WO2020207150A1 - 一种仿生控冰材料及含有该仿生控冰材料的冷冻保存液 - Google Patents

一种仿生控冰材料及含有该仿生控冰材料的冷冻保存液 Download PDF

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WO2020207150A1
WO2020207150A1 PCT/CN2020/077472 CN2020077472W WO2020207150A1 WO 2020207150 A1 WO2020207150 A1 WO 2020207150A1 CN 2020077472 W CN2020077472 W CN 2020077472W WO 2020207150 A1 WO2020207150 A1 WO 2020207150A1
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Prior art keywords
ice
solution
pva
cryopreservation
water
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PCT/CN2020/077472
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English (en)
French (fr)
Inventor
王健君
金晟琳
吕健勇
严杰
乔杰
闫丽盈
李蓉
Original Assignee
中国科学院化学研究所
北京大学第三医院（北京大学第三临床医学院）
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Priority claimed from CN201910282422.5A external-priority patent/CN111790327B/zh
Priority claimed from CN201910282418.9A external-priority patent/CN111795909B/zh
Application filed by 中国科学院化学研究所, 北京大学第三医院（北京大学第三临床医学院） filed Critical 中国科学院化学研究所
Priority to US17/594,340 priority Critical patent/US20220192179A1/en
Priority to EP20786705.2A priority patent/EP3928862A4/en
Priority to AU2020256938A priority patent/AU2020256938B2/en
Priority to KR1020217036485A priority patent/KR20210142751A/ko
Priority to JP2021560635A priority patent/JP7389138B2/ja
Priority to SG11202110868XA priority patent/SG11202110868XA/en
Publication of WO2020207150A1 publication Critical patent/WO2020207150A1/zh
Priority to JP2023195336A priority patent/JP2024023322A/ja

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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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/12—Hydrolysis
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N13/00—Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0227—Investigating particle size or size distribution by optical means using imaging; using holography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/18—Water
    • G01N33/1873—Ice or snow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/50—Molecular design, e.g. of drugs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/60—In silico combinatorial chemistry
    • G16C20/64—Screening of libraries
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C60/00—Computational materials science, i.e. ICT specially adapted for investigating the physical or chemical properties of materials or phenomena associated with their design, synthesis, processing, characterisation or utilisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G01N15/0211—Investigating a scatter or diffraction pattern
    • G01N2015/0222—Investigating a scatter or diffraction pattern from dynamic light scattering, e.g. photon correlation spectroscopy
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C10/00—Computational theoretical chemistry, i.e. ICT specially adapted for theoretical aspects of quantum chemistry, molecular mechanics, molecular dynamics or the like

Definitions

  • the invention belongs to the technical field of biomedical materials, and in particular relates to a bionic ice control material and a cryopreservation liquid containing the bionic ice control material.
  • 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.
  • the most commonly used cryopreservation method is vitrification.
  • the vitrification technology uses permeable or non-permeable cryoprotective agents, although the liquid inside and outside the cell can be directly vitreous during the rapid freezing process to avoid the damage caused by the formation of ice crystals during the freezing process.
  • the existing cryopreservation reagents cannot effectively control the growth of ice crystals, thereby damaging cells. Since the ice control mechanism of antifreeze proteins and bionic ice control materials at the molecular level is still controversial, the research and development of bionic ice control materials can only rely on the "trial and error method" to gradually try the ice control effect of certain ice control materials, which requires a lot of work. The chance of success is low.
  • the currently commonly used cryopreservation reagents do not have the ability to effectively control the growth of ice crystals during the rewarming process, and the reagents are highly toxic.
  • the present invention provides a molecular design method for bionic ice control materials and a screening method for ice control materials, which can guide people to purposefully synthesize and screen bionic ice control materials.
  • the present invention also provides The bionic ice control materials obtained by the method and the cryopreservation reagents containing these materials.
  • a molecular design method of an ice control material which includes the following steps:
  • step (2) the ice-controlling molecules with the required ice affinity and water affinity are selected.
  • the main chain of the ice-controlling molecule is a carbon chain or a peptide chain structure.
  • 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, etc.; exemplary Preferably, the hydrophilic group may be selected from at least one of hydroxyl (-OH), amino (-NH 2 ), carboxylic acid (-COOH), amide (-CONH 2 ), etc., or, for example, From proline (L-Pro), arginine (L-Arg), lysine (L-Lys) and other hydrophilic amino acids, gluconolactone (GDL), carbohydrates and other compounds 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, etc.; illustratively
  • 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, selected from Ice-philic amino acids such as glutamine (L-Gln), threonine (L-Thr), aspartic acid (L-Asn), benzene ring (C 6 H 6 ), pyrrolidine (C 4 H 9 N) And other compounds or molecular fragments.
  • the ice-controlling material may be formed by covalently bonding a hydrophilic group and an ice-philic group.
  • the ice-controlling material can be formed by a non-covalent bond between a hydrophilic group and an ice-philic group, such as an ionic bond.
  • the method further includes the step (4): the step of synthesizing the ice-controlling molecule (ice-controlling material), for example, by a known chemical synthesis method, such as a polymerization reaction, a condensation reaction, or genetically engineered bacteria. Synthesis by fermentation and other methods.
  • a known chemical synthesis method such as a polymerization reaction, a condensation reaction, or genetically engineered bacteria. Synthesis by fermentation and other methods.
  • the present invention also provides an ice control material obtained according to the molecular design method of the first aspect.
  • a method for screening ice control materials including the following steps: (a) measuring the affinity of the ice control material with water; (b) measuring the ice control material at the ice-water interface The spreading performance.
  • the step (a) can be determined by measuring the solubility, hydration constant, dispersion size, diffusion coefficient, etc. of the ice-controlling material in water, and/or calculating the ice-controlling material and water molecules.
  • Intermolecular hydrogen bond number and other methods specifically, for example, molecular dynamics simulation (Molecular dynamics simulation, MD) is used to determine the number of intermolecular hydrogen bonds formed by the ice-controlling material molecules and water molecules, or dynamic light scattering
  • MD molecular dynamics simulation
  • the spreading performance of the material at the ice-water interface can be obtained by measuring the adsorption content of the ice-controlling material on the ice surface at the ice-water interface, and/or calculating the Methods such as the number of intermolecular hydrogen bonds formed by the ice-controlling material and ice-water molecules are used to determine the affinity between the material and ice; specifically, for example, MD simulations are used to determine the intermolecular formation between the ice-controlling molecules and ice-water molecules. The number of hydrogen bonds, or the adsorption amount of the ice-controlling material molecules on the ice surface measured at the ice-water interface using an ice adsorption experiment.
  • the ice adsorption experiment includes measuring the adsorption amount of the ice control material on the ice surface.
  • the adsorption amount of the ice control material on the ice surface (the mass m 1 of the ice control material adsorbed on the ice surface / the total mass m 2 of the ice control material in the original solution containing the ice control material) ⁇ 100%.
  • the ice adsorption experiment includes the following steps:
  • the temperature control rod is a rod made of a thermally conductive material.
  • the rod may be solid or hollow.
  • the temperature control rod is hollow, there is cooling liquid flowing in the hollow cavity, and the temperature of the temperature control rod can be controlled by controlling the temperature of the cooling liquid, thereby controlling the growth rate of ice.
  • the temperature control rod may be pre-cooled by one of liquid nitrogen, dry ice, ultra-low temperature refrigerator freezing and the like.
  • the supercooling degree and the adsorption time are kept unchanged so that the surface area of the ice obtained on the surface of the temperature control rod remains unchanged within the error tolerance range.
  • the applicable concentration range of the same ice-controlling material in specific applications can be evaluated.
  • the ice control material in step S1 may be pre-fluorescently labeled, for example, labeled with fluorescein, and the fluorescein may be selected from fluorescein isothiocyanate (FITC), tetraethyl rhodan At least one of amine (RB200), rhodamine tetramethyl isothiocyanate (TRITC), propidium iodide (PI), and the like.
  • fluorescein fluorescein isothiocyanate
  • RB200 tetraethyl rhodan At least one of amine (RB200), rhodamine tetramethyl isothiocyanate (TRITC), propidium iodide (PI), and the like.
  • FITC fluorescein isothiocyanate
  • RB200 tetraethyl rhodan At least one of amine
  • TRITC rhodamine tetramethyl isothi
  • the function of the fluorescent label is to measure the amount of the ice control material, therefore, if the adsorption amount of the ice control material can be accurately measured by other means, or the material itself has ultraviolet or fluorescence spectra Absorption characteristics do not require fluorescent labeling.
  • step S3 includes:
  • the concentration c in the S3b, can be measured by methods known in the art, such as ultraviolet-visible spectroscopy, fluorescence spectroscopy, and the like.
  • the method is used to control the screening of ice crystal growth materials, such as PVA, polyamino acid, antifreeze protein, polypeptide and the like.
  • the method further includes step (c) after step (a) and/or (b): evaluating the affinity of the material with water and the spreading performance at the ice-water interface, a material with strong spreading ability Has good ice control performance.
  • the supercooling temperature refers to the temperature below the freezing point of water but still does not solidify or crystallize. At room temperature 25°C, the supercooling temperature is generally in the range of -0.01 to -0.5°C, for example -0.1°C.
  • the present invention also provides an ice adsorption experimental device, which includes a multi-layer liquid storage cavity, a temperature control rod and a temperature controller.
  • the multi-layer liquid storage cavity sequentially includes an ice adsorption cavity, a warm bath cavity, and a cooling liquid storage cavity from the inside to the outside.
  • the temperature control rod is placed in the ice adsorption cavity, and the temperature of the temperature control rod and the liquid storage cavity is controlled by a temperature controller.
  • the temperature control rod is a hollow structure made of a thermally conductive material, and the hollow structure of the temperature control rod is provided with a liquid inlet and a liquid outlet;
  • the temperature controller is a fluid temperature control
  • the temperature controller is provided with a cooling liquid outflow end and a return end; both ends of the cooling liquid storage cavity are provided with a liquid inlet and a liquid outlet; the cooling liquid outflow end and the temperature control rod of the temperature controller
  • the liquid inlet, the liquid outlet of the temperature control rod, the liquid inlet of the cooling liquid storage tank, the liquid outlet of the cooling liquid storage tank, and the return end of the temperature controller are connected in sequence through a pipeline in which cooling liquid flows.
  • the multi-layer liquid storage cavity is provided with a cover.
  • the ice adsorption chamber When in use, the ice adsorption chamber contains an aqueous solution of ice-controlling materials, and the middle-layer warm bath chamber contains a warm bath medium of a predetermined temperature, such as a water bath, an ice bath or an oil bath, etc.; after the temperature of the cooling liquid reaches the set temperature, the temperature controller Flow out and flow into the hollow structure of the temperature control rod to control the temperature of the temperature control rod, then flow out from the outlet of the temperature control rod, and then flow into the outer coolant storage cavity to keep the temperature of the bath medium at a predetermined level, and then pass the coolant
  • the discharge port of the storage tank flows through the return end of the temperature controller into the temperature controller cycle.
  • the molecular design method of the ice control material and the screening method of the ice control material of the present invention can be carried out independently of each other or in combination.
  • the present invention provides a whole-process method for designing and screening ice-control materials, which in turn includes: the molecular design step described in the first aspect and the step of screening ice-control materials described in the second aspect.
  • the method includes the following steps:
  • step (2) screen out ice-controlling molecules with required ice affinity and water affinity
  • the step (7) includes the step (c) of further evaluating the spreading performance, evaluating the affinity of the material with water and the spreading performance at the ice-water interface, and the spreading ability is strong The material has good ice control performance.
  • hydroxyl stereoregularity has an effect on the ability of polyvinyl alcohol PVA to control the growth of ice crystals
  • a specific syndiotactic polyvinyl alcohol (PVA) has Very excellent ability to control the growth of ice crystals
  • the PVA syndiotacticity r is 45%-60%
  • the molecular weight is 10kDa-500kDa; preferably, the PVA syndiotacticity r is 50% to 55 %, the molecular weight is 10-30kDa.
  • the inventors of the present invention have also designed and synthesized a variety of peptide compounds, such as dipeptides, tripeptides, peptoids, and glycopeptide compounds, which have a very excellent ability to control the growth of ice crystals.
  • the peptide compounds are composed of ice-philic amino acids, such as: threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc. and other hydrophilic amino acids or glucose Ester (GDL) or sugars are reacted, and the other hydrophilic amino acids can be selected from arginine, proline, alanine and the like.
  • the peptide compound is composed of amino acids containing ice-philic groups and amino acids containing hydrophilic groups.
  • the amino acids composing the peptide compound are two or more amino acids, or one or more amino acids and glucose lactone or sugars.
  • the present invention also found that some specific amino acids or polyamino acids have very excellent ability to control the growth of ice crystals.
  • the amino acid is an amino acid containing an ice-philic group and a hydrophilic group
  • the polyamino acid is an amino acid homopolymer.
  • the amino acid is selected from the group consisting of arginine, threonine, proline, lysine, and Homopolymers of amino acids such as acid, glutamic acid, aspartic acid, and glycine; preferably the degree of polymerization is 2-40, more preferably the degree of polymerization is 2-20, for example, the degree of polymerization is 6, 8, 15, 20, etc., For example, it is one or a combination of two or more of poly-L-proline and poly-L-arginine.
  • the amino acid is selected from one or two of arginine, threonine, proline, lysine, histidine, glutamic acid, aspartic acid, glycine, etc.; for example, The combination of arginine and threonine.
  • a cryopreservation solution comprising the ice control material designed by the method of the first aspect, or the ice control material screened by the method of the second aspect.
  • the ice control material is polyvinyl alcohol (PVA), amino acid or polyamino acid, and/or one or a combination of peptide compounds; the cryopreservation solution also includes polyol, water-soluble Sugar (or its derivatives, such as water-soluble cellulose) and buffers.
  • the cryopreservation solution includes a peptide compound, and per 100 mL of cryopreservation solution, it contains 0.1-50 g of the peptide compound, 0-6.0 g PVA, 0-9.0 g polyamino acid, and 0 -15mL of DMSO, 5-45mL of polyol, 0.1-1.0mol L -1 of water-soluble sugar, 0-30mL of serum, the balance is buffer.
  • the cryopreservation solution includes polyvinyl alcohol, and per 100 mL of cryopreservation solution, it contains 0.01-6.0g of PVA, 0-50g of the peptide compound, 0-9.0g of polyamino acid, and 0 -15mL of DMSO, 5-45mL of polyol, 0.1-1.0mol L -1 of water-soluble sugar, 0-30mL of serum, the balance is buffer.
  • the cryopreservation solution includes amino acids or polyamino acids, and per 100 mL of cryopreservation solution, it contains 0.1-50g amino acids or polyamino acids, 0-6.0g PVA, 0-15mL DMSO, 5- 45mL of polyol, 0.1-1.0mol L -1 of water-soluble sugar, 0-30mL of serum, and the remainder is buffer.
  • the content of the amino acid and/or polyamino acid per 100 mL of cryopreservation solution is 0.5-50g, preferably 1.0-35g, for example, when amino acids are contained, the content may be 5.0-35g, preferably 15-25g; When polyamino acid is contained, its content may be 0.5-9.0g, preferably 1.0-5.0g.
  • the polyol may be a polyol with 2-5 carbon atoms, preferably a diol and/or triol with 2-3 carbon atoms, such as ethylene glycol, propylene glycol, glycerol Any of them; preferably ethylene glycol.
  • the water-soluble sugar may be at least one of non-reducing disaccharides, water-soluble polysaccharides, water-soluble cellulose, and sugar anhydrides, for example selected from sucrose, trehalose, hydroxypropyl methylcellulose, Ficoll; 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 serum can be selected from human serum albumin or its substitutes for human-derived cryopreserved objects, such as sodium lauryl sulfonate, and for non-human-derived cryopreserved objects, fetal bovine serum or bovine serum can be selected. albumin.
  • the DMSO content in each 100 mL cryopreservation solution is 0-10 mL, preferably the DMSO content is 1.0-7.5 mL, such as 1.5-5 mL; as another embodiment of the present invention, the DMSO content per 100 mL cryopreservation solution The DMSO content is zero.
  • the serum content per 100 mL cryopreservation solution is 0.1-30 mL, for example 5.0-20 mL, 10-15 mL; as another embodiment of the present invention, the serum content per 100 mL cryopreservation solution is 0.
  • cryopreservation solution per 100mL of said 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; specifically, for example, 0.25mol L - 1 , 0.5mol L -1 , 1.0mol L -1 .
  • the content of the polyol per 100 mL of cryopreservation solution is 5.0-40 mL, for example 6.0-20 mL, 9-15 mL.
  • the pH of the cryopreservation solution is 6.5-7.6, for example, 6.9-7.2.
  • the peptide compound or amino acid or polyamino acid has the meaning described above.
  • 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 cryopreservation solution contains the following components per 100 mL: 0.5-50 g amino acids, 5.0-45 mL polyols, 0-10 mL DMSO, 0.1-30 mL serum, and 0.1-1.0 water-soluble sugars.
  • mol L -1 the buffer balance.
  • the cryopreservation solution is calculated per 100mL and contains the following components: L-Arg 2.0-20g, L-Thr 1.0-10g, ethylene glycol 5.0-15mL, DMSO 5.0-10mL, serum 5.0-20mL, sucrose 0.1 -1.0mol L -1, the remaining amount of DPBS.
  • the cryopreservation solution contains the following components per 100mL volume: 0.5-9.0g polyamino acid, 5.0-45mL polyol, 0-10mL DMSO, 5.0-20mL serum, water-soluble sugar 0.1-1.0mol L -1 , buffer balance.
  • the cryopreservation solution contains the following components per 100mL volume: poly-L-proline or poly-L-arginine 1.0-8.0g, ethylene glycol 5-45mL, DMSO 0.1-10mL, Serum 5.0-20mL, sucrose 0.1-1.0mol L -1 , DPBS balance.
  • the cryopreservation solution includes the following components per 100 mL volume: PVA 0.01-6.0 g, polyol 5.0-45 mL, serum 0.1-30 mL, water-soluble sugar 0.1-1.0 mol L -1 , the buffer balance.
  • the cryopreservation solution includes the following components per 100 mL volume: 0.01-6.0 g of PVA, 5.0-30 mL of ethylene glycol, 10-20 mL of serum, 0.1-0.6 mol L -1 of sucrose, and the balance of DPBS.
  • the cryopreservation solution includes the following components per 100 mL volume: PVA 1.0-5.0 g, polyol 5.0-20 mL, DMSO 0.1-10 mL, serum 0.1-20 mL, water-soluble Sugar 0.2-0.8mol L -1, buffer balance.
  • the cryopreservation solution is calculated per 100mL volume and includes the following components: PVA 1.0-4.0g, ethylene glycol 5.0-15mL, DMSO 4-10mL, serum 10-20mL, sucrose 0.2-0.6mol L -1 , DPBS balance.
  • the cryopreservation solution is composed of the following components per 100 mL volume: PVA 0.1-6.0 g, polyol 10-45 mL, water-soluble sugar 0.2-1.0 mol L -1 , buffer Liquid balance.
  • the cryopreservation solution is composed of the following components per 100 mL volume: PVA 1.0-5.0 g, ethylene glycol 5.0-20 mL, sucrose 0.2-0.6 mol L -1 , and the balance of DPBS.
  • the cryopreservation solution contains the following components per 100mL volume: 0.5-9.0g polyamino acid, 5.0-45mL polyol, 0.1-6g PVA, 0-20mL serum, water-soluble Sugar 0.1-1.0mol L -1 , buffer balance.
  • the cryopreservation solution contains the following components per 100mL volume: poly-L-proline or poly-L-arginine 1.0-8.0g, ethylene glycol 5-45mL, PVA 0.1-6g, Serum 5.0-20mL, sucrose 0.1-1.0mol L -1 , DPBS balance.
  • the present invention also provides a preparation method of the above-mentioned cryopreservation solution, which includes the following steps:
  • step (3) After the solutions of step (1) and step (2) are cooled to room temperature, they are mixed, the pH is adjusted and the volume is adjusted to a predetermined volume with a buffer solution to obtain the cryopreservation solution.
  • the serum is added when the cryopreservation solution is used.
  • a warm bath is used for heating and stirring, for example, a water bath or an oil bath is used; for example, the temperature of the water bath is 65-85°C, 70-80°C; the stirring is mechanical stirring such as magnetic force Stir.
  • the dissolution of the water-soluble sugar is ultrasonic assisted dissolution.
  • the cryopreservation solution of the present invention can be used in combination with a freezing balance solution.
  • the present invention provides a frozen balance solution, which contains 5.0-45 mL of polyol and a buffer balance per 100 mL.
  • the freezing balance solution according to the present invention optionally further includes DMSO 0-15 mL, serum 0-30 mL, and/or PVA 0-5.0 g.
  • the content of the polyol is 6.0-28 mL, for example 7.0-20 mL, 10-15 mL.
  • the content of DMSO is 0.1-15 mL, for example, 1.0-10 mL, 5.0-7.5 mL; as an embodiment of the present invention, the content of DMSO is zero.
  • the serum content is 0.1-30 mL, for example 5.0-20 mL, 10-15 mL; as an embodiment of the present invention, the serum content is zero.
  • the PVA content is 0.1-5.0 g, for example, 0.1 g, 0.5 g, 1.0 g, 2.0 g; as an embodiment of the present invention, the PVA content is zero.
  • the polyol, serum, and buffer can be selected from the same species as in the cryopreservation solution.
  • PVA is added to the freezing balance solution.
  • the freezing balance solution contains 5.0-7.5 mL of polyol, 5.0-7.5 mL of DMSO, 10-20 mL of serum, and a buffer balance per 100 mL.
  • the freezing balance solution contains 7.5-15 mL of polyol, 10-20 mL of serum, and a buffer balance per 100 mL.
  • the freezing balance solution is based on 100 mL, containing 1.0-5.0 g of PVA, 7.5-15 mL of polyol, and a buffer balance.
  • the present invention also provides a preparation method of the above-mentioned freezing balance solution, which includes dissolving each component in a buffer solution, storing the serum separately, and adding it during use.
  • a reagent for cryopreservation includes the above-mentioned freezing balance solution and the above-mentioned freezing preservation solution, and the balance solution and the preservation solution exist independently of each other.
  • cryopreservation reagent of the present invention when the cryopreservation solution does not contain serum, PVA is added to the freezing balance solution.
  • the freezing balance solution when the content of DMSO in the cryopreservation solution is 0, the freezing balance solution contains 0-5.0 g of PVA, 7.5-15 mL of polyol, 10-20 mL of serum, and a buffer balance per 100 mL; When the contents of DMSO and serum in the cryopreservation solution are both 0, the freezing balance solution contains 1.0-5.0 g of PVA, 7.5-15 mL of polyols, and a buffer balance per 100 mL.
  • the cryopreservation solution or cryopreservation balance solution of the present invention or a combination of the two can be used for cryopreservation of various types of cells, tissues, and organs.
  • Various types of cells include, but are not limited to, germ cells, such as oocytes, sperm, and various stem cells, such as umbilical cord mesenchymal stem cells;
  • various tissues include, but are not limited to, ovarian tissue, embryonic tissue, and fertilized eggs;
  • various organs include, but not Limited to ovaries or other mammalian organs.
  • the present invention provides the application of the above cryopreservation solution or cryopreservation balance solution or a combination of the two in cryopreservation of cells, tissues, and organs.
  • the aforementioned cryopreservation solution or cryopreservation balance solution or a combination of both are used for cryopreservation of oocytes; in one embodiment, the aforementioned cryopreservation solution or cryopreservation balance solution or a combination of both is used for Embryo cryopreservation; in one embodiment, the above-mentioned cryopreservation solution or cryopreservation balance solution or a combination of the two are used for cryopreservation of ovarian tissue or ovarian organs; in one embodiment, the above-mentioned cryopreservation solution or cryopreservation balance solution or The combination of the two is used for cryopreservation of stem cells.
  • the present invention further provides a method for freezing and thawing cells or embryos, including:
  • the cells or embryos are put in a balance solution for balance before being placed in the cryopreservation solution.
  • the present invention further provides a method for cryopreservation of stem cells using a droplet method.
  • the method for cryopreservation of stem cells includes the following steps: adding the cryopreservation solution to the stem cells, pipetting and dispersing to prepare a stem cell suspension, and removing the stem cell suspension Placed on a frozen slide, stored in liquid nitrogen (-196°C).
  • the thawing of cryopreserved stem cells includes placing a frozen slide with stem cells in a-MEM medium and thawing at 37°C.
  • the stem cells are various stem cells known in the art that have differentiation functions, such as totipotent stem cells, pluripotent stem cells or multipotent stem cells, including but not limited to embryonic stem cells and various types of mesenchymal stem cells ( For example, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.), hematopoietic stem cells, etc.
  • differentiation functions such as totipotent stem cells, pluripotent stem cells or multipotent stem cells, including but not limited to embryonic stem cells and various types of mesenchymal stem cells ( For example, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, etc.), hematopoietic stem cells, etc.
  • the present invention also provides a cryopreservation method for organs and/or tissues, which includes: balancing the organs and/or tissues in a cryopreservation solution, then putting the organs and/or tissues in the cryopreservation solution, and then putting the organs and/or tissues Place it on a frozen slide and store in liquid nitrogen.
  • the organ and/or tissue is an ovarian tissue or an ovarian organ, which may be an ovarian tissue section or a complete ovarian tissue.
  • cryopreservation and “cryopreservation” have the same meaning and can be used interchangeably. It refers to the preservation of certain substances or cells, tissues, and organs at low temperatures to maintain their original physical and chemical and/or biological activities. Physiological and biochemical functions.
  • ice control molecule or “ice control material” have the same meaning, and refer to a compound that has the ability to inhibit the growth of ice crystals in an aqueous solution.
  • the ice-controlling molecules have good spreading properties at the ice-water interface, which can reduce the size of ice crystals, or the ice-controlling molecules have no thermal hysteresis or have a sufficiently small thermal hysteresis, which can significantly reduce the The formation of ice crystals.
  • the present invention finds for the first time that the ice-controlling molecules control the growth of ice crystals in the ice-water mixed phase.
  • the ice-controlling material needs to have good affinity with both ice and water.
  • the affinity of ice-controlling molecules with ice can ensure that they are better adsorbed on the ice surface; the affinity of molecules with water can ensure that they spread better at the ice-water interface, so as to achieve the smallest amount of material possible. Cover the largest ice surface area.
  • the idea of designing ice-controlling molecules with both ice-friendly and hydrophilic properties is proposed, which provides a new method for the synthesis of ice-controlling materials.
  • the present invention introduces MD simulation into the molecular structure design of ice-controlling materials for the first time, and evaluates the ice affinity and water affinity of the designed ice-controlling molecules through molecular dynamics simulation, and predicts the ice-controlling performance of the ice-controlling materials , And can achieve structural optimization.
  • the present invention solves the limitation that the current ice control material research and development process can only use the "experimental trial and error method" to perform performance analysis and screening of existing materials. Provides a new idea of molecular structure design, which greatly promotes the development and application of ice-control materials.
  • the cryopreservation solution provided by the present invention has a wide range of sources, good biocompatibility, low toxicity, high safety, and greatly reduces the amount of DMSO. Even without DMSO, it can achieve commercialization of more than 15% of the existing DMSO The cryopreservation solution has the same or even higher cell survival rate.
  • the cryopreservation solution of the present invention has simple composition, convenient source of raw materials, low cost, and can be widely used for cryopreservation of various types of cells and tissues, such as oocytes, embryos, stem cells, ovarian tissues, ovarian organs, etc. Preservation can maintain good biological activity.
  • Figure 1 Schematic diagram of the molecular structure of ice-controlling materials
  • Figure 2 MD simulation of the aggregation state of atactic polyvinyl alcohol (a-PVA) and isotactic polyvinyl alcohol (i-PVA) at the ice-water interface;
  • a-PVA atactic polyvinyl alcohol
  • i-PVA isotactic polyvinyl alcohol
  • FIG. 4 Hydrogen nuclear magnetic resonance spectra of PBVE and i-PVA synthesized in Example 1, A is PBVE and B is i-PVA;
  • Figure 5 GPC curve of PBVE synthesized in Example 1;
  • Figure 6 The dispersion size of a-PVA(A) and i-PVA(B) in water at different concentrations in the DLS experiment;
  • Figure 7 Optical micrographs of ice crystal growth of two PVA PBS solutions, A is a-PVA, B is i-PVA, C is the relationship between the maximum ice crystal size and concentration of the two PVA PBS solutions relative to PBS;
  • Figure 8 The effect of a-PVA (Figure A, B) and i-PVA ( Figure C, D) modified ice crystal morphology in pure water;
  • Figure 9 Molecular structure models of two PVAs simulated by MD
  • Figure 10 The contact surface area of two PVA molecules with water molecules and ice water molecules at the ice-water interface at 240K simulated by MD.
  • the upper part of the picture is the result of a-PVA molecule 3 times, and the lower part is the i-PVA molecule 3 Second result
  • Figure 11 The aggregation probability of two kinds of PVA in aqueous solution calculated by MD simulation
  • Figure 12 MD simulation calculates the number of intermolecular hydrogen bonds between two PVA and water in aqueous solution at 240K, and the number of intermolecular hydrogen bonds formed with water molecules and ice-water molecules at the ice-water interface.
  • Figure 13 Optical micrographs of GDL-L-Thr (compound of formula (6)) for inhibiting the growth of ice crystals and statistics of the size of ice crystals.
  • Figure 14 GDL-L-Thr modified ice crystal morphology in pure water.
  • Fig. 15 Microscopic optical image of GDL-L-Ser (compound of formula (7)) inhibiting the growth of ice crystals. Statistics of the size of ice crystals.
  • Fig. 16 The microscopic optical image of GDL-L-Val (the compound of formula (8)) inhibiting the growth of ice crystals. The statistics of the size of ice crystals.
  • Figure 17 Optical micrographs of the ice crystal growth inhibitory activity of the TR short-chain peptide prepared in Example 3 and a statistical diagram of the size of the ice crystals.
  • Figure 18 The effect of modifying the morphology of ice crystals with the TR short-chain peptide prepared in Example 3 in pure water.
  • FIG. 20 Example 8 peptides (A) R-COOH, (B) R-CH 3 and (C) R-CH 2 CH 3 modified ice crystal morphology in pure water.
  • Figure 21 Schematic diagram of ice adsorption experiment and its device
  • Figure 23 Optical micrographs of ice crystal growth of two kinds of PVA in DPBS solution, A is a-PVA, B is i-PVA.
  • Figure 24 Stained image of fresh (unfrozen) ovarian organ slices of 3 days old mice;
  • Figure 26 Application Example 13: Images of sections of frozen ovarian organs after thawing
  • Figure 27 Application Example 14: Images of sections of frozen ovarian organs after thawing
  • Figure 28 Application example 15 frozen ovarian organs after thawing section staining pictures
  • Figure 29 Stained image of fresh (unfrozen) ovarian tissue sections of sexually mature mice
  • Figure 30 Comparative Example 9: Images of section staining after thawing of frozen ovarian tissue
  • Figure 31 Image of section staining after thawing of frozen ovarian tissue in Application Example 16;
  • Figure 32 Application example 17 frozen ovarian tissue section stained pictures after thawing
  • Figure 33 Application Example 18: The sectioned stained picture of the frozen ovarian tissue after thawing;
  • Figure 34 Application example 26: The frozen section of ovarian tissue after thawing and staining
  • Figure 35 Application Example 27: The sectioned stained picture of the frozen ovarian tissue after thawing
  • Figure 36 Application example 28, the frozen section of ovarian tissue after thawing and staining pictures
  • Figure 37 Application Example 29: The sectioned stained picture of the frozen ovarian tissue after thawing
  • Figure 38 Application example 30 frozen ovarian tissue slices stained pictures after thawing
  • Figure 39 Application Example 31: The sectioned stained picture of the frozen ovarian tissue after thawing;
  • Figure 40 Application example 37, the frozen section of ovarian tissue after thawing and staining pictures
  • Figure 41 Application example 38, the frozen section of ovarian tissue after thawing.
  • the core molecule of the ice-controlling material of the present invention can be designed as various groups having affinity with water and groups having affinity with ice connected by covalent bonds or non-covalent bonds such as ionic bonds.
  • the molecular design method of the ice control material of the present invention includes the following steps:
  • the main chain of the ice-controlling molecule is a carbon chain or a peptide chain structure.
  • the MD simulation in step (2) can be performed by GROMACS, AMBER, CHARMM, NAMD, or LAMMPS.
  • the water molecule model in the step (2) MD simulation, can be selected from TIP3P, TIP4P, TIP4P/2005, SPC, TIP3P, TIP5P, or SPC/E, preferably TIP4P/2005 water molecule model.
  • the force field parameters are provided by one of GROMOS, ESFF, MM morphological force fields, AMBER, CHARMM, COMPASS, UFF, CVFF and other force fields.
  • the interaction between the ice-controlling molecules, the interaction between the ice-controlling molecules and water molecules, and the interaction between the ice-controlling molecules and ice-water molecules are simulated and calculated.
  • the effects include whether to form a hydrogen bond, van der Waals interaction, electrostatic interaction, hydrophobic interaction, ⁇ - ⁇ interaction and the like.
  • step (2) MD simulation when the simulation calculates molecular interaction, temperature and pressure are adjusted.
  • a V-rescale (modified Berendsen) temperature controller and a pressure controller are used to adjust the temperature and pressure.
  • the potential energy parameter is selected to maintain the molecular configuration of the compound molecule.
  • the selected potential energy parameters allow the molecular configuration of the compound molecule to be maintained at a higher temperature.
  • step (2) when simulating an aqueous solution system, periodic boundary conditions are used in the three directions of x, y, and z; when simulating an ice-water mixed system, periodic boundary conditions are used in both directions of x and y. .
  • a cubic or octahedral water box is selected, preferably a cubic water box of 3.9 ⁇ 3.6 ⁇ 1.0 nm 3 .
  • a V-rescale (modified Berendsen) temperature controller and a pressure controller adjust the temperature and pressure.
  • the main criterion for judging the existence of hydrogen bonds is the energy criterion or the geometric criterion, preferably the geometric criterion; when the distance between oxygen atoms is less than 0.35nm and the angle HO...H is less than 30 degrees, two hydroxyl groups or hydroxyl groups Form hydrogen bonds with water molecules.
  • the ice-controlling material may be a compound with a carbon chain structure as the main chain and substituted by an ice-philic group and a hydrophilic group; the ice-controlling material may contain a hydrophilic and ice-philic dual affinity Sexual groups, such as hydroxyl and amino, can also contain ice-philic groups and hydrophilic groups, respectively.
  • the molecular structure of the ice control material is designed to have repeating units of -[CH 2 -CHOH]-.
  • the ice control material molecule is polyvinyl alcohol (PVA).
  • PVA is selected from one or a combination of two or more of isotactic PVA, syndiotactic PVA, and random PVA, for example, the syndiotacticity of the PVA is 15%-65%, specifically, 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 may 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 ice control material molecule is a peptide compound.
  • the peptide compounds are composed of ice-philic amino acids, such as: threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc. and other hydrophilic amino acids or glucose Ester (GDL) or sugars are reacted, and the other hydrophilic amino acids can be selected from arginine, proline, alanine and the like.
  • the peptide compound is a peptide formed by two or more amino acid units, such as: 2-8 amino acid units, specifically 2-5, such as 2, 3, 4, 5, 6 Amino acid unit; each amino acid unit is different.
  • the molar ratio of ice-philic amino acids such as threonine to other hydrophilic amino acids in the peptide compound is (0.1-3):1, preferably (0.5-2):1.
  • the arrangement of ice-philic amino acids and other hydrophilic amino acids in the peptide compound is not particularly limited, and amino acid linking groups or chemical bonds known in the art can be used.
  • ice-philic amino acids and hydrophilic amino acids can be arranged individually and sequentially.
  • the peptide compound 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), at least one of L-Thr-L-Cys-L-Thr (TCT).
  • the peptide compound is a GDL-L-amino acid, such as GDL-L-Thr, GDL-L-Ser, GDL-L-Val.
  • the peptide compound has any structure shown in formula (1)-formula (8):
  • the method for preparing the peptide compound can be synthesized by a peptide synthesis method known in the art, for example, by a solid phase synthesis method.
  • the preparation method according to the present invention includes the following steps: resin swelling, an amino-protected amino acid is covalently attached to the swollen resin, deprotection, addition of another amino-protected amino acid, condensation reaction, deprotection, cleavage, and purification.
  • the glycopeptide derivatives can be prepared by the method of reacting amino acids and sugars known in the present invention.
  • the glycopeptide derivatives can be prepared by reacting glucosactone or other sugars with amino acids in an organic solvent, or by solid phase synthesis.
  • the method is to prepare the glycopeptide derivative, dissolve glucolactone (GDL) in an organic solvent, add amino acid and alkaline catalyst into the organic solvent, and add to the solution of glucose lactone after being completely dissolved, 55-60 After the reaction was completed, the white precipitate was filtered off, and the filtrate was evaporated to dryness to obtain a crude product.
  • GDL glucolactone
  • the organic solvent may be selected from methanol, ethanol and the like.
  • solid-phase synthesis is used to prepare glycopeptide derivatives, including: resin swelling, covalent bonding of an amino-protected amino acid to the swollen resin, deprotection, and addition of carbohydrate compounds (such as ring opening The subsequent glucose lactone) condensation reaction, cleavage, and purification.
  • the synthesis method of GDL-L-Val and GDL-L-Ser refers to the synthesis method of GDL-L-Thr.
  • the present invention also provides peptide compounds represented by formula (9):
  • R is selected from substituted or unsubstituted alkyl, and the substituent may be selected from -OH, -NH 2 , -COOH, -CONH 2 etc., for example, R is substituted or unsubstituted C1-6 alkyl, Preferably, R is -CH 3 , -CH 2 CH 3 , or -CH 2 CH 2 COOH; 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 1-100. In some embodiments of the present invention, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the compound represented by formula (9) has any of the following structures:
  • the compound represented by formula (9) is prepared by the following synthetic route:
  • the ice control material molecule is an amino acid or polyamino acid.
  • the present invention also provides the application of the above-mentioned ice-controlling material molecules, such as PVA, peptide compounds, amino acids and polyamino acids for controlling the growth of ice crystals in aqueous solutions, and the application of the above-mentioned peptide compounds for preparing cell or tissue cryopreservation liquid.
  • the ice-controlling materials designed and prepared according to the present invention such as PVA, peptide compounds, amino acids and polyamino acids, are used to prepare cryopreservation solutions for cryopreservation of cells, tissues, organs, etc.
  • a compound molecule containing repeating units of -[CH 2 -CHOH]- is designed to obtain a molecular structure library including random and isotactic polyvinyl alcohol molecular models.
  • the MD simulation experiment predicts the difference in affinity between random polyvinyl alcohol and isotactic polyvinyl alcohol for ice and water.
  • MD simulations are all completed by GROMACS 5.1, the water model uses TIP4P/2005, and its melting point is about 252.5K.
  • the interaction parameters of the PVA molecules are provided by the GROMOS54A7 force field, using the leapfrog integration algorithm, and the integration step is 2fs.
  • the electrostatic interaction is calculated by the PME method, and the cut-off radius of the Coulomb potential and the L-J potential are both 1.0nm.
  • the temperature and pressure are adjusted by the V-rescale (modified Berendsen) temperature controller and pressure controller.
  • the time constant is set to 0.1ps.
  • the system of aqueous solutions of molecules is studied.
  • the pressure is 1 atm
  • the temperature is 240K, 250K, 260K, 270K, 300K, 330K.
  • the topology file of the PVA molecule is generated by ATB, and the topology file is directly used.
  • the potential energy parameter is selected as 50kcal/mol, which can ensure that even at a higher temperature, the two PVA molecules The molecular configuration of can also be maintained.
  • a-PVA matches the ice crystal lattice size with three times the distance between adjacent OHs, so it can effectively hydrogen bond with the ice surface and be adsorbed on the ice surface. Since i-PVA only changes the direction of the hydroxyl group and does not change the distance between adjacent OHs, it can ensure that i-PVA and a-PVA have similar adsorption capabilities with ice. At the same time, according to the MD simulation results, the number of intermolecular hydrogen bonds formed by a-PVA and water molecules is more than the number of intermolecular hydrogen bonds formed by i-PVA and water molecules, so the affinity of a-PVA and water is higher than that of i- PVA is stronger.
  • the MD simulation shows that the contact area of the two PVA and water molecules at the ice-water interface at 240K, the contactable surface area of a-PVA is larger than that of i-PVA, further verifying that the spreading performance of a-PVA at the ice-water interface is better than that of i- PVA (see Figure 10).
  • the aggregation probability of the two kinds of PVA in the aqueous solution calculated by MD, i-PVA is significantly higher than that of a-PVA ( Figure 11); and at 240K, at the ice-water interface, the number of hydrogen bonds formed between the two kinds of PVA and ice-water molecules It is equivalent, but the number of intermolecular hydrogen bonds formed between a-PVA and water at the ice-water interface and aqueous solution is significantly more than that of i-PVA; therefore, a-PVA can spread better at the ice-water interface, while i-PVA aggregates ( Figure 12).
  • PBVE poly-tert-butyl vinyl ether
  • HBr dry hydrogen bromide gas
  • PBr 3 phosphorus tribromide
  • Aladdin phosphorus tribromide
  • the generated gas sequentially passes through carbon tetrachloride (CCl 4 ), red phosphorus (P, Alfa Aesar) and calcium chloride (CaCl 2 ) to obtain dry HBr gas.
  • CCl 4 carbon tetrachloride
  • P red phosphorus
  • CaCl 2 calcium chloride
  • i-PVA isotactic-PVA
  • PBVE dry toluene solution
  • dry argon was continuously introduced to remove oxygen in the solution.
  • the dry HBr gas prepared in step b was passed into the toluene solution of the oxygen-free PBVE. After about 5.0 minutes, a light yellow precipitate was formed, and the dry HBr gas was continuously introduced until no precipitate continued to form.
  • the above reaction solution was poured into a 2.0 M methanol solution of 200 mL ammonia.
  • a-PVA molecular weight is about 13 ⁇ 23kDa
  • syndiotacticity r (diad syndiotacticity) is about 55% (Sigma-Aldrich)
  • i-PVA molecular weight is about 14 ⁇ 26kDa
  • isotacticity m (isotacticity is about 84%)
  • the particle size distribution in 25°C aqueous solution is measured by dynamic light scattering (DLS) experiment.
  • a-PVA and i-PVA aqueous solutions with concentrations of 1.0 mg mL -1 , 4.0 mg mL -1 , 10 mg mL -1 , and 20 mg mL -1 respectively; fill about 1.0 mL of PVA solution into a 12 mm disposable Measure with polystyrene cuvette.
  • Ice crystal recrystallization inhibition (IRI) activity adopts the "sputter freezing method" to dissolve and disperse the sample in DPBS solution, and drop 10-30 ⁇ L of the above solution at a height of 1.0m or more onto the clean silicon wafer surface precooled at -60°C , Use a cold and hot table to slowly heat up to -6°C at a speed of 10°C min -1 , and anneal at this temperature for 30 minutes. Use a polarizing microscope and a high-speed camera to observe and record the size of ice crystals. The cold and hot table is sealed to ensure that the internal humidity is within Around 50%. Each sample is repeated at least three times. Nano Measurer 1.2 is used to calculate the size of ice crystals. The error of the statistical results is the standard deviation.
  • the ice crystal morphology (DIS) observation and thermal hysteresis (TH) measurement adopt a nano-osmometer.
  • the capillary is melted by the outer flame of an alcohol lamp, and the capillary with very fine pores is produced by stretching at the same time.
  • the capillary is connected with the micro-injector Connected.
  • the higher viscosity immersion lens oil is injected into the micron aperture disc, and the water solution with dissolved material is injected into the micropores using a micro-injector.
  • the droplets freeze quickly and slowly heat up to obtain single crystal ice.
  • the temperature is slowly lowered with an accuracy of 0.01°C.
  • the ice crystal morphology and TH test are observed using a microscope equipped with a high-speed camera.
  • the minimum is about 20% of the maximum ice crystal size of DPBS; i-PVA of different molecular weights relative to the MLGS of DPBS increases with the increase in concentration, and reaches the minimum at 10 mg mL -1 , and the minimum is only about 50 of the maximum ice crystal size of DPBS %, and the concentration continued to increase with 20mg mL -, MLGS slightly increased and did not decrease.
  • the i-PVA with a degree of polymerization greater than 333 (M w 14 ⁇ 26kD) is difficult to dissolve when the concentration is greater than 30 mg mL -1 .
  • the IRI activity of i-PVA is optimally 10 mg mL -1 when 50 % Of DPBS MLGS; and the IRI activity of a-PVA is optimally 2.0 mg mL -1 when 20% of DPBS MLGS.
  • a-PVA is easier to spread at the ice-water interface than i-PVA in the MD simulation.
  • the easier spreading performance enables a-PVA to be able to be used at a lower dosage than i-PVA. Achieve better suppression of ice crystal growth.
  • GDL-L-Thr prepared by solid-phase synthesis has higher purity and is more conducive to product separation.
  • Experimental results show that GDL-L-Thr prepared by solid-phase synthesis has higher purity and maintains a good ability to inhibit ice crystal growth (Figure 13) .
  • the compounds represented by formulas (7) and (8) can be obtained by solid phase synthesis.
  • R is a peptoid of -CH 3 , -CH 2 CH 3 and -CH 2 CH 2 COOH. Mass Spectrum 444.6 R is -CH 3 of [M + H] +, 528.8 R is -CH [M + H] 2 CH 3 + and 792.1 R is -CH 2 CH2COOH the [M + H] +.
  • Ice crystal recrystallization inhibition (IRI) activity adopts the "sputter freezing method" to dissolve and disperse the sample in DPBS solution, and drop 10-30 ⁇ L of the above solution at a height of 1.0m or more onto the clean silicon wafer surface precooled at -60°C , Use a cold and hot stage to slowly heat up to -6°C at a rate of 10°C/min, and anneal at this temperature for 30 minutes, use a polarizing microscope and a high-speed camera to observe and record the size of the ice crystals, and seal the hot and cold stage to ensure that the internal humidity is at 50 %about. Repeat for each sample at least three times, use Nano Measurer 1.2 to count the size of ice crystals, and the error of the statistical results is the standard deviation.
  • the ice crystal morphology (DIS) observation and thermal hysteresis (TH) measurement adopt a nano-osmometer.
  • the capillary is melted by the outer flame of an alcohol lamp, and the capillary with very fine pores is produced by stretching at the same time.
  • the capillary is connected with the micro-injector Connected.
  • the higher viscosity immersion lens oil is injected into the micron aperture disc, and the water solution with dissolved material is injected into the micropores using a micro-injector.
  • the droplets freeze quickly and slowly heat up to obtain single crystal ice.
  • the temperature is slowly lowered with an accuracy of 0.01°C.
  • the ice crystal morphology and TH test are observed using a microscope equipped with a high-speed camera.
  • TR has a weak effect of modifying the morphology of ice crystals (subcooling degree -0.1°C, -0.4 ⁇ 0.01°C), such as Shown in Figure 18. And the thermal hysteresis was not measured.
  • the prepared peptide compounds have the activity of inhibiting the growth of ice crystals and the effect of modifying the morphology of ice crystals.
  • the compound of formula (9) in which R is -CH 3 , -CH 2 CH 3 has an excellent effect on modifying the morphology of ice crystals. And there is no thermal hysteresis, can realize the function of controlling the growth of ice crystals, and can be used for cryopreservation liquid.
  • the adsorption amount of the ice control material on the ice surface (the mass m 1 of the ice control material adsorbed on the ice surface / the total mass m 2 of the ice control material in the original solution containing the ice control material) ⁇ 100%.
  • the ice adsorption experiment includes the following steps:
  • the device shown in Figure 21 is used to conduct an ice adsorption experiment, including a multi-layer liquid storage chamber, a temperature control rod, and a temperature controller.
  • the multi-layer liquid storage chamber includes an ice adsorption chamber, a warm bath chamber, and a cooling liquid storage from the inside to the outside.
  • the temperature control rod is placed in the ice adsorption chamber, and the temperature of the temperature control rod and the liquid storage chamber is controlled by a temperature controller.
  • the temperature control rod is a hollow structure made of a thermally conductive material, and the hollow structure of the temperature control rod is provided with a liquid inlet and a liquid outlet;
  • the temperature controller is a fluid temperature controller, and the temperature controller is provided with Cooling liquid outflow end and return end;
  • the cooling liquid storage cavity is provided with a liquid inlet and a liquid outlet at both ends; the cooling liquid outflow end of the temperature controller, the liquid inlet of the temperature control rod, and the discharge port of the temperature control rod
  • the liquid port, the liquid inlet of the cooling liquid storage tank, the liquid discharge port of the cooling liquid storage tank, and the return end of the temperature controller are sequentially connected through a pipeline, and the cooling liquid flows in the pipeline.
  • the multi-layer liquid storage cavity is provided with a cover.
  • the ice adsorption chamber When in use, the ice adsorption chamber contains an aqueous solution of ice-controlling materials, and the middle-layer warm bath chamber contains a warm bath medium of a predetermined temperature, such as a water bath, an ice bath or an oil bath, etc.; after the temperature of the cooling liquid reaches the set temperature, the temperature controller Flow out and flow into the hollow structure of the temperature control rod to control the temperature of the temperature control rod, then flow out from the outlet of the temperature control rod, and then flow into the outer coolant storage cavity to keep the temperature of the bath medium at a predetermined level, and then pass the coolant
  • the discharge port of the storage tank flows through the return end of the temperature controller into the temperature controller cycle.
  • a-PVA molecular weight is about 13 ⁇ 23kDa, syndiotacticity r (diad syndiotacticity) is about 55% (Sigma-Aldrich);
  • i-PVA The molecular weight is about 14 ⁇ 26kDa, and the isotacticity is about 84%.
  • the ice adsorption experiment is used to determine the adsorption amount of PVA on the ice surface.
  • the experimental device is shown in Figure 21.
  • the FITC-labeled PVA aqueous solution is continuously magnetically stirred for 1.0 hour at a supercooling temperature of -0.1°C until the PVA is gradually adsorbed to the surface of the ice. All adsorption experiments keep the degree of subcooling and adsorption time constant to ensure that the surface area of the resulting ice is almost unchanged within the error tolerance.
  • the adsorption amount of PVA on the ice surface is obtained by the mass of solute PVA in the ice cube than the mass of solute PVA in the original solution.
  • the concentration of PVA solution is determined by UV-visible spectrophotometry, and the volume is determined by pipette and graduated cylinder.
  • the ice adsorption experiment shows that the adsorption capacity of a-PVA and i-PVA at each concentration is shown in Figure 22.
  • the adsorption capacity of a-PVA on the ice surface increased from 16.3% at a concentration of 0.2 mg mL -1 to 1.0 mg mL -1 28.7%, and after the concentration is greater than 1.0 mg mL -1 , the adsorption capacity of a-PVA on the ice surface reaches saturation, and the adsorption capacity at saturation is about 36.5%.
  • the concentration of i-PVA is less than 1.0 mg mL -1 , the ice adsorption capacity is 0% to 19.3%, which is lower than the adsorption capacity of a-PVA on the ice surface at the same concentration.
  • the adsorption of the two kinds of PVA on ice is not saturated, and the ice surface area covered by i-PVA is lower than that of a-PVA.
  • Ice crystal recrystallization inhibition (IRI) activity adopts the "sputter freezing method" to dissolve and disperse the above two kinds of PVA into DPBS solution separately, and add 10-30 ⁇ L of the solution at a height of 1.0m or more to -60°C to pre-cool
  • Use a cold and hot stage to slowly heat up to -6°C at a speed of 10°C min -1 , and anneal at this temperature for 30 minutes.
  • Use a polarizing microscope and a high-speed camera to observe and record the size of ice crystals. Ensure that the internal humidity is around 50%. Each sample was repeated at least three times.
  • Nano Measurer 1.2 was used to calculate the size of ice crystals. The error of the result was the standard deviation.
  • Example 9 it can be seen that the affinity of i-PVA with water is weaker than that of a-PVA. Therefore, i-PVA tends to exist in an aggregated state at the aqueous solution and ice-water interface, while a-PVA can spread well at the aqueous solution and ice-water interface.
  • the amount of i-PVA required is much higher than that of a-PVA. Therefore, compared with i-PVA, a-PVA is a better ice control material, and a lower concentration can achieve a better effect of inhibiting the growth of ice crystals.
  • Cryopreservation Solution A Each 100mL contains the following components:
  • Liquid preparation steps Heat 2.0 g of PVA in a water bath at 80°C and dissolve it in 25 mL of DPBS with magnetic stirring. After all PVA is dissolved and cooled to room temperature, adjust the pH to 7.0, which is solution 1; 17g (0.05mol) of sucrose (The final concentration of sucrose in the cryopreservation solution is 0.5mol L -1 ) Dissolve it in 25mL of DPBS ultrasonically. After the sucrose is completely dissolved, add 10mL of ethylene glycol and 10mL of DMSO to form solution 2, and wait for solution 1 and solution 2. Return to room temperature, then mix the two solutions, adjust the pH value and make up the balance to 80% of the total volume at a constant volume, and store 20 mL of serum separately to be added when the preservation solution is used.
  • Cryopreservation Solution B Each 100ml contains the following components:
  • Liquid preparation steps Heat 2.0g of PVA in a water bath at 80°C and dissolve it in 20mL of DPBS with magnetic stirring, adjust the pH to 7.1, which is solution 1; dissolve 8.0g of L-Arg and 4.0g of L-Thr In 20mL DPBS, adjust the pH to 7.1, which is solution 2; 17g (0.05mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5mol L -1 ) is ultrasonically dissolved in 20mL of DPBS, and after the sucrose is completely dissolved Add 10 mL of ethylene glycol to make solution 3; after solution 1, solution 2 and solution 3 return to room temperature, mix the three solutions evenly, adjust the pH value and make up the remaining volume to 80% of the total volume. When adding 20mL of serum.
  • Cryopreservation Solution C Each 100ml contains the following components:
  • Liquid preparation steps Heat 2.0g of PVA in a water bath at 80°C and dissolve it in 25mL of DPBS with magnetic stirring, adjust the pH to 6.9, to be solution 1; 17g (0.05mol) of sucrose (the final concentration of sucrose in the cryopreservation solution 0.5mol L -1 ) was dissolved in 25mL of DPBS ultrasonically. After the sucrose was completely dissolved, 10mL of ethylene glycol was added to form solution 2. After solution 1 and solution 2 were restored to room temperature, the two solutions were mixed and adjusted. The pH value and constant volume make up the balance to 80% of the total volume, and 20 mL of serum is stored separately to be added when the preservation solution is used.
  • Cryopreservation Solution C1 Each 100ml contains the following components:
  • the liquid preparation procedure is the same as the cryopreservation liquid C.
  • Cryopreservation Solution D Each 100ml contains the following components:
  • Liquid preparation steps Heat 2.0g of PVA in a water bath at 80°C and dissolve it in 30mL of DPBS with magnetic stirring, adjust the pH to 7.0, to be solution 1; 17g (0.05mol) of sucrose (final concentration of sucrose in the cryopreservation solution 0.5mol L -1 ) was dissolved in 25mL of DPBS ultrasonically. After the sucrose was completely dissolved, 10mL of ethylene glycol was added to form solution 2. After solution 1 and solution 2 were restored to room temperature, the two solutions were mixed and adjusted. pH value and constant volume to make up the balance to a total volume of 100mL, ready for use.
  • Cryopreservation Solution E Each 100ml contains the following components:
  • Liquid preparation steps Heat 2.0 g of PVA in a water bath at 80°C and dissolve it in 25 mL of DPBS with magnetic stirring, adjust the pH to 7.0, to be solution 1; add 1.5 g of poly-L-proline (polymerization degree 15) 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 is 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 evenly, adjust the pH value and make up the balance to a total volume of 100 mL ,spare.
  • Cryopreservation Solution F Each 100ml contains the following components:
  • the liquid preparation procedure is the same as the cryopreservation solution E, and the serum is added during use.
  • Frozen balance solution a Add 7.5 mL of ethylene glycol and 7.5 mL of DMSO to 65 mL of DPBS, mix well, and add 20 mL of serum during use.
  • Freezing balance solution b Dissolve 7.5 mL of ethylene glycol in 72.5 mL of DPBS, mix well, and add 20 mL of serum when using.
  • Frozen balance solution c Heat 2.0 g of PVA in a water bath at 80°C and dissolve it in 50 mL of DPBS with magnetic stirring. After all the PVA is dissolved, adjust the pH to 7.0, add 7.5 mL of ethylene glycol, mix well, adjust the pH and Make up the remaining volume to 100mL and set aside.
  • Frozen balance solution a each 1mL contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS;
  • Cryopreservation solution 1# each 1mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, the balance For DPBS.
  • Frozen Balance 2# Each 1mL contains 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS;
  • Cryopreservation Solution 2# Each 1 mL contains 10% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, and the balance is DPBS.
  • Cryopreservation Solution 3# Each 1mL contains 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance is medium a-MEM (USA, Invitrogen, C12571500BT)
  • Example 10 There are three types of thawing solution formulations used in Example 10 and Comparative Examples as follows:
  • Thaw solution 1# Thaw solution I (containing 1.0mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw solution II (containing 0.5mol L -1 sucrose, 20% serum, the balance is DPBS) ; Thaw Solution III (containing 0.25mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw Solution IV (20% serum, the balance is DPBS).
  • Thaw solution 2# Thaw solution I (contains 1.0mol L -1 sucrose, 20mg mL -1 PVA, the balance is DPBS); Thaw solution II (contains 0.5mol L -1 sucrose, 20mg mL -1 PVA, The remainder is DPBS); Thaw Solution III (contains 0.25mol L -1 sucrose, 20 mg mL -1 PVA, the balance is DPBS); Thaw Solution IV (20 mg mL -1 PVA, the remainder is DPBS).
  • I thawing solution (containing 1.0mol L -1 of sucrose, 20mg mL PVA -1 and polyproline 10mg mL -1 of balance DPBS); II thawed liquid (containing 0.5mol L - 1 sucrose, 20mg mL PVA -1 and polyproline 5.0mg mL -1, with the balance of DPBS); iii thawed liquid (containing 0.25mol L -1 sucrose, 20mg mL PVA -1's, 2.5mg mL - 1 polyproline, the balance is DPBS); Thaw Solution IV (20mg mL -1 PVA, the balance is DPBS).
  • the mouse oocytes are first placed in the cryopreservation solution to equilibrate for 5 minutes; then placed in the prepared cryopreservation solution for 1 minute, the oocytes that have been equilibrated in the cryopreservation solution are placed on the freezing rod, and then quickly Put it into liquid nitrogen (-196°C), and keep the rod closed and continue to store; when thawing, put the frozen oocytes in the thawing solution I at 37°C for 5 minutes, and then in the thawing solution II-IV in turn Incubate the thawed oocytes for 3 minutes and observe the number of viable cells after culturing for 2 hours, and calculate the survival rate (see Table 1).
  • the mouse embryos are first placed in the freezing balance solution for 5 minutes, and then placed in the cryopreservation solution prepared by the formula in the above example or comparative example for 50 seconds, and the embryos that have been equilibrated in the freezing solution are placed on the freezing carrier. Then quickly put it into liquid nitrogen (-196°C), and close the carrier rod and continue to save; when thawing, put the embryo in 37°C Thawing Solution I for 3 minutes, and then in the thawing solution II-IV each for 3 Minutes; culture the thawed embryos for 2 hours, observe the number of surviving embryos, and calculate the survival rate (see Table 2).
  • cryopreservation solution can achieve a survival rate of more than 90% or even 100%, which can reach or far exceed the cryopreservation recovery rate of the commercial cryopreservation solution containing 15% DMSO commonly used in clinical practice.
  • Application example 1 Containing 10% DMSO
  • Comparative Example 2 containing 7.5% DMSO
  • Comparative Example 1 that is, the comparison of commercialized oocyte cryopreservation solution (containing 15% DMSO)
  • PVA significantly improves
  • the survival rate of oocytes application examples 2-3 also show that the present invention can achieve a higher survival rate of oocytes or embryos by adding less DMSO or cryopreservation solutions that do not contain DMSO.
  • cryopreservation solution has high DMSO concentration and great damage to cells; and application examples 5 and 7-9 show that it can also be achieved without adding DMSO and serum to the freezing solution, balance solution and thawing solution. Higher oocyte or embryo survival rate.
  • the cryopreservation solution without DMSO or serum solves the problems of short shelf life and the introduction of parasitic biological contaminants caused by the commercial cryopreservation solution commonly used in clinical practice because it contains serum.
  • cryopreservation solutions of the above examples and comparative examples were used to perform cryopreservation of human umbilical cord-filled mesenchymal stem cells according to the protocol in Table 3.
  • Cryopreservation of human umbilical cord mesenchymal stem cells by microdrop method After digesting the human umbilical cord mesenchymal stem cells on a culture dish with 25% trypsin for 2 minutes, put them in an equal volume of culture medium (10% FBS+a-MEM medium) , Gently pipette until all the stem cells fall off, add a 1.5mL centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant (to separate the cells from the culture medium), add 10uL of freezing solution to the bottom of the centrifuge tube, gently pipette to disperse the stem cell mass, this 10uL The freezing solution with stem cells is placed on a frozen slide and stored in liquid nitrogen (-196°C).
  • the survival rate of stem cells can reach 92.4% and 72.2% even without DMSO (application examples 12 and 10), even when DMSO and serum are not added at all, they survive The rate can reach 77.1%, reaching the survival rate level of the existing freezing reagents, indicating that the freezing reagents can not only achieve the effectiveness of conventional freezing liquid freezing dry cells, but also reach or even far exceed the commonly used freezing containing 10% DMSO.
  • the cryopreservation recovery rate of the preservation solution (Comparative Example 7), based on PVA, was significantly better than that of Comparative Example 6 without PVA.
  • the freezing balance solution and cryopreservation solution of the above-mentioned examples and comparative examples were used to freeze-preserve mouse ovarian organs and ovarian tissue sections of sexually mature mice within 3 days of newborn according to the schemes in Table 4 and Table 5.
  • the cryopreservation solution prepared with the PVA-type bionic ice control material as the main component of the present invention has a good effect of inhibiting the growth of ice crystals, and can reduce the amount of DMSO in the preservation system, even without DMSO, and can maintain a good biological phase. It is compatible and can be used for cryopreservation of oocytes, embryos, stem cells, reproductive organs and tissues at the same time, and can achieve good cell survival rate and biological activity.
  • Cryopreservation Solution G Each 100ml contains the following components:
  • Liquid preparation steps total volume 100mL: Dissolve 16g of L-Arg and 8g of L-Thr in 25mL of DPBS, adjust the pH to 6.9, which is solution 1; add 17g (0.05mol) of sucrose (sucrose in cryopreservation solution) The final concentration is 0.5mol L -1 ) Dissolve in 25mL DPBS by ultrasound, add 10mL ethylene glycol and 10mL DMSO after the sucrose is completely dissolved, which is solution 2. After solution 1 and solution 2 return to room temperature, Mix the two solutions evenly, adjust the pH to 6.9, and use DPBS to make up the balance to 80% of the total volume. 20 mL of fetal bovine serum is stored separately and added before the cryopreservation solution is used.
  • Cryopreservation Solution H Each 100ml contains the following components:
  • Liquid preparation steps ultrasonically dissolve 1.5g of poly-L-proline (polymerization degree 15) in 25mL of DPBS, adjust the pH to 6.8, which is solution 1; ultrasonically dissolve 17g (0.05mol) of sucrose in 25mL After the sucrose is completely dissolved, add 10 mL of ethylene glycol and 10 mL of DMSO in sequence to form solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions evenly, adjust the pH to 7.0, and use DPBS Make up the remaining volume to 80% of the total volume at a constant volume, and store 20 mL of serum separately and add it before using the cryopreservation solution.
  • Cryopreservation Solution I Each 100ml contains the following components:
  • Liquid preparation steps The total volume is 100mL, 1.5g of poly-L-arginine (polymerization degree is 8) is ultrasonically dissolved in 25mL of DPBS, the pH is adjusted to 7.0, which is solution 1; 17g (0.05mol) of sucrose Dissolve it in 20mL of DPBS by ultrasonic, add 10mL of ethylene glycol and 10mL of DMSO to solution 2 after the sucrose is completely dissolved. After solution 1 and solution 2 return to room temperature, mix the two solutions and adjust the pH to 7.0, and use DPBS to make up the balance to 80% of the total volume. 20mL of serum is stored separately and added before the cryopreservation solution is used.
  • Cryopreservation Solution J Each 100ml contains the following components:
  • the liquid preparation procedure is the same as the cryopreservation liquid I.
  • Cryopreservation Solution K Each 100ml contains the following components:
  • the liquid preparation procedure is the same as the cryopreservation liquid I.
  • Cryopreservation Solution L Each 100ml contains the following components:
  • the liquid preparation procedure is the same as the cryopreservation liquid G.
  • Frozen balance solution a Add 7.5 mL of ethylene glycol and 7.5 mL of DMSO to 65 mL of DPBS, mix well, and add 20 mL of serum during use.
  • Frozen balance solution b 7.5 mL of ethylene glycol was added to 72.5 mL of DPBS, mixed well, and 20 mL of serum was added during use.
  • Frozen balance solution a each 1mL contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS;
  • Cryopreservation solution 1# each 1mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, the balance For DPBS.
  • Cryopreservation Solution 3# Each 1mL contains 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance is medium a-MEM (USA, Invitrogen, C12571500BT)
  • Example 11 The thawing liquid formula used in Example 11 and Comparative Example 2 is as follows:
  • Thaw solution 1# Thaw solution I (containing 1.0mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw solution II (containing 0.5mol L -1 sucrose, 20% serum, the balance is DPBS) ; Thaw Solution III (containing 0.25mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw Solution IV (20% serum, the balance is DPBS).
  • Example 11 and Comparative Example 2 were used to perform cryopreservation of oocytes and embryos according to the protocols in Table 6 and Table 7.
  • the freezing and thawing methods are the same as in Application Example 1.
  • the survival rate of oocytes can reach more than 95%, and the survival rate of embryos
  • the survival rate can reach 100%, which can reach or even be much higher than the cryopreservation recovery rate of the commercial cryopreservation solution containing 15% DMSO (Comparative Examples 10-11) commonly used in clinical practice, and the amino acid bionic ice control material is added
  • the cryopreservation effect is significantly better than the cryopreservation solution without bionic ice control material.
  • Example 11 and Comparative Example 2 were used for cryopreservation of human umbilical cord-filled mesenchymal stem cells according to the protocol in Table 8. See Application Example 2 for freezing and thawing methods.
  • the survival rate of the stem cells can reach more than 80% even if no or only a small amount of DMSO (7.5%) is used (for example, application examples 23-25), indicating that the freezing is used
  • DMSO fetal sulfate
  • the reagent not only achieves the effectiveness of conventional freezing liquid freeze-dried cells, it is even much higher than the cryopreservation recovery rate of the commonly used cryopreservation solution containing 10% DMSO (Comparative Example 13), and the freezing of the amino acid bionic ice control material is added.
  • the preservation effect is significantly better than Comparative Examples 14 and 15 without adding amino acid-based ice control materials.
  • Example 11 Using the freezing balance solution and cryopreservation solution of the above-mentioned Example 11 and Comparative Example 2 according to the schemes in Table 9 and Table 10, the complete ovarian organs of mice within 3 days of newborn and the ovarian tissue sections of sexually mature mice were cryopreserved. .
  • Application Example 3 for the freezing and thawing methods of ovarian organs and ovarian tissues of sexually mature mice.
  • Cryopreservation Solution M Each 100ml contains the following components:
  • Liquid preparation steps total volume 100mL, ultrasonically dissolve 28g TR in 25mLDPBS, adjust pH to 7.0, which is solution 1; ultrasonically dissolve 0.05mol sucrose in 25mL DPBS, add 10mL ethylene glycol and 7.5 after sucrose is completely dissolved mL DMSO is solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions, adjust the pH and dilute to 80% of the total volume with DPBS. Finally, add 20 mL of serum before use.
  • Cryopreservation Solution N Each 100ml contains the following components:
  • Preparation steps total volume 100mL, ultrasonically dissolve 28g TPT in 25mLDPBS, adjust the pH to 7.0, as solution 1; ultrasonically dissolve 0.05mol sucrose in 25mLDPBS, add 10mL ethylene glycol and 7.5mL DMSO after all sucrose is dissolved, It is solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions, adjust the pH and use DPBS to make the volume to 80% of the total volume. Finally, add 20 mL of serum before use.
  • Cryopreservation Solution O Each 100ml contains the following components:
  • Liquid preparation steps Volume 100mL, ultrasonically dissolve 28g TR in 25mLDPBS, adjust pH to 7.0, as solution 1; ultrasonically dissolve 0.05mol sucrose in 25mL DPBS, add 10mL ethylene glycol after all the sucrose is dissolved, and the solution is 2. After solution 1 and solution 2 are restored to room temperature, mix the two solutions, adjust the pH and use DPBS to make the volume to 80% of the total volume. Finally, add 20 mL of serum before use.
  • Frozen balance solution a Add 7.5 mL of ethylene glycol and 7.5 mL of DMSO to 65 mL of DPBS, mix well, and add 20 mL of serum during use.
  • Frozen balance solution a each 1mL contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, and the balance is DPBS;
  • Cryopreservation solution 1# each 1mL contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, the balance For DPBS.
  • Cryopreservation Solution 3# Each 1mL contains 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance is medium a-MEM (USA, Invitrogen, C12571500BT)
  • Example 12 The thawing liquid formula used in Example 12 and Comparative Example 3 is as follows:
  • Thaw solution 1# Thaw solution I (containing 1.0mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw solution II (containing 0.5mol L -1 sucrose, 20% serum, the balance is DPBS) ; Thaw Solution III (containing 0.25mol L -1 sucrose, 20% serum, the balance is DPBS); Thaw Solution IV (20% serum, the balance is DPBS).
  • Example 13 and Comparative Example 2 were used for cryopreservation of oocytes and embryos according to the protocols in Table 11 and Table 12.
  • the freezing and thawing methods are the same as in Application Example 1.
  • the data in Table 11 and Table 12 show that the polypeptide of the present invention is used for the cryopreservation of oocytes and embryos, and only a small amount of DMSO (7.5%) can achieve the oocytes and oocytes of the existing commercial cryopreservation solution (DMSO content 15%).
  • the embryo survival rate, and the data of Application Example 32 and Application Example 34 show that the TR polypeptide has a more excellent effect in cryopreservation of oocytes and embryos.
  • Example 12 The cryopreservation solutions of Example 12 and Comparative Example 3 were used for cryopreservation of human umbilical cord-filled mesenchymal stem cells according to the protocol in Table 13. See Application Example 2 for freezing and thawing methods.
  • the cryopreservation solution of the present invention does not add DMSO or only adds a small amount of DMSO (7.5%), can achieve a level of cell survival equivalent to that of the cryopreservation solution with 10% DMSO added in the prior art It greatly reduces the dosage of DMSO, reduces the damage and toxicity of DMSO to cells, and can greatly improve the passage stability and cell viability of stem cells after freezing.
  • Example 3 Using the freezing balance solution and cryopreservation solution of the above-mentioned Example 12 and Comparative Example 3, the complete ovarian organs of mice within 3 days of newborn and the ovarian tissue sections of sexually mature mice were cryopreserved according to the protocols in Table 14 and Table 15. . Refer to Application Example 3 for the freezing and thawing methods of ovarian organs and ovarian tissues of sexually mature mice.
  • Fig. 29, Fig. 30 and Fig. 41 compared with the fresh and unfrozen adult mouse ovarian tissue of Comparative Example 22, compared with the scheme of Application Example 38, the structure of growth phase and antral follicles is relatively complete. It can be seen that the cryopreservation solution of the present invention is used The cryopreservation of ovarian tissue also has better results than the prior art.
  • cryopreservation solution prepared with the peptide bionic ice control material as the main component of the present invention can be simultaneously applied to cryopreservation of oocytes, embryos, stem cells, reproductive organs and tissues, and can achieve good cell survival and Biological activity.

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Abstract

提供了一种仿生控冰材料，通过构建化合物分子结构库，所述化合物分子含有亲水基团和亲冰基团；采用分子动力学模拟(MD模拟)评估各化合物分子在冰水两相界面的铺展性能；筛选出所需冰亲和性和水亲和性的化合物分子。首次提出控冰材料亲冰性和亲水性的机制，将MD模拟引入到控冰材料的分子结构设计中，通过分子动力学模拟评估所设计的控冰材料的冰亲和性和水亲和性，预测控冰材料的控冰性能，并可实现结构优化。所述的方法设计和筛选的仿生控冰材料有良好的抑制冰晶生长的能力，其用于冷冻保存试剂时，即便不加DMSO也可以达到良好的冷冻保存效果。

Description

一种仿生控冰材料及含有该仿生控冰材料的冷冻保存液
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2019102824189、2019102824225、2019102824174、201910282416X、2019102819867的在先申请的优先权。前述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，具体涉及一种仿生控冰材料及含有该仿生控冰材料的冷冻保存液。
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玻璃化冷冻。玻璃化冷冻技术采用渗透性或非渗透性低温保护剂，虽然在快速冷冻过程中可使细胞内外的液体直接成为玻璃态而避免了冷冻过程中因冰晶形成而导致的损伤。但是，在复温过程中，现有的冷冻保存试剂不能有效的控制冰晶的生长，从而损害细胞。由于抗冻蛋白和仿生控冰材料在分子层面的控冰机制仍有争议，仿生控冰材料的研发只能依赖“试误法”逐步尝试某种控冰材料的控冰效果，工作量大，成功几率低。目前常用的冷冻保存试剂不具备复温过程中有效控制冰晶生长的能力，而且试剂毒性大。
发明内容
为改善现有技术的上述缺陷，本发明提供一种仿生控冰材料的分子设计方法和控冰材料的筛选方法，可以指导人们有目的的合成和筛选仿生控冰材料，本发明还提供了基于该方法得到的仿生控冰材料以及含有这些材料的冷冻保存试剂。
本发明提供以下技术方案:
本发明的第一方面，提供一种控冰材料的分子设计方法，包括如下步骤：
(1)构建化合物分子结构库，所述化合物分子含有亲水基团和亲冰基团；
(2)采用分子动力学模拟(Molecular dynamics simulation,MD)，模拟评估各化合物分子在冰水两相界面的铺展性能；
(3)根据步骤(2)筛选出所需冰亲和性和水亲和性的控冰分子。
根据本发明，所述控冰分子的主链为碳链或者肽链结构。
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作用等；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)等中的至少一种，或，例如选自脯氨酸(L-Pro)、精氨酸(L-Arg)、赖氨酸(L-Lys)等亲水氨基酸，葡萄糖酸内酯(GDL)，糖类等化合物或其分子片段。
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作用等；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，例如选自谷氨酰胺(L-Gln)、苏氨酸(L-Thr)、天冬氨酸(L-Asn)等亲冰氨基酸，苯环(C 6H 6)，吡咯烷(C 4H 9N)等化合物或其分子片段。
根据本发明，所述控冰材料可以为亲水基团与亲冰基团通过共价键键合而成。
根据本发明，所述控冰材料可以为亲水基团与亲冰基团通过非共价键，如：离子键作用而成。
根据本发明，所述方法还包括步骤(4)：合成所述控冰分子(控冰材料)的步骤，例如可通过已知的化学合成方法，例如聚合反应、缩合反应，或者基因工程菌生物发酵等方法合成。
本发明还提供根据第一方面所述分子设计方法所得的控冰材料。
本发明的第二方面，提供一种筛选控冰材料的方法，包括如下步骤：(a)测得控冰材料与水的亲和性；(b)测得所述控冰材料在冰水界面的铺展性能。
作为本发明的实施方案，所述步骤(a)可以通过测定所述控冰材料在水中的溶解度、水合常数、分散尺寸、扩散系数等、和/或计算所述控冰材料与水分子形成的分子间氢键数等方法测定；具体地，例如采用分子动力学模拟(Molecular dynamics simulation,MD)测定所述控冰材料分子与水分子形成的分子间氢键数，或者采用动态光散射测定所述控冰材料在水溶液中的分散尺寸。
作为本发明的实施方案，所述步骤(b)可以通过测定在冰水界面处所述控冰材料在冰表面的吸附含量得到所述材料在冰水界面的铺展性能，和/或计算所述控冰材料与冰-水分子形成的分子间氢键数等方法测定所述材料与冰的亲和性；具体地，例如采用MD模拟测定所述控冰分子与冰-水分子形成的分子间氢键数，或者采用冰吸附实验于冰水界面处测得所述控冰材料分子在冰表面的吸附量。
根据本发明，所述冰吸附实验包括测得所述控冰材料在冰表面的吸附量。
根据本发明，所述控冰材料在冰表面的吸附量＝(冰表面所吸附的控冰材料质量m 1/含有控冰材料的原溶液中控冰材料的总质量m 2)×100％。
作为本发明的实施方案，所述冰吸附实验包括以下步骤：
S1:取质量为m 2的控冰材料，配制所述控冰材料的水溶液，降温至过冷温度；
S2:将预冷的控温棒置于所述水溶液中诱导冰层在控温棒表面生长，持续搅拌水溶液，以待控冰材料逐渐吸附于冰层表面，保持水溶液、控温棒温度在过冷温度；
S3:测定控冰材料在冰表面的吸附量m 1。
根据本发明的实施方案，所述控温棒为导热材料制得的棒状物。所述棒状物可以是实心的或者中空的。当所述控温棒为中空时，其中空内腔有冷却液流动，可以通过控制冷却液的温度控制控温棒的温度，从而控制冰块的生长速度。
根据本发明的实施方案，所述控温棒可经液氮、干冰、超低温冰箱冷冻等方式中的一种预冷。
根据本发明的实施方案，所述冰吸附实验过程中，保持过冷度以及吸附时间不变以使控温棒表面所得冰的表面积在误差允许范围内保持不变。
根据本发明的实施方案，配置不同浓度的控冰材料的水溶液，进行冰吸附实验，可以评估同一控冰材料在具体应用时的适用浓度范围。
根据本发明的实施方案，所述步骤S1中控冰材料可以为预先荧光标记的，例如用荧光素进行标记，所述荧光素可以选自异硫氰酸荧光素(FITC)、四乙基罗丹明(RB200)、四甲基异硫氰酸罗丹明(TRITC)、碘化丙啶(PI)等中的至少一种。本领域技术人员应当理解，所述荧光标记的作用在于测得所述控冰材料的量，因此，如果所述控冰材料的吸附量可以通过其他方式准确测量，或者材料本身具有紫外或荧光光谱吸收特性，则无需进行荧光标记。
根据本发明的实施方案，步骤S3包括：
S3a:将吸附完成的冰块取出，纯水淋洗冰表面，融化冰块得到控冰材料吸附溶液；
S3b:测定融化的控冰材料吸附溶液的体积V，测定所述吸附溶液中控冰材料的质量/体积浓度c，通过公式m 1＝cV计算得到冰表面所吸附的控冰材料质量m 1。
根据本发明的实施方案，所述S3b中，所述浓度c可通过本领域已知的方法测得，例如紫外可见光谱法、荧光光谱法等。
根据本发明，所述方法用于控制冰晶生长材料的筛选，例如PVA、聚氨基酸、抗冻蛋白、多肽等。
根据本发明，所述方法在步骤(a)和/或(b)之后还包括步骤(c)：评估所述材料与水的亲和性和在冰水界面的铺展性能，铺展能力强的材料具有良好的控冰性能。
作为本发明步骤(c)的一个具体评估方案，覆盖一定冰表面积所需控冰材料的用量越小，则说明其铺展性能越好，即满足铺展系数S＞0，其中S＝γ I-W-(γ IRIA-I+γ IRIA-W)，γ I-W为常数，即，冰水界面能γ I-W大于材料与冰以及材料与水的界面能之和γ IRIA-I+γ IRIA-W(γ IRIA-I：材料与冰的界面能；γ IRIA-W：材料与水的界面能)。
本发明中，过冷温度是指低于水的凝固点但仍不凝固或结晶的温度，在室温25℃时，所述过冷温度一般在-0.01～-0.5℃范围内，例如-0.1℃。本发明还提供一种冰吸附实验装置，包括多层储液腔、控温棒和温度控制器，所述多层储液腔由里到外依次包括冰吸附腔、温浴腔、冷却液储存腔，所述控温棒置于冰吸附腔内，所述控温棒和储液腔的温度由温度控制器控制。
根据本发明的冰吸附实验装置，所述控温棒为导热材料制成的中空结构，所述控温棒的中空结构设置有进液口和排液口；所述温度控制器为流体温控器，所述温度控制器设置有冷却液流出端和回流端；所述冷却液储存腔两端设置有进液口和排液口；所述温度控制器的冷却液流出端、控温棒的进液口、控温棒的排液口、冷却液储存箱的进液口、冷却液储存箱的排液口以及温度控制器的回流端依次经管道连通，所述管道内流动冷却液。
根据本发明的冰吸附实验装置，所述多层储液腔设有盖子。
使用时，所述冰吸附腔内盛放控冰材料的水溶液，中层温浴腔装有预定温度的温浴介质例如水浴、冰浴或者油浴等；冷却液温度达到设定温度后，经温度控制器流出，流入中空的控温棒中空结构，对控温棒温度实现控制，随后从控温棒排液口流出，再流入外层冷却液储存腔保持温浴介质的温度在预定水平，再经冷却液储存箱的排液口流经温度控制器的回流端进入温度控制器循环。
本发明的控冰材料的分子设计方法和控冰材料筛选方法，可以彼此独立地进行，也可以结合进行。在一个实施方案中，本发明提供一种设计和筛选控冰材料的全流程方法，依次包括：第一方面所述的分子设计步骤、第二方面所述的筛选控冰材料的步骤。
具体来说，所述方法包括如下步骤：
(1)构建化合物分子结构库，所述化合物分子含有亲水基团和亲冰基团；
(2)采用分子动力学模拟(Molecular dynamics simulation,MD)，模拟评估各化合物分子在冰水两相界面的铺展性能；
(3)根据步骤(2)的结果筛选出所需冰亲和性和水亲和性的控冰分子；
(4)合成筛选出的具有冰亲和性和水亲和性的控冰分子(控冰材料)；
(5)测得控冰材料与水的亲和性；
(7)测得所述控冰材料在冰水界面的铺展性能。
根据本发明的技术方案，所述步骤(7)之后包括进一步评估所述铺展性能的步骤(c)，评估所述材料与水的亲和性和在冰水界面的铺展性能，铺展能力强的材料具有良好的控冰性能。
作为本发明步骤(c)的一个具体评估方案，覆盖一定冰表面积所需控冰材料的用量越小，则说明其铺展性能越好，即满足铺展系数S＞0，其中S＝γ I-W-(γ IRIA-I+γ IRIA-W)，γ I-W为常数，即，冰水界面能γ I-W大于材料与冰以及材料与水的界面能之和γ IRIA-I+γ IRIA-W(γ IRIA-I：材料与冰的界面能；γ IRIA-W：材料与水的界面能)。
根据上述分子设计方法和筛选方法，本发明的发明人发现羟基立构规整度对聚乙烯醇PVA控制冰晶生长能力存在影响，并进一步发现一种特定间同规整度的聚乙烯醇(PVA)具有非常优异的控制冰晶生长的能力，所述PVA间同规整度(diad syndiotacticity)r为45％～60％，分子量为10kDa–500kDa；优选地，所述PVA间同规整度r为50％～55％，分子量为10-30kDa。
本发明的发明人还设计并合成了多种肽类化合物，例如二肽、三肽、类肽、糖肽化合物，具有非常优异的控制冰晶生长的能力。
所述肽类化合物由亲冰性氨基酸，如：苏氨酸(L-Thr)、谷氨酰胺(L-Gln)、天冬氨酸(L-Asn)等与其他亲水性氨基酸或葡萄糖内酯(GDL)或糖类反应得到，所述其他亲水性氨基酸可选自精氨酸、脯氨酸、丙氨酸等。所述肽类化合物为含有亲冰基团的氨基酸和含有亲水基团的氨基酸组成。在一个实施方案中，组成所述肽类化合物的氨基酸为两种以上的氨基酸，或者一种以上氨基酸与葡萄糖内酯或糖类。
本发明还发现一些特定的氨基酸或者聚氨基酸具有非常优异的控制冰晶生长的能力。
所述氨基酸为含有亲冰基团和亲水基团的氨基酸，所述聚氨基酸为氨基酸均聚物，例如所述氨基酸选自精氨酸、苏氨酸、脯氨酸、赖氨酸、组氨酸、谷酰胺酸、天冬氨酸、甘氨酸等氨基酸的均聚物；优选聚合度为2-40，更优选聚合度为2-20，例如聚合度为6、8、15、20等，例如为聚-L-脯氨酸、聚-L-精氨酸等中的一种或两种以上的组合。
示例性地，所述氨基酸选自精氨酸、苏氨酸、脯氨酸、赖氨酸、组氨酸、谷酰胺酸、天冬氨酸、甘氨酸等中的一种或两种；例如为精氨酸与苏氨酸的组合。
本发明的第三方面，提供一种冷冻保存液，包括如第一方面的方法所设计的控冰材料，或者第二方面所述方法筛选的控冰材料。在一个实施方案中，所述控冰材料为聚乙烯醇(PVA)，氨基酸或聚氨基酸，和/或肽类化合物的一种或几种的组合；所述冷冻保存液还包括多元醇、水溶性糖(或其衍生 物，如水溶性纤维素)和缓冲液。
在一个实施方案中，所述冷冻保存液包括肽类化合物，以每100mL冷冻保存液计，含有0.1-50g所述肽类化合物，0-6.0g的PVA，0-9.0g的聚氨基酸，0-15mL的DMSO，5-45mL的多元醇，0.1-1.0mol L -1的水溶性糖，0-30mL的血清，余量为缓冲液。
在一个实施方案中，所述冷冻保存液包括聚乙烯醇，以每100mL冷冻保存液计，含有PVA0.01-6.0g，0-50g所述肽类化合物，0-9.0g的聚氨基酸，0-15mL的DMSO，5-45mL的多元醇，0.1-1.0mol L -1的水溶性糖，0-30mL的血清，余量为缓冲液。
在一个实施方案中，所述冷冻保存液包括氨基酸或聚氨基酸，以每100mL冷冻保存液计，含有0.1-50g的氨基酸或聚氨基酸，0-6.0g的PVA，0-15mL的DMSO，5-45mL的多元醇，0.1-1.0mol L -1的水溶性糖，0-30mL的血清，余量为缓冲液。根据本发明，每100mL冷冻保存液中所述氨基酸和/或聚氨基酸含量为0.5-50g，优选1.0-35g，例如，当含有氨基酸时，其含量可以为5.0-35g，优选为15-25g；当含有聚氨基酸时，其含量可以为0.5-9.0g，优选为1.0-5.0g。
根据本发明，所述多元醇可以为碳原子数为2-5的多元醇，优选碳原子数2-3的二元醇、和/或三元醇，例如乙二醇，丙二醇，丙三醇中的任一种；优选乙二醇。
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、水溶性纤维素、糖酐中的至少一种，例如选自蔗糖、海藻糖、羟丙基甲基纤维素、聚蔗糖；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，或其他细胞培养缓冲液中的至少一种。
根据本发明，所述血清针对人源性冷冻保存对象可选人血清白蛋白或其替代物，例如十二烷基磺酸钠，针对非人源性冷冻保存对象可选胎牛血清或牛血清白蛋白。
根据本发明，每100mL冷冻保存液中所述DMSO含量为0-10mL，优选DMSO含量为1.0-7.5mL，例如1.5-5mL；作为本发明的另一实施方案，每100mL冷冻保存液中所述DMSO含量为0。
根据本发明，每100mL冷冻保存液中所述血清含量为0.1-30mL，例如5.0-20mL，10-15mL；作为本发明的另一实施方案，每100mL冷冻保存液中所述血清含量为0。
根据本发明，每100mL冷冻保存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；具体地，例如0.25mol L -1，0.5mol L -1，1.0mol L -1。
根据本发明，每100mL冷冻保存液中所述多元醇，含量为5.0-40mL，例如6.0-20mL，9-15mL。
根据本发明，所述冷冻保存液的pH为6.5-7.6，例如为6.9-7.2。根据本发明，所述肽类化合物或氨基酸、聚氨基酸具有上文所述的含义。
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％。
作为本发明的一个实施方案，所述冷冻保存液以每100mL计，含有如下组分：氨基酸0.5-50g，多元醇5.0-45mL，DMSO 0-10mL，血清0.1-30mL，水溶性糖0.1-1.0mol L -1，缓冲液余量。优选：所述冷冻保存液以每100mL计，含有如下组分：L-Arg 2.0-20g，L-Thr 1.0-10g，乙二醇5.0-15mL，DMSO 5.0-10mL，血清5.0-20mL，蔗糖0.1-1.0mol L -1，DPBS余量。
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，含有如下组分：聚氨基酸0.5-9.0g，多元醇5.0-45mL，DMSO 0-10mL，血清5.0-20mL，水溶性糖0.1-1.0mol L -1，缓冲液余量。优选：所述冷冻保存液以每100mL体积计，含有如下组分：聚-L-脯氨酸或聚-L-精氨酸1.0-8.0g，乙二醇5-45mL，DMSO 0.1-10mL，血清5.0-20mL，蔗糖0.1-1.0mol L -1，DPBS余量。
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，包括如下组分：PVA0.01-6.0g，多元醇5.0-45mL，血清0.1-30mL，水溶性糖0.1-1.0mol L -1，缓冲液余量。
优选，所述冷冻保存液以每100mL体积计，包括如下组分：，PVA 0.01-6.0g，乙二醇5.0-30mL， 血清10-20mL，蔗糖0.1-0.6mol L -1，DPBS余量。
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，包括如下组分：，PVA1.0-5.0g，多元醇5.0-20mL，DMSO 0.1-10mL，血清0.1-20mL，水溶性糖0.2-0.8mol L -1，缓冲液余量。优选：所述冷冻保存液以每100mL体积计，包括如下组分：PVA 1.0-4.0g，乙二醇5.0-15mL，DMSO 4-10mL，血清10-20mL，蔗糖0.2-0.6mol L -1，DPBS余量。
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，由如下组分组成：PVA0.1-6.0g，多元醇10-45mL，水溶性糖0.2-1.0mol L -1，缓冲液余量。优选：所述冷冻保存液以每100mL体积计，由如下组分组成：PVA 1.0-5.0g，乙二醇5.0-20mL，蔗糖0.2-0.6mol L -1，DPBS余量。
作为本发明的一个实施方案，所述冷冻保存液以每100mL体积计，含有如下组分：，聚氨基酸0.5-9.0g，多元醇5.0-45mL，PVA 0.1-6g，血清0-20mL，水溶性糖0.1-1.0mol L -1，缓冲液余量。优选：所述冷冻保存液以每100mL体积计，含有如下组分：聚-L-脯氨酸或聚-L-精氨酸1.0-8.0g，乙二醇5-45mL，PVA 0.1-6g，血清5.0-20mL，蔗糖0.1-1.0mol L -1，DPBS余量。
本发明还提供上述冷冻保存液的制备方法，包括如下步骤：
(1)分别量取所述氨基酸或聚氨基酸、聚乙烯醇、肽类化合物中的任一种或多种，分别溶解于部分缓冲液，调节pH后形成溶液；
(2)将水溶性糖溶解于另一部分的缓冲液中，待水溶性糖全部溶解后加入除血清外的其他组分，制得溶液；
(3)待步骤(1)和步骤(2)的溶液冷却至室温后混合，调节pH并用缓冲液定容至预定体积，得到所述冷冻保存液。
根据本发明冷冻保存液的制备方法，当所述冷冻保存液含有血清时，所述血清在所述冷冻保存液使用时添加。
根据本发明的制备方法，所述PVA溶解时，采用温浴加热并进行搅拌，例如采用水浴或油浴加热；例如水浴温度为65-85℃、70-80℃；所述搅拌为机械搅拌例如磁力搅拌。
根据本发明的制备方法，所述水溶性糖的溶解为超声辅助溶解。
本发明的冷冻保存液可与冷冻平衡液配合使用。在一个实施方案中，本发明提供一种冷冻平衡液，以每100mL计，含有多元醇5.0-45mL，缓冲液余量。
根据本发明的冷冻平衡液，还任选地包括DMSO 0-15mL，血清0-30mL，和/或PVA 0-5.0g。
根据本发明的冷冻平衡液，所述多元醇含量为6.0-28mL，例如7.0-20mL，10-15mL。
根据本发明的冷冻平衡液，所述DMSO含量为0.1-15mL，例如1.0-10mL，5.0-7.5mL；作为本发明的一个实施方案，所述DMSO的含量为0。
根据本发明的冷冻平衡液，所述血清含量为0.1-30mL，例如5.0-20mL，10-15mL；作为本发明的一个实施方案，所述血清的含量为0。
根据本发明的冷冻平衡液，所述PVA含量为0.1-5.0g，例如0.1g、0.5g、1.0g、2.0g；作为本发明的一个实施方案，所述PVA的含量为0。
本发明冷冻平衡液中，所述多元醇、血清、缓冲液可选自与所述冷冻保存液中相同的种类。在一个实施方案中，当所述冷冻保存液不含有血清时，所述冷冻平衡液中加入PVA。
作为本发明的一个实施方案，所述冷冻平衡液以每100mL计，含有多元醇5.0-7.5mL，DMSO5.0-7.5mL,血清10-20mL，缓冲液余量。
作为本发明的一个实施方案，所述冷冻平衡液以每100mL计，含有多元醇7.5-15mL，血清10-20mL，缓冲液余量。
作为本发明的一个实施方案，所述冷冻平衡液以100mL计，含有PVA 1.0-5.0g，多元醇7.5-15mL，缓冲液余量。
本发明还提供上述冷冻平衡液的制备方法，包括将各组分溶解于缓冲液中，血清单独存放，在使用时添加。
一种冷冻保存用试剂，包括上述冷冻平衡液和上述冷冻保存液，所述平衡液和保存液彼此独立存在。
根据本发明的冷冻保存用试剂，当所述冷冻保存液不含有血清时，所述冷冻平衡液中加入PVA。
具体来说，所述冷冻保存液中DMSO含量为0时，所述冷冻平衡液以每100mL计，含有PVA 0-5.0 g，多元醇7.5-15mL,血清10-20mL，缓冲液余量；所述冷冻保存液中DMSO和血清含量都为0时，所述冷冻平衡液以每100mL计，含有PVA 1.0-5.0g，多元醇7.5-15mL，缓冲液余量。
本发明的上述冷冻保存液或冷冻保存平衡液或者两者的组合可用于各类细胞、组织、器官冷冻保存。各类细胞包括但不限于生殖细胞，例如卵母细胞、精子，各种干细胞，例如脐带间充质干细胞；各类组织包括但不限于卵巢组织、胚胎组织、受精卵；各类器官包括但不限于卵巢或其他哺乳动物器官。
进一步地，本发明提供上述冷冻保存液或者冷冻保存平衡液或者两者的组合在细胞、组织、器官冷冻保存中的应用。在一个实施方式中，上述冷冻保存液或者冷冻保存平衡液或者两者的组合用于卵母细胞冷冻保存；在一个实施方式中，上述冷冻保存液或者冷冻保存平衡液或者两者的组合用于胚胎冷冻保存；在一个实施方式中，上述冷冻保存液或者冷冻保存平衡液或者两者的组合用于卵巢组织或卵巢器官冷冻保存；在一个实施方式中，上述冷冻保存液或者冷冻保存平衡液或者两者的组合用于干细胞冷冻保存。
本发明进一步提供一种细胞或胚胎的冷冻和复苏方法，包括：
(1)将细胞或胚胎置于本发明的冷冻保存液中，制成细胞悬浮液，进行冷冻；
(2)将冷冻的细胞或胚胎放入解冻液中进行解冻复苏。
本发明所述的冷冻和复苏方法，所述细胞或胚胎在置于冷冻保存液中之前，先放入平衡液中进行平衡。
本发明进一步提供一种干细胞冷冻保存的方法，采用微滴法，例如所述干细胞冷冻保存的方法包括如下步骤:将冷冻保存液加入干细胞中，吹打分散，制成干细胞悬液，将干细胞悬液置于冷冻载片上，液氮(-196℃)冷冻保存。
根据本发明的实施方案，冷冻保存的干细胞的解冻包括将置有干细胞的冷冻载片置于a-MEM培养基中，37℃解冻。
根据本发明的实施方案，所述干细胞为本领域已知的具有分化功能的各种干细胞，例如全能干细胞、多能干细胞或者专能干细胞，包括但不限于胚胎干细胞、各类间充质干细胞(例如脐带间充质干细胞、脂肪间充质干细胞、骨髓间充质干细胞等)、造血干细胞等。
本发明还供器官和/或组织的冷冻保存方法，包括：将器官和/或组织在冷冻平衡液中平衡，然后将器官和/或组织放入冷冻保存液中，再将器官和/或组织置于冷冻载片上，液氮冷冻保存。
在一个实施方案中，所述器官和/或组织为卵巢组织或者卵巢器官，可以为卵巢组织切片或者完整的卵巢组织。
本发明中“冻存”和“冷冻保存”具有相同含义，可互换使用，指通过低温对某种物质或者细胞、组织、器官进行保存，使其保持其本来的理化和/或生物活性、生理生化功能。
本发明中“控冰分子”或“控冰材料”具有相同含义，指具有抑制水溶液中冰晶生长能力的化合物。在一个实施方案中，所述控冰分子在冰水界面具有良好的铺展性能，可以减小冰晶尺寸，或者所述控冰分子不具有热滞后或具有足够小的热滞后，可以显著减少水溶液中冰晶的形成。
有益效果
1.本发明首次发现控冰分子在冰水混合相中控制冰晶生长的机制，控冰材料需与冰和水均具有良好的亲和性。控冰分子与冰具有亲和性可以保证其较好的吸附在冰表面上；分子与水具有亲和性可以确保其在冰水界面更好地铺展，以尽可能实现用最少量的材料，覆盖最大的冰表面积。基于这一控冰机理，提出设计兼具亲冰性能和亲水性能的控冰分子设计思路，为控冰材料的合成提供新方法。
2.本发明首次将MD模拟引入到控冰材料的分子结构设计中，通过分子动力学模拟评估所设计的控冰分子的冰亲和性和水亲和性，预测控冰材料的控冰性能，并可实现结构优化。
3.本发明通过将控冰机制和MD模拟相结合的方式，很好地解决了目前控冰材料研发过程中仅能通过“实验试误法”对现有材料进行性能分析筛选材料的局限，提供了一种分子结构设计的新思路，对控冰材料的开发和应用具有极大促进作用。
4.本发明提供的冷冻保存液来源广泛，生物相容性好，毒性小，安全性高，并且大大减少DMSO的用量，甚至不加DMSO时也能达到与现有含DMSO15％以上的商业化冷冻保存液同等甚至更高的细胞存活率。本发明的冷冻保存液组成简单，且原料来源方便，成本低廉，可广泛应用于各类细胞、组织的冷冻保存，例如可广泛用于卵母细胞、胚胎、干细胞、卵巢组织、卵巢器官等冷冻保存，均可 保持良好的生物活性。
附图说明
图1：控冰材料的分子结构示意图；
图2：MD模拟无规立构聚乙烯醇(a-PVA)与全同立构聚乙烯醇(i-PVA)在冰水界面的聚集状态；
图3：实施例1合成的a-PVA的氢核磁共振谱图；
图4：实施例1合成的PBVE和i-PVA的氢核磁共振谱图，A为PBVE，B为i-PVA；
图5：实施例1合成的PBVE的GPC曲线；
图6：DLS实验中不同浓度时a-PVA(A)与i-PVA(B)在水中的分散尺寸；
图7：两种PVA的PBS溶液的冰晶生长的光学显微图片，A为a-PVA，B为i-PVA，C为两种PVA的PBS溶液相对于PBS的最大冰晶尺寸与浓度关系；
图8：a-PVA(图A、B)与i-PVA(图C、D)在纯水中修饰冰晶形貌效果；
图9：MD模拟的两种PVA的分子结构模型；
图10：MD模拟的两种PVA分子在240K时在冰水界面处与水分子以及冰水分子的可接触表面积，其中图片上部为a-PVA分子3次的结果，下部为i-PVA分子3次的结果；
图11：MD模拟计算的两种PVA在水溶液中的聚集概率；
图12：MD模拟计算240K时两种PVA在水溶液中与水形成分子间氢键数目，在冰水界面处与水分子以及冰-水分子形成的分子间氢键数目。
图13：GDL-L-Thr(式(6)的化合物)抑制冰晶生长活性的光学显微图片和冰晶尺寸大小的统计图。
图14：GDL-L-Thr在纯水中修饰冰晶形貌效果。
图15：GDL-L-Ser(式(7)的化合物)抑制冰晶生长活性的显微镜光学图片冰晶尺寸大小的统计图。
图16：GDL-L-Val(式(8)的化合物)抑制冰晶生长活性的显微镜光学图片冰晶尺寸大小的统计图。
图17：实施例3制备的TR短链肽抑制冰晶生长活性的光学显微图片和冰晶尺寸大小的统计图。
图18：实施例3制备的TR短链肽在纯水中修饰冰晶形貌效果。
图19：实施例8类肽R-COOH,R-CH 3以及R-CH 2CH 3抑制冰晶生长活性效果。
图20：实施例8类肽(A)R-COOH,(B)R-CH 3以及(C)R-CH 2CH 3在纯水中修饰冰晶形貌效果。
图21：冰吸附实验及其装置示意图；
图22：实施例9两种PVA的冰吸附量随浓度变化图；
图23：两种PVA在DPBS溶液中冰晶生长的光学显微图片，A为a-PVA，B为i-PVA。
图24：新生3天的小鼠新鲜(未冷冻)卵巢器官切片染色图片；
图25：对比实例8冻存的卵巢器官解冻后切片染色图片；
图26：应用实例13冻存的卵巢器官解冻后切片染色图片；
图27：应用实例14冻存的卵巢器官解冻后切片染色图片；
图28：应用实例15冻存的卵巢器官解冻后切片染色图片；
图29：性成熟的小鼠新鲜(未冷冻)卵巢组织切片染色图片；
图30：对比实例9冻存的卵巢组织解冻后切片染色图片；
图31：应用实例16冻存的卵巢组织解冻后切片染色图片；
图32：应用实例17冻存的卵巢组织解冻后切片染色图片；
图33：应用实例18冻存的卵巢组织解冻后切片染色图片；
图34：应用实例26冻存的卵巢组织解冻后切片染色图片；
图35：应用实例27冻存的卵巢组织解冻后切片染色图片；
图36：应用实例28冻存的卵巢组织解冻后切片染色图片；
图37：应用实例29冻存的卵巢组织解冻后切片染色图片；
图38：应用实例30冻存的卵巢组织解冻后切片染色图片；
图39：应用实例31冻存的卵巢组织解冻后切片染色图片；
图40：应用实例37冻存的卵巢组织解冻后切片染色图片；
图41：应用实例38冻存的卵巢组织解冻后切片染色图片。
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。
A.控冰材料分子设计
本发明的控冰材料核心分子，可以设计为各类与水有亲和性的基团以及与冰有亲和性的基团通过共价键或非共价键例如离子键连接而成。
本发明的控冰材料的分子设计方法，包括如下步骤：
(1)构建化合物分子结构库，所述化合物分子含有亲水基团和亲冰基团；
(2)采用分子动力学模拟(Molecular dynamics simulation,MD)，模拟评估各化合物分子在冰水两相界面的铺展性能；
(3)筛选出所需冰亲和性和水亲和性的控冰分子。
根据本发明，所述控冰分子的主链为碳链或者肽链结构。
根据本发明，所述步骤(2)的MD模拟可以通过GROMACS、AMBER、CHARMM、NAMD、或LAMMPS进行。
根据本发明，所述步骤(2)MD模拟中，水分子模型可以选自TIP3P、TIP4P、TIP4P/2005、SPC、TIP3P、TIP5P、或SPC/E，优选TIP4P/2005水分子模型。
根据本发明，所述步骤(2)MD模拟中，力场参数由GROMOS、ESFF、MM形态力场、AMBER、CHARMM、COMPASS、UFF、CVFF等力场中的一种提供。
根据本发明，所述步骤(2)MD模拟中，模拟计算控冰分子之间的相互作用，控冰分子与水分子的相互作用，控冰分子与冰－水分子的相互作用。所述作用包括是否形成氢键、范德华尔斯作用、静电作用、疏水作用、π-π作用等。
根据本发明，所述步骤(2)MD模拟中，所述模拟计算分子相互作用时，对温度和压强进行调节。在本发明的一个实施方式中，采用V-rescale(modified Berendsen)温度控制器和压强控制器调节温度和压强。
根据本发明，所述步骤(2)MD模拟中，通过选择势能参数以保持化合物分子的分子构型。优选，所选择的势能参数使化合物分子的分子构型在较高温度下得以保持。
根据本发明，所述步骤(2)中，模拟水溶液体系时，x，y，z三个方向均采用周期性边界条件；模拟冰水混合体系时，x，y两个方向采用周期性边界条件。
根据本发明，所述步骤(2)MD模拟中，选用立方体或八面体水盒子，优选3.9×3.6×1.0nm 3的立方体水盒子。
作为本发明的具体实施方案，所述MD模拟在分子动力学计算过程中，V-rescale(modified Berendsen)温度控制器和压强控制器调节温度和压强。
在所述MD模拟计算中，判断氢键存在的主要准则为能量准则或几何准则，优选几何准则；当氧原子间距小于0.35nm，角HO...H小于30度时，两个羟基或者羟基与水分子间形成氢键。
作为本发明的具体实施方式，所述控冰材料可以为碳链结构作为主链并被亲冰基团和亲水基团取代的化合物；所述控冰材料可以含有亲水和亲冰双重亲和性的基团，例如羟基、氨基，还可以分别含有亲冰基团和亲水基团。例如，所述控冰材料分子结构设计为具有-[CH 2-CHOH]-的重复单元。
在本发明的实施方案中，所述控冰材料分子为聚乙烯醇(PVA)。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。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。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％。
在本发明的实施方案中，所述控冰材料分子为肽类化合物。所述肽类化合物由亲冰性氨基酸，如：苏氨酸(L-Thr)、谷氨酰胺(L-Gln)、天冬氨酸(L-Asn)等与其他亲水性氨基酸或葡萄糖内酯(GDL)或糖类反应得到，所述其他亲水性氨基酸可选自精氨酸、脯氨酸、丙氨酸等。
所述肽类化合物为两个以上的氨基酸单元形成的肽类，如：2-8个氨基酸单元，具体地可以为2-5个，例如2个、3个、4个、5个、6个氨基酸单元；每个氨基酸单元不同。所述肽类化合物中亲冰氨基酸如苏氨酸与其他亲水氨基酸的摩尔比为(0.1-3)：1，优选(0.5-2)：1。所述肽类化合物中亲冰氨基酸与其他亲水氨基酸的排列方式没有特别的限定，可采用本领域已知的氨基酸连接基团或化学键连接，例如亲冰氨基酸和亲水氨基酸可以单个间次排列，也可以多个亲冰氨基酸或者多个亲水氨基酸相连，形成亲冰氨基酸片段或亲水氨基酸片段，再分别与亲水氨基酸(或片段)、亲冰氨基酸(或片段)连接。
在本发明的实施方案中，所述肽类化合物为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)中的至少一种。
在另一实施方案中，所述肽类化合物为GDL-L-氨基酸，例如GDL-L-Thr、GDL-L-Ser、GDL-L-Val。
在又一实施方案中，所述肽类化合物具有式(1)-式(8)所示任一结构：
Figure PCTCN2020077472-appb-000001
Figure PCTCN2020077472-appb-000002
上述肽类化合物的制备方法，可以采用本领域已知的多肽合成方法合成，例如采用固相合成法合成。
根据本发明的制备方法，包括如下步骤:树脂溶胀、一种氨基保护的氨基酸共价连接在溶胀的树脂上、脱保护、加入另一种氨基保护的氨基酸缩合反应、脱保护、切割、纯化。
可以用本发明已知的氨基酸与糖类反应的方法制备糖肽衍生物，例如可以将葡萄糖内酯或其他糖类与氨基酸在有机溶剂中反应制备所述糖肽衍生物，或者采用固相合成方法制备所述糖肽衍生物，将葡萄糖内酯(GDL)溶解在有机溶剂中，并将氨基酸和碱性催化剂加入到有机溶剂里，完全溶解后加入到葡萄糖内酯的溶液中，55-60℃反应，反应结束后，过滤掉白色沉淀物，把滤液蒸干得到粗产物。
根据本发明的制备方法，所述有机溶剂可以选自甲醇、乙醇等。
在一个实施方案中，采用固相合成法制备糖肽衍生物，包括：树脂溶胀、将一种氨基保护的氨基酸共价键连接在溶胀的树脂上、脱保护、加入糖类化合物(例如开环后的葡萄糖内酯)缩合反应、切割、纯化。GDL-L-Val和GDL-L-Ser的合成方法参照GDL-L-Thr的合成方法。
本发明还提供式(9)所示的肽类化合物：
Figure PCTCN2020077472-appb-000003
其中，R选自取代或未取代的烷基，所述取代基可以选自-OH、-NH 2、-COOH、-CONH 2等，例如，R为取代或未取代的C1-6烷基，优选R为-CH 3、-CH 2CH 3、-CH 2CH 2COOH；n为大于等于1而小于等于1000的整数，例如可以为1～100范围内的整数。在本发明的一些实施方式中，n为2、3、4、5、 6、7、8、9、10的整数。
作为本发明的一个实施方案，所述式(9)所示化合物具有如下任一所示的结构：
Figure PCTCN2020077472-appb-000004
根据本发明，所述式(9)所示的化合物采用如下合成路线制备：
Figure PCTCN2020077472-appb-000005
在本发明的实施方案中，所述控冰材料分子为氨基酸或聚氨基酸。本发明还提供上述控冰材料分子，例如PVA、肽类化合物、氨基酸和聚氨基酸用于控制水溶液中冰晶生长的应用、上述肽类化合物用于制备细胞或组织冷冻保存液的应用。
根据本发明所设计并制备的控冰材料例如PVA、肽类化合物、氨基酸和聚氨基酸用于制备冷冻保存液，用于细胞、组织、器官等的冷冻保存。
【实施例1】
(1)化合物分子结构设计：
设计含有具有-[CH 2-CHOH]-的重复单元的化合物分子，得到包括无规和等规的聚乙烯醇分子模型的分子结构库。
(2)MD模拟实验
通过MD模拟实验预测无规聚乙烯醇和等规聚乙烯醇对于冰和水的亲和性差异。
a.MD模拟均由GROMACS 5.1完成，水模型选用TIP4P/2005，其熔点约为252.5K。PVA分子的相互作用参数由GROMOS54A7力场提供，采用leapfrog积分算法，积分步长为2fs。静电相互作用通过PME方法计算，库伦作用势和L-J作用势的截断半径均为1.0nm。由V-rescale(modified Berendsen)温度控制器和压强控制器调节温度和压强。时间常数设为0.1ps。
b.模拟选取含有7个重复单元的化合物分子链进行研究。PVA分子的拓扑文件通过ATB生成，且为了保持两种PVA分子的立构规整度，需对分子碳链的二面角势函数做相应的调整。
c.模拟PVA的水溶液体系时，x，y，z三个方向均采用周期性边界条件；模拟冰水混合体系时，x，y两个方向采用周期性边界条件。所有体系均模拟120ns,取后60ns的数据进行分析。
首先研究了分子的水溶液的体系，在只有一条PVA链的体系中，共有1491个水分子，压强为1atm，温度取240K、250K、260K、270K、300K、330K。
在研究PVA分子与冰的相互作用的体系中，在3.9×3.6×1.0nm 3的水盒子中放入6条PVA分子链，把含有1100个水分子的冰块在240K下平衡10ns，把冰快沿z轴方向放置在水盒子下方。把混合体系z方向的大小增大到10nm，并把冰水混合体系置于水盒子中央。
PVA分子的拓扑文件通过ATB生成，直接使用该拓扑文件，且为了保持两种PVA分子的立构 规整度，选择势能参数为50kcal/mol，能够保证即使在较高的温度下，两种PVA分子的分子构型也能被保持住。
MD模拟的两种PVA的分子结构模型参见图9。
(3)评估模拟结果
a-PVA因三倍相邻OH的距离与冰的晶格尺寸匹配，故可以有效地与冰表面发生氢键作用而吸附在冰面上。i-PVA因只改变羟基方向而不改变相邻OH的距离，故可保证i-PVA与a-PVA具有相似的与冰发生吸附作用的能力。同时，根据MD模拟结果可知，a-PVA与水分子形成的分子间氢键数多于i-PVA与水分子形成的分子间氢键数，故a-PVA与水的亲和性较i-PVA更强。此外，MD模拟6条PVA分子链在冰水界面的状态显示，a-PVA因与冰和水都具有良好的亲和性而更倾向于在冰水界面铺展；而i-PVA因与水的亲和性较弱，在冰水界面倾向聚集(图2)。
表1
Figure PCTCN2020077472-appb-000006
MD模拟显示，240K时冰水界面处两种PVA与水分子的可接触面积，a-PVA的可接触表面积大于i-PVA，进一步验证了a-PVA在冰水界面的铺展性能优于i-PVA(参见图10)。MD计算的两种PVA在水溶液中的聚集概率，i-PVA明显高于a-PVA(图11)；而且240K时，在冰水界面处，两种PVA与冰-水分子形成的氢键数目相当，但a-PVA在冰水界面处以及水溶液中与水形成分子间氢键数要明显多于i-PVA；故a-PVA可在冰水界面更好的铺展，而i-PVA则聚集(图12)。
因此，通过MD模拟的多项结果均显示，a-PVA由于其分子结构与水分子具有更好的亲和性，因此在冰水界面有更好的铺展性能，相比于i-PVA，应当具有更好的控冰效果。
(4)合成所设计的PVA分子
(4.1)制备无规立构聚乙烯醇a-PVA：分子量约为13～23kDa，间同规整度r(diad syndiotacticity)约为55％
氩气氛围保护下，在250mL圆底烧瓶中将已除去抑制剂的乙酸乙烯酯(vinyl acetate,VAc,Sigma-Aldrich)溶解在100mL的溶剂中(甲醇)以获得25％～45％的VAc溶液。将上述溶液冷却到-5℃后，将80mM的2,2'-偶氮二(2-甲基丙腈)(2,2’-Azobis(2-methylpropionitrile,Sigma-Aldrich)小心滴加到反应溶液中。将上述溶液放置室温后继续搅拌15小时后，将反应溶液用1L的丙酮溶解并滴加到甲醇中以获得白色沉淀。上述沉淀用甲醇清洗过滤后，在60℃烘箱真空干燥6.0小时获得白色固体。将白色固体溶解到甲醇溶液中(10wt.％)，并通氩气以除去溶液中的氧气，并将25％的氢氧化钾的甲醇溶液滴加到上述溶液中，持续搅拌4小时后，将反应溶液溶解在2M的盐酸溶液中，并在2.0M的氨的甲醇溶液中沉淀以获得无规立构聚乙烯醇(a-PVA)。氢核磁共振谱(图3)显示所得化合物为水解完全a-PVA。
(4.2)制备全同立构聚乙烯醇i-PVA：分子量约为14～26kDa，全同规整度m(isotacticity)约为84％
a.聚叔丁基乙烯基醚的制备(poly-tert-butyl vinyl ether,PBVE)。氩气氛围保护下，在250mL圆底烧瓶中将叔丁基乙烯基醚(tert-butyl vinyl ether,t-BVE,Sigma-Aldrich)溶解在100mL的干燥甲苯中以获得2.5％的t-BVE的甲苯溶液。将上述溶液冷却到-78℃后，将0.2mM的三氟化硼乙醚(BF 3·OEt 2,Sigma-Aldrich)小心滴加到已冷却的溶液中，并于2.0小时后补加0.2mM的BF 3·OEt 2。将上述溶液在-78℃继续搅拌3.0小时后，用少量甲醇终止反应。并将反应溶液滴加到快速搅拌的2.0L的甲醇中以获得淡黄色沉淀。上述沉淀用甲醇清洗过滤后，在60℃烘箱真空干燥6.0小时获得淡黄色固体粉末，氢核磁共振谱(图4A)显示所得化合物为PBVE。通过调控三氟化硼乙醚与叔丁基 乙烯基醚的浓度来控制所合成的PBVE的分子量。凝胶渗透色谱(GPC采用四氢呋喃(THF)体系测得，流速为1mL min -1)显示成功合成获得具有不同分子量的PBVE(图5)。
b.干燥溴化氢气体的制备(HBr)；在一个100mL的两口烧瓶中，将5.0～30mL的三溴化磷(PBr 3,阿拉丁)逐滴加入到10mL 48％的溴化氢(HBr,Alfa Aesar)水溶液中。所生成的气体依次通过四氯化碳(CCl 4)，红磷(P,Alfa Aesar)以及氯化钙(CaCl 2)以得到干燥的HBr气体。
c.全同立构聚乙烯醇(isotactic-PVA,i-PVA)的制备。在氩气氛围保护下，将0.5g的PBVE溶解到15mL干燥甲苯溶液中，并持续通入干燥氩气以除去溶液中的氧气。在0℃下，将b步骤中制备的干燥HBr气体通入到上述无氧的PBVE的甲苯溶液中。约5.0分钟后，淡黄色沉淀生成，持续通入干燥的HBr气体直到没有沉淀继续生成。将上述反应溶液倒入2.0M的200mL的氨的甲醇溶液中。所得沉淀用甲醇清洗过滤后，在60℃烘箱真空干燥6.0小时以获得淡黄色固体粉末。氢核磁共振谱(图4B)显示PBVE水解完全得固体i-PVA。
(5)所合成的PVA控冰效果验证
(5.1)动态光散射DLS实验
两种PVA(a-PVA:分子量约为13～23kDa，间同规整度r(diad syndiotacticity)约为55％(Sigma-Aldrich)；i-PVA:分子量约为14～26kDa，全同规整度m(isotacticity)约为84％)在25℃水溶液中的粒径分布采用动态光散射(DLS)实验测量，实验仪器为带有恒温腔室和4mW He-Ne激光(λ＝632.8nm)的Nano ZS(Malvern Instruments)，其中散射角为173°。首先，分别配制浓度为1.0mg mL -1、4.0mg mL -1、10mg mL -1、20mg mL -1的a-PVA和i-PVA水溶液；将约1.0mL的PVA溶液装入12mm的一次性用聚苯乙烯比色皿进行测量。
动态光散射(DLS)结果显示，相同浓度的a-PVA在水溶液中的分散尺寸远小于i-PVA(图6)。即，相比于a-PVA，i-PVA在水溶液中更倾向于以聚集状态存在。这与MD模拟中a-PVA的分子内氢键数少于i-PVA的分子内氢键数，a-PVA与水分子的分子间氢键数多于i-PVA与水分子的分子间氢键数的结果一致。
(5.2)冰晶重结晶抑制(IRI)活性测量
冰晶重结晶抑制(IRI)活性采用“溅射冷冻法”，将样品溶解分散到DPBS溶液中，将10～30μL上述溶液在1.0m以上的高度滴加到-60℃预冷的干净硅片表面，利用冷热台以10℃min -1的速度缓慢升温到-6℃，并在该温度下退火30min，利用偏光显微镜以及高速摄像机观察记录冰晶的大小，冷热台密封，保证内部的湿度在50％左右。每一个样品重复至少三次，使用Nano Measurer 1.2统计冰晶的尺寸，统计结果的误差为标准偏差。
(5.3)冰晶形貌(DIS)观察和热滞后(TH)测量
冰晶形貌(DIS)观察和热滞后(Thermal Hysteresis，TH)测量采用纳升渗透压仪，首先用酒精灯外焰熔化毛细管，并同时拉伸产生极细孔径毛细管，将毛细管与微量进样器相连。将黏度较高的浸镜油注入微米孔径圆片中，利用微量进样器将溶解了材料的水溶液注入微孔中。通过控制温度，使液滴快速结冰，并缓慢升温以获得单晶冰，以0.01℃的精度缓慢降温，利用配备了高速摄像机的显微镜观察冰晶形貌以及TH测试。
a-PVA(M w 13～23kD)抑制冰晶生长的能力远远优于相应分子量的i-PVA(M w 14～26kD)(图7)。图7A中可见，a-PVA冰晶尺寸明显小于同浓度时i-PVA的冰晶尺寸；图7B中可见，a-PVA相对于DPBS的最大冰晶尺寸(MLGS)在2.0mg mL -1后达到最小，最小约为DPBS最大冰晶尺寸的20％；不同分子量的i-PVA相对于DPBS的MLGS随着浓度的增加有所增加，并在10mg mL -1达到最小，最小仅约为DPBS最大冰晶尺寸的50％，且随着浓度继续增加到20mg mL -，MLGS不再减少且有略微增加。聚合度大于333的i-PVA(M w 14～26kD)在浓度大于30mg mL -1时溶解困难，故因i-PVA溶解度的限制，i-PVA的IRI活性最优为10mg mL -1时50％的DPBS的MLGS；而a-PVA的IRI活性最优为2.0mg mL -1时20％的的DPBS的MLGS。这与MD模拟中，a-PVA比i-PVA更容易在冰水界面处铺展的结果一致，所述更容易铺展的性能，使a-PVA在比i-PVA更低的用量情况下就能实现更好的抑制冰晶生长的效果。
从以上MD模拟以及实际验证实验结果可见，两者的结果一致性好，MD模拟可以准确预测控冰 材料的控冰性能，可有效实现控冰材料分子设计。
采用同样的分子设计方法设计了式(1)-式(9)的化合物，并合成，研究其控冰效果。
【实施例2】式(1)化合物的合成
(1)将2-氯三苯甲基氯树脂(2-Chlorotrityl Chloride Resin)放入反应管中，加DCM(20mL g -1)，振荡30分钟。砂芯抽滤除去溶剂，加入三倍摩尔过量Fmoc-L-Pro-OH，再加入8倍摩尔过量的DIEA,最后加入DMF溶解，振荡30分钟。甲醇封头30分钟。
(2)除去溶剂DMF，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液。取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(3)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后，向反应管中加入用尽量少的DMF溶解的Fmoc-L-Thr(tBu)-OH；两倍过量，HBTU两倍过量。之后，立刻加入8倍过量的DIEA，反应30分钟。
(4)抽掉溶液后，取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，无色为阴性反应，即反应完全。
(5)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后除去溶剂，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液，取少量树脂，用乙醇清洗后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(6)将上述反应得到产物用依次用DMF(15mL g-1，两次)、甲醇(15mL g -1，两次)以及DCM(15mL g -1，两次)清洗后抽干树脂。
(7)使用切割液(15mL g -1，TFA：水：EDT：Tis＝95:1:2:2,V/V)将产物切割90分钟。并将切割液用氮气吹干，之后冻干，得到多肽粗品。
(8)用HPLC纯化多肽并转盐或脱盐，HPLC：tR＝6.1mins(纯化柱子型号：Kromasil100-5C18,4.6mm*250mm；梯度洗脱液：0.1％TFA乙腈溶液和0.1％TFA水溶液,0mins-1:99，20mins-1:9)。将纯化后的溶液冻干，既得到成品L-Thr-L-Pro(记为TP)。产率约为80％。质谱鉴定217.3为[M+H]+。
【实施例3】式(2)化合物的合成
(1)将2-氯三苯甲基氯树脂放入反应管中，加DCM(20mL g -1)，振荡30分钟。砂芯抽滤除去溶剂，加入三倍摩尔过量Fmoc-L-Thr(tBu)-OH，再加入8倍摩尔过量的DIEA,最后加入DMF溶解，振荡30分钟。甲醇封头30分钟。
(2)除去溶剂DMF，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液。取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(3)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后，向反应管中加入用尽量少的DMF溶解的Fmoc-Arg(Pbf)-OH；两倍过量，HBTU两倍过量。之后，立刻加入8倍过量的DIEA，反应30分钟。
(4)抽掉溶液后，取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，无色为阴性反应，即反应完全。
(5)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后除去溶剂，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液，取少量树脂，用乙醇清洗后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(6)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DCM(15mL g -1，两次)清洗后抽干树脂。
(7)使用切割液(15mL g -1，TFA：水：EDT：Tis＝95:1:2:2,V/V)将产物切割90分钟。并将切割液用氮气吹干，之后冻干，得到多肽粗品。
(8)用HPLC纯化多肽并转盐或脱盐，HPLC：tR＝4.8mins(纯化柱子型号：Kromasil 100-5C18,4.6mm*250mm；梯度洗脱液：0.1％TFA乙腈溶液和0.1％TFA水溶液,0mins-1:99，20mins-1:4)。将纯化后的溶液冻干，既得到成品L-Thr-L-Arg(TR)。产率约为80％。质谱鉴定276.2 为[M+H]+。
【实施例4】式(3)化合物的合成
(1)将2-氯三苯甲基氯树脂放入反应管中，加DCM(20mL g -1)，振荡30分钟。砂芯抽滤除去溶剂，加入三倍摩尔过量Fmoc-L-Thr(tBu)-OH，再加入8倍摩尔过量的DIEA,最后加入DMF溶解，振荡30分钟。甲醇封头30分钟。
(2)除去溶剂DMF，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液。取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(3)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后，向反应管中加入用尽量少的DMF溶解的Fmoc-Arg(Pbf)-OH；两倍过量，HBTU两倍过量。之后，立刻加入8倍过量的DIEA，反应30分钟。
(4)抽掉溶液后，取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，无色为阴性反应，即反应完全。
(5)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后除去溶剂，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液，取少量树脂，用乙醇清洗后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(6)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后抽干树脂。
(7)重复操作(3)～(5)，链接氨基酸Fmoc-L-Thr(tBu)-OH。反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DCM(15mL g -1，两次)清洗后抽干树脂。
(8)使用切割液(15mL g -1，TFA：水：EDT：Tis＝95:1:2:2,V/V)将产物切割90分钟。并将切割液用氮气吹干，之后冻干，得到多肽粗品。
(9)用HPLC纯化多肽并转盐或脱盐，HPLC：tR＝3.9mins(纯化柱子型号：Kromasil100-5C18,4.6mm*250mm；梯度洗脱液：0.1％TFA乙腈溶液和0.1％TFA水溶液,0mins-1:99，20mins-1:4)。将纯化后的溶液冻干，既得到成品L-Thr-L-Arg-L-Thr(TRT)。产率约为75％。质谱鉴定377.4为[M+H]+。
【实施例5】式(4)化合物的合成
(1)将2-氯三苯甲基氯树脂放入反应管中，加DCM(20mL g -1)，振荡30分钟。砂芯抽滤除去溶剂，加入三倍摩尔过量Fmoc-L-Thr(tBu)-OH，再加入8倍摩尔过量的DIEA,最后加入DMF溶解，振荡30分钟。甲醇封头30分钟。
(2)除去溶剂DMF，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液。取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(3)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后，向反应管中加入用尽量少的DMF溶解的Fmoc-L-Pro-OH；两倍过量，HBTU两倍过量。之后，立刻加入8倍过量的DIEA，反应30分钟。
(4)抽掉溶液后，取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，无色为阴性反应，即反应完全。
(5)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后除去溶剂，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液，取少量树脂，用乙醇清洗后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(6)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后抽干树脂。
(7)重复操作(3)～(5)，链接氨基酸Fmoc-L-Thr(tBu)-OH。反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DCM(15mL g -1，两次)清洗后抽干树脂。
(8)使用切割液(15mL g-1，TFA：水：EDT：Tis＝95:1:2:2,V/V)将产物切割90分钟。并将切割液用氮气吹干，之后冻干，得到多肽粗品。
(9)用HPLC纯化多肽并转盐或脱盐，HPLC：tR＝7.6mins(纯化柱子型号：Kromasil100-5C18,4.6mm*250mm；梯度洗脱液：0.1％TFA乙腈溶液和0.1％TFA水溶液,0mins-1:99，20mins-2:8)。将纯化后的溶液冻干，既得到成品L-Thr-L-Pro-L-Thr(TPT)。产率约为70％。质谱鉴定318.3为[M+H]+
【实施例6】式(5)化合物的合成
(1)将2-氯三苯甲基氯树脂放入反应管中，加DCM(20mL g -1)，振荡30分钟。砂芯抽滤除去溶剂，加入三倍摩尔过量Fmoc-L-Thr(tBu)-OH，再加入8倍摩尔过量的DIEA,最后加入DMF溶解，振荡30分钟。甲醇封头30分钟。
(2)除去溶剂DMF，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液。取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(3)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后，向反应管中加入用尽量少的DMF溶解的Fmoc-L-Ala-OH；两倍过量，HBTU两倍过量。之后，立刻加入8倍过量的DIEA，反应30分钟。
(4)抽掉溶液后，取少量树脂，用乙醇清洗三次后，加入茚三酮试剂，105～110℃加热5分钟，无色为阴性反应，即反应完全。
(5)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后除去溶剂，加20％哌啶/DMF溶液(10mL g -1)，5分钟后除去溶剂，再加入20％哌啶/DMF溶液(10mL g -1)，15分钟后除去哌啶溶液，取少量树脂，用乙醇清洗后，加入茚三酮试剂，105～110℃加热5分钟，变深蓝色为阳性反应。
(6)将上述反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DMF(15mL g -1，两次)清洗后抽干树脂。
(7)重复操作(3)～(5)，链接氨基酸Fmoc-L-Ala-OH。反应得到产物用依次用DMF(15mL g -1，两次)、甲醇(15mL g -1，两次)以及DCM(15mL g -1，两次)清洗后抽干树脂。
(8)使用切割液(15mL g -1，TFA：水：EDT：Tis＝95:1:2:2,V/V)将产物切割90分钟。并将切割液用氮气吹干，之后冻干，得到多肽粗品。
(9)用HPLC纯化多肽并转盐或脱盐，HPLC：tR＝7.9mins(纯化柱子型号：Kromasil100-5C18,4.6mm*250mm；梯度洗脱液：0.1％TFA乙腈溶液溶液乙腈溶液和0.1％TFA水溶液水溶液,0mins-1:99，20mins-1:9)。将纯化后的溶液冻干，既得到成品L-Ala-L-Ala-L-Thr(AAT)。产率约为70％。质谱鉴定260.1为[M-8H]+。
【实施例7】式(6)、式(7)和式(8)化合物的合成
制备式(6)所示的化合物：
(1)通过固相合成的方法制备GDL-L-Thr。
(2)HPLC纯化，HPLC：tR＝3.4mins(纯化柱子型号：SHIMADZU Intertsil ODS-SP(4.6mm*250mm*5μm)；梯度洗脱液：0.1％TFA乙腈溶液和0.1％TFA水溶液,0.01-20mins-1:99，20-30mins-21:79,30-40mins-100:0,40-50mins-1:99),产率约为50％，质谱鉴定296.099为[M-H]+。
固相合成方法制备的GDL-L-Thr纯度更高，更利于产物分离，实验结果表明，固相合成制备的GDL-L-Thr纯度更高，且保持良好的抑制冰晶生长能力(图13)。
分子式(7)、(8)所示的化合物皆可用固相合成方法得到。
【实施例8】式(9)化合物的合成
(1)将Dichlorodimethylsilane的DCM溶液倒入多肽合成管中，静至30分钟后将合成管晾干备用。
(2)取用100mg树脂于合成管中，加入2mL的DMF，通氮气，溶胀树脂10分钟后抽滤。
(3)加入1mL的4-甲基哌啶/DMF溶液去保护，5分钟后除去，再加1mL的4-甲基哌啶/DMF溶液，15分钟后除去，鼓泡，抽滤。
(4)DMF冲洗5次，鼓泡，抽滤。
(5)依次加入2M,0.5mL的溴乙酸/DMF溶液，N,N-二异丙基碳二亚胺/DMF溶液,鼓泡20分钟，抽滤，DMF冲洗3次。
(6)加入1M,1mL的伯胺/DMF溶液，鼓泡30分钟，DMF冲洗，二氯甲烷冲洗(x3)。
(7)重复步骤5,6，直至所需的分子量。
(8)加入4mL的裂解液，充分摇匀，通氮气吹干，最后冷冻干燥，纯化处理，得到最终成品。
R为-CH 3，-CH 2CH 3以及-CH 2CH 2COOH的类肽。质谱鉴定444.6是R为-CH 3的[M+H] +、528.8是R为-CH 2CH 3的[M+H] +、792.1是R为-CH 2CH2COOH的[M+H] +。
Figure PCTCN2020077472-appb-000007
【冰晶重结晶抑制实验】
冰晶重结晶抑制(IRI)活性采用“溅射冷冻法”，将样品溶解分散到DPBS溶液中，将10～30μL上述溶液在1.0m以上的高度滴加到-60℃预冷的干净硅片表面，利用冷热台以10℃/min的速度缓慢升温到-6℃，并在该温度下退火30min，利用偏光显微镜以及高速摄像机观察记录冰晶的大小，冷热台密封，保证内部的湿度在50％左右。每一个样品重复至少三次，使用Nano Measurer 1.2统计冰晶的尺寸，统计结果的误差为标准偏差。
冰晶形貌(DIS)观察和热滞后(Thermal Hysteresis，TH)测量采用纳升渗透压仪，首先用酒精灯外焰熔化毛细管，并同时拉伸产生极细孔径毛细管，将毛细管与微量进样器相连。将黏度较高的浸镜油注入微米孔径圆片中，利用微量进样器将溶解了材料的水溶液注入微孔中。通过控制温度，使液滴快速结冰，并缓慢升温以获得单晶冰，以0.01℃的精度缓慢降温，利用配备了高速摄像机的显微镜观察冰晶形貌以及TH测试。
对上述实施例3中制备的TR的DPBS溶液20μL利用“溅射冷冻法”进行IRI活性测试。测得的相对于DPBS的最大冰晶尺寸(％)如图17所示。通过化学键结合的TR的最大冰晶尺寸明显小于相同浓度下精氨酸的DPBS溶液和苏氨酸的DPBS溶液的最大冰晶尺寸。
取上述实施例3中制备的TR的去离子水溶液，利用纳升进行冰晶形貌观察发现，TR有微弱的修饰冰晶形貌的效果(过冷度-0.1℃,-0.4～0.01℃)，如图18所示。且并未测得热滞后。
对上述实施例7中制备的GDL-L-Thr、GDL-L-Ser和GDL-L-Val的DPBS溶液20μL利用“溅射冷冻法”进行IRI活性测试。测得的相对于DPBS的最大冰晶尺寸(％)如图13、15-16所示。通过化学键结合的GDL-L-Thr、GDL-L-Ser和GDL-L-Val的最大冰晶尺寸明显小于相同浓度下GDL的DPBS溶液和氨基酸的DPBS溶液的最大冰晶尺寸，且小于同浓度GDL和氨基酸混合的DPBS溶液的最大冰晶尺寸。
取上述实施例7中制备的GDL-L-Thr的去离子水溶液，利用纳升进行冰晶形貌观察发现，GDL-L-Thr有微弱的修饰冰晶形貌的效果(过冷度-0.1℃,-0.4～0.01℃)，如图14所示。且并未测得热滞后。
对上述实施例8中制备的化合物的DPBS溶液20μL利用“溅射冷冻法”进行IRI活性测试。测得的相对于DPBS的最大冰晶尺寸(％)如图19所示。
取上述实施例8中制备的三种类肽的去离子水溶液，利用纳升进行冰晶形貌观察发现R为-CH 3以及-CH 2CH 3的类肽有较为明显的修饰冰晶形貌的效果，R为-CH 2CH 2COOH的类肽无修饰冰晶形貌的效果(过冷度-0.1℃,-0.4～0.01℃)所得形貌如图20所示，且三种类肽皆未并未测得热滞后。
上述结果表明，所制备的肽类化合物具有抑制冰晶生长的活性，修饰冰晶形貌的效果，其中R为-CH 3，-CH 2CH 3的式(9)化合物修饰冰晶形貌的效果优异，且没有热滞后，能实现控制冰晶生长的作用，可以用于冷冻保存液。
B.控冰材料控冰性能评估与筛选
控冰材料在冰表面的吸附量＝(冰表面所吸附的控冰材料质量m 1/含有控冰材料的原溶液中控冰材 料的总质量m 2)×100％。
在一个实施方案中，所述冰吸附实验包括以下步骤：
S1:取质量为m2的控冰材料，配制所述控冰材料的水溶液，降温至过冷温度；
S2:将预冷的控温棒置于所述水溶液中诱导冰层在控温棒表面生长，持续搅拌水溶液，以待控冰材料逐渐吸附于冰层表面，保持水溶液、控温棒温度在过冷温度；
S3:测定控冰材料在冰表面的吸附量。
采用图21所示的装置进行冰吸附实验，包括多层储液腔、控温棒和温度控制器，所述多层储液腔由里到外依次包括冰吸附腔、温浴腔、冷却液储存腔，所述控温棒置于冰吸附腔内，所述控温棒和储液腔的温度由温度控制器控制。所述控温棒为导热材料制成的中空结构，所述控温棒的中空结构设置有进液口和排液口；所述温度控制器为流体温控器，所述温度控制器设置有冷却液流出端和回流端；所述冷却液储存腔两端设置有进液口和排液口；所述温度控制器的冷却液流出端、控温棒的进液口、控温棒的排液口、冷却液储存箱的进液口、冷却液储存箱的排液口以及温度控制器的回流端依次经管道连通，所述管道内流动冷却液。多层储液腔设有盖子。使用时，所述冰吸附腔内盛放控冰材料的水溶液，中层温浴腔装有预定温度的温浴介质例如水浴、冰浴或者油浴等；冷却液温度达到设定温度后，经温度控制器流出，流入中空的控温棒中空结构，对控温棒温度实现控制，随后从控温棒排液口流出，再流入外层冷却液储存腔保持温浴介质的温度在预定水平，再经冷却液储存箱的排液口流经温度控制器的回流端进入温度控制器循环。
【实施例9】
a-PVA:分子量约为13～23kDa，间同规整度r(diad syndiotacticity)约为55％(Sigma-Aldrich)；
i-PVA:分子量约为14～26kDa，全同规整度m(isotacticity)约为84％。
(1)测定两种PVA在冰水界面的铺展性能
采用冰吸附实验测定PVA在冰表面的吸附量，实验装置如图21所示。
a.将a-PVA和i-PVA用FITC Isomer I荧光标记。
b.将FITC标记的不同浓度PVA水溶液(40mL)置于烧杯中，将烧杯置于循环冷槽中并将溶液温度以及控温棒冷却到-0.1℃。
c.将控温棒插入到事先冷却的FITC标记的PVA水溶液中之前，先将控温棒插入液氮中预冷1.0分钟。之后，再快速的将控温棒插入已经预冷的FITC标记的PVA水溶液里，以便在控温棒表面诱导一层极薄的冰层以诱导冰的生长。
d.FITC标记的PVA水溶液在过冷温度-0.1℃下持续磁力搅拌1.0小时，以待PVA逐渐吸附到冰的表面。所有吸附实验保持过冷度以及吸附时间不变以此确保所得冰的表面积在误差允许范围内几乎不变。
e.将形成的冰块从溶液中取出，并用纯水淋洗表面以除去附着在表面的溶液。将冰块融化。
f.PVA在冰表面的吸附量由冰块中溶质PVA的质量比原始溶液中溶质PVA的质量获得，PVA溶液的浓度由紫外可见分光光度法确定，体积由移液枪以及量筒确定。
冰吸附实验显示各浓度的a-PVA和i-PVA吸附量如图22所示，a-PVA在冰表面的吸附量由0.2mg mL -1浓度时的16.3％增加到1.0mg mL -1的28.7％，并且在浓度大于1.0mg mL -1后，a-PVA在冰表面的吸附量达到饱和，饱和时吸附量约为36.5％。i-PVA在浓度小于1.0mg mL -1时冰吸附量为0％～19.3％，低于同浓度下a-PVA在冰表面的吸附量。在低浓度时，两种PVA对冰的吸附皆未达到饱和，i-PVA覆盖的冰表面积低于a-PVA。
当i-PVA浓度≥1.2mg mL -1吸附在冰面上的量高于a-PVA，并在2.0mg mL -1时i-PVA在冰面上的吸附量达到饱和，饱和时的吸附量为56.5％。进一步说明，两种PVA对相同大小的冰表面吸附覆盖达到饱和时，所需i-PVA的量远大于a-PVA。也就是说a-PVA可以更有效的覆盖冰的表面。
(2)冰晶重结晶抑制(IRI)活性测量
冰晶重结晶抑制(IRI)活性采用“溅射冷冻法”，将上述两种PVA分别溶解分散到DPBS溶液中，将10～30μL所述溶液在1.0m以上的高度滴加到-60℃预冷的干净硅片表面，利用冷热台以10℃min -1的速度缓慢升温到-6℃，并在该温度下退火30min，利用偏光显微镜以及高速摄像机观察记录 冰晶的大小，冷热台密封，保证内部的湿度在50％左右。每一个样品重复至少三次，使用Nano Measurer 1.2统计冰晶的尺寸，结果的误差为标准偏差。
结果如图23所示，a-PVA冰晶尺寸明显小于同浓度时i-PVA的冰晶尺寸，说明a-PVA抑制冰晶生长的能力远远优于i-PVA。
根据实施例9的结果可以看出，i-PVA与水的亲和性较a-PVA弱。因此，i-PVA在水溶液以及冰水界面倾向于以聚集状态存在，而a-PVA在水溶液以及冰水界面可以很好的铺展。两种PVA对相同大小的冰表面吸附覆盖达到饱和时，所需i-PVA的量远高于a-PVA。因此，a-PVA与i-PVA相比，是更好的控冰材料，较低浓度即能起到更好的抑制冰晶生长的效果。
C.冷冻保存液配方和制备、应用实例
【实施例10】制备含PVA为控冰材料的冷冻保存液
1.制备冷冻保存液：按以下配方配制冷冻保存液
冷冻保存液A:每100mL中含有如下组分：
物质 含量
PVA(g) 2.0
乙二醇(mL) 10
DMSO(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于25mL的DPBS中，待PVA全部溶解并冷却到室温后调节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、10mL的DMSO，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值并定容补齐余量至总体积的80％，20mL血清单独存放待保存液使用时添加。
冷冻保存液B：每100ml中含有如下组分：
物质 含量
L-Arg(g) 8.0
L-Thr(g) 4.0
PVA(g) 2.0
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：将2.0g的PVA在80℃水浴中加热并磁力搅拌溶于20mL的DPBS中，调节pH为7.1，为溶液1；将8.0g的L-Arg和4.0g的L-Thr溶于20mL的DPBS中，调节pH为7.1，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)超声溶解于20mL的DPBS中，待蔗糖全部溶解后加入10mL的乙二醇，为溶液3；待溶液1、溶液2及溶液3恢复至室温，再将三种溶液混均，调节pH值并定容补齐余量至总体积的80％，使用时加入20mL的血清。
冷冻保存液C:每100ml中含有以下组分：
物质 用量
PVA(g) 2.0
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：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6.9，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值并定容补齐余量至总体积的80％，20mL血清单独存放待保存液使用时添加。
冷冻保存液C1：每100ml中含有以下组分：
物质 用量
PVA(g) 1.0
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
血清(mL) 20
DPBS(ml) 余量
配液步骤同冷冻保存液C。
冷冻保存液D：每100ml中含有以下组分：
物质 用量
PVA(g) 2.0
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
DPBS(mL) 余量
配液步骤：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值并定容补齐余量至总体积100mL，备用。
冷冻保存液E:每100ml中含有以下组分：
物质 用量
聚-L-脯氨酸(g) 1.5
PVA(g) 2.0
乙二醇(ml) 10
蔗糖(mol L -1) 0.5
DPBS(ml) 余量
配液步骤：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(聚合度15)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3种溶液混均，调节pH值并定容补齐余量至总体积100mL，备用。
冷冻保存液F:每100ml中含有以下组分：
物质 用量
聚-L-精氨酸(g,聚合度为8) 4.0
PVA(g) 1.0
乙二醇(ml) 10
蔗糖(mol L -1) 0.5
血清(ml) 20
DPBS(ml) 余量
配液步骤同冷冻保存液E，血清在使用时加入。
2.制备冷冻平衡液：按以下配方配制冷冻平衡液
冷冻平衡液a：将7.5mL的乙二醇、7.5mL的DMSO加入65mL的DPBS中，混匀，使用时加入20m L的血清。
冷冻平衡液b：将7.5mL的乙二醇溶于72.5mL的DPBS中，混匀，使用时加20mL的血清。
冷冻平衡液c: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值并定容补齐余量至100mL，备用。
对比例：
冷冻平衡液a：每1mL中含有7.5％(v/v)的DMSO,7.5％(v/v)的乙二醇，20％(v/v)的胎牛血清，余量为DPBS；
冷冻保存液1#：每1mL中含有15％(v/v)的DMSO,15％(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2#：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；
冷冻保存液2#：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。
冷冻保存液3#：每1mL中含有10％(v/v)的DMSO，15％(v/v)的胎牛血清，余量为培养基a-MEM(USA,Invitrogen,C12571500BT)
实施例10和对比例采用的解冻液配方有如下三种：
解冻液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(20％的血清，余量为DPBS)。
解冻液2#：解冻液Ⅰ(含有1.0mol L -1的蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅱ(含有0.5mol L -1蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅲ(含有0.25mol L -1蔗糖，20mg mL -1的PVA，余量为DPBS)；解冻液Ⅳ(20mg mL -1的PVA，余量为DPBS)。
解冻液3#：解冻液Ⅰ(含有1.0mol L -1的蔗糖，20mg mL -1的PVA，10mg mL -1的聚脯氨酸，余量为DPBS)；解冻液Ⅱ(含有0.5mol L -1蔗糖，20mg mL -1的PVA，5.0mg mL -1的聚脯氨酸，余量为DPBS)；解冻液Ⅲ(含有0.25mol L -1蔗糖，20mg mL -1的PVA，2.5mg mL -1的聚脯氨酸，余量为DPBS)；解冻液Ⅳ(20mg mL -1的PVA，余量为DPBS)。
【应用例1】卵母细胞和胚胎冷冻保存采用上述实施例及对比例的冷冻平衡液以及冷冻保存液按表1和表2中的方案分别进行卵母细胞和胚胎冷冻保存。本发明实施例中存活率均为3-12次重复实验的存活率平均值。
1.卵母细胞冷冻保存
小鼠卵母细胞先置于冷冻平衡液中平衡5分钟；然后置于所制备冷冻保存液中1分钟，将已在冷冻保存液中平衡后的卵母细胞放置于冷冻载杆上，然后快速投入液氮中(-196℃)中，并封闭载杆后继续保存；解冻时，将冻存的卵母细胞置于37℃的解冻液Ⅰ中平衡5分钟，再依次在解冻液Ⅱ-Ⅳ中各平衡3分钟；将解冻完毕的卵母细胞培养2小时后观察存活细胞数量，计算存活率(参见表1)。
2.胚胎冷冻保存
小鼠胚胎先置于冷冻平衡液平衡5分钟，然后置于上述实施例或对比例中的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；解冻时，将胚胎置于37℃的解冻液Ⅰ中平衡3分钟，再依次在解冻液Ⅱ-Ⅳ中各平衡3分钟；将解冻完毕胚胎培养2小时，观察存活胚胎数量，计算存活率(参见表2)。
表1小鼠卵母细胞冷冻保存存活率
编号 平衡液 冷冻保存液 解冻液 冻卵总数 2小时后存活率
应用实例1 a A 解冻液1# 67 100.0％
应用实例2 b B 解冻液1# 109 94.8％
应用实例3 b C 解冻液1# 90 97.7％
应用实例4 c D 解冻液1# 50 93.4％
应用实例5 c D 解冻液2# 53 96.5％
应用实例6 c E 解冻液1# 39 89.7％
应用实例7 c E 解冻液3# 60 98.6％
对比实例1 a 冷冻液1# 解冻液1# 146 95.0％
对比实例2 平衡液2# 冷冻液2# 解冻液1# 96 81.9％
对比实例3 平衡液2# 冷冻液2# 解冻液2# 44 94.7％
表2小鼠胚胎冷冻保存存活率
编号 平衡液 冷冻保存液 解冻液 冻胚胎总数 2小时后存活率
应用实例8 c D 解冻液1# 41 95.8％
应用实例9 c E 解冻液1# 42 95.2％
对比实例4 a 冷冻液1# 解冻液1# 38 94.3％
对比实例5 平衡液2# 冷冻液2# 解冻液1# 39 82.4％
以上数据表明该冷冻保存液可以达到90％以上甚至100％的存活率，能达到或远远超过目前临床普遍使用的含有15％DMSO的商业化冷冻保存液的冷冻保存复苏率，应用实例1(含有10％的DMSO)和对比实例2(含有7.5％的DMSO)以及对比实例1，即商业化的卵母细胞冻存液(含有15％的DMSO)的对比可以看出，PVA的加入显著提高了卵母细胞的存活率；应用实例2-3也表明，本发明少加DMSO或者不含有DMSO的冷冻保存液，也能达到较高的卵母细胞或胚胎存活率解决了目前临床普遍使用的商业化冷冻保存液DMSO浓度高，对细胞损害大的问题；并且，应用实例5、7-9表明，在冷冻液，平衡液以及解冻液中均不添加DMSO和血清的情况下，也可实现较高的卵母细胞或胚胎存活率。既无DMSO也无血清的冷冻保存液，解决了目前临床普遍使用的商业化冷冻保存液因含有血清而造成的保质期短、可带入寄生性生物污染物质等问题。
【应用例2】人脐带充间质干细胞冷冻保存
采用上述实施例及对比例的冷冻保存液按表3中的方案分别进行人脐带充间质干细胞的冷冻保存。
微滴法冷冻保存人脐带充间质干细胞：将培养皿上的人脐带间充质干细胞用25％胰酶消化2分钟后，放入等体积培养液(10％FBS+a-MEM培养基)，轻柔吹打至干细胞全部脱落，加入1.5mL离心管，1000rmp离心5分钟，弃上清(将细胞与培养基分离)，将10uL冷冻液加入离心管底部，轻柔吹打使干细胞团分散，将此10uL带有干细胞的冷冻液置于冷冻载片上，置于液氮(-196℃)冻存。解冻时，将带有细胞及冷冻液的冷冻载杆直接放入37℃培养基中进行解冻。解冻后，台盼蓝染色察看其存活率，并使用仪器JIMBIO-FIL计数细胞数量，存活率＝活细胞数/细胞总数(参见表3)。
表3人脐带充间质干细胞冷冻保存存活率
编号 冷冻保存液 冷冻保存方法 存活率
应用实例10 C1 微滴法 72.2％
应用实例11 D 微滴法 77.1％
应用实例12 F 微滴法 92.4％
对比实例6 冷冻液1# 微滴法 63.9％
对比实例7 冷冻液3# 微滴法 76.6％
本发明的冷冻保存液进行人脐带充间质干细胞冷冻保存时，即使不使用DMSO干细胞存活率可达92.4％和72.2％(应用实例12和10)，甚至在完全不添加DMSO和血清时，存活率可达到77.1％，达到现有冷冻试剂的存活率水平，表明该冷冻用试剂不仅能达到常规冷冻液冷冻干细胞的有效性，达到或甚至远高于目前普遍使用的含有10％的DMSO的冷冻保存液(对比实例7)的冷冻保存复苏率，基于PVA的冷冻保存效果显著优于不添加PVA的对比实例6。
【应用例3】卵巢器官与卵巢组织冻存
采用上述实施例及对比例的冷冻平衡液以及冷冻保存液按表4、表5中的方案分别对新生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。
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％CO 2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24-33所示，图24为新鲜未冷冻的卵巢器官切片照片，图29为新鲜未冷冻的卵巢组织切片照片。
表4卵巢器官冷冻保存方案
编号 平衡液 冷冻保存液 解冻液 形态
应用实例13 c D 解冻液2# 图26
应用实例14 b C1 解冻液1# 图27
应用实例15 b F 解冻液1# 图28
对比实例8 a 冷冻液1# 解冻液1# 图25
表5卵巢组织冷冻保存方案
编号 平衡液 冷冻保存液 解冻液 形态
应用实例16 c D 解冻液2# 图31
应用实例17 b C1 解冻液1# 图32
应用实例18 b F 解冻液1# 图33
对比实例9 a 冷冻液1# 解冻液1# 图30
根据图24-图28可知，与使用不添加聚乙烯醇的对比实例8以及新鲜未冷冻的卵巢器官相比，应用实例13-15中原始卵泡结构相对完整，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。可见，应用实例13-15组对于卵巢器官的冻存效果更好。
根据图29-图33可知，应用实例16-18的方案和对比实例9以及新鲜未冷冻的卵巢组织相比，窦卵泡结构相对完整，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，可见本发明的冻存液用于冻存卵巢组织也比现有技术具有更好的效果。
由此可见，本发明以PVA类仿生控冰材料为主要成分制备的冷冻保存液具有良好的抑制冰晶生长的效果，可以减少保存体系中DMSO的用量，甚至不加DMSO，可保持良好的生物相容性，并且可以同时适用于卵母细胞、胚胎、干细胞、生殖器官和组织的冷冻保存，均可达到良好的细胞存活率和生物活性。
【实施例11】制备氨基酸类控冰材料的冷冻保存液
冷冻保存液G:每100ml中含有以下组分:
物质 含量
L-Arg(g) 16.0
L-Thr(g) 8.0
DMSO(mL) 10
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：总体积100mL：将16g的L-Arg与8g的L-Thr溶于25mL的DPBS中，调节pH为6.9，为溶液1；将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，待蔗糖全部溶解后依次加入10mL的乙二醇、10mL的DMSO，为溶液2，待溶液1及溶液2恢复至室温，再将2种溶液混均，调节pH至6.9，并用DPBS定容补齐余量至总体积的80％，20mL的胎牛血清单独存放，在冷冻保存液使用前加入。
冷冻保存液H:每100ml中含有以下组分:
物质 含量
聚-L-脯氨酸(g，聚合度15) 1.5
DMSO(mL) 10
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：将1.5g的聚-L-脯氨酸(聚合度为15)超声溶解于25mL的DPBS中，调节pH为6.8，为溶液1；将17g(0.05mol)的蔗糖超声溶解于25mL的DPBS中，待蔗糖全部溶解后依次加入10mL的乙二醇、10mL的DMSO，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至7.0，并用DPBS定容补齐余量至总体积的80％，20mL的血清单独存放，在冷冻保存液使用前加入。
冷冻保存液I：每100ml中含有以下组分:
物质 含量
聚-L-精氨酸(g，聚合度8) 1.5
DMSO(mL) 10
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：总体积100mL，将1.5g的聚-L-精氨酸(聚合度为8)超声溶解于25mL的DPBS中，调节pH为7.0，为溶液1；将17g(0.05mol)的蔗糖超声溶解于20mL的DPBS中，待蔗糖全部溶解后依次加入10mL的乙二醇、10mL的DMSO，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至7.0，并用DPBS定容补齐余量至总体积的80％，20mL的血清单独存放，在冷冻保存液使用前加入。
冷冻保存液J：每100ml中含有以下组分:
物质 含量
聚-L-精氨酸(g，聚合度8) 4.0
DMSO(mL) 7.5
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤同冷冻保存液I。
冷冻保存液K：每100ml中含有以下组分:
物质 含量
聚-L-脯氨酸(g，聚合度8) 4.0
DMSO(mL) 7.5
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤同冷冻保存液I。
冷冻保存液L：每100ml中含有以下组分:
物质 含量
L-Arg(g) 16.0
L-Thr(g) 8.0
DMSO(mL) 7.5
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤同冷冻保存液G。
制备冷冻平衡液：按以下配方配制冷冻平衡液
冷冻平衡液a：将7.5mL的乙二醇、7.5mL的DMSO加入65mL的DPBS中，混匀，使用时加入20m L的血清。
冷冻平衡液b：7.5mL的乙二醇加入72.5mL的DPBS中，混匀，使用时加入20m L的血清。
对比例2：
冷冻平衡液a：每1mL中含有7.5％(v/v)的DMSO,7.5％(v/v)的乙二醇，20％(v/v)的胎牛血清，余量为DPBS；
冷冻保存液1#：每1mL中含有15％(v/v)的DMSO,15％(v/v)的乙二醇，20％(v/v)的胎牛血清，0.5M蔗糖，余量为DPBS。
冷冻保存液3#：每1mL中含有10％(v/v)的DMSO，15％(v/v)的胎牛血清，余量为培养基a-MEM(USA,Invitrogen,C12571500BT)
实施例11和对比例2采用的解冻液配方有如下一种：
解冻液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(20％的血清，余量为DPBS)。
【应用例4】卵母细胞和胚胎冷冻保存
采用上述实施例11及对比例2的冷冻平衡液以及冷冻保存液按表6和表7中的方案分别进行卵母细胞和胚胎冷冻保存。冷冻和解冻方法与应用例1相同。
表6小鼠卵母细胞冷冻保存存活率
编号 平衡液 冷冻保存液 解冻液 冻卵总数(颗) 2小时后存活率
应用实例19 a G 解冻液1# 67 98.5％
应用实例20 a H 解冻液1# 109 96.3％
应用实例21 a I 解冻液1# 67 95.5％
对比实例10 a 冷冻液1# 解冻液1# 146 95.0％
表7小鼠胚胎冷冻保存存活率
编号 平衡液 冷冻保存液 解冻液 冻胚胎总数(颗) 2小时后存活率
应用实例22 a J 解冻液1# 25 100.00％
对比实例11 a 冷冻液1# 解冻液1# 38 94.30％
由表6和表7的数据可以看出，本发明的冷冻保存液在降低DMSO和EG用量的后进行卵母细胞和胚胎冷冻保存时，卵母细胞存活率可达95％以上，胚胎存活率可以达到存活率100％，能达到甚至远高于目前临床普遍使用的含有15％DMSO的商业化冷冻保存液(对比实例10-11)的冷冻保存复苏率，且添加了氨基酸仿生控冰材料的冷冻保存效果显著优于未添加仿生控冰材料的冷冻保存液。
【应用例5】人脐带充间质干细胞的冷冻保存
采用上述实施例11及对比例2的冷冻保存液按表8中的方案分别进行人脐带充间质干细胞的冷冻保存。冷冻和解冻方法参见应用例2。
表8人脐带充间质干细胞冷冻保存存活率
编号 冷冻保存液 冷冻保存方法 存活率
应用实例23 J 微滴法 81.2％
应用实例24 K 微滴法 82.6％
应用实例25 L 微滴法 80.5％
对比实例12 冷冻液1# 微滴法 63.9％
对比实例13 冷冻液3# 微滴法 76.6％
本发明的冷冻保存液进行人脐带充间质干细胞冷冻保存时，即使不使用或仅使用少量DMSO(7.5％)干细胞存活率可达80％以上(例如应用实例23-25)，表明该冷冻用试剂不仅能达到常规冷冻液冷冻干细胞的有效性，甚至远高于目前普遍使用的含有10％的DMSO的冷冻保存液(对比实例13)的冷冻保存复苏率，添加了氨基酸仿生控冰材料的冷冻保存效果显著优于不添加氨基酸类控冰材料的对比实例14、15。
【应用例6】卵巢器管和卵巢组织的冷冻保存
采用上述实施例11及对比例2的冷冻平衡液以及冷冻保存液按表9、表10中的方案分别对新生3天内的小鼠完整的卵巢器官和性成熟小鼠的卵巢组织切片进行冷冻保存。卵巢器官和性成熟小鼠的卵巢组织的冷冻和解冻方法参见应用例3。
表9卵巢器官冷冻保存方案
编号 平衡液 冷冻保存液 解冻液 形态
应用实例26 a J 解冻液1# 图34
应用实例27 a L 解冻液1# 图35
应用实例28 a K 解冻液1# 图36
对比实例14 a 冷冻液1# 解冻液1# 图25
表10卵巢组织冷冻保存方案
编号 平衡液 冷冻保存液 解冻液 形态
应用实例29 a J 解冻液1# 图37
应用实例30 a L 解冻液1# 图38
应用实例31 a K 解冻液1# 图39
对比实例15 a 冷冻液1# 解冻液1# 图30
【实施例11】制备肽类化合物控冰材料的冷冻保存液
冷冻保存液M:每100ml中含有以下组分:
物质 含量
TR(g) 28
DMSO(mL) 7.5
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：总体积100mL，将28g TR超声溶于25mLDPBS中，调节pH为7.0，为溶液1；将0.05mol蔗糖超声溶解于25mL DPBS中，待蔗糖全部溶解后依次加入10mL乙二醇、7.5mLDMSO，为溶液2，待溶液1及溶液2恢复至室温，再将两种溶液混匀，调节pH并采用DPBS定容至总体积的80％，最后，使用前加20mL血清。
冷冻保存液N：每100ml中含有以下组分:
物质 含量
TPT(g) 28
DMSO(mL) 7.5
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：总体积100mL，将28gTPT超声溶于25mLDPBS中，调节pH为7.0，为溶液1；将0.05mol蔗糖超声溶解于25mLDPBS中，待蔗糖全部溶解后依次加入10mL乙二醇、7.5mLDMSO，为溶液2，待溶液1及溶液2恢复至室温，再将两种溶液混匀，调节pH并采用DPBS定容至总体积的80％，最后，使用前加20mL血清。
冷冻保存液O：每100ml中含有以下组分:
物质 含量
TR(g) 28
乙二醇(mL) 10
蔗糖(mol L -1) 0.5
胎牛血清(mL) 20
DPBS(mL) 余量
配液步骤：体积100mL，将28g TR超声溶于25mLDPBS中，调节pH为7.0，为溶液1；将0.05mol蔗糖超声溶解于25mL DPBS中，待蔗糖全部溶解后依次加入10mL乙二醇，为溶液2，待溶液1及溶液2恢复至室温，再将两种溶液混匀，调节pH并采用DPBS定容至总体积的80％，最后，使用前加20mL血清。
制备冷冻平衡液：按以下配方配制冷冻平衡液
冷冻平衡液a：将7.5mL的乙二醇、7.5mL的DMSO加入65mL的DPBS中，混匀，使用时加入20m L的血清。
对比例3：
冷冻平衡液a：每1mL中含有7.5％(v/v)的DMSO,7.5％(v/v)的乙二醇，20％(v/v)的胎牛血清，余量为DPBS；
冷冻保存液1#：每1mL中含有15％(v/v)的DMSO,15％(v/v)的乙二醇，20％(v/v)的胎牛血清，0.5M蔗糖，余量为DPBS。
冷冻保存液3#：每1mL中含有10％(v/v)的DMSO，15％(v/v)的胎牛血清，余量为培养基a-MEM(USA,Invitrogen,C12571500BT)
实施例12和对比例3采用的解冻液配方有如下一种：
解冻液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(20％的血清，余量为DPBS)。
【应用例7】卵母细胞和胚胎冷冻保存
采用上述实施例13及对比例2的冷冻平衡液以及冷冻保存液按表11和表12中的方案分别进行卵母细胞和胚胎冷冻保存。冷冻和解冻方法与应用例1相同。
表11小鼠卵母细胞冷冻保存存活率
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32 a M 解冻液1# 93 96.2％
应用实例33 a N 解冻液1# 48 90％
对比实例16 a 冷冻液1# 解冻液1# 146 95％
表12小鼠胚胎冷冻保存存活率
编号 平衡液 冷冻液 解冻液 胚胎总数 2小时后存活率
应用实例34 a M 解冻液1# 41 95.9％
对比实例17 a 冷冻液1# 解冻液1# 38 94.3％
表11和表12的数据表明，本发明多肽用于卵母细胞和胚胎冷冻保存，仅添加少量DMSO(7.5％)就能达到现有商业化冻存液(DMSO含量15％)的卵母细胞和胚胎存活率，并且应用实例32和应用实例34的数据表明，TR多肽用于卵母细胞和胚胎冻存具有更加优异的效果。
【应用例8】人脐带充间质干细胞的冷冻保存
采用上述实施例12及对比例3的冷冻保存液按表13中的方案分别进行人脐带充间质干细胞的冷冻保存。冷冻和解冻方法参见应用例2。
表13人脐带充间质干细胞冷冻保存存活率
编号 冷冻保存液 冷冻保存方法 存活率
应用实例35 M 微滴法 87.8％
应用实例36 O 微滴法 75.1％
对比实例18 冷冻液3# 微滴法 76.6％
根据表13的结果可以看出，本发明的冷冻保存液不加入DMSO或仅加入少量DMSO(7.5％)时，就可以达到与现有技术中加入10％DMSO的冷冻保存液相当水平的细胞存活率，大大减少了DMSO的用量，减少了DMSO对于细胞的损伤和毒性，可以大大提高冷冻后干细胞的传代稳定性和细胞活性。
【应用例9】卵巢器管和卵巢组织的冷冻保存
采用上述实施例12及对比例3的冷冻平衡液以及冷冻保存液按表14、表15中的方案分别对新生3天内的小鼠完整的卵巢器官和性成熟小鼠的卵巢组织切片进行冷冻保存。卵巢器官和性成熟小鼠的卵巢组织的冷冻和解冻方法参见应用例3。
表14卵巢器官冷冻保存方案
编号 平衡液 冷冻保存液 解冻液 形态
应用实例37 a M 解冻液1# 图40
对比实例19 a 冷冻液1# 解冻液1# 图25
表15卵巢组织冷冻保存方案
编号 平衡液 冷冻保存液 解冻液 形态
应用实例38 a M 解冻液1# 图41
对比实例20 a 冷冻液1# 解冻液1# 图30
根据图24、25和图40可知，与使用不添加肽类仿生控冰材料的对比实例相比(图25、图30)，采用应用实例37冷冻保存液冻存的卵巢器官解冻后的切片照片中显示卵泡结构相对完整，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。可见，应用实例37对于卵巢器官的冻存效果更好。
根据图29、图30和图41可知，应用实例38的方案和对比实例22新鲜未冷冻的成年小鼠卵巢组织相比，生长期卵泡及窦卵泡结构相对完整，可见本发明的冻存液用于冻存卵巢组织也比现有技术具有更好的效果。
由此可见，本发明以肽类仿生控冰材料为主要成分制备的冷冻保存液可以同时适用于卵母细胞、胚胎、干细胞、生殖器官和组织的冷冻保存，均可达到良好的细胞存活率和生物活性。
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 (46)

  1. 一种控冰材料的分子设计方法，其特征在于，包括如下步骤：
    (1)构建化合物分子结构库，所述化合物分子含有亲水基团和亲冰基团；
    (2)采用分子动力学模拟评估各化合物分子在冰水两相界面的铺展性能；
    (3)筛选出所需冰亲和性和水亲和性的化合物分子。
  2. 根据权利要求1所述的分子设计方法，其特征在于，所述步骤(2)的MD模拟通过GROMACS、AMBER、CHARMM、NAMD、或LAMMPS进行；
    优选地，所述步骤(2)MD模拟中，水分子模型选自TIP3P、TIP4P、TIP4P/2005、SPC、TIP3P、TIP5P、或SPC/E，优选TIP4P/2005水分子模型；
    优选地，所述步骤(2)MD模拟中，所述力场参数由GROMOS、ESFF、MM形态力场、AMBER、CHARMM、COMPASS、UFF、CVFF等力场中的一种提供。
  3. 根据权利要求1或2任一项所述的分子设计方法，其特征在于，所述步骤(2)MD模拟中，模拟计算化合物分子之间的相互作用，化合物分子与水分子的相互作用，化合物分子与冰－水分子的相互作用；例如所述作用包括是否形成氢键、范德华尔斯作用、静电作用、疏水作用、π-π作用等。
  4. 根据权利要求1-3任一项所述的分子设计方法，其特征在于，所述步骤(2)MD模拟中，所述模拟计算分子相互作用时，对温度和压强进行调节；优选地，采用V-rescale温度控制器和压强控制器调节温度和压强；
    优选地，所述步骤(2)MD模拟中，通过选择势能参数以保持化合物分子的分子构型；
    优选地，所述步骤(2)中，模拟水溶液体系时，x，y，z三个方向均采用周期性边界条件；模拟冰水混合体系时，x，y两个方向采用周期性边界条件；
    优选地，所述步骤(2)MD模拟中，选用立方体或八面体水盒子，优选3.9×3.6×1.0nm 3的水盒子。
  5. 根据权利要求1-4任一项所述的分子设计方法，其特征在于，所述化合物分子的主链为碳链或者肽链结构。
  6. 根据权利要求1-5任一项所述的分子设计方法，其特征在于，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；例如，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，例如选自脯氨酸(L-Pro)、精氨酸(L-Arg)、赖氨酸(L-Lys)等亲水氨基酸，葡萄糖酸内酯(GDL)、糖类等化合物分子或其分子片段；
    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作用等；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)等，或，例如选自谷氨酰胺(L-Gln)、苏氨酸(L-Thr)、天冬氨酸(L-Asn)等亲冰氨基酸，苯环(C 6H 6)，吡咯烷(C 4H 9N)等化合物分子或其分子片段。
  7. 根据权利要求1-6任一项所述的分子设计方法，其特征在于，所述控冰材料为含有亲水基团的嵌段与含有亲冰基团的嵌段共价键键合而成，或者为含有亲水基团的分子与含有亲冰基团的分子通过离子键结合。
  8. 根据权利要求1-7任一项所述的分子设计方法，其特征在于，所述方法还包括合成所述化合物分子的步骤，例如聚合反应、脱水缩合反应，或者基因工程菌生物发酵等方法合成。
  9. 根据权利要求1-8任一项所述的分子设计方法所得的控冰材料。
  10. 权利要求9所述的控冰材料，其特征在于，所述控冰材料为间同规整度r为45％～60％的PVA，分子量为10kDa–500kDa；优选地，所述PVA间同规整度r为50％～55％，分子量为10-30kDa。
  11. 一种筛选控冰材料的方法，其特征在于，包括如下步骤：(1)测得所述控冰材料与水的亲和性；(2)测得所述控冰材料在冰水界面的铺展性能。
  12. 根据权利要求11所述的筛选控冰材料的方法，其特征在于，所述步骤(1)通过测定所述控冰材料在水中的溶解度、水合常数、分散尺寸、和/或所述控冰材料分子与水分子形成的分子间氢键数的方法测定。
  13. 根据权利要求11或12所述的筛选控冰材料的方法，其特征在于，所述步骤(2)为采用冰吸附实验测得所述控冰材料在冰表面的吸附量，
    所述控冰材料在冰表面的吸附量＝(冰表面所吸附的控冰材料质量m 1/含有控冰材料的原溶液中控 冰材料的总质量m 2)╳100％。
  14. 根据权利要求11-13任一项所述的筛选控冰材料的方法，其特征在于，所述冰吸附实验包括：
    S1:配制所述控冰材料的水溶液，降温至过冷温度；
    S2:将预冷的控温棒置于所述水溶液中诱导冰层在控温棒表面生长，持续搅拌水溶液，以待控冰材料逐渐吸附于冰层表面，保持控温棒、水溶液温度在过冷温度；
    S3:测定控冰材料在冰表面的吸附量；
    优选地，所述控温棒经液氮、干冰、或超低温冰箱冷冻中的任一种进行预冷。
  15. 根据权利要求11-14任一项所述的筛选控冰材料的方法，其特征在于，所述冰吸附实验过程中，保持过冷度以及吸附时间恒定以此确保所得冰的表面积在误差允许范围内保持不变。
  16. 根据权利要求14-15任一项所述的筛选控冰材料的方法，其特征在于，所述步骤S1中控冰材料为预先荧光标记的，例如用荧光素进行标记；
    优选地，当所述控冰材料自身具有紫外或荧光光谱吸收特性时，不进行荧光标记。
  17. 根据权利要求14-16任一项所述的筛选控冰材料的方法，其特征在于，步骤S3包括:
    S3a:将吸附完成的冰块取出，纯水洗涤，融化得到控冰材料吸附溶液；
    S3b:测定融化的控冰材料吸附溶液的体积V，测定所述吸附溶液中控冰材料的质量/体积浓度c，通过公式m 1＝cV计算得到冰表面所吸附的控冰材料质量m 1。
  18. 根据权利要求17所述的筛选控冰材料的方法，其特征在于，所述S3b中，所述浓度c通过紫外可见光谱法测得。
  19. 根据权利要求11-18任一项所述的筛选控冰材料的方法，其特征在于，所述方法用于控制冰晶生长材料的筛选。
  20. 根据权利要求11-19任一项所述的筛选控冰材料的方法，其特征在于，所述方法还包括步骤(3)：评估所述材料与水的亲和性和与冰的亲和性，水亲和性和冰亲和性都强的材料具有良好的控冰性能。
  21. 一种用于权利要求13或14所述方法的冰吸附实验的装置，包括多层储液腔、控温棒和温度控制器，所述多层储液腔由里到外依次包括冰吸附腔、温浴腔、冷却液储存腔，所述控温棒置于冰吸附腔内，所述控温棒和储液腔的温度由温度控制器控制。
  22. 根据权利要求21的冰吸附实验装置，所述控温棒为导热材料制成的中空结构，所述控温棒的中空结构设置有进液口和排液口；所述温度控制器为流体温控器，所述温度控制器设置有冷却液流出端和回流端；所述冷却液储存腔两端设置有进液口和排液口；所述温度控制器的冷却液流出端、控温棒的进液口、控温棒的排液口、冷却液储存箱的进液口、冷却液储存箱的排液口以及温度控制器的回流端依次经管道连通，所述管道内流动冷却；
    优选，所述多层储液腔设有盖子；
    优选，所述冰吸附实验装置在使用时，所述冰吸附腔内盛放控冰材料的水溶液，中层温浴腔装有预定温度的温浴介质例如水浴、冰浴或者油浴等；冷却液温度达到设定温度后，经温度控制器流出，流入中空的控温棒中空结构，对控温棒温度实现控制，随后从控温棒排液口流出，再流入外层冷却液储存腔保持温浴介质的温度在预定水平，再经冷却液储存箱的排液口流经温度控制器的回流端进入温度控制器循环。
  23. 一种冷冻保存液，包括权利要求9或10所述的仿生控冰材料。
  24. 根据权利要求23所述的冷冻保存液，所述仿生控冰材料为聚乙烯醇(PVA)，氨基酸或聚氨基酸，和/或肽类化合物的一种或几种的组合；所述冷冻保存液还包括多元醇、水溶性糖(或其衍生物，如水溶性纤维素)和缓冲液。
  25. 根据权利要求24所述的冷冻保存液，所述冷冻保存液包括肽类化合物，以每100mL冷冻保存液计，含有0.1-50g所述肽类化合物，0-6.0g的PVA，0-9.0g的聚氨基酸或氨基酸，0-15mL的DMSO，5-45mL的多元醇，0.1-1.0mol L -1的水溶性糖，0-30mL的血清，余量为缓冲液。
  26. 根据权利要求24所述的冷冻保存液，所述冷冻保存液包括聚乙烯醇，以每100mL冷冻保存液计，含有PVA 0.01-6.0g，0-50g所述肽类化合物，0-9.0g的聚氨基酸或氨基酸，0-15mL的DMSO，5-45mL的多元醇，0.1-1.0mol L -1的水溶性糖，0-30mL的血清，余量为缓冲液。
  27. 根据权利要求24所述的冷冻保存液，所述冷冻保存液包括氨基酸或聚氨基酸，以每100mL冷冻保存液计，含有0.1-50g的氨基酸或聚氨基酸，0-6.0g的PVA，0-15mL的DMSO，5-45mL的多元醇，0.1-1.0mol L -1的水溶性糖，0-30mL的血清，余量为缓冲液。
  28. 根据权利要求27所述的冷冻保存液，每100mL冷冻保存液中所述氨基酸和/或聚氨基酸含量为0.5-50g，优选1.0-35g，例如，当含有氨基酸时，其含量可以为5.0-35g，优选为15-25g；当含有聚氨基酸时，其含量可以为0.5-9.0g，优选为1.0-5.0g。
  29. 根据权利要求24-28任一项所述的冷冻保存液，所述多元醇可以为碳原子数为2-5的多元醇，优选碳原子数2-3的二元醇、和/或三元醇，例如乙二醇，丙二醇，丙三醇中的任一种；优选乙二醇；优选每100mL冷冻保存液中所述多元醇，含量为5.0-40mL，例如6.0-20mL，9-15mL。
  30. 根据权利要求24-28任一项所述的冷冻保存液，所述水溶性糖为非还原性双糖、水溶性多糖、水溶性纤维素、糖酐中的至少一种，例如选自蔗糖、海藻糖、羟丙基甲基纤维素、聚蔗糖。
  31. 根据权利要求24-28任一项所述的冷冻保存液，所述缓冲液可选自DPBS或hepes-buffered HTF，或其它细胞培养缓冲液中的至少一种。
  32. 根据权利要求24-28任一项所述的冷冻保存液，每100mL冷冻保存液中所述DMSO含量为0-10mL，例如DMSO含量为1.0-7.5mL；
    每100mL冷冻保存液中所述血清含量为0.1-30mL，例如5.0-20mL；
    每100mL冷冻保存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；
    每100mL冷冻保存液中所述多元醇含量为5.0-40mL，例如6.0-20mL。
  33. 根据权利要求23-32任一项所述的冷冻保存液，所述冷冻保存液的pH为6.5-7.6。
  34. 根据权利要求24-32任一项所述的冷冻保存液，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％，优选所述PVA的间同规整度为45％-65％的PVA；
  35. 根据权利要求34所述的冷冻保存液，所述PVA可选自分子量为10-500kDa或者更高分子量的PVA。
  36. 根据权利要求24-32任一项所述的冷冻保存液，所述肽类化合物由亲冰性氨基酸，如：苏氨酸(L-Thr)、谷氨酰胺(L-Gln)、天冬氨酸(L-Asn)等与其他亲水性氨基酸或葡萄糖内酯(GDL)或糖反应得到，所述其他亲水性氨基酸可选自精氨酸、脯氨酸、丙氨酸。
  37. 根据权利要求35所述的冷冻保存液，所述肽类化合物为两个以上的不同种类的氨基酸单元形成的肽类，如：2-8个氨基酸单元。
  38. 根据权利要求35-37任一项所述的冷冻保存液，所述肽类化合物具有式(1)-式(9)所示任一结构：
    Figure PCTCN2020077472-appb-100001
    Figure PCTCN2020077472-appb-100002
    其中，式(9)中R选自取代或未取代的烷基，所述取代基可以选自-OH、-NH 2、-COOH、-CONH 2等，例如，R为取代或未取代的C1-6烷基，优选R为-CH 3、-CH 2CH 3、-CH 2CH 2COOH；n为大于等于 1而小于等于1000的整数。
  39. 根据权利要求24-32任一项所述的冷冻保存液，所述氨基酸为含有亲冰基团和亲水基团的氨基酸，所述聚氨基酸为含有亲冰基团的氨基酸和含有亲水基团的氨基酸组成的聚氨基酸，所述聚氨基酸优选聚合度为2-40，例如聚合度为6、8、15、20等，例如为聚-L-脯氨酸、聚-L-精氨酸等中的一种或两种以上的组合；所述氨基酸选自精氨酸、苏氨酸、脯氨酸、赖氨酸、组氨酸、谷酰胺酸、天冬氨酸、甘氨酸等中的一种或两种，例如为精氨酸与苏氨酸的组合；或者由上述氨基酸组成的聚氨基酸。
  40. 一种冷冻平衡液，以每100mL计，含有多元醇5.0-45mL，缓冲液余量。
  41. 根据权利要求40所述的冷冻平衡液，其还任选地包括DMSO 0-15mL，血清0-30mL，和/或PVA0-5.0g。
  42. 一种冷冻保存试剂，包括权利要求23-39任一项所述的冷冻保存液和权利要求40-41任一项所述的冷冻平衡液。
  43. 权利要求23-39任一项所述的冷冻保存液或者权利要求40-41任一项所述的冷冻平衡液或者权利要求42所述的冷冻保存试剂在细胞、组织、器官冷冻保存中的应用。
  44. 根据权利要求43所述的应用，所述细胞为生殖细胞或者干细胞，例如生殖细胞选自卵母细胞、精子；所述干细胞选自胚胎干细胞、各类间充质干细胞(例如脐带间充质干细胞、脂肪间充质干细胞、骨髓间充质干细胞等)、造血干细胞。
  45. 根据权利要求43所述的应用，所述组织为卵巢组织或胚胎组织。
  46. 根据权利要求43所述的应用，所述器官为卵巢组器官。
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