DMSO-free cryopreservation solution and preparation method and use thereof

Patent metadata

Field	Value
Publication number	US-12408659-B2
Application number	US-202017594202-A
Country	US
Kind code	B2
Filing date	Mar 2, 2020
Priority date	Apr 9, 2019
Publication date	Sep 9, 2025
Grant date	Sep 9, 2025

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.

First claim

The invention claimed is:

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 −1 , 0-30 mL of serum and the balance of a buffer, wherein the biomimetic ice growth inhibition material is selected from an atactic polyvinyl alcohol (PVA) having a syndiotacticity of 15%-60% and a molecular weight of from great than 10 kDa to 500 kDa, or a combination of said PVA with 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 a polyamino acid with a degree of polymerization ≥2, an amino acid, a peptide compound, and mixtures thereof.

3. The cryopreservation solution according to claim 2 , wherein the peptide compound is a polypeptide, a glycopeptide derivative, or a compound of formula (I):

wherein R is selected from substituted or unsubstituted alkyl, and the substituent is selected from —OH, —NH 2 , —COOH, and —CONH 2 , and n is an integer greater than or equal to 1 and less than or equal to 1000.

4. The cryopreservation solution according to claim 3 , wherein R is substituted or unsubstituted C 1-6 alkyl.

5. The cryopreservation solution according to claim 3 , wherein R is —CH 3 , —CH 2 CH 3 , or —CH 2 CH 2 COOH.

6. The cryopreservation solution according to claim 1 , wherein the ice growth inhibition material is PVA, and the content of the PVA is 0.1-6.0 g.

7. The cryopreservation solution according to claim 1 , wherein the ice growth inhibition material is a combination of the PVA and an amino acid, the PVA and a polyamino acid, and the PVA with a mixture of amino acid and polyamino acid.

8. The cryopreservation solution according to claim 7 , wherein ice growth inhibition material 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.

9. The cryopreservation solution according to claim 1 , wherein the PVA has a syndiotacticity of 50%-60%.

10. The cryopreservation solution according to claim 9 , wherein
the PVA is a PVA having a degree of hydrolysis of greater than 80%; and/or
the polyamino acid is selected from lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine, and mixtures thereof; and/or
the polypeptide is a peptide consisting of 2 to 8 different amino acids; and/or
the glycopeptide derivative is synthesized by a saccharide and an amino acid.

11. The cryopreservation solution according to claim 10 , wherein
the polypeptide is selected from 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), L-Ala-L-Ala-L-Thr (AAT), and mixtures thereof; and/or
the glycopeptide derivative is selected from GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Val, GDL-L-Ser, and mixtures thereof.

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

13. The cryopreservation solution according to claim 12 , wherein the polyalcohol is C 2-5 polyalcohol; and/or
the water-soluble saccharide is selected from a non-reducing disaccharide, a water-soluble polysaccharide, a glycoside, and mixtures thereof; and/or
the buffer is selected from DPBS, hepes-buffered HTF buffer, a cell buffer, and mixtures thereof and/or
the serum is human serum albumin or a substitute thereof for a human-derived cryopreservation object, or is fetal bovine serum or bovine serum albumin for a non-human-derived cryopreservation object.

14. The cryopreservation solution according to claim 13 , wherein
the polyalcohol is ethylene glycol, propylene glycol, or glycerol; and/or
the water-soluble saccharide is selected from sucrose, water-soluble cellulose, trehalose, and polysucrose; and/or
the serum is sodium dodecyl sulfate.

15. A DMSO-free cryopreservation reagent comprising the cryopreservation solution according to claim 1 and a freezing equilibration solution, wherein the cryopreservation solution and the freezing equilibration solution are independently present; and 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.

16. A preparation method of the cryopreservation solution according to claim 1 , comprising: dissolving the ice growth inhibition material in the buffer, cooling to room temperature before adjusting the pH, dissolving other components in the rest buffer, and mixing after cooling.

17. The preparation method according to claim 16 , wherein the preparation method comprises:
dissolving the PVA in a portion of the buffer and cooling to room temperature before adjusting the pH to form a solution 1;
optionally, dissolving the polyamino acid or amino acid in a portion of the buffer and cooling to room temperature before adjusting the pH to form a solution 2;
dissolving the water-soluble saccharide in a portion of the buffer and adding other components except serum after the water-soluble saccharide is completely dissolved to form a solution 3;
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 obtaining the cryopreservation solution; and
optionally, adding serum to the cryopreservation solution.

18. A method of cryopreservation of biological tissues comprising utilizing the cryopreservation solution according to claim 1 and a freezing equilibration solution, wherein 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.

19. The method according to claim 18 , wherein the biological tissue is selected from at least one of an oocyte, an embryo, a stem cell, and an organ and tissue.

20. The cryopreservation solution according to claim 18 , wherein
the polypeptide is a dipeptide, a tripeptide, or a tetrapeptide; and/or
the glycopeptide derivative is a molecule consisting of gluconolactone (GDL) and an ice-philic amino acid through chemical bonding.

Assignees

• Univ Peking Third Hospital
• Inst Chemistry Cas

Inventors

• Yan Jie
• Qiao Jie
• Yan Liying
• Li Rong
• Wang Jianjun
• Jin Shenglin
• Lv Jianyong

Classifications

• C07K5/081

  the side chain containing O or S as heteroatoms, e.g. Cys, Ser · CPC title

• C07K5/0806

  the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala · CPC title

• C07K5/06165

  and Pro-amino acid; Derivatives thereof · CPC title

• C07K5/06095

  Arg-amino acid · CPC title

• C07K5/0606 Primary

  the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr · CPC title