolved in 20 mL of DPBS, and the pH was adjusted to 7.1 to give a solution 2; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L.sup.−1) was ultrasonically dissolved in 20 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 3; after returning to room temperature, the solution 1, the solution 2 and the solution 3 were well mixed, the pH value was adjusted to 7.1, and the volume was made up to 80% of the total volume with DPBS; 20 mL of serum was added when the cryopreservation solution was used.

[0154] A cryopreservation solution C

[0155] comprises, per 100 mL, the following components:

TABLE-US-00003 Substances Content PVA (g) 2.0 Ethylene glycol (mL) 10 Sucrose (mol .Math. L.sup.−1) 0.5 Fetal bovine serum (mL) 20 DPBS (mL) Balance

[0156] 2.0 g of a PVA was dissolved in 25 mL of DPBS in a water bath at 80° C. by heating magnetic stirring, and the pH was adjusted to 6.9 to give a solution 1; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L.sup.−1) was ultrasonically dissolved in 25 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 2; after returning to room temperature, the solution 1 and the solution 2 were well mixed, the pH was adjusted, and the volume was made up to 80% of the total volume; 20 mL of serum was stored separately and added when the cryopreservation solution was used.

[0157] A cryopreservation solution D

[0158] comprises, per 100 mL, the following components:

TABLE-US-00004 Substances Content PVA (g) 7.0 Ethylene glycol (mL) 10 Sucrose (mol .Math. L.sup.−1) 0.5 DPBS (mL) Balance

[0159] 2.0 g of a PVA was dissolved in 30 mL of DPBS in a water bath at 80° C. by heating and magnetic stirring, and the pH was adjusted to 7.0 to give a solution 1; 17 g (0.05 mol) of sucrose (the final concentration of the sucrose in the cryopreservation solution was 0.5 mol L.sup.−1) was ultrasonically dissolved in 25 mL of DPBS, and after the sucrose was completely dissolved, 10 mL of ethylene glycol was added to give a solution 2; after returning to room temperature, the solution 1 and the solution 2 were well mixed, the pH was adjusted, and the volume was made up to a total volume of 100 mL to give the cryopreservation solution D for later use.

[0160] 2. Preparation of freezing equilibration solutions: the freezing equilibration solutions were prepared according to the following formulations

[0161] Freezing equilibration solution a: 2.0 g of a PVA was dissolved in 50 mL of DPBS in a water bath at 80° C. by heating magnetic stirring, the pH was adjusted to 7.0 after the PVA was completely dissolved, 7.5 mL of ethylene glycol was added, all the components were well mixed, and the volume was made up to 100 mL with DPBS to give the freezing equilibration solution a for later use. Freezing equilibration solution b (total volume: 100 mL): 7.5 mL of ethylene glycol was dissolved in 72.5 mL of DPBS and well mixed, and 20 mL of serum was added when the freezing equilibration solution was used.

Comparative Example 1

[0162] A freezing equilibration solution 1# comprises, per 1 mL, 7.5% (v/v) of DMSO, 7.5% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum and the balance of DPBS.

[0163] A cryopreservation solution 1# comprises, per 1 mL, 15% (v/v) of DMSO, 15% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum, 0.5 M sucrose and the balance of DPBS.

[0164] Freezing equilibration solution b comprises, per 1 mL, 7.5% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum and the balance of DPBS.

[0165] A cryopreservation solution 2# comprises, per 1 mL, 10% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum, 0.5 M sucrose and the balance of DPBS.

[0166] The three formulations of the thawing solutions used in the Example 1 and Comparative Example 1 disclosed herein were as follows:

[0167] A thawing solution 1# comprises a thawing solution I (comprising sucrose at 1.0 mol L.sup.−1, 20% of serum, and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol.sup.L−1, 20% of serum, and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol L.sup.−1, 20% of serum, and the balance of DPBS), and a thawing solution IV (20% of serum, and the balance of DPBS).

[0168] A thawing solution 2# comprises a thawing solution I (comprising sucrose at 1.0 mol.Math.L.sup.−1, a PVA at 20 mg.Math.mL.sup.−1 and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol.Math.L.sup.−1, a PVA at 20 mg.Math.mL.sup.4 and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol.Math.L.sup.−1, a PVA at 20 mg mL.sup.−1 and the balance of DPBS), and a thawing solution IV (comprising a PVA at 20 mg.Math.mL.sup.−1 and the balance of DPBS).

[0169] A thawing solution 3# comprises a thawing solution I (comprising sucrose at 1.0 mol L.sup.−1, a PVA at 20 mg polyproline at 10 mg mL.sup.−1, and the balance of DPBS), a thawing solution If (comprising sucrose at 0.5 mol L.sup.−1, a PVA at 20 mg mL.sup.−1, polyproline at 5.0 mg mL.sup.−1, and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol mL.sup.−1, a PVA at 20 mg mL.sup.−1, polyproline at 2.5 mg mL.sup.−1, and the balance of DPBS), and a thawing solution IV (a PVA at 20 mg mL.sup.−1 and the balance of DPBS).

Application Example 1

[0170] The freezing equilibration solutions and the cryopreservation solutions of the example and comparative example described above were used to cryopreserve oocytes and embryos according to the schemes in Table 1 and. Table 2, respectively.

1. Cryopreservation of Oocytes

[0171] Mouse oocytes were firstly equilibrated in a freezing equilibration solution for 5 min, and then equilibrated in the prepared cryopreservation solution for 1 min. The oocytes equilibrated in the cryopreservation solution were loaded onto straws, and the straws were quickly put into liquid nitrogen (−196° C.) and then closed for cryopreservation. At the time of thawing, the cryopreserved oocytes were equilibrated in the thawing solution I at 37° C. for 5 min, and then equilibrated in the thawing solutions II-IV in sequence for 3 min each. After the thawed oocytes were incubated for 21 h, the number of the survived cells was observed, and the survival rates were calculated (see Table 1).

2. Cryopreservation of Embryos

[0172] Mouse embryos were firstly equilibrated in a freezing equilibration solution for 5 min, and then equilibrated in the prepared cryopreservation solution for 50 s. The embryos equilibrated in the cryopreservation solution were loaded onto straws, and the straws were quickly put into liquid nitrogen (−196° C.) and closed for cryopreservation. At the time of thawing, the frozen embryos were equilibrated in the thawing solution I at 37° C. for 3 min, and then equilibrated in the thawing solutions II-IV in sequence for 3 min each. After the thawed embryos were incubated for 2 h, the number of survived embryos was observed, and the survival rates were calculated (see Table 2).

TABLE-US-00005 TABLE 1 Survival rates of cryopreserved mouse oocytes Total number Survival Equilibration Cryopreservation Thawing of frozen rates after No. solution solution solution oocytes 2 h Application a A Thawing 39 89.7% Embodiment 1 solution 1# Application a A Thawing 60 98.6% Embodiment 2 solution 3# Application b B Thawing 109 94.8% Embodiment 3 solution 1# Application b C Thawing 90 97.7% Embodiment 4 solution 1# Application a D Thawing 50 93.4% Embodiment 5 solution 1# Application a D Thawing 53 96.5% Embodiment 6 solution 2# Comparative Equilibration Freezing solution Thawing 146 95   Embodiment 1 solution 1# 1# solution 1# Comparative b Freezing solution Thawing 96 81.9% Embodiment 2 2# solution 1#

TABLE-US-00006 TABLE 2 Survival rates of cryopreserved mouse embryos Survival Equilibration Cryopreservation Thawing Total number of rates after No. solution solution solution frozen embryos 2 h Application a A Thawing 42 95.2% Embodiment 7 solution 1# Application a D Thawing 41 95.8% Embodiment 8 solution 1# Comparative Equilibration Freezing Thawing 38 94.3% Embodiment 3 solution 1# solution 1# solution 1# Comparative b Freezing Thawing 39 82.4% Embodiment 4 solution 2# solution 1#

[0173] According to the data of each of Example 1 and Comparative Example 1, it can he seen that although no DMSO is added, the DMSO-free cryopreservation solution and freezing equilibration solution disclosed herein, through the synergy of all the components, still have good cryopreservation effects on oocytes and embryos, and overcome the defect that existing cryopreservation solutions are toxic to cells or embryos due to the addition of DMSO at a relatively high concentration. Moreover, as can be seen from Application Embodiments 2 and 6-8, when the equilibration solution, the freezing solution and the thawing solution all are free of serum and DMSO, the survival rates of cryopreserved oocytes and embryos can be superior to those of existing commercial cryopreservation solutions under the combined action of the biomimetic ice growth inhibition material disclosed herein, a permeable protectant, namely ethylene glycol, and the like, The problems are further solved that the commercial cryopreservation solutions commonly used in clinical practice at present are short in shelf life and prone to introduce parasitic biological contaminants due to the presence of serum.

Example 2. Cryopreservation of Human Umbilical Cord Mesenchymal Stem Cells

[0174] 1. Preparation of cryopreservation solutions: cryopreservation solutions were prepared according to the following formulations.

[0175] A cryopreservation solution E (total volume: 100 mL) comprises 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L.sup.−1), 4.0 g of poly-L-arginine (with a degree of polymerization of 8), 1.0 g of a PVA, and the balance of DPBS.

[0176] A cryopreservation solution F (total volume: 100 mL) comprises 20 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L.sup.−1), 16 g of L-Arg, 8.0 g of L-Thr, and the balance of DPBS.

[0177] A cryopreservation solution G (total volume: 100 mL) comprises 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L.sup.−1), 2.0 g of a PVA, and the balance of DPBS.

[0178] A cryopreservation solution H (total volume: 100 mL) comprises 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L.sup.−1), 28 g of TR, and the balance of DPBS.

[0179] A cryopreservation solution I (total volume: 100 mL) comprises 10 mL of ethylene glycol, 17 g of sucrose (0.5 mol L.sup.−1), 2.0 g of a PVA, and the balance of DPBS.

[0180] The formulating methods of the cryopreservation solutions were same as those in Example 1.

[0181] A method of preparing TR is as follows:

[0182] (1) 2-chlorotrityl chloride resin was placed into a reaction tube, and added with DCM (20 mL.Math.g.sup.−1). The resulting mixture was shaken for 30 min. With the use of a sand-core funnel, the solvent was removed with suction. The residue was added with a three-fold molar excess of Fmoc-L-Thr(tBu)-OH and an eight-fold molar excess of DIEA, and finally added with DMF to dissolve. The resulting mixture was shaken for 30 min, Methanol was used for end-capping for 30 min.

[0183] (2) The solvent MIT was removed. 20% piperidine/DMF solution (10 mL.Math.g.sup.−1) was added, and the solvent was removed after 5 min; 20% piperidine/DMF solution (10 mL.Math.g.sup.−1) was added again, and the piperidine solution was removed after 15 min. A small amount of resin was taken and washed with ethanol three times, added with a ninhydrin reagent, and heated at 105-110° C. for 5 min. The color turned dark blue, which suggested a positive reaction.

[0184] (3) After the product obtained by the above reaction was sequentially washed with DMF (15 mL.Math.g.sup.−1, twice), methanol (15 mL.Math.g.sup.−1, twice) and DMF (15 mL.Math.g.sup.−1, twice), a two-fold excess of Fmoc-Arg(Pbf)-OH that was dissolved in as small an amount of DMF as possible was added to a reaction tube; a two-fold excess of HBTU was added. Immediately thereafter, an eight-fold excess of DIEA was added and reacted for 30 min.

[0185] (4) After the solution was removed with suction, a small amount of resin was taken and washed with ethanol three times, added with a ninhydrin reagent, and heated at 105-110° C. for 5 min. The colorless mixture suggested a negative reaction, that is, the reaction was complete.

[0186] (5) After the product obtained by the above reaction was sequentially washed with DMF (15 mL.Math.g.sup.−1, twice methanol (15 mL.Math.g.sup.−1, twice) and DMF (15 mL.Math.g.sup.−1, s.sup.−1, twice), the solvent was removed. 20% piperidine/DMF solution (10 mL.Math.g.sup.−1) was added, and the solvent was removed after 5 min; 20% piperidine/DMF solution (10 mL.Math.g.sup.−1) was added again, and the piperidine solution was removed after 15 min. A small amount of resin was taken and washed with ethanol, added with a ninhydrin reagent, and heated at 105-110° C. for 5 min. The color turned dark blue, which suggested a positive reaction.

[0187] (6) After the product obtained by the above reaction was sequentially washed with DMF (15 mL.Math.g.sup.−1, twice), methanol (15 mL.Math.g.sup.−1, twice) and DCM (15 mL.Math.g.sup.−1, twice), the resin was dried with suction.

[0188] (7) The product was cut using a cleaving liquid (15 mL.Math.g.sup.−1, TFA:water:EDT:Tis=95:1:2:2, v/v) for 90 min, The cleaving liquid was blown to dryness with nitrogen, and then lyophilized to give a crude product of polypeptide.

[0189] (8) The polypeptide was purified by HPLC and subjected to salt-conversion or desalting. HPLC: tR=4.8 mins (purification column model: Kromasil 100-5C18, 4.6 mm*250 mm; gradient eluent: acetonitrile with 0.1% TFA and aqueous solution with 0.1% TFA, 0 mins-1:99, 20 mins-1:4). The purified solution was lyophilized to give a finished product L-Thr-L-Arg (TR). The yield was about 80%. The mass spectrum presents [M+H].sup.+ at 276.2.

Comparative Example 2

[0190] A cryopreservation solution 3# comprises, per 1 mL: 10% (v/v) of DMSO, 15% (v/v) of fetal bovine serum, and the balance of a-MEM medium (C12571500BT, Invitrogen, USA).

[0191] Human umbilical cord mesenchymal stem cells were cryopreserved using the cryopreservation solutions described above according to the scheme in Table 3. The cryopreservation method of the human umbilical cord stem cells is specifically a microdroplet method, namely: human umbilical cord mesenchymal stem cells on a culture dish were digested using 25% pancreatin for 2 min, put into a culture solution (10% FBS+a-MEM culture medium) of the same volume, and gently pipetted until the stem cells completely fell off; the cells were added into a 1.5 mL, centrifuge tube for centrifuging for 5 min at 1000 rpm, and the supernatant was discarded to separate the cells from the culture medium; 10 μL of freezing solution was added to the bottom of the centrifuge tube, the stem cells were gently pipetted to disperse stem cell dusters, and 10 μL of freezing solution with the stem cells was placed on a freezing slide and then cryopreserved in liquid nitrogen (−196° C.). At the time of thawing, the freezing slide with the cells and the freezing solution was placed directly in the a-MFM medium at 37° C. for thawing. After thawing, cells were stained with trypan blue to observe the survival rates, and the number of cells was counted using an instrument JIMBIO-FIL, survival rate=number of live cells/total number of cells (see Table 3).

TABLE-US-00007 TABLE 3 Survival rates of cryopreserved human umbilical cord mesenchymal stem cells Cryopreservation Cryopreservation No. solution method Survival rates Application Cryopreservation Microdroplet 92.4% Embodiment 9 solution E method Application Cryopreservation Microdroplet 71.0% Embodiment 10 solution F method Application Cryopreservation Microdroplet 72.2% Embodiment 11 solution G method Application Cryopreservation Microdroplet 75.1% Embodiment 12 solution H method Application Cryopreservation Microdroplet 77.1% Embodiment 13 solution I method Comparative Cryopreservation Microdroplet 76.6% Embodiment 5 solution 3# method Comparative Cryopreservation Microdroplet 63.9% Embodiment 6 solution 1# method

[0192] When the cryopreservation solution disclosed herein is used for cryopreservation of the human umbilical cord mesenchymal stem cells, the survival rate of the stem cells can reach 92.4% (Application Embodiment 9) although no DMSO is added and can reach 77.1% (Application Embodiment 13) even when no DMSO and serum are added. This means that the cryopreservation reagent can achieve the same effectiveness as a conventional freezing solution in freezing stem cells, and has a post-thaw survival rate even far higher than that of a cryopreservation solution (Comparative Embodiment 5) comprising 10% of DMSO commonly used at present, and the cryopreservation effect of the PVA-based cryopreservation solution is remarkably superior to that of Comparative Embodiment 6 without PVA.

Example 3. Cryopreservation of Intact Ovarian Organs and Ovarian Tissue Slices

[0193] A cryopreservation solution J (total volume: 100 mL) comprises 10 mL of ethylene glycol, 17 sucrose (0.5 mol L.sup.−1), 2.0 of a PVA, and the balance of DPBS.

[0194] A cryopreservation solution K (total volume: 100 mL) comprises 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L.sup.−1), 1.0 g of a PVA, and the balance of DPBS.

[0195] A cryopreservation solution L comprises 10 mL of ethylene glycol, 20 mL of serum, 17 g of sucrose (0.5 mol L.sup.−1), 4.0 g of poly L-arginine (with a degree of polymerization of 8), 1.0 g of a PVA, and the balance of DPBS.

[0196] Comparative Example 3: a cryopreservation solution comprises, per 1 mL, 15% (v/v) of DMSO, 15% (v/v) of ethylene glycol, 20% (v/v) of serum, 0.5 M sucrose, and the balance of DPBS.

[0197] Freezing equilibration solution a: 2.0 g of a PVA was dissolved in 50 mL of DPBS in a water bath at 80° C. by heating magnetic stirring, the pH was adjusted to 7.0 after the PVA was completely dissolved, 7.5 mL of ethylene glycol was added, all the components were well mixed, the pH was adjusted, and the volume was made up to 100 mL to give the freezing equilibration solution a for later use.

[0198] Freezing equilibration solution b: 7.5 mL of ethylene glycol was added into 72.5 mL of DPBS and well mixed, and 20 mL of serum was added when the freezing equilibration solution was used.

Comparative Example 3:

[0199] A freezing equilibration solution 14 comprises, per 1 mL, 7.5% (v/v) of DMSO, 7.5% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum and the balance of DPBS.

[0200] A cryopreservation solution 14 comprises, per 1 mL: 15% (v/v) of DMSO, 15% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum, sucrose at 0.5 mol L.sup.−1, and the balance of DPBS.

[0201] A thawing solution 14 comprises a thawing solution I (comprising sucrose at 1.0 mol L.sup.−1, 20% of serum and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol L.sup.−1, 20% of serum and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol L.sup.−1, 20% of serum and the balance of DPBS), and a thawing solution IV (comprising 20% of serum and the balance of DPBS).

[0202] A thawing solution 2# comprises a thawing solution I (comprising sucrose at 1.0 mol L.sup.−1; a PVA at 20 mg.Math.mL.sup.−1 and the balance of DPBS), a thawing solution II (comprising sucrose at 0.5 mol.Math.L.sup.−1, a PVA at 20 mg.Math.mL.sup.−1 and the balance of DPBS), a thawing solution III (comprising sucrose at 0.25 mol.Math.L.sup.−1, a PVA at 20 mg mL.sup.−1 and the balance of DPBS), and a thawing solution IV (comprising a PVA at 20 mg.Math.mL.sup.−1 and the balance of DPBS).

[0203] The intact ovarian organs of mice newly born within 3 days and the ovarian tissue slices of sexually mature mice were cryopreserved using the cryopreservation solutions described above and the freezing equilibration solutions and cryopreservation solutions of the comparative example according to the schemes in Table 4 and Table 5.

[0204] The whole ovarian organs or ovarian tissue slices were firstly equilibrated in an equilibration solution at room temperature for 25 min, then equilibrated in the prepared cryopreservation solution for 15 min, and then loaded onto straws; the straws were put into liquid nitrogen for preservation. After thawing, the intact ovarian organs or ovarian tissue slices were incubated in a culture solution (10% FBS+a-MEM) in an incubator at 37° C./5% CO.sub.2 for 2 h for further thawing, and then fixed with 4% paraformaldehyde, embedded in paraffin and stained with HE for morphological observation. The results are shown in FIGS. 1-10. FIG. 1 is a picture of a slice of a fresh unfrozen ovarian organ, and FIG. 6 is a picture of a slice of a fresh unfrozen ovarian tissue.

TABLE-US-00008 TABLE 4 Ovarian organ cryopreservation scheme Equilibration Cryopreservation Thawing Mor- No. solution solution solution phology Application a J Thawing FIG. 3 Embodiment 14 solution 2# Application b K Thawing FIG. 4 Embodiment 15 solution 1# Application b L Thawing FIG. 5 Embodiment 16 solution 1# Comparative Equilibration Freezing Thawing FIG. 2 Embodiment 7 solution 1# solution 1# solution 1#

TABLE-US-00009 TABLE 5 Ovarian tissue cryopreservation scheme Equilibration Cryopreservation Thawing Mor- No. solution solution solution phology Application a J Thawing FIG. 8 Embodiment 17 solution 2# Application b K Thawing FIG. 9 Embodiment 18 solution 1# Application b L Thawing FIG. 10 Embodiment 19 solution 1# Comparative Equilibration Freezing Thawing FIG. 7 Embodiment 8 solution 1# solution 1# solution 1#

[0205] As can be seen from FIGS. 1-5, compared with Comparative Embodiment 7 free of the amino acid biomimetic ice growth inhibition material and fresh unfrozen ovarian organs, Examples 14-16 are characterized in that: the original follicle structure is relatively intact, the interstitial structure is relatively intact, the cytoplasm of cells is homogeneous and lightly stained in a relatively large amount, and nucleus shrinkage and deep staining are relatively mild; the structure of the vascular wall is intact, lumen collapse is mild, the cytoplasm of endothelial cells is homogeneous and lightly stained in a relatively large amount, and nucleus shrinkage and deep staining are relatively mild. It can be seen that Examples 14-16 have better cryopreservation effect on ovarian organs.

[0206] As can be seen from FIGS. 6-10, as compared with Comparative Embodiment 8 and fresh unfrozen ovarian tissue, the schemes of Examples 17-19 are characterized in that: the antral follicle structure is relatively intact, the interstitial structure is relatively intact, the cytoplasm of cells is homogeneous and lightly stained in a relatively large amount, and nucleus shrinkage and deep staining are relatively mild. It can be seen that the cryopreservation solutions disclosed herein have better effect on cryopreservation of ovarian tissues than the prior art.

[0207] The examples of the present invention have been described above. However, the present invention is not limited to the above examples. Any modification, equivalent, improvement and the like made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.