Use of a DMSO-free cryopreservation solution in organ and tissue cryopreservation

Abstract

This invention discloses the application of a DMSO-free cryopreservation solution in the cryopreservation of organs and / or tissues. The cryopreservation solution, per 100 mL volume, contains 0.01-50.0 g of a biomimetic ice-controlling material, 5.0-30 mL of polyol, 1-30 g of water-soluble sugar, 0-30 mL of serum, with the remainder being buffer solution. The biomimetic ice-controlling material is selected from PVA and / or amino acid-based biomimetic ice-controlling materials. The cryopreservation solution and cryoequilibration solution of this invention do not contain DMSO, exhibiting high preservation efficiency when used for cryopreservation of ovarian tissue or organs. Furthermore, the DMSO- and serum-free cryopreservation solution further solves the problems of poor stability and the potential introduction of parasitic biological contaminants caused by the presence of serum in currently widely used commercial cryopreservation solutions. The cryopreservation solution of this invention has a simple composition, readily available raw materials, and low cost, and can be widely used for the cryopreservation of various organs or tissues.

Description

[0001] This application claims priority to two earlier applications filed with the China National Intellectual Property Administration on April 9, 2019: Patent Application No. 201910281978.2, entitled "A DMSO-free cryopreservation solution and its preparation method," and Patent Application No. 201910281986.7, entitled "A peptide compound and a cryopreservation solution containing the compound." The full text of both earlier applications is incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedical materials technology, specifically relating to the application of a DMSO-free cryopreservation solution in the cryopreservation of organs and tissues. Background Technology

[0003] Since its inception, cryopreservation technology has become an indispensable research method in the natural sciences and has been widely adopted. With the improvement of living standards and the development of medical technology, the cryopreservation of human reproductive cells (sperm, oocytes), gonadal tissues, etc., has become an important means of preserving fertility. Furthermore, with the increasing aging of the world's population, the demand for cryopreservation of donated human cells, tissues, or organs that can be used for regenerative medicine and organ transplantation is also increasing rapidly. Therefore, how to efficiently cryopreserve precious cell, tissue, and organ resources has become an important issue in the life sciences.

[0004] Vitrification is currently the most commonly used cryopreservation method. While vitrification allows the intracellular and extracellular fluids to become glassy during rapid freezing, avoiding damage caused by ice crystal formation, existing cryopreservation reagents cannot effectively control ice crystal growth during thawing, thus damaging cells. Dimethyl sulfoxide (DMSO) is a commonly used solubilizer and permeabilizing cell cryopreservation agent in in vitro cell culture; however, DMSO has produced adverse side effects in clinical trials. Furthermore, DMSO is highly cytotoxic, and different cell types have varying sensitivities to DMSO concentrations, limiting the application of cryopreservation reagents with DMSO as the primary cryoprotectant. Currently, vitrification typically uses high concentrations (≥15%) of DMSO, severely impacting the survival rate of cryopreserved subjects after thawing, and even the safety and functional expression of (progeny) cells. In summary, currently used cryopreservation reagents lack the ability to effectively control ice crystal growth during thawing and also suffer from high reagent toxicity. Summary of the Invention

[0005] To overcome the aforementioned deficiencies of the prior art, the present invention provides a DMSO-free cryopreservation solution and its preparation method.

[0006] This invention provides the following technical solution:

[0007] A DMSO-free cryopreservation solution, per 100 mL volume, contains 0.01-50.0 g of biomimetic ice-controlling material, 5.0-45 mL of polyol, and 0.1-1 mol / L of water-soluble sugar. -1 The solution contains 0-30 mL of serum, with the remainder being buffer solution. The biomimetic ice-controlling material is selected from polyvinyl alcohol (PVA) and / or amino acid-based biomimetic ice-controlling materials. The cryopreservation solution does not contain dimethyl sulfoxide (DMSO).

[0008] According to the present invention, the amino acid-based biomimetic ice-controlling material is selected from one or more of polyamino acids (degree of polymerization ≥2, preferably 2 to 40, such as 6, 8, 15, 20, etc.), amino acids, and peptide compounds.

[0009] According to the present invention, the peptide compound is a polypeptide (preferably a peptide composed of 2-8 amino acids, such as dipeptide, tripeptide, tetrapeptide), a glycopeptide derivative, or a compound of formula (I).

[0010]

[0011] Wherein, R is selected from substituted or unsubstituted alkyl groups, and the substituted group can be selected from -OH, -NH2, -COOH, -CONH2, etc. For example, R is a substituted or unsubstituted C 1-6 Alkyl group, preferably R is -CH3, -CH2CH3, or -CH2CH2COOH; n is an integer greater than or equal to 1 and less than or equal to 1000, for example, an integer in the range of 1 to 100. In some embodiments of the present invention, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0012] According to the present invention, the polyol may be a C2-5 polyol, preferably a C2-C3 diol or triol, such as any one of ethylene glycol, propylene glycol, and glycerol.

[0013] According to the present invention, the water-soluble sugar may be at least one of non-reducing disaccharides, water-soluble polysaccharides, and sugar anhydrides, for example selected from sucrose, trehalose, water-soluble cellulose (e.g., hydroxypropyl methylcellulose), and polysucrose; preferably sucrose or hydroxypropyl methylcellulose. The water-soluble sugar can protect the cell membrane and prevent cell sedimentation.

[0014] According to the present invention, the buffer may be at least one of DPBS, hepes-buffered HTF buffer, or other cell culture buffers.

[0015] According to the present invention, the serum may be human serum albumin or its alternative, such as sodium dodecyl sulfate (SDS), for human cryopreservation subjects, and fetal bovine serum or bovine serum albumin may be selected for non-human cryopreservation subjects.

[0016] According to the cryopreservation solution of the present invention, the biomimetic ice-controlling material can be PVA, and the PVA content is 0.1-6.0g, for example 0.5-5.0g; specifically, it can be 1.0g, 2.0g, 3.0g, or 4.0g.

[0017] According to the cryopreservation solution of the present invention, the biomimetic ice-controlling material can be polyamino acid or amino acid, and the content of the polyamino acid or amino acid is 0.01-50g, for example 1.5-50g; specifically, it can be 8.0g, 10g, 15g, 20g, 30g, or 40g.

[0018] According to the cryopreservation solution of the present invention, the ice-controlling material can be a combination of PVA and polyamino acids, for example, composed of 0.1-5.0g of PVA and 1.0-9.0g of polyamino acids.

[0019] According to the cryopreservation solution of the present invention, the ice-controlling material can be a combination of PVA and amino acids, for example, composed of 0.1-5.0g of PVA and 8.0-35g of amino acids.

[0020] According to the cryopreservation solution of the present invention, the polyol content is 6.0-28 mL, for example 7.0-20 mL, or 10-15 mL per 100 mL.

[0021] According to the cryopreservation solution of the present invention, the serum content is 0.1-30 mL per 100 mL, for example 5.0-20 mL, 10-15 mL.

[0022] According to the cryopreservation solution of the present invention, the cryopreservation solution preferably has a serum content of 0 per 100 mL;

[0023] According to the cryopreservation solution of the present invention, the content of water-soluble sugar in each 100 mL of cryopreservation solution is 0.1-1.0 mol / L. -1 For example, 0.1-0.8 mol L -1 0.2-0.6 mol L -1 Specifically, for example, 0.25 mol L -1 0.5 mol L -1 1.0 mol L -1 .

[0024] The cryopreservation solution according to the present invention has a pH of 6.5-7.6, for example 6.9-7.2.

[0025] As one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL volume:

[0026]

[0027] Preferably, the cryopreservation solution consists of the following components per 100 mL volume:

[0028]

[0029] As one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL volume:

[0030]

[0031] Preferably, per 100 mL volume, it consists of the following components:

[0032]

[0033] As one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL volume:

[0034]

[0035] Preferably, the cryopreservation solution consists of the following components per 100 mL volume:

[0036]

[0037] As one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL volume:

[0038]

[0039]

[0040] Preferably, the cryopreservation solution consists of the following components per 100 mL volume:

[0041]

[0042] The present invention also provides a method for preparing the cryopreservation solution, comprising the following steps: dissolving the biomimetic ice-controlling material in a buffer solution, cooling to room temperature and adjusting the pH, dissolving the other components except serum in the remaining buffer solution, cooling and mixing, reconfirming or adjusting the pH and replenishing the remaining buffer solution, and adding serum when in use.

[0043] The preparation method according to the present invention includes the following steps:

[0044] (1) Dissolve PVA in a portion of the buffer solution, cool to room temperature and adjust the pH to obtain solution 1;

[0045] (2) Optionally, dissolve a polyamino acid or amino acid in a portion of a buffer solution, cool to room temperature, and adjust the pH to form solution 2.

[0046] (3) Dissolve the water-soluble sugar in another part of the buffer solution. After all the water-soluble sugar has dissolved, add other components except serum to prepare solution 3.

[0047] (4) After the solutions 1, 2, and 3 have cooled to room temperature, they are mixed, the pH value is adjusted, and the remaining buffer solution is added to the predetermined volume to obtain the cryopreservation solution.

[0048] The preparation method according to the present invention includes the following steps:

[0049] (1) Dissolve polyamino acids or amino acids in a portion of buffer solution, cool to room temperature and adjust pH to form solution 1;

[0050] (2) Optionally, PVA is dissolved in a portion of the buffer solution, cooled to room temperature, and the pH is adjusted to obtain solution 2;

[0051] (3) Dissolve the water-soluble sugar in another part of the buffer solution. After all the water-soluble sugar has dissolved, add the other components except serum to prepare solution 3.

[0052] (4) After the solutions 1, 2, and 3 have cooled to room temperature, they are mixed, the pH value is adjusted, and the remaining buffer solution is added to the predetermined volume to obtain the cryopreservation solution.

[0053] According to the preparation method of the present invention, when the cryopreservation solution contains serum, the serum is added when the cryopreservation solution is used.

[0054] According to the preparation method of the present invention, PVA dissolution involves dissolving PVA in a heated bath, such as a water bath or oil bath; for example, the water bath temperature is 60-95°C, preferably 80°C. Preferably, the dissolution of PVA includes a stirring step. In one embodiment, the dissolution of the polyamino acid or amino acid is ultrasound-assisted dissolution.

[0055] This invention provides a DMSO-free cryogenic equilibration solution, which contains 0-5.0g PVA, 0-15g polyamino acids, 5.0-45mL polyol, 0-30mL serum, and the remainder of buffer solution per 100mL volume.

[0056] According to the freezing equilibration solution of the present invention, the PVA content is 0.1-5.0g, preferably 0.5-2.5g, for example 0.1g, 0.5g, 1.0g, 2.0g.

[0057] According to the freezing equilibration solution of the present invention, the polyamino acid content is 0-15g, preferably 0.5-8g, for example 0.1g, 0.5g, 1.0g, 8.0g.

[0058] According to the cryoequilibration solution of the present invention, the polyol content is 6.0-28 mL, for example 7.0-20 mL, 10-15 mL.

[0059] According to the cryoequilibration solution of the present invention, the serum content is 0.1-30 mL, for example 5.0-20 mL, 10-15 mL. As one embodiment of the present invention, the serum content is 0.

[0060] As one embodiment of the present invention, the cryoequilibration solution contains 7.5-15 mL of polyol, 10-20 mL of serum, and the remainder of DPBS per 100 mL volume.

[0061] As one embodiment of the present invention, the cryoequilibration solution contains 1.0-5.0 g of PVA, 7.5-15 mL of polyol, and the remainder of buffer solution per 100 mL volume.

[0062] In the cryoequilibration solution of the present invention, the PVA, polyamino acids, polyols and serum can be selected from the types of corresponding components of the cryopreservation solution.

[0063] The present invention also provides a method for preparing the above-mentioned cryoequilibration solution, which includes dissolving each component in a buffer solution, storing the serum separately, and adding it when using.

[0064] The present invention further provides a DMSO-free cryopreservation reagent, comprising the above-mentioned cryoequilibration solution and the above-mentioned cryopreservation solution, wherein the cryoequilibration solution and the cryopreservation solution exist independently.

[0065] According to the cryopreservation reagent of the present invention, the serum content of the cryopreservation solution is 0, and the cryoequilibration solution contains 1.0-5.0 g of PVA, 7.5-15 mL of polyol, and the balance of buffer solution per 100 mL volume; preferably, the cryoequilibration solution contains 0.5-2.5 g of PVA, 7.5-15 mL of polyol, and the balance of buffer solution per 100 mL volume.

[0066] The cryopreservation reagent according to the present invention, wherein the cryoequilibration solution comprises, per 100 mL volume, the following components:

[0067]

[0068] The cryopreservation solution, based on a total volume of 100 mL, comprises the following components:

[0069]

[0070] According to the present invention, the PVA is selected from one or more combinations of isotactic PVA, anaregular PVA and random PVA, for example, the anaregularity of the PVA is 15%-60%, preferably 45%-60%, for example 50%-55%.

[0071] According to the present invention, the PVA may be selected from PVA with a molecular weight of 10-500kDa or higher, such as 10-30kDa, 30-50kDa, 80-90kDa, or 200-500kDa.

[0072] According to the present invention, the PVA may be selected from PVA with a degree of hydrolysis greater than 80%, for example, with a degree of hydrolysis of 80%-99%, 82-87%, 87%-89%, 89%-99%, or 98%-99%.

[0073] According to the present invention, the polyamino acid may be selected from at least one homopolymer (degree of polymerization ≥ 2) of lysine, arginine, proline, threonine, histidine, glutamine, aspartic acid, glycine, etc. For example, it may be one or a combination of two or more of poly-L-proline and poly-L-arginine. The polyamino acid preferably has a degree of polymerization of 2-40, more preferably 2-20, for example, a degree of polymerization of 6, 8, 15, 20, etc.

[0074] According to the present invention, the polypeptide is composed of two or more different amino acids, selected from one 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). The polypeptide can be prepared by polypeptide synthesis methods known in the art, such as solid-phase synthesis.

[0075] According to the present invention, the glycopeptide derivative is a molecule composed of gluconolactone (GDL) or other sugars and ionophilic amino acids through chemical bonding, such as GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Val, GDL-L-Ser, etc. The glycopeptide derivative can be synthesized by reaction methods of sugar compounds and amino acids known in the art, for example, by solid-phase synthesis or by reacting sugar compounds and amino acids in an organic solvent.

[0076] According to the present invention, the peptide compound has any of the structures shown in formula (1)-(8):

[0077]

[0078]

[0079] According to the present invention, the compound represented by formula (I) has any of the following structures:

[0080]

[0081] In the cryopreservation solution and cryoequilibration solution of this invention, the amount of each component is based on a total solution volume of 100 mL, with the remainder being buffer solution.

[0082] The present invention also provides the application of the above-mentioned cryopreservation solution or cryoequilibration solution or cryopreservation reagent for the cryopreservation of cells, tissues or organs; for example, for the cryopreservation of oocytes, sperm or stem cells, ovarian tissue, embryos or ovarian organs.

[0083] Preferably, the present invention provides the application of the cryopreservation solution in the cryopreservation of organs and / or tissues.

[0084] This invention provides the application of the cryoequilibration solution in the cryopreservation of organs and / or tissues.

[0085] This invention provides the application of the cryopreservation reagent in the cryopreservation of organs and / or tissues.

[0086] Furthermore, the application of the cryopreservation solution, cryoequilibration solution, or cryopreservation reagent described in this invention includes: equilibrating the organ and / or tissue in the cryoequilibration solution, then placing the organ and / or tissue in the cryopreservation solution, and then placing the organ and / or tissue on a cryocarp for cryopreservation in liquid nitrogen.

[0087] In one embodiment, the organ and / or tissue is ovarian tissue or an ovarian organ, such as a slice of ovarian tissue or a whole ovarian tissue.

[0088] Beneficial effects

[0089] The cryopreservation solution and cryoequilibration solution provided by this invention do not contain DMSO. When used for cryopreservation of mouse oocytes and embryos, they achieve cell and tissue viability and functional expression stability equal to or even higher than those of commercial cryopreservation solutions (containing 15% DMSO by volume), demonstrating high preservation efficiency. Furthermore, the cryopreservation solution, which contains neither DMSO nor serum, further solves the problems of poor stability and susceptibility to parasitic biological contaminants caused by the presence of serum in currently widely used commercial cryopreservation solutions. The cryopreservation solution of this invention has a simple composition, readily available raw materials, and low cost, making it widely applicable for the cryopreservation of tissues and organs. Attached Figure Description

[0090] Figure 1 A stained photograph of a fresh ovarian section from a 3-day-old newborn mouse.

[0091] Figure 2 For comparison, see stained section photographs of thawed intact ovarian organs from Example 7;

[0092] Figure 3 A stained section of a frozen, intact ovarian organ after thawing, as shown in Application Example 14.

[0093] Figure 4 A stained section of a frozen, intact ovarian organ after thawing, as shown in Application Example 15.

[0094] Figure 5 A stained section of a frozen, intact ovarian organ after thawing, as shown in Application Example 16.

[0095] Figure 6 A stained photograph of a fresh ovarian tissue section from a sexually mature mouse.

[0096] Figure 7 For comparison, here are stained images of thawed sections of frozen ovarian tissue from Example 8;

[0097] Figure 8 Photographs of stained sections of thawed frozen ovarian tissue from Application Example 17;

[0098] Figure 9 Photographs of stained sections of thawed frozen ovarian tissue from Application Example 18;

[0099] Figure 10 This is a stained photograph of a section of thawed ovarian tissue from Application Example 19. Detailed Implementation

[0100] The preparation method of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0101] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0102] The PVA used in the embodiments of the present invention has a syndiotactic degree of 50%-55%, a molecular weight of 13-23 kDa, and a degree of hydrolysis of 98%.

[0103] In this embodiment of the invention, the cryogenic fluid uses poly-L-proline with a degree of polymerization of 8 or 15 and a molecular weight of 795 and 1475; poly-L-arginine has a degree of polymerization of 8 and a molecular weight of 1267. The thawing fluid uses poly-L-proline with a degree of polymerization of 8 and a molecular weight of 795.

[0104] In this embodiment of the invention, the survival rate is the average survival rate of 3-12 repeated experiments.

[0105] Example 1. Cryopreservation of mouse oocytes and embryos

[0106] 1. Preparation of cryopreservation solution: Prepare the cryopreservation solution according to the following formula.

[0107] Cryopreservation solution A: Each 100ml contains the following components:

[0108] substance Dosage Poly-L-proline (g) 1.5 PVA(g) 2.0 Ethylene glycol (mL) 10 <![CDATA[sucrose (mol L -1 )]]> 0.5 DPBS (mL) margin

[0109] Dissolve 2.0 g of PVA in 25 mL of DPBS by heating and magnetic stirring in an 80 °C water bath, adjusting the pH to 7.0; this is solution 1. Dissolve 1.5 g of poly-L-proline in another 20 mL of DPBS by sonication, adjusting the pH to 7.0; this is solution 2. Dissolve 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5 mol / L). -1 Dissolve the sucrose in 25 mL of DPBS by sonication. After the sucrose is completely dissolved, add 10 mL of ethylene glycol to form solution 3. After solutions 1, 2 and 3 have returned to room temperature, mix the three solutions well, adjust the pH to 7.0, and bring the remaining volume to 100 mL with DPBS.

[0110] Cryopreservation solution B: Each 100ml contains the following components:

[0111]

[0112]

[0113] Cryopreservation solution preparation steps: Dissolve 2.0 g of PVA in 20 mL of DPBS by heating and magnetic stirring in an 80°C water bath, and adjust the pH to 7.1 to obtain solution 1; dissolve 8.0 g of L-Arg and 4.0 g of L-Thr in 20 mL of DPBS, and adjust the pH to 7.1 to obtain solution 2; add 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5 mol L). -1 Dissolve the sucrose in 20 mL of DPBS by sonication. After the sucrose is completely dissolved, add 10 mL of ethylene glycol to make solution 3. After solutions 1, 2 and 3 have returned to room temperature, mix the three solutions thoroughly, adjust the pH to 7.1 and add DPBS to bring the remaining volume to 80% of the total volume. Add 20 mL of serum before use.

[0114] Cryopreservation solution C:

[0115] Each 100ml contains the following components:

[0116] substance Dosage PVA(g) 2.0 Ethylene glycol (mL) 10 <![CDATA[Sucrose (mol L -1 )]]> 0.5 Fetal bovine serum (mL) 20 DPBS (mL) margin

[0117] 2.0 g of PVA was dissolved in 25 mL of DPBS by heating and magnetic stirring in an 80 °C water bath, and the pH was adjusted to 6.9 to form Solution 1; 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution was 0.5 mol / L) was added. -1 Dissolve the sucrose in 25 mL of DPBS by sonication. After the sucrose is completely dissolved, add 10 mL of ethylene glycol to make solution 2. After solutions 1 and 2 have returned to room temperature, mix the two solutions thoroughly, adjust the pH value, and make up the remaining volume to 80% of the total volume. Store 20 mL of serum separately and add it when the preservation solution is used.

[0118] Cryopreservation solution D:

[0119] Each 100ml contains the following components:

[0120] substance Dosage PVA(g) 2.0 Ethylene glycol (mL) 10 <![CDATA[Sucrose (mol L -1 )]]> 0.5 DPBS (mL) margin

[0121] 2.0 g of PVA was dissolved in 30 mL of DPBS by heating and magnetic stirring in an 80 °C water bath, and the pH was adjusted to 7.0 to form solution 1; 17 g (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution was 0.5 mol / L) was added. -1 Dissolve the sucrose in 25 mL of DPBS by sonication. After the sucrose is completely dissolved, add 10 mL of ethylene glycol to make solution 2. After solutions 1 and 2 have returned to room temperature, mix the two solutions thoroughly, adjust the pH value, and make up the remaining volume to a total volume of 100 mL. Set aside for later use.

[0122] 2. Preparation of freezing equilibrium solution: Prepare the freezing equilibrium solution according to the following formula.

[0123] Freezing equilibration solution a: Dissolve 2.0 g of PVA in 50 mL of DPBS by heating and magnetic stirring in an 80 °C water bath. Once the PVA is completely dissolved, adjust the pH to 7.0, add 7.5 mL of ethylene glycol, mix well, and bring the volume to 100 mL with DPBS.

[0124] Freezing equilibration solution b: Total volume 100 mL. Dissolve 7.5 mL of ethylene glycol in 72.5 mL of DPBS, mix well, and add 20 mL of serum before use.

[0125] Comparative Example 1:

[0126] Freezing equilibration solution #1: Each 1 mL contains 7.5% (v / v) DMSO, 7.5% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, and the balance is DPBS;

[0127] Cryopreservation solution #1: Each 1 mL contains 15% (v / v) DMSO, 15% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, 0.5 M sucrose, and the balance is DPBS.

[0128] Freezing equilibration solution b: Each 1 mL contains 7.5% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, and the remainder is DPBS;

[0129] Cryopreservation solution #2: Each 1 mL contains 10% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, 0.5 M sucrose, and the balance is DPBS.

[0130] The thawing solution formulations used in Example 1 and Comparative Example 1 of this invention are as follows:

[0131] Thawing solution 1#: Thawing solution I (containing 1.0 mol L) -1 Sucrose, 20% serum, balance DPBS); Thawing solution II (containing 0.5 mol / L... -1 Sucrose, 20% serum, balance DPBS); Thawing solution III (containing 0.25 mol / L... -1 Sucrose (20% serum, balance DPBS); Thawing solution IV (20% serum, balance DPBS).

[0132] Thawing solution 2#: Thawing solution I (containing 1.0 mol L) -1 sucrose, 20 mg / mL -1 PVA (the remainder being DPBS); Thawing solution II (containing 0.5 mol / L) -1 Sucrose, 20 mg / mL -1PVA (the remainder being DPBS); Thawing solution III (containing 0.25 mol / L) -1 Sucrose, 20 mg / mL -1 PVA (the remainder is DPBS); thawing solution IV (20 mg / mL) -1 PVA, with the remainder being DPBS).

[0133] Thawing solution 3#: Thawing solution I (containing 1.0 mol L) -1 sucrose, 20 mg / mL -1 PVA, 10 mg / mL -1 Polyproline (the remainder being DPBS); Thawing solution II (containing 0.5 mol / L) -1 Sucrose, 20 mg / mL -1 PVA, 5.0 mg / mL -1 Polyproline (the remainder being DPBS); Thawing solution III (containing 0.25 mol L...) -1 Sucrose, 20 mg / mL -1 PVA, 2.5 mg / mL -1 Polyproline (the remainder being DPBS); Thawing solution IV (20 mg / mL) -1 PVA, with the remainder being DPBS).

[0134] Application Example 1:

[0135] Oocytes and embryos were cryopreserved according to the freezing equilibrium solution and cryopreservation solution used in the above embodiments and comparative examples, as shown in Tables 1 and 2.

[0136] 1. Cryopreservation of oocytes

[0137] Mouse oocytes were first equilibrated in cryopreservation solution for 5 minutes; then placed in the prepared cryopreservation solution for 1 minute. The equilibrated oocytes were then placed on a cryopreservation rod and quickly immersed in liquid nitrogen (-196℃). The rod was then sealed and the cells were stored. During thawing, the frozen oocytes were equilibrated in thawing solution I at 37℃ for 5 minutes, and then equilibrated in thawing solutions II-IV for 3 minutes each. After 2 hours of culturing, the number of surviving cells was observed and the survival rate was calculated (see Table 1).

[0138] 2. Embryo cryopreservation

[0139] Mouse embryos were first equilibrated in cryopreservation solution for 5 minutes, then placed in the prepared cryopreservation solution for 50 seconds. The equilibrated embryos were then placed on a cryopreservation rack and quickly immersed in liquid nitrogen (-196℃). The rack was then sealed and the embryos were stored. During thawing, the frozen embryos were equilibrated in thawing solution I at 37℃ for 3 minutes, and then in thawing solutions II-IV for 3 minutes each. The thawed embryos were cultured for 2 hours, and the number of surviving embryos was observed and the survival rate was calculated (see Table 2).

[0140] Table 1. Survival rate of mouse oocytes after cryopreservation

[0141] serial number Balanced fluid cryopreservation solution thawing fluid Total number of frozen eggs Survival rate after 2 hours Application Example 1 a A Thawing solution #1 39 89.7％ Application Example 2 a A Thawing fluid #3 60 98.6％ Application Example 3 b B Thawing solution #1 109 94.8％ Application Example 4 b C Thawing solution #1 90 97.7％ Application Example 5 a D Thawing solution #1 50 93.4％ Application Example 6 a D Thawing solution #2 53 96.5％ Comparison Example 1 Balanced fluid #1 Coolant #1 Thawing solution #1 146 95.0％ Comparison Example 2 b Coolant #2 Thawing solution #1 96 81.9％

[0142] Table 2 Survival rate of mouse embryos after cryopreservation

[0143]

[0144]

[0145] Data from Examples 1 and 1 (Comparative Example 1) show that the cryopreservation reagents used in Examples 1-6 and 7-8 achieve excellent oocyte and embryo survival rates due to the addition of special biomimetic ice-controlling materials such as polyamino acids and / or PVA. Specifically, data from Example 3 and 2 (Comparative Example 2) indicate that the cryopreservation solution of this invention, without DMSO, still achieves better cryopreservation results than existing commercial cryopreservation solutions (containing 15% DMSO). Data from Examples 2, 6-8 further demonstrate that even without DMSO and serum added to the equilibration solution, freezing solution, and thawing solution, the biomimetic ice-controlling materials and permeability protectant ethylene glycol used in this invention still achieve cryopreservation oocyte and embryo survival rates superior to existing commercial cryopreservation solutions, and show better results than solutions containing serum in the thawing solution (Examples 1 and 5). This invention provides DMSO-free cryopreservation solutions and cryoequilibration solutions. Through the synergistic effect of each component, these solutions still provide good preservation results for oocytes and embryos even without the addition of DMSO. This solves the defect of existing cryopreservation solutions that cause toxicity to cells or embryos due to the addition of high concentrations of DMSO. Furthermore, it addresses the problems of short shelf life and easy introduction of parasitic biological contaminants caused by the presence of serum in currently widely used commercial cryopreservation solutions in clinical practice.

[0146] Example 2: Cryopreservation of human umbilical cord mesenchymal stem cells

[0147] 1. Preparation of cryopreservation solution: Prepare the cryopreservation solution according to the following formula.

[0148] Cryopreservation solution E: Total volume 100 mL, containing 10 mL ethylene glycol, 20 mL serum, and 17 g sucrose (0.5 mol / L). -1 ), poly-L-arginine (degree of polymerization 8) 4.0g, PVA 1.0g, DPBS balance.

[0149] Cryopreservation solution F: Total volume 100 mL, containing 25 mL ethylene glycol, 20 mL serum, and 17 g sucrose (0.5 mol / L). -1 L-Arg 16g, L-Thr 8g, and the remainder of DPBS.

[0150] Cryopreservation solution G: Total volume 100 mL, containing 10 mL ethylene glycol, 20 mL serum, and 17 g sucrose (0.5 mol / L). -1 ), PVA 2.0g, DPBS balance.

[0151] Cryopreservation solution H: Total volume 100 mL, containing 10 mL ethylene glycol, 20 mL serum, and 17 g sucrose (0.5 mol / L). -1 ), TR28g, and the remainder of DPBS.

[0152] Cryopreservation solution I: Total volume 100 mL, containing 10 mL ethylene glycol and 17 g sucrose (0.5 mol / L). -1 ), PVA 2.0g, DPBS balance.

[0153] The method for preparing the cryopreservation solution is the same as in Example 1.

[0154] The preparation method of TR is as follows:

[0155] (1) Place 2-chlorotriphenylmethyl chloro resin into a reaction tube and add DCM (20 mL g). -1 Shake for 30 minutes. Filter the solution through a sand filter to remove the solvent, add three molar excess of Fmoc-L-Thr(tBu)-OH, then add eight molar excess of DIEA, and finally add DMF to dissolve. Shake for 30 minutes. Seal the solution with methanol for 30 minutes.

[0156] (2) Remove the solvent DMF, add 20% piperidine / DMF solution (10 mL g) -1 After 5 minutes, remove the solvent and add 20% piperidine / DMF solution (10 mL g). -1 After 15 minutes, remove the piperidine solution. Take a small amount of resin, wash it three times with ethanol, add ninhydrin reagent, and heat at 105-110℃ for 5 minutes. A deep blue color indicates a positive reaction.

[0157] (3) The product obtained from the above reaction was successively treated with DMF (15 mL g) -1(twice), methanol (15 mL g) -1 (twice) and DMF (15 mL g) -1 After washing twice, add Fmoc-Arg(Pbf)-OH dissolved in as little DMF as possible to the reaction tube; add a double excess of HBTU. Then, immediately add an 8-fold excess of DIEA and react for 30 minutes.

[0158] (4) After removing the solution, take a small amount of resin, wash it three times with ethanol, add ninhydrin reagent, heat at 105-110℃ for 5 minutes. If there is no color, it is a negative reaction, that is, the reaction is complete.

[0159] (5) The product obtained from the above reaction was successively treated with DMF (15 mL g) -1 (twice), methanol (15 mL g) -1 (twice) and DMF (15 mL g) -1 After washing twice to remove the solvent, add 20% piperidine / DMF solution (10 mL g) -1 After 5 minutes, remove the solvent and add 20% piperidine / DMF solution (10 mL g). -1 After 15 minutes, remove the piperidine solution, take a small amount of resin, wash it with ethanol, add ninhydrin reagent, heat at 105-110℃ for 5 minutes, and the reaction is positive when it turns dark blue.

[0160] (6) The product obtained from the above reaction was successively treated with DMF (15 mL g) -1 (twice), methanol (15 mL g) -1 (twice) and DCM (15mL g) -1 After washing twice, the resin is dried.

[0161] (7) Use cutting fluid (15 mL g) -1 The product was cleaved for 90 minutes using a ratio of TFA:water:EDT:Tis = 95:1:2:2 (V / V). The cleavage solution was then dried with nitrogen and freeze-dried to obtain the crude peptide.

[0162] (8) The peptide was purified by HPLC and then converted or desalted. HPLC: tR = 4.8 mins (Purification column: Kromasil 100-5C18, 4.6 mm * 250 mm; Gradient elution buffer: 0.1% TFA acetonitrile solution and 0.1% TFA aqueous solution, 0 mins - 1:99, 20 mins - 1:4). The purified solution was lyophilized to obtain the product L-Thr-L-Arg(TR). The yield was approximately 80%. Mass spectrometry identification at 276.2 was [M+H]. + .

[0163] Comparative Example 2:

[0164] Cryopreservation solution #3: Each 1 mL contains 10% (v / v) DMSO, 15% (v / v) fetal bovine serum, and the remainder is a-MEM medium (USA, Invitrogen, C12571500BT).

[0165] Human umbilical cord mesenchymal stem cells were cryopreserved using the cryopreservation solutions described above, following the protocols in Table 3. The specific cryopreservation method for human umbilical cord mesenchymal stem cells was the microdroplet method: Human umbilical cord mesenchymal stem cells on a culture dish were digested with 25% trypsin for 2 minutes, then placed in an equal volume of culture medium (10% FBS + α-MEM medium). The cells were gently pipetted until completely detached, then transferred to a 1.5 ml centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were then separated from the culture medium, and 10 μL of cryopreservation solution was added to the bottom of the centrifuge tube. The cells were gently pipetted to disperse the stem cell clusters. This 10 μL of cryopreservation solution containing the stem cells was placed on a cryostat and cryopreserved in liquid nitrogen (-196°C). For thawing, the cryostat containing the cells and cryopreservation solution was directly placed in α-MEM medium at 37°C. After thawing, trypan blue staining was used to check the viability, and the cell count was performed using the JIMBIO-FIL instrument. Viability = number of live cells / total number of cells (see Table 3).

[0166] Table 3. Survival rate of cryopreserved human umbilical cord mesenchymal stem cells

[0167]

[0168]

[0169] When human umbilical cord mesenchymal stem cells are cryopreserved using the cryopreservation solution of the present invention, the stem cell survival rate can still reach 92.4% even without the addition of DMSO (Application Example 9). In fact, the survival rate can reach 77.1% even without the addition of DMSO and serum. This shows that the cryopreservation reagent can not only achieve the effectiveness of conventional cryopreservation solutions for freezing stem cells, but also far exceed the cryopreservation recovery rate of the commonly used cryopreservation solution containing 10% DMSO and culture medium (Comparative Example 5). The cryopreservation effect based on PVA is significantly better than that of Comparative Example 6 without the addition of PVA.

[0170] Example 3: Cryopreservation of intact ovarian organs and ovarian tissue sections

[0171] Cryopreservation solution J: Total volume 100mL, containing 10mL ethylene glycol and 17g sucrose (0.5mol / L). -1 ), PVA 2.0g, DPBS balance.

[0172] Cryopreservation solution K: Total volume 100 mL, containing 10 mL ethylene glycol, 20 mL serum, and 17 g sucrose (0.5 mol / L). -1 ), PVA 1.0g, DPBS balance.

[0173] Cryopreservation solution L: Total volume 100mL, containing 10mL ethylene glycol, 20mL serum, and 17g sucrose (0.5mol / L). -1 ), poly-L-arginine (degree of polymerization 8) 4.0g, PVA 1.0g, DPBS balance.

[0174] Freezing equilibration solution a: Dissolve 2.0 g of PVA in 50 mL of DPBS by heating and magnetic stirring in an 80 °C water bath. Once the PVA is completely dissolved, adjust the pH to 7.0, add 7.5 mL of ethylene glycol, mix well, adjust the pH value, and bring the volume to 100 mL.

[0175] Freezing equilibration solution b: Add 7.5 mL of ethylene glycol to 72.5 mL of DPBS, mix well, and add 20 mL of serum before use;

[0176] Comparative Example 3:

[0177] Freezing equilibration solution #1: Each 1 mL contains 7.5% (v / v) DMSO, 7.5% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, and the balance is DPBS;

[0178] Cryopreservation solution #1: Each 1 mL contains 15% (v / v) DMSO, 15% (v / v) ethylene glycol, 20% (v / v) fetal bovine serum, and 0.5 mol / L... -1 Sucrose, with the remainder being DPBS.

[0179] Thawing solution 1#: Thawing solution I (containing 1.0 mol L) -1 Sucrose, 20% serum, balance DPBS); Thawing solution II (containing 0.5 mol / L... -1 Sucrose, 20% serum, balance DPBS); Thawing solution III (containing 0.25 mol / L... -1 Sucrose (20% serum, balance DPBS); Thawing solution IV (20% serum, balance DPBS).

[0180] Thawing solution 2#: Thawing solution I (containing 1.0 mol L) -1 sucrose, 20 mg / mL -1 PVA (the remainder being DPBS); Thawing solution II (containing 0.5 mol / L) -1 Sucrose, 20 mg / mL -1PVA (the remainder being DPBS); Thawing solution III (containing 0.25 mol / L) -1 Sucrose, 20 mg / mL -1 PVA (the remainder is DPBS); thawing solution IV (20 mg / mL) -1 PVA, with the remainder being DPBS).

[0181] Using the above-mentioned cryopreservation solution and the comparative cryopreservation equilibrium solution, as well as the cryopreservation solution according to the schemes in Tables 4 and 5, complete ovarian organs of newborn mice within 3 days and ovarian tissue sections of sexually mature mice were cryopreserved.

[0182] Whole ovarian organs or ovarian tissue sections were first equilibrated at room temperature in cryoequilibration buffer for 25 minutes, then placed in the prepared cryopreservation solution for 15 minutes. Afterward, the intact ovarian organs or ovarian tissue sections were placed on a cryostat and immersed in liquid nitrogen for preservation. After thawing, the intact ovarian organs or ovarian tissue sections were placed in culture medium (10% FBS + α-MEM) and incubated at 37°C in a 5% CO2 incubator for 2 hours. They were then fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (HE) for morphological observation. The results are as follows: Figure 1-10 As shown, Figure 1 Photographs of fresh, unfrozen, intact ovarian organs. Figure 6 Photograph of a section of fresh, unfrozen ovarian tissue.

[0183] Table 4. Cryopreservation Protocol for Complete Ovarian Organs

[0184] serial number Balanced fluid cryopreservation solution thawing fluid form Application Example 14 a J Thawing solution #2 Figure 3 Application Example 15 b K Thawing solution #1 Figure 4 Application Example 16 b L Thawing solution #1 Figure 5 Comparison Example 7 Balanced fluid #1 Coolant #1 Thawing solution #1 Figure 2

[0185] Table 5. Ovarian tissue cryopreservation protocol

[0186] serial number Balanced fluid cryopreservation solution thawing fluid form Application Example 17 a J Thawing solution #2 Figure 8 Application Example 18 b K Thawing solution #1 Figure 9 Application Example 19 b L Thawing solution #1 Figure 10 Comparison Example 8 Balanced fluid #1 Coolant #1 Thawing solution #1 Figure 7

[0187] according to Figure 1-5 It can be seen that, compared with Comparative Example 7, which used biomimetic cryopreservation materials without added amino acids or PVA, and fresh, unfrozen ovarian organs, the primordial follicles in Application Examples 14-16 showed relatively intact structures and stromal structures, with more homogeneous and lightly stained cytoplasm and fewer shrunken and darkly stained nuclei; the vascular walls showed intact structures and less lumen collapse, with more homogeneous and lightly stained cytoplasm and fewer shrunken and darkly stained nuclei. Therefore, Application Examples 14-16 showed better cryopreservation results for ovarian organs compared to Comparative Example 7.

[0188] according to Figure 6-10 It can be seen that, compared with the schemes of Examples 17-19 and Comparative Example 8 and fresh, unfrozen adult mouse ovarian tissue, the structures of follicles and antral follicles in the growth period are relatively intact. It is evident that the cryopreservation solution of the present invention has better effect on cryopreserving ovarian tissue than the prior art.

[0189] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a DMSO-free cryopreservation solution in organ and / or tissue cryopreservation, characterized in that, The cryopreservation solution contains 0.01-50 g of biomimetic ice-controlling material, 5.0-45 mL of polyol, 0.1-1.0 mol / L of water-soluble sugar, 0-30 mL of serum, and the rest is buffer, per 100 mL volume, the biomimetic ice-controlling material is selected from PVA, or PVA and amino acid biomimetic ice-controlling material; The amino acid biomimetic ice-controlling material is selected from polyamino acid with polymerization degree ≥2, or one or more than two kinds of amino acid; The polyamino acid is poly-L-arginine or poly-L-proline; The amino acid is L-arginine or L-threonine; The PVA is random PVA, the para-regularity of the PVA is 50-55%, the molecular weight is 13-500 kDa, and the hydrolysis degree is greater than 80%.

2. The application according to claim 1, the ice-controlling material is PVA, and the PVA content is 0.1-6.0 g.

3. The application according to claim 1, the polyol is C2-5 polyol.

4. The application according to claim 3, the polyol is any one of ethylene glycol, propylene glycol, and glycerol.

5. The application according to any one of claims 1-3, the water-soluble sugar is at least one of non-reducing disaccharide, water-soluble polysaccharide, and glycothiol.

6. The application according to claim 5, the water-soluble sugar is at least one of sucrose, trehalose, hydroxypropyl methyl cellulose, and polysucrose.

7. The application according to any one of claims 1-3, the buffer is at least one of DPBS, hepes-buffered HTF buffer, or other cell culture buffer.

8. The application according to any one of claims 1-3, the ice-controlling material is a combination of PVA and amino acid and / or polyamino acid.

9. The application according to claim 8, the ice-controlling material is composed of 0.1-5.0 g of PVA and 8.0-35 g of amino acid and / or 1.0-9.0 g of polyamino acid.

10. The application according to any one of claims 1-3, the polyamino acid polymerization degree is 2-40.

11. The application according to claim 10, the polyamino acid polymerization degree is 2-20.

12. The application according to claim 11, the polyamino acid polymerization degree is 6, 8, 15, or 20.

13. Use according to any one of claims 1 to 3, characterized in that, The polyol content is 6.0-28 mL per 100 mL.

14. The application according to any one of claims 1-3, the serum content is 0.

15. The application according to any one of claims 1-3, the water-soluble sugar content is 0.2-0.5 mol / L.

16. The application according to any one of claims 1-3, the pH of the cryopreservation solution is 6.5-7.

6.

17. The application according to any one of claims 1-3, the cryopreservation solution is prepared by the following steps: dissolving the ice-controlling material in the buffer, adjusting the pH after cooling to room temperature, dissolving other components in the remaining buffer, and mixing after cooling.

18. The application according to claim 17, the preparation of the cryopreservation solution comprises the following steps: (1) dissolving PVA in a part of the buffer, adjusting pH after cooling to room temperature to obtain solution 1; (2) optionally, dissolving polyamino acid or amino acid in a part of the buffer, adjusting pH after cooling to room temperature to form solution 2; (3) dissolving water-soluble sugar in a part of the buffer, adding other components except serum after the water-soluble sugar is completely dissolved to prepare solution 3; (4) mixing solution 1, optionally solution 2, and solution 3 after cooling to room temperature, adjusting pH and using buffer to constant volume to the predetermined volume to obtain the cryopreservation solution; Optionally, when the cryopreservation solution contains serum, the serum is added when the cryopreservation solution is used.

19. The use according to any one of claims 1-3, wherein the organ is an ovary organ; and the tissue is an ovary tissue section.

20. Use of a DMSO-free cryo-balancing solution in organ and / or tissue cryopreservation, characterized in that, The cryobalancing solution contains PVA 0.1-5.0 g, polyamino acid 0-15 g, polyol 5.0-45 mL, serum 0-30 mL, and the balance of buffer per 100 mL. The PVA is random PVA, the meso-isotacticity of the PVA is 50-55%, the molecular weight of the PVA is 13-500 kDa, and the degree of hydrolysis of the PVA is greater than 80%. The polyamino acid has a polymerization degree of ≥2, and the polyamino acid is poly-L-arginine.

21. The use according to claim 20, wherein the PVA content is 0.1-4.0 g.

22. The use according to claim 20 or 21, wherein the organ is an ovary organ; and the tissue is an ovary tissue section.

23. The use according to claim 20 or 21, wherein the cryobalancing solution contains PVA 0.5-3.5 g, polyamino acid 0-8 g, polyol 7.5-15 mL, and the balance of buffer per 100 mL.

24. Use of a reagent for cryopreservation free of DMSO for the cryopreservation of organs and / or tissues, characterized in that, The cryopreservation solution and the cryobalancing solution independently exist.

25. The use according to claim 24, wherein the serum content of the cryopreservation solution is 0, and the cryobalancing solution contains PVA 0.5-2.5 g, polyol 7.5-15 mL, and the balance of buffer per 100 mL.

26. Use according to claim 24 or 25, which use comprises: The organ and / or tissue are balanced in the cryobalancing solution, then the organ and / or tissue are placed in the cryopreservation solution, and then the organ and / or tissue are placed on a freezing slide for liquid nitrogen cryopreservation.

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