Serum-free cryopreservation solution and preparation method and application thereof

Patent metadata

Field	Value
Publication number	US-12453345-B2
Application number	US-202017600987-A
Country	US
Kind code	B2
Filing date	Mar 2, 2020
Priority date	Apr 9, 2019
Publication date	Oct 28, 2025
Grant date	Oct 28, 2025

Abstract

A serum-free cryopreservation solution contains a biomimetic ice control material, a polyol, a water-soluble sugar, and a buffer solution margin. The bionic ice control material can be polyvinyl alcohol or an amino acid compound. The cryopreservation liquid of the present invention uses the bionic ice control material as the main component, does not contain serum, has low toxicity, and can achieve a cell survival rate that is the same as, or higher than, existing cryopreservation solutions.

First claim

The invention claimed is:

1. A serum-free cryopreservation solution comprising, per 100 mL in volume, 0.01-50.0 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 , 0-15 mL of DMSO and the balance of a buffer, wherein the biomimetic ice growth inhibition material is a combination of an atactic polyvinyl alcohol (PVA) having a syndiotacticity of 15%-60% and a degree of hydrolysis of greater than 80% with an amino acid biomimetic ice growth inhibition material selected from GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Val, or GDL-L-Ser, and wherein the polyalcohol differs from the atactic PVA.

2. The cryopreservation solution according to claim 1 , wherein:
the polyalcohol is a polyalcohol with 2-5 carbon atoms;
the water-soluble saccharide is selected from a non-reducing disaccharide, a water-soluble polysaccharide, a glycoside, and mixtures thereof; and
the buffer is selected from DPBS, hepes-buffered HTF buffer, cell buffers, and mixtures thereof.

3. The cryopreservation solution according to claim 1 , wherein:
the content of DMSO is 1.0-10 mL per 100 mL of the cryopreservation solution;
the content of the water-soluble saccharide is 0.1-0.8 mol L −1 per 100 mL of the cryopreservation solution; and
the content of the polyalcohol is 5.0-45 mL per 100 mL of the cryopreservation solution.

4. The cryopreservation solution according to claim 1 , comprising, per 100 mL in volume, 0.1-50 g of the amino acid biomimetic ice growth inhibition material, 5.0-45 mL of the polyalcohol, 0-15 mL of DMSO, the water-soluble saccharide at 0.1-1 mol L −1 , and the balance of the buffer.

5. The cryopreservation solution according to claim 1 , comprising, per 100 mL in volume, 0.01-6.0 g of the atactic PVA, 5.0-45 mL of the polyalcohol, the water-soluble saccharide at 0.1-1.0 mol L −1 , 0-15 mL of DMSO, and the balance of the buffer.

6. A preparation method of the cryopreservation solution according to claim 1 , comprising the following steps:
(1) dissolving the amino acid biomimetic ice growth inhibition material in a portion of the buffer, and adjusting the pH to form a solution 1; optionally, dissolving the atactic PVA in another portion of the buffer, and adjusting the pH to give a solution 2;
(2) dissolving the sucrose in a third portion of the buffer, and adding other components after the sucrose is completely dissolved to prepare a solution 3; and
(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 give the cryopreservation solution.

7. An amino acid cryopreservation reagent, comprising the cryopreservation solution according to claim 1 and a freezing equilibrium solution, wherein the freezing equilibrium solution comprises, per 100 mL in volume, 5.0-45 mL of the polyalcohol, the balance of the buffer, and optionally, 0-15 mL of DMSO, and 0.1-5.0 g of the atactic PVA having a syndiotacticity of 15%-60% and a degree of hydrolysis of greater than 80%, wherein the polyalcohol differs from the atactic PVA, wherein the freezing equilibrium solution and the preservation solution are present independently of each other.

8. A process for cryopreservation of a biological tissue, comprising placing the biological tissue in the cryopreservation solution according to claim 1 to prepare a cell suspension, and freezing the cell suspension.

9. The process according to claim 8 , wherein the biological tissue is selected from an oocyte, an embryo, a stem cell, an organ, tissue, and mixtures thereof.

10. The cryopreservation solution according to claim 2 , wherein:
the polyalcohol is selected from ethylene glycol, propylene glycol, and glycerol; or
the water-soluble saccharide is selected from sucrose and trehalose.

11. The cryopreservation solution according to claim 1 , wherein:
the polyalcohol is selected from ethylene glycol, propylene glycol, and glycerol; or
the water-soluble saccharide is selected from sucrose, trehalose, water-soluble cellulose, polysucrose, and mixtures thereof.

Assignees

• Inst Chemistry Cas
• Univ Peking Third Hospital
• Inst Of Chemistry Chinese Academic Of Sciences

Inventors

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

Classifications

• C12N5/0682

  Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells (oocytes C12N5/0609) · CPC title

• C12N5/0604

  Whole embryos; Culture medium therefor · CPC title

• C12N5/0609

  Oocytes, oogonia (fertilised oocytes C12N5/0604) · CPC title

• C12N5/0665

  Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood · CPC title

• A01N1/125 Primary

  Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators · CPC title