WO2020207153A1 - 一种解冻液及其制备方法与应用 - Google Patents

一种解冻液及其制备方法与应用 Download PDF

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WO2020207153A1
WO2020207153A1 PCT/CN2020/077475 CN2020077475W WO2020207153A1 WO 2020207153 A1 WO2020207153 A1 WO 2020207153A1 CN 2020077475 W CN2020077475 W CN 2020077475W WO 2020207153 A1 WO2020207153 A1 WO 2020207153A1
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Prior art keywords
thawing
pva
ice
solution
water
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PCT/CN2020/077475
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English (en)
French (fr)
Inventor
乔杰
严杰
闫丽盈
李蓉
王健君
金晟琳
吕健勇
Original Assignee
北京大学第三医院（北京大学第三临床医学院）
中国科学院化学研究所
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Priority claimed from CN201910282421.0A external-priority patent/CN111789103B/zh
Priority claimed from CN201910282415.5A external-priority patent/CN111789102B/zh
Application filed by 北京大学第三医院（北京大学第三临床医学院）, 中国科学院化学研究所 filed Critical 北京大学第三医院（北京大学第三临床医学院）
Priority to SG11202110901QA priority Critical patent/SG11202110901QA/en
Priority to JP2021560654A priority patent/JP7459130B2/ja
Priority to KR1020217033906A priority patent/KR20210142698A/ko
Priority to AU2020256873A priority patent/AU2020256873B2/en
Priority to EP20787891.9A priority patent/EP3939429A4/en
Priority to US17/594,345 priority patent/US20220192180A1/en
Publication of WO2020207153A1 publication Critical patent/WO2020207153A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0278—Physical preservation processes
    • A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time

Definitions

  • the invention belongs to the technical field of biomedical materials, and specifically relates to a thawing solution for cryopreservation and a thawing method.
  • Cryopreservation refers to the preservation of biological materials in an ultra-low temperature state to slow down or stop cell metabolism and division. Once the normal physiological temperature is restored, it can continue to develop. Since its inception, this technology has become one of the indispensable research methods in the natural sciences and has been widely adopted. In recent years, with the increase in life pressure, human fertility has been declining year by year. The preservation of fertility has received more and more attention. The cryopreservation of human germ cells (sperm, oocyte) and gonadal tissue has become preservation An important means of fertility. In addition, with the aging of the world population, the demand for cryopreservation of donated human-derived cells, tissues or organs that can be used in regenerative medicine and organ transplantation is also rapidly increasing. Therefore, how to efficiently cryopreserve precious cells, tissues and organ resources for emergency needs has become an urgent scientific and technological problem.
  • cryopreservation method The most commonly used cryopreservation method is freezing. During the thawing process, care should be taken to prevent freezing and thawing damage such as ice crystals, cold shock, solute effects, fragmentation damage, recrystallization, osmotic shock, etc.
  • the rapid rewarming method is to place high osmotic pressure blastocysts or oocytes in a culture medium containing a certain concentration gradient of cryoprotectant, so that the osmotic pressure gap between inside and outside the cell is gradually reduced, and the speed of cell volume change is slowed down , To avoid cell or embryo damage caused by the thawing process.
  • most of the thawing reagents widely used clinically are mainly composed of sucrose, serum and buffer.
  • the above-mentioned thawing solution cannot effectively control the growth of ice crystals during the rewarming process, thereby damaging the cells.
  • the current thawing liquid has unclear ingredients and contains perishable serum, resulting in short shelf life and the introduction of parasitic biological contaminants.
  • the present invention provides a thawing solution for cryopreservation and a thawing method.
  • a thawing solution containing 0.1-50g of a bionic ice-controlling material, 0-1.0mol L -1 of water-soluble sugar, and a buffer balance per 100mL, wherein the bionic ice-controlling material is a material with an ice-philic group and a hydrophilic group Ice control material.
  • the hydrophilic group is a functional group that can form a non-covalent interaction with water molecules, for example, can form a hydrogen bond with water, van der Waals interaction, electrostatic interaction, hydrophobic interaction or ⁇ - ⁇ interaction;
  • the hydrophilic group may be selected from at least one of hydroxyl (-OH), amino (-NH 2 ), carboxylic acid group (-COOH), amide group (-CONH 2 ), or, for example, Amino acid (L-Pro), arginine (L-Arg), lysine (L-Lys), histidine (L-His), glycine (L-Gly), gluconolactone (GDL), Compound molecules such as sugars or their molecular fragments.
  • the ice-philic group is a functional group that can form a non-covalent interaction with ice, for example, can form a hydrogen bond with ice, van der Waals interaction, electrostatic interaction, hydrophobic interaction or ⁇ - ⁇ interaction;
  • the ice-philic group can be selected from hydroxyl (-OH), amino (-NH 2 ), phenyl (-C 6 H 5 ), pyrrolidinyl (-C 4 H 8 N), or, for example, Aminoamide (L-Gln), threonine (L-Thr), aspartic acid (L-Asn), benzene ring (-C 6 H 6 ), pyrrolidine (-C 4 H 9 N) and other compound molecules Or its molecular fragments.
  • the bionic ice control material is selected from at least one or a combination of two or more of polyvinyl alcohol (PVA), amino acids, polypeptides, and polyamino acids.
  • PVA polyvinyl alcohol
  • the amino acid may be selected from one or more of arginine, threonine, proline, lysine, histidine, glutamine, aspartic acid, glycine, etc.
  • the polyamino acid is a homopolymer that can be selected from at least one of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine, etc.
  • the degree of polymerization is ⁇ 2, preferably the degree of polymerization is 2-40, for example, the degree of polymerization is 6, 8, 15, 20, etc.).
  • the polypeptide is a polypeptide composed of two or more of the above-mentioned amino acids or a glycopeptide derivative produced by the reaction of an amino acid and a carbohydrate (for example, glucolactone), for example, the polypeptide is 2-8 different amino acids
  • the constituent polypeptides such as dipeptides, tripeptides, tetrapeptides, etc.
  • the polypeptide is 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) , One or more of L-Thr-L-Cys-L-Thr (TCT).
  • the content of the bionic ice control material is 1.0-50g, 2.0-20g, 5.0-10g; in one embodiment, the content of the bionic ice control material is 3.0g, 4.0g, 5.0g, 10g, 25g , 30g.
  • the bionic ice control material includes PVA 1.0-6.0 g.
  • the bionic ice control material includes 1.0-30 g of amino acids.
  • the amino acid is a combination of arginine and threonine, for example, it contains 1.0-20g, 1.0-10g, 1.0-5g of arginine, and 1.0-10g, 1.0-5.0g, 1.0 of threonine. -2.5g.
  • the bionic ice control material includes 0.1-9.0 g of polyamino acid, such as 1-5.0 g.
  • the amino acid is poly-L-proline and/or poly-L-arginine.
  • the bionic ice control material includes 1.0-50g of polypeptide; for example, 1.0-25g, 1.0-13g, 1.0-10g, 1.0-5.0g.
  • the bionic ice control material is a combination of PVA and the above-mentioned amino acids, polypeptides, and/or polyamino acids.
  • each of the liquid thawing 100mL water-soluble sugar content is 0.1-1.0mol L -1, e.g. 0.1-0.8mol L -1, 0.2-0.6mol L -1; e.g. 0.25mol L -1, 0.5mol L -1 , 1.0mol L -1 .
  • a thawing reagent includes thawing liquid I, thawing liquid II, thawing liquid III and thawing liquid IV, and the thawing liquid I-IV has the composition of the thawing liquid as described above.
  • the concentration gradient of the bionic ice control material in the thawing liquids I-IV is not particularly limited, and the concentration of the bionic ice control material in each thawing liquid can be the same or different; as an exemplary solution, In the thawing liquid I-IV, the concentration of the bionic ice control material is different.
  • the content of the bionic ice control material in the thawing liquid II is 50%-100% of the thawing liquid I
  • the bionic ice control material in the thawing liquid III The content of the material is 50%-100% in the thawing liquid II.
  • the thawing liquid I-IV contains 1.0-6.0 g of PVA; preferably, the PVA concentration in the thawing liquid I-IV is the same.
  • the water-soluble sugar concentration in the thawing liquid II is 50%-100% in the thawing liquid I
  • the water-soluble sugar concentration in the thawing liquid III is 50%-100% in the thawing liquid II
  • the sugar concentration is 0.
  • the thawing liquid I contains 1.0-50g of amino acids, 1.0-5.0g of PVA, 1.0mol L -1 of water-soluble sugar, and a buffer balance per 100 mL;
  • the thawing solution II contains 1.0-25 g amino acids, 1.0-5.0 g PVA, 0.5 mol L -1 of water-soluble sugar, and the remaining amount of buffer solution per 100 mL;
  • the thawing liquid III per 100 mL, contains 1.0-12.5 g amino acids, 1.0-5.0 g PVA, 0.25 mol L -1 of water-soluble sugar, and the remaining amount of buffer;
  • the thawing liquid IV contains 0-6.25g of amino acids, 1.0-5.0g of PVA, and a buffer balance per 100mL.
  • the thawing liquid I per 100 mL, contains 1.0-5.0 g of PVA, 1.0 mol L -1 of water-soluble sugar, and a buffer balance;
  • the thawing solution II contains 1.0-5.0 g of PVA, 0.5 mol L -1 of water-soluble sugar, and the balance of the buffer solution per 100 mL;
  • the thawing liquid III contains 1.0-5.0 g of PVA, 0.25 mol L -1 of water-soluble sugar, and the balance of the buffer solution per 100 mL;
  • the IV of the thawing solution is calculated per 100 mL, containing 1.0-5.0 g of PVA and the remaining amount of the buffer.
  • the thawing solution I per 100 mL, contains 0.1-9.0 g of polyamino acid, 1.0-5.0 g of PVA, 1.0 mol L -1 of water-soluble sugar, and a buffer balance;
  • the thawing solution II per 100 mL, contains 0.1-4.5 g of polyamino acid, 1.0-5.0 g of PVA, 0.5 mol L -1 of water-soluble sugar, and a buffer balance;
  • the thawing liquid III per 100 mL, contains 0.1-2.3g of polyamino acid, 1.0-5.0g of PVA, 0.25mol L -1 of water-soluble sugar, and the remaining amount of buffer;
  • the thawing solution IV contains 0-1.2 g of polyamino acid, 1.0-5.0 g of PVA, and a buffer balance per 100 mL.
  • the thawing solution I per 100 mL, contains 1.0-50 g of polypeptide, 1.0-5.0 g of PVA, 1.0 mol L -1 of water-soluble sugar, and a buffer balance;
  • the thawing solution II contains 1.0-25 g of polypeptide, 1.0-5.0 g of PVA, 0.5 mol L -1 of water-soluble sugar, and the remaining amount of buffer solution per 100 mL;
  • the thawing liquid III contains 1.0-12.5 g of polypeptide, 1.0-5.0 g of PVA, 0.25 mol L -1 of water-soluble sugar, and the remaining amount of buffer solution per 100 mL;
  • the thawing solution IV contains 0-6.25 g of polypeptide, 1.0-5.0 g of PVA, and a buffer balance per 100 mL.
  • the water-soluble sugar can be at least one of non-reducing disaccharides, water-soluble polysaccharides, and sugar anhydrides, for example selected from sucrose and trehalose; preferably sucrose.
  • the water-soluble sugar can protect the cell membrane and prevent cell sedimentation.
  • the buffer can be selected from at least one of DPBS or hepes-buffered HTF buffer or other cell culture buffers.
  • the PVA is selected from one or a combination of two or more of isotactic PVA, syndiotactic PVA and random PVA.
  • the syndiotacticity of the PVA is 15%-65%, specifically, for example, 40%-60%, 53%-55%. It is preferably a random PVA, for example, the PVA whose syndiotacticity is 45%-65%.
  • the PVA may be selected from PVA with a molecular weight of 10-500 kDa or higher, for example, a molecular weight of 10-30 kDa, 30-50 kDa, 80-90 kDa, 200-500 kDa.
  • the PVA can be selected from PVA with a degree of hydrolysis greater than 80%, for example, the degree of hydrolysis is 80%-99%, 82-87%, 87%-89%, 89%-99%, 98%-99% .
  • the thawing liquid of the present invention can be prepared by methods known in the art, for example:
  • the bionic ice control material is dissolved in a part of the buffer, the pH is adjusted, the water-soluble sugar is dissolved in a part of the buffer, and the two solutions are mixed after cooling to room temperature.
  • a thawing method for cryopreserving cells or tissues or organs includes the following steps:
  • thawing solution I Put the frozen cells or tissues or organs into the thawing solution I at 37°C for 3-5 minutes and then put them in the thawing solution II, thawing solution III, and thawing solution IV for 3-5 minutes at room temperature.
  • the above-mentioned thawing liquid and thawing reagent can be used for the recovery and thawing of cryopreserved oocytes and embryos, tissues or organs.
  • the tissue or organ is ovarian tissue or ovarian organ.
  • the present invention provides the application of the above-mentioned thawing solution and thawing reagent, specifically the application in the recovery and thawing of cryopreserved oocytes and embryos, tissues or organs, for example, the tissue or organ is ovarian tissue or Ovarian organs.
  • the thawing solution and thawing reagent prepared by the bionic ice-controlling material with the characteristics of ice-philicity and hydrophilicity can effectively control the growth of ice crystals and significantly improve the damage to cells or tissues caused by temperature changes during the resuscitation process; and the biocompatibility is good, It does not contain animal serum and has low toxicity. Compared with traditional thawing liquid containing serum, it reduces the risk of short shelf life and the introduction of parasitic biological contaminants, and is more conducive to maintaining the stability of cells or tissues. In addition, the present invention has simple ingredients, low cost and good application prospects.
  • Figure 1 is a stained photo of fresh and unfrozen ovarian organ sections
  • Fig. 2 is a photograph of section staining of an ovarian organ after being thawed with the thawing solution of Example 1;
  • Figure 3 is a stained photo of fresh unfrozen ovarian tissue sections
  • Fig. 4 is a photograph of section staining of ovarian tissue after being thawed with the thawing solution of Example 1.
  • the PVA used in the embodiment of the present invention has a syndiotacticity of 50%-55%, a molecular weight of 13-23 kDa, and a degree of hydrolysis of 98%.
  • the poly-L-proline used in the cryopreservation solution has a polymerization degree of 15 and a molecular weight of 1475.
  • the degree of polymerization of poly-L-proline in the thawing solution is 8, and the molecular weight is 795.
  • Cryopreservation Solution A The total volume is 100mL, 2.0g of PVA is heated in a water bath at 80°C and magnetically stirred and dissolved in 30mL of DPBS, the pH is adjusted to 7.0, and it is solution 1; 17g (0.05mol) of sucrose (sucrose in freezing The final concentration in the preservation solution is 0.5mol L -1 ) Dissolve in 25mL of DPBS by ultrasonic, add 10mL of ethylene glycol after the sucrose is completely dissolved, which is solution 2. After solution 1 and solution 2 return to room temperature, add the two Mix the solution evenly, adjust the pH value and use DPBS to make up the balance to a total volume of 100 mL, and set aside.
  • Frozen balance solution a Total volume 100mL. Heat 2.0g of PVA in a water bath at 80°C and dissolve it in 50mL DPBS with magnetic stirring. After all PVA is dissolved, adjust the pH to 7.0, add 7.5mL ethylene glycol, and mix well. Adjust the pH value and use DPBS to make up the remaining volume to a total volume of 100 mL for use.
  • Cryopreservation solution B The total volume is 100mL, 2.0g of PVA is heated in a water bath at 80°C and magnetically stirred and dissolved in 25mL of DPBS, the pH is adjusted to 7.0, which is solution 1; 1.5g of poly-L-proline Dissolve in another 20mL of DPBS ultrasonically, adjust the pH to 7.0, to be solution 2; 17g (0.05mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5mol L -1 ) ultrasonically dissolve in 25mL of DPBS, wait After all the sucrose has been dissolved, add 10 mL of ethylene glycol to form solution 3. After solution 1, solution 2 and solution 3 return to room temperature, mix the three solutions, adjust the pH value and make up the balance to the total volume with DPBS. 100mL, spare.
  • Frozen balance solution b the same composition as freezing balance solution a, with a total volume of 100mL. Heat 2.0g of PVA in a water bath at 80°C and dissolve it in 40mL of DPBS with magnetic stirring. When all PVA is dissolved, adjust the pH to 7.0 and add 7.5 mL of ethylene glycol, mix well, adjust the pH value and make up the balance to a total volume of 100 mL with DPBS for use.
  • Cryopreservation Solution C Each 1 mL contains 10% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, and the balance is DPBS.
  • Frozen balance solution c each 1 mL contains 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS.
  • Thaw liquid I contains the following components per 100mL
  • Thaw Solution II contains the following components per 100mL
  • the thawing solution III contains the following components per 100mL
  • the thawing solution IV contains the following components per 100mL
  • Thaw liquid I contains the following components per 100mL
  • Thaw Solution II contains the following components per 100mL
  • the thawing solution III contains the following components per 100mL
  • the thawing solution IV contains the following components per 100mL
  • Thawing solution I contains 1.0mol L -1 sucrose, 20% serum, and the balance is DPBS;
  • Thawing Solution II Contains 0.5mol L -1 sucrose, 20% serum, and the balance is DPBS;
  • Thawing solution III Contains 0.25mol L -1 sucrose, 20% serum, and the balance is DPBS;
  • Thawing solution IV contains 0mol L -1 sucrose, 20% serum, and the balance is DPBS.
  • the method for cryopreservation of oocytes used in the present invention is specifically that the oocytes are first placed in a cryopreservation solution to balance for 5 minutes, and then placed in the above cryopreservation solution for 45 seconds, and the oocytes that have been balanced in the cryopreservation solution are placed Put it on the freezing rod, then quickly put it into liquid nitrogen (-196°C), and close the rod and continue to store.
  • mice were thawed using the formulas in Examples 1 and 2 and the thawing reagents prepared in Comparative Example 1.
  • the specific method of thawing oocytes is to quickly remove the oocytes from liquid nitrogen and place them at 37°C. In the thawing solution I for 3 to 5 minutes, then put them in the thawing solution II, thawing solution III, and thawing solution IV for 3 minutes at room temperature, then put the oocytes in the culture medium, and place it at 37°C, 5% carbon dioxide Incubate in an incubator for 2 hours and observe the survival rate of oocytes (Table 1).
  • the embryo cryopreservation method used in the present invention specifically includes that the embryos are first placed in a freezing balance solution for 8 minutes, then placed in the cryopreservation solution prepared by the above formula for 50 seconds, and the embryos that have been equilibrated in the freezing solution are placed on the freezing rod , Then quickly put it into liquid nitrogen (-196°C), and close the carrier rod and continue to store.
  • the mouse embryos were thawed using the formulas in the above Examples 1 and 2 and the thawing reagents prepared in Comparative Example 1.
  • the embryo thawing method is specifically to quickly remove the embryos from liquid nitrogen and put them in 37°C Thawing Solution I 3 to After 5 minutes, put the thawing solution II, thawing solution III, and thawing solution IV for 3 minutes in sequence at room temperature, then put the embryos in the culture medium and incubate them in a 37°C, 5% carbon dioxide incubator for 2 hours. Observe Embryo survival rate (Table 2).
  • the survival rate in the embodiment of the present invention is the average survival rate of 3-12 repeated experiments.
  • the survival rate of thawed oocytes of the thawing reagent of the present invention reached more than 94%, which can be as high as 98.6%, and the survival rate of thawed embryos reached more than 97%, which was much higher than that of Comparative Example 1 (commercialized The embryo thawing survival rate of the thawing solution) shows that the thawing reagent is better than conventional vitrification thawing solution in thawing oocytes and embryos.
  • the thawing reagent of the present invention does not add serum, which reduces the risk of parasitic biological contamination and is more conducive to maintaining the passage stability of cells or tissues.
  • the combination of the above freezing balance solution a and the cryopreservation solution A was used to freeze preservation of mouse ovarian organs and ovarian tissue sections of sexually mature mice within 3 days of newborn, and then use the thawing solution of Example 1 above for thawing.
  • the freezing and thawing process is as follows: the whole ovarian organ or ovarian tissue section is placed in the balance solution at room temperature for 25 minutes, and then placed in the prepared cryopreservation solution for 15 minutes, and then the whole ovarian organ or ovarian tissue section is placed on the freezing rod Put it into liquid nitrogen for storage.
  • FIG. 4 is the photo of the section stained after thawing the thawing solution of Example 1 of the ovarian tissue. It can be seen that after the thawing solution of Example 1 is thawed, the follicular structure of the ovarian organs or ovarian tissue is relatively complete, the interstitial structure is relatively complete, the cell cytoplasm is homogeneous, relatively lightly stained, and the nuclei are shrunk and darkly stained.
  • the structure of the vessel wall is complete, the lumen is less collapsed, the cytoplasm of endothelial cells is homogeneous, and the nucleus is relatively less stained, and the nucleus is relatively less collapsed and dark stained, achieving a good recovery effect.

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Abstract

本发明公开了一种冷冻保存用解冻液及其制备方法与应用，其中解冻液包括：仿生控冰材料0.1-50g、水溶性糖0-1.0mol L-1、缓冲液余量，仿生控冰材料为具有亲冰基团和亲水基团的控冰材料；亲水基团为可与水分子形成非共价作用的官能团，亲冰基团为可与冰形成非共价作用的官能团。本发明采用具有亲冰和亲水特性的仿生控冰材料制备的解冻液及解冻试剂，能有效控制冰晶生长，显著改善复苏过程中冰晶不可控生长对于细胞或组织的损害；且生物相容性好，不含动物血清，毒性小，相比于传统含有血清的解冻液，降低了保质期短、带入寄生性生物污染物质等风险，更有利于维持细胞或组织的传代稳定性。且本发明成分简单，成本低廉，具有良好的应用前景。

Description

一种解冻液及其制备方法与应用
本申请要求2019年4月9日向中国国家知识产权局提交的专利申请号为2019102824210，发明名称为“一种冷冻保存用解冻液及解冻方法”，以及专利申请号为2019102824155，发明名称为“一种解冻液在冷冻保存的卵母细胞或胚胎解冻中的应用”的在先申请的优先权。该两件在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于生物医用材料技术领域，具体涉及一种冷冻保存用解冻液及解冻方法。
背景技术
冷冻保存是指将生物材料保存于超低温状态下，使细胞新陈代谢和分裂速度减慢或者停止，一旦恢复正常生理温度又能继续发育。该技术自问世以来，成为自然科学领域不可缺少的研究方法之一，已被广泛采用。近年来，随着生活压力的增加，人类生育力呈逐年下降的趋势，生育力保存越来越受到人们的重视，人类生殖细胞(精子、卵母细胞)、性腺组织等的冷冻保存就成为保存生育力的重要手段。另外，随着世界人口老龄化加剧，对捐赠的可用于再生医学和器官移植的人源性细胞、组织或器官的冷冻保存的需求也极速增加。因此，如何高效的冷冻保存珍贵的细胞、组织以及器官资源以备不时之需成为亟待解决的科学技术问题。
目前最常用的冷冻保存方法为冷冻。在解冻过程中，需要注意防止冰晶、冷休克、溶质效应、破碎损伤、重结晶、渗透性休克等冷冻融解损伤，基于提高胚胎、卵母细胞复苏存活率的考虑，通常冷冻融解方案为：采用快速复温的方法，将高渗透压的囊胚或卵母细胞置于含有一定浓度梯度的冷冻保护剂的培养液中，从而使得细胞内外的渗透压差距逐渐减小，减缓细胞体积的变化速度，避免解冻过程中造成的细胞或胚胎损伤。目前临床广泛使用的解冻试剂大多以蔗糖、血清和缓冲液为主要成分。但上述解冻液在复温过程中不能有效的控制冰晶的生长，从而损害细胞。除此之外，目前解冻液因使用成分不明确且包含易变质的血清，导致解冻液存在保质期短、带入寄生性生物污染物质等问题。
发明内容
为改善现有技术的上述缺陷，本发明提供一种冷冻保存用解冻液及解冻方法。
本发明通过以下技术方案实现:
一种解冻液，每100mL含有仿生控冰材料0.1-50g、水溶性糖0-1.0mol L -1、缓冲液余量，所述仿生控冰材料为具有亲冰基团和亲水基团的控冰材料。
根据本发明，所述亲水基团为可与水分子形成非共价作用的官能团，例如可与水形成氢键、范德华尔斯作用、静电作用、疏水作用或者π-π作用；示例性地，所述亲水基团可以选自羟基(-OH)、氨基(-NH 2)、羧酸基(-COOH)、酰胺基(-CONH 2)中的至少一种，或，选自例如脯氨酸(L-Pro),精氨酸(L-Arg),赖氨酸(L-Lys)，组氨酸(L-His)，甘氨酸(L-Gly)，葡萄糖酸内酯(GDL)，糖类等化合物分子或其分子片段。
根据本发明，所述亲冰基团为可与冰形成非共价作用的官能团，例如可与冰形成氢键、范德华尔斯作用、静电作用、疏水作用或者π-π作用；示例性地，所述亲冰基团可以选自羟基(-OH),氨基(-NH 2),苯基(-C 6H 5)，吡咯烷基(-C 4H 8N)，或，选自例如谷氨酰胺(L-Gln),苏氨酸(L-Thr),天冬氨酸(L-Asn)，苯环(-C 6H 6)，吡咯烷(-C 4H 9N)等化合物分子或其分子片段。
根据本发明，所述仿生控冰材料选自聚乙烯醇(PVA)、氨基酸、多肽、聚氨基酸中的至少一种或两种以上的组合。
根据本发明，所述氨基酸可以选自精氨酸、苏氨酸、脯氨酸、赖氨酸、组氨酸、谷氨酰胺、天冬氨酸、甘氨酸等中的一种或两种以上的组合；所述聚氨基酸为可选自赖氨酸、精氨酸、脯氨酸、苏氨酸、组氨酸、谷酰胺酸、天冬氨酸、甘氨酸等中至少一种的均聚物(聚合度≥2，优选聚合度为2～40，例如聚合度为6、8、15、20等)。
示例性地，所述多肽为上述氨基酸中的两种以上组成的多肽或者为氨基酸与糖类(例如葡萄糖内酯)反应生成的糖肽衍生物，例如所述多肽是2-8个不同的氨基酸组成的多肽，再例如二肽、三肽、四肽等。在本发明的实施方案中，所述多肽为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)，L-Thr-L-Cys-L-Thr(TCT)中的一种或两种以上。
根据本发明，所述仿生控冰材料含量为1.0-50g、2.0-20g、5.0-10g；在一个实施方案中，所述仿生控冰材料含量为3.0g、4.0g、5.0g、10g、25g、30g。
作为本发明的一个实施方案，所述仿生控冰材料包括PVA 1.0-6.0g。
作为本发明的一个实施方案，所述仿生控冰材料包括氨基酸1.0-30g。示例性地，所述氨基酸为精氨酸与苏氨酸组合而成，例如含有精氨酸1.0-20g、1.0-10g、1.0-5g，含有苏氨酸 1.0-10g、1.0-5.0g、1.0-2.5g。
作为本发明的一个实施方案，所述仿生控冰材料包括聚氨基酸0.1-9.0g，例如1-5.0g。示例性地，所述氨基酸为聚-L-脯氨酸和/或聚-L-精氨酸。
作为本发明的一个实施方案，所述仿生控冰材料包括多肽1.0-50g；例如1.0-25g、1.0-13g、1.0-10g、1.0-5.0g。
作为本发明的一个实施方案，所述仿生控冰材料是PVA与上述氨基酸、多肽、和/或聚氨基酸的组合。
根据本发明，每100mL解冻液中所述水溶性糖含量为0.1-1.0mol L -1，例如0.1-0.8mol L -1，0.2-0.6mol L -1；例如0.25mol L -1，0.5mol L -1，1.0mol L -1。
一种解冻试剂，包括解冻液I、解冻液II、解冻液III和解冻液IV，所述解冻液I-IV具有如上所述解冻液的组成。
根据本发明的解冻试剂，所述解冻液I—IV中，仿生控冰材料的浓度梯度没有特别的限定，各解冻液中仿生控冰材料的浓度可以相同或不同；作为一个示例性方案，所述解冻液I—IV中，仿生控冰材料的浓度不同，例如所述解冻液II中仿生控冰材料的含量为解冻液I中的50％-100％，所述解冻液III中仿生控冰材料的含量为解冻液II中的50％-100％。
根据本发明的解冻试剂，所述解冻液I-IV中，含有PVA 1.0-6.0g；优选地，所述解冻液I-IV中，PVA浓度相同。
根据本发明的解冻试剂，所述解冻液II中水溶性糖浓度为解冻液I中的50％-100％，所述解冻液III中水溶性糖浓度为解冻液II中的50％-100％，所述解冻液IV中，糖浓度为0。
作为本发明解冻试剂的一个实施方案，所述解冻液I以每100mL计，含有氨基酸1.0-50g，PVA 1.0-5.0g，水溶性糖1.0mol L -1，缓冲液余量；
所述解冻液II以每100mL计，含有氨基酸1.0-25g，PVA 1.0-5.0g，水溶性糖0.5mol L -1，缓冲液余量；
所述解冻液III以每100mL计，含有氨基酸1.0-12.5g，PVA 1.0-5.0g，水溶性糖0.25mol L -1，缓冲液余量；
所述解冻液IV以每100mL计，含有氨基酸0-6.25g，PVA 1.0-5.0g，缓冲液余量。
作为本发明解冻试剂的一个实施方案，所述解冻液I以每100mL计，含有PVA 1.0-5.0g，水溶性糖1.0mol L -1，缓冲液余量；
所述解冻液II以每100mL计，含有PVA 1.0-5.0g，水溶性糖0.5mol L -1，缓冲液余量；
所述解冻液III以每100mL计，含有PVA 1.0-5.0g，水溶性糖0.25mol L -1，缓冲液余量；
所述解冻液IV以每100mL计，含有PVA 1.0-5.0g，缓冲液余量。
作为本发明解冻试剂的一个实施方案，所述解冻液I以每100mL计，含有聚氨基酸0.1-9.0g，PVA 1.0-5.0g，水溶性糖1.0mol L -1，缓冲液余量；
所述解冻液II以每100mL计，含有聚氨基酸0.1-4.5g，PVA 1.0-5.0g，水溶性糖0.5mol L -1，缓冲液余量；
所述解冻液III以每100mL计，含有聚氨基酸0.1-2.3g，PVA 1.0-5.0g，水溶性糖0.25mol L -1，缓冲液余量；
所述解冻液IV以每100mL计，含有聚氨基酸0-1.2g，PVA 1.0-5.0g，缓冲液余量。
作为本发明解冻试剂的一个实施方案，所述解冻液I以每100mL计，含有多肽1.0-50g，PVA 1.0-5.0g，水溶性糖1.0mol L -1，缓冲液余量；
所述解冻液II以每100mL计，含有多肽1.0-25g，PVA 1.0-5.0g，水溶性糖0.5mol L -1，缓冲液余量；
所述解冻液III以每100mL计，含有多肽1.0-12.5g，PVA 1.0-5.0g，水溶性糖0.25mol L -1，缓冲液余量；
所述解冻液IV以每100mL计，含有多肽0-6.25g，PVA 1.0-5.0g，缓冲液余量。
根据本发明，所述水溶性糖可以为非还原性双糖、水溶性多糖、糖酐中的至少一种，例如选自蔗糖、海藻糖；优选蔗糖。所述水溶性糖可以起到保护细胞膜和避免细胞沉降的作用。
根据本发明，所述缓冲液可选自DPBS或hepes-buffered HTF缓冲液或其它细胞培养缓冲液中的至少一种。
根据本发明，所述PVA选自等规PVA、间规PVA和无规PVA的一种或两种以上的组合，例如所述PVA的间同规整度为15％-65％，具体地，例如40％-60％、53％-55％。优选无规PVA，例如所述PVA的间同规整度为45％-65％的PVA。
根据本发明，所述PVA可选自分子量为10-500kDa或者更高分子量的PVA，例如分子量为10-30kDa、30-50kDa、80-90kDa、200-500kDa。
根据本发明，所述PVA可选自水解度大于80％的PVA，例如水解度为80％-99％、82-87％、87％-89％、89％-99％、98％-99％。
本发明的解冻液可采用本领域的已知方法配制，例如：
将仿生控冰材料溶解于一部分缓冲液中，调节pH，将水溶性糖溶解于一部分缓冲液中， 冷却至室温后将两种溶液混合。
一种冷冻保存细胞或组织或器官的解冻方法，包括如下步骤：
将冷冻的细胞或组织或器官放入37℃所述解冻液I中复苏3-5分钟，然后常温下依次放入所述解冻液II、解冻液III、解冻液IV中各3-5分钟。
上述解冻液和解冻试剂可用于冷冻保存的卵母细胞和胚胎、组织或器官的复苏和解冻。例如所述组织或器官为卵巢组织或卵巢器官。
进一步地，本发明提供上述解冻液和解冻试剂的应用，具体而言是在冷冻保存的卵母细胞和胚胎、组织或器官的复苏和解冻中的应用，例如所述组织或器官为卵巢组织或卵巢器官。
有益效果
本发明采用具有亲冰和亲水特性的仿生控冰材料制备的解冻液及解冻试剂，能有效控制冰晶生长，显著改善复苏过程中温度变化对于细胞或组织的损害；且生物相容性好，不含动物血清，毒性小，相比于传统含有血清的解冻液，降低了保质期短、带入寄生性生物污染物质等风险，更有利于维持细胞或组织的传代稳定性。且本发明成分简单，成本低廉，具有良好的应用前景。
附图说明
图1为新鲜未冷冻的卵巢器官切片染色照片；
图2为卵巢器官经实施例1的解冻液解冻后切片染色的照片；
图3为新鲜未冷冻的卵巢组织切片染色照片；
图4为卵巢组织经实施例1的解冻液解冻后切片染色的照片。
具体实施方式
下文将结合具体实施例对本发明的制备方法做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
本发明实施例中采用的PVA，间同规整度为50％-55％，分子量为13-23kDa，水解度98％。
本发明实施例中冷冻保存液中采用聚-L-脯氨酸聚合度为15，分子量为1475。解冻液中聚-L-脯氨酸聚合度为8，分子量为795。
本发明实施例中采用如下冷冻平衡液和冷冻保存液：
冷冻保存液A：总体积100mL，将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于30mL的DPBS中，调节pH为7.0，为溶液1；17g(0.05mol)的蔗糖(蔗糖在冷冻保存液中终浓度为0.5mol L -1)超声溶解于25mL的DPBS中，待蔗糖全部溶解后加入10mL的乙二醇，为溶液2，待溶液1及溶液2恢复至室温，再将两种溶液混均，调节pH值并用DPBS定容补齐余量至总体积100mL，备用。
冷冻平衡液a：总体积100mL，将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于50mL的DPBS中，待PVA全部溶解，调节pH为7.0，加入7.5mL乙二醇，混匀，调节pH值并用DPBS定容补齐余量至总体积100mL，备用。
冷冻保存液B：总体积100mL，将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于25mL的DPBS中，调节pH为7.0，为溶液1；将1.5g的聚-L-脯氨酸超声溶于另外20mL的DPBS中，调节pH为7.0，为溶液2；17g(0.05mol)的蔗糖(蔗糖在冷冻保存液中终浓度为0.5mol L -1)超声溶于25mL的DPBS中，待蔗糖全部溶解后依次加入10mL乙二醇，为溶液3，待溶液1、溶液2及溶液3恢复至室温，再将三种溶液混匀，调节pH值并用DPBS定容补齐余量至总体积100mL，备用。
冷冻平衡液b：与冷冻平衡液a组成相同，总体积100mL，将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于40mL的DPBS中，待PVA全部溶解，调节pH为7.0，加入7.5mL的乙二醇，混匀，调节pH值并用DPBS定容补齐余量至总体积100mL，备用。
冷冻保存液C：每1mL中含有10％(v/v)的乙二醇，20％(v/v)的胎牛血清，0.5M蔗糖，余量为DPBS。
冷冻平衡液c：每1mL中含有7.5％(v/v)的乙二醇，20％(v/v)的胎牛血清，余量为DPBS。
实施例1
解冻液Ⅰ每100mL含有如下组分
物质 含量
PVA(mg mL -1) 20
蔗糖(mol L -1) 1.0
DPBS(V) 余量
解冻液Ⅱ每100mL含有如下组分
物质 含量
PVA(mg mL -1) 20
蔗糖(mol L -1) 0.5
DPBS(V) 余量
解冻液Ⅲ每100mL含有如下组分
物质 含量
PVA(mg mL -1) 20
蔗糖(mol L -1) 0.25
DPBS(V) 余量
解冻液Ⅳ每100mL含有如下组分
物质 含量
PVA(mg mL -1) 20
DPBS(V) 余量
实施例2
解冻液Ⅰ每100mL含有如下组分
物质 含量
聚-L-脯氨酸/(mg mL -1) 10
PVA(mg mL -1) 20
蔗糖(mol L -1) 1.0
DPBS(V) 余量
解冻液Ⅱ每100mL含有如下组分
物质 含量
聚-L-脯氨酸/(mg mL -1) 5.0
PVA(mg mL -1) 20
蔗糖(mol L -1) 0.5
DPBS(V) 余量
解冻液Ⅲ每100mL含有如下组分
物质 含量
聚-L-脯氨酸/(mg mL -1) 2.5
PVA(mg mL -1) 20
蔗糖(mol L -1) 0.25
DPBS(V) 余量
解冻液Ⅳ每100mL含有如下组分
物质 含量
PVA(mg mL -1) 20
DPBS(V) 余量
对比例1：
解冻液Ⅰ：含有1.0mol L -1蔗糖，20％的血清，余量为DPBS；
解冻液Ⅱ：含有0.5mol L -1蔗糖，20％的血清，余量为DPBS；
解冻液Ⅲ：含有0.25mol L -1蔗糖，20％的血清，余量为DPBS；
解冻液Ⅳ：含有0mol L -1蔗糖，20％的血清，余量为DPBS。
应用例1冷冻卵母细胞的解冻
本发明所用卵母细胞冷冻保存方法具体为卵母细胞先置于冷冻平衡液中平衡5分钟，然后置于上述冷冻保存液中平衡45秒，将已在冷冻保存液中平衡的卵母细胞放置于冷冻载杆上，然后快速投入液氮(-196℃)中，并封闭载杆后继续保存。
采用上述实施例1、2中的配方和对比例1所制备的解冻用试剂对小鼠的卵母细胞进行解冻，卵母细胞解冻方法具体为从液氮中快速取出卵母细胞放入37℃解冻液Ⅰ中3至5分钟，后常温下依次放入解冻液Ⅱ、解冻液Ⅲ、解冻液Ⅳ中各3分钟，然后将卵母细胞放入培养基中，置于37℃、5％二氧化碳培养箱中培养2小时，观察卵母细胞存活率(表1)。
应用例2冷冻胚胎的解冻
本发明所用胚胎冷冻保存方法具体为胚胎先置于冷冻平衡液中平衡8分钟，然后置于上述配方所制备冷冻保存液50秒，将已在冷冻保存液中平衡的胚胎放置于冷冻载杆上，然后快速投入液氮(-196℃)中，并封闭载杆后继续保存。
采用上述实施例1、2中的配方和对比例1所制备的解冻用试剂对小鼠的胚胎进行解冻，胚胎解冻方法具体为从液氮中快速取出胚胎放入37℃解冻液Ⅰ中3至5分钟，后常温下依次放入解冻液Ⅱ、解冻液Ⅲ、解冻液Ⅳ中各3分钟，然后将胚胎放入培养基中，置于37℃、5％二氧化碳培养箱中培养2小时，观察胚胎存活率(表2)。
本发明实施例中存活率为3-12次重复实验的存活率平均值。
表1解冻液解冻小鼠卵母细胞的存活率对比
编号 平衡液 冷冻液 解冻液 冻卵总数 2小时后存活率％
应用实例1 a A 实施例1 53 96.5
应用实例2 b B 实施例2 60 98.6
应用实例3 c C 实施例1 44 94.7
对比实例1 a A 对比例1 50 93.4
对比实例2 b B 对比例1 39 89.7
对比实例3 c C 对比例1 96 81.9
表2解冻液解冻小鼠胚胎的存活率对比
编号 平衡液 冷冻液 解冻液 胚胎总数 2小时后存活率％
对比实例4 c C 对比例1 39 82.0
应用实例4 b B 实施例2 39 97.4
应用实例5 a A 实施例1 37 97.1
由表1和表2数据可以看出，本发明解冻试剂解冻卵母细胞存活率均达到94％以上，可高达98.6％，解冻胚胎存活率达97％以上，远高于对比例1(商业化解冻液)的胚胎解冻存活率，表明该解冻试剂优于常规玻璃化解冻液解冻卵母细胞和胚胎的有效性。而且本发明的解冻试剂不加入血清，减少了寄生性生物污染物质等风险，更有利于维持细胞或组织的传代稳定性。
应用例3完整卵巢器官或卵巢组织切片冷冻后的解冻
采用上述冷冻平衡液a以及冷冻保存液A的组合分别对新生3天内的小鼠卵巢器官和性成熟小鼠的卵巢组织切片进行冷冻保存，然后采用上述实施例1的解冻液进行解冻。冷冻和解冻过程如下：整个卵巢器官或者卵巢组织切片先置于平衡液室温平衡25分钟，然后置于所制备的冷冻保存液中15分钟，之后将完整卵巢器官或卵巢组织切片放置于冷冻载杆上，投入液氮中保存。解冻后，完整卵巢器官或卵巢组织切片放入培养液(10％FBS+a-MEM)后置于37℃、5％CO2培养箱中复苏培养2小时后使用4％多聚甲醛固定、石蜡包埋、HE染色观察形态。
结果如图1-4所示，其中图1为新鲜未冷冻的卵巢器官切片染色照片，图2为卵巢器官 经实施例1的解冻液解冻后切片染色的照片；图3为新鲜未冷冻的卵巢组织切片染色照片，图4为卵巢组织经实施例1的解冻液解冻后切片染色的照片。可见，经实施例1的解冻液解冻后，卵巢器官或卵巢组织的卵泡结构相对完整，间质结构相对完整，细胞胞浆均质、淡染相对较多，胞核皱缩、深染相对较少；血管管壁结构完整，管腔塌陷较少，内皮细胞胞浆均质、淡染相对较多，胞核皱缩、深染相对较少，达到良好的复苏效果。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (11)

  1. 一种解冻液，其特征在于：每100mL解冻液含有仿生控冰材料0.1-50g、水溶性糖0-1.0mol L -1、缓冲液余量，所述仿生控冰材料为具有亲冰基团和亲水基团的控冰材料；所述亲水基团为可与水分子形成非共价作用的官能团或分子，所述亲冰基团为可与冰形成非共价作用的官能团或分子。
  2. 根据权利要求1所述的解冻液，其特征在于，所述亲水基团与水形成氢键、范德华尔斯作用、静电作用、疏水作用或者π-π作用；示例性地，所述亲水基团选自羟基(-OH)、氨基(-NH 2)、羧酸基(-COOH)、酰胺基(-CONH 2)中的至少一种，或，选自例如脯氨酸(L-Pro),精氨酸(L-Arg),赖氨酸(L-Lys)，葡萄糖酸内酯(GDL)，糖类等化合物分子或其分子片段；
    优选，所述亲冰基团与冰形成氢键、范德华尔斯作用、静电作用、疏水作用或者π-π作用；示例性地，所述亲冰基团选自羟基(-OH),氨基(-NH 2),苯基(-C 6H 5)，吡咯烷基(-C 4H 8N)，或，选自例如谷氨酰胺(L-Gln),苏氨酸(L-Thr),天冬氨酸(L-Asn)，组氨酸(L-His)，甘氨酸(L-Gly)，苯环(C 6H 6)，吡咯烷(C 4H 9N)等化合物分子或其分子片段。
  3. 根据权利要求1或2所述的解冻液，其特征在于，所述仿生控冰材料选自聚乙烯醇PVA、氨基酸、多肽、聚氨基酸中的至少一种或两种以上的组合；
    优选，所述氨基酸选自精氨酸、苏氨酸、脯氨酸、赖氨酸、组氨酸、谷氨酰胺、天冬氨酸、甘氨酸等中的一种或两种以上的组合；所述聚氨基酸为选自赖氨酸、精氨酸、脯氨酸、苏氨酸、组氨酸、谷酰胺酸、天冬氨酸、甘氨酸等中至少一种的均聚物(聚合度≥2，优选聚合度为2～40，例如聚合度为6、8、15、20等)；
    优选，所述多肽为两种以上的氨基酸组成或者为氨基酸与糖类组成的糖肽衍生物，例如为2-8个不同的氨基酸组成，优选地所述多肽为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)，L-Thr-L-Cys-L-Thr(TCT)中的一种或两种以上；
    优选，所述PVA选自等规PVA、间规PVA和无规PVA的一种或两种以上的组合，例如所述PVA的间同规整度为15％-65％，优选间同规整度45％-65％；
    优选，所述PVA选自分子量为10-500kDa或者更高分子量的PVA；优选PVA水解度为80％-99％、82-87％、87％-89％、89％-99％、98％-99％。
  4. 根据权利要求1-3任一项所述的解冻液，其特征在于，所述仿生控冰材料含量为1.0-30g；
    优选，所述仿生控冰材料包括PVA 1.0-6.0g；
    优选，所述仿生控冰材料包括氨基酸1.0-30g，更优选，所述氨基酸为精氨酸与苏氨酸组合而成；
    优选，所述仿生控冰材料包括聚氨基酸0.1-9.0g；
    优选，所述仿生控冰材料包括多肽1.0-50g；
    优选，所述仿生控冰材料是PVA与所述氨基酸、多肽、和/或聚氨基酸的组合。
  5. 根据权利要求1-4任一项所述的解冻液，其特征在于，每100mL解冻液中所述水溶性糖含量为0.1-1.0mol L -1；
    优选，所述水溶性糖可以为非还原性双糖、水溶性多糖、糖酐中的至少一种，例如选自蔗糖、海藻糖、水溶性纤维素(例如羟丙基甲基纤维素)、聚蔗糖；
    优选，所述缓冲液可选自DPBS或hepes-buffered HTF缓冲液或其他细胞缓冲液中的至少一种。
  6. 一种解冻试剂，其特征在于，包括解冻液I、解冻液II、解冻液III和解冻液IV，所述解冻液I-IV具有如权利要求1-5任一项所述解冻液的组成。
  7. 根据权利要求6所述的解冻试剂，其特征在于，优选，所述解冻液II中水溶性糖浓度为解冻液I中的50％-100％，所述解冻液III中水溶性糖浓度为解冻液II中的50％-100％，所述解冻液IV中，水溶性糖浓度为0；优选，所述解冻液I-IV中，例如仿生控冰材料的浓度相同或不同，例如解冻液II中仿生控冰材料浓度为解冻液I中的50％-100％，所述解冻液III中仿生控冰材料浓度为解冻液II中的50％-100％，所述解冻液IV中，仿生控冰材料浓度为0；
    优选，所述解冻液I-IV中，含有PVA 1.0-6.0g；优选地，所述解冻液I-IV中，PVA浓度相同；
  8. 根据权利要求6或7所述的解冻试剂，其特征在于，所述解冻试剂中，所述解冻液I以每100mL计，含有氨基酸、或者多肽中的至少一种1.0-50g，PVA 1.0-5.0g，水溶性糖1.0mol L -1，缓冲液余量；
    所述解冻液II以每100mL计，含有氨基酸、或者多肽中的至少一种1.0-25g，PVA 1.0-5.0g，水溶性糖0.5mol L -1，缓冲液余量；
    所述解冻液III以每100mL计，含有氨基酸、或者多肽中的至少一种1.0-12.5g，PVA1.0-5.0g，水溶性糖0.25mol L -1，缓冲液余量；
    所述解冻液IV以每100mL计，含有氨基酸、或者多肽中的至少一种0-6.25g，PVA1.0-5.0g，缓冲液余量。
  9. 根据权利要求6或7所述的解冻试剂，其特征在于，所述解冻液I以每100mL计，含有PVA 1.0-5.0g，水溶性糖1.0mol L -1，缓冲液余量；
    所述解冻液II以每100mL计，含有PVA 1.0-5.0g，水溶性糖0.5mol L -1，缓冲液余量；
    所述解冻液III以每100mL计，含有PVA 1.0-5.0g，水溶性糖0.25mol L -1，缓冲液余量；
    所述解冻液IV以每100mL计，含有PVA 1.0-5.0g，缓冲液余量；
    优选，所述解冻液I以每100mL计，含有聚氨基酸0.1-9.0g，PVA 1.0-5.0g，水溶性糖1.0mol L -1，缓冲液余量；
    所述解冻液II以每100mL计，含有聚氨基酸0.1-4.5g，PVA 1.0-5.0g，水溶性糖0.5mol L -1，缓冲液余量；
    所述解冻液III以每100mL计，含有聚氨基酸0.1-2.3g，PVA 1.0-5.0g，水溶性糖0.25mol L -1，缓冲液余量；
    所述解冻液IV以每100mL计，含有聚氨基酸0-1.2g，PVA 1.0-5.0g，缓冲液余量。
  10. 一种冷冻保存细胞或组织的解冻方法，包括如下步骤：
    将冷冻的细胞或组织放入37℃如权利要求6-9任一项所述解冻液I中复苏3-5分钟，然后常温下依次放入权利要求6-9任一项所述解冻液II、解冻液III、解冻液IV中各3-5分钟。
  11. 权利要求1-5任一项所述的解冻液或者权利要求6-9任一项所述解冻试剂的应用，其应用于冷冻保存的卵母细胞和胚胎、组织或器官的复苏和解冻，例如所述组织或器官为卵巢组织或卵巢器官。
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