CN111793109A - Peptide compound and cryopreservation liquid containing same - Google Patents

Abstract

The invention discloses a peptide compound and a cryopreservation liquid containing the same. The peptide compound consists of the ice-philic amino acid and the hydrophilic group, has good capability of inhibiting the growth of ice crystals and modifying the appearance of the ice crystals in aqueous solution, has no thermal hysteresis and good biocompatibility, and is an ideal ice control material. The cryopreservation liquid containing the peptide compound contains the peptide compound, polyhydric alcohol, water-soluble sugar and other components, can keep good survival rate when used for preserving embryos or oocytes, and has simple components and stable performance.

Description

Peptide compound and cryopreservation liquid containing same

The present application claims the priority of the prior application entitled "a peptide compound and a cryopreservation solution containing the same" filed on the national intellectual property office of china with patent application number 201910281986.7 filed on 9/4/9/2019. The entire content of this prior application is incorporated by reference into this application.

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a peptide compound and cryopreservation liquid containing the peptide compound.

Background

Cryopreservation refers to keeping the biological material at ultralow temperature to slow down or stop cell metabolism and division, and to continue to develop once normal physiological temperature is restored. Since the advent, this technique has become one of indispensable research methods in the field of natural science, and has been widely adopted. In recent years, with the increasing pressure of life, human fertility tends to decrease year by year, and fertility preservation is receiving more and more attention, and cryopreservation of human germ cells (sperm and oocyte), gonadal tissue, and the like is an important means for fertility preservation. In addition, as the world population ages, the need for cryopreservation of donated human cells, tissues or organs available for regenerative medicine and organ transplantation is also increasing dramatically. Therefore, how to efficiently store precious cells, tissues and organ resources in a freezing way becomes a scientific and technical problem to be solved urgently.

The most common cryopreservation method currently used is vitrification freezing. The vitrification freezing technology can ensure that liquid inside and outside cells is in a vitrification state in the rapid freezing process, thereby avoiding damage in the freezing process. However, prior art cryopreservation reagents are not effective in controlling the growth of ice crystals during the rewarming process, thereby damaging the cells. The high concentration (more than or equal to 15 percent) of toxic organic solvents used by the prior vitrification freezing method, such as: DMSO causes toxic and side effects of the cryopreservation reagent on cells, and seriously influences survival rate and even (offspring) safety and functional expression of the cryopreserved subjects after resuscitation. In conclusion, the currently adopted cryopreservation reagent does not have the capability of effectively controlling the growth of ice crystals in the rewarming process, and simultaneously has the problem of poor safety of the reagent.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a peptide compound, a synthetic method thereof and a cryopreservation solution containing the peptide compound.

The invention is realized by the following technical scheme:

a peptide compound consisting of an ice-philic amino acid, such as: threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc. with other hydrophilic amino acids selected from arginine, proline, alanine, etc., or Gluconolactone (GDL) or saccharides.

According to the invention, the peptide compound is a polypeptide compound formed by more than two amino acid units, such as: 2-8 amino acid units, specifically 2, 3, 4, 5, 6 amino acid units; the polypeptide compound is composed of more than two different amino acids.

As an exemplary technical scheme, the molar ratio of the ice-philic amino acid to other hydrophilic amino acids in the peptide compound is (0.1-3): 1, preferably (0.5-2): 1.

according to the present invention, the arrangement of the hydrophilic amino acid and the hydrophilic amino acid in the peptide compound is not particularly limited, and the peptide compound may be connected by amino acid linking groups or chemical bonds known in the art, for example, the hydrophilic amino acid and the hydrophilic amino acid may be arranged in single intervals, or a plurality of hydrophilic amino acids may be connected to form hydrophilic amino acid fragments or hydrophilic amino acid fragments, and then the hydrophilic amino acid fragments or hydrophilic amino acid fragments are respectively connected to the hydrophilic amino acid (or fragment) and the hydrophilic amino acid (or fragment).

According to the invention, the peptidic compound is at least one of L-Thr-L-Arg (TR), L-Thr-L-Pro (TP), L-Arg-L-Thr (RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), L-Ala-L-Ala-L-Thr (AAT), L-Thr-L-Cys-L-Thr (TCT).

According to the invention, the peptide compound is a molecule formed by chemically bonding glucolactone or other saccharides and an ice-philic amino acid, such as: GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, -GDL-L-Val, GDL-L-Ser.

According to the invention, the peptide compound has any one structure shown in formula (1) to formula (8):

the peptide compounds can be prepared by peptide synthesis methods known in the art, such as solid phase synthesis.

The preparation method comprises the following steps of resin swelling, covalent connection of amino acid protected by one amino group to the swelled resin, deprotection, condensation reaction by adding amino acid protected by another amino group, deprotection, cutting and purification.

According to the preparation method of the present invention, the glycopeptide derivative may be prepared by a known method of the present invention, for example, by reacting gluconolactone or other saccharides with an amino acid in an organic solvent, or by a solid phase synthesis method. In one embodiment, the glycopeptide derivative is synthesized by dissolving Gluconolactone (GDL) in an organic solvent, and adding an amino acid and a basic catalyst to the organic solvent to perform a reaction.

According to the preparation method of the present invention, the organic solvent may be selected from methanol, ethanol, and the like.

According to the preparation method, the GDL-L-Thr is prepared by adopting the following synthetic route:

the invention also provides application of the peptide compound in controlling ice crystal growth in an aqueous solution and application of the peptide compound in preparing a cell or tissue cryopreservation solution.

In one embodiment, the glycopeptide derivative is prepared using a solid phase synthesis method comprising: swelling resin, covalently linking an amino acid protected by an amino group to the swollen resin, deprotecting, adding a saccharide compound (such as gluconolactone) for condensation reaction, cleavage, and purification. Methods for the synthesis of GDL-L-Val and GDL-L-Ser refer to the method for the synthesis of GDL-L-Thr.

The present invention also provides a peptide compound represented by formula (9):

wherein R is selected from substituted or unsubstituted alkyl, and the substituent can be selected from-OH, -NH₂、-COOH、-CONH₂Etc., e.g., R is substituted or unsubstituted C_1-6Alkyl, preferably R is-CH₃、-CH₂CH₃、-CH₂CH₂COOH; n is 1 or more and 1000 or lessThe integer of (b) may be, for example, an integer in the range of 1 to 100. In some embodiments of the invention, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.

As one embodiment of the present invention, the compound represented by the formula (9) has a structure represented by any one of the following:

according to the invention, the compound of formula (9) is prepared using the following synthetic route:

the invention also provides application of the compound of the formula (9) in controlling ice crystal growth in aqueous solution, and application of the compound of the formula (9) in preparing a cell or tissue cryopreservation solution.

According to the application of the invention, the peptide compound can be combined with other ice control materials, and the ice control materials are selected from at least one of PVA, amino acid, polyamino acid and the like.

The invention also provides a cryopreservation solution containing the peptide compound.

The cryopreservation solution according to the present invention contains 0.1 to 50g of the peptide compound per 100 mL.

The cryopreservation solution of the present invention may further contain one or more of PVA, amino acid, polyamino acid, DMSO, and polyhydric alcohol.

The cryopreservation solution of the present invention may further contain serum.

The cryopreservation solution comprises 0.1-50g of the peptide compound, 0-6.0g of PVA, 0-9.0g of polyamino acid, 0-15mL of DMSO, 5-45mL of polyalcohol and 0.1-1.0mol of L per 100mL^-10-30mL of serum, and the balance of buffer solution.

In one embodiment of the present invention, the cryopreservation solution contains 0.1-50g of the peptide compound, 0.1-15mL of DMSO, 5.0-45mL of polyol, 0.1-1.0mol L^-1Water-soluble sugar, 0-30mL of serum, no PVA and polyamino acid, and the balance of buffer solution.

In one embodiment of the present invention, the cryopreservation solution comprises, per 100mL, 0.1 to 50g of the peptide compound, 0.1 to 6.0g of PVA, 0 to 9.0g of the polyamino acid, 0 to 15mL of DMSO, 5.0 to 45mL of the polyol, and 0.1 to 1.0mol of L^-10-30mL of serum, and the balance of buffer solution.

In one embodiment of the present invention, the cryopreservation solution comprises, per 100mL, 0.1 to 50g of the peptide compound, 0.1 to 6.0g of PVA, 0.1 to 9.0g of the polyamino acid, 0.1 to 7.5mL of DMSO, 5.0 to 45mL of the polyol, and 0.1 to 1.0mol L of the polyol^-1The balance being buffer solution.

In one embodiment, the cryopreservation solution contains 0.1-10mL of DMSO, e.g., 0.1-7.5mL of DMSO.

In one embodiment, the cryopreservation solution contains 0.1 to 6.0g of PVA, for example 0.1 to 4.0g of PVA.

In one embodiment, the cryopreservation solution contains 5 to 30mL of the polyol, e.g., 8.0 to 25mL, 20mL, 15 mL.

In one embodiment, the cryopreservation solution contains 0.1 to 0.8mol L^-1Water-soluble sugars of (2), e.g. 0.2-0.6mol L^-1The sucrose of (1).

In one embodiment, the cryopreservation solution contains 5.0-30mL of serum, such as 5.0-20mL of fetal bovine serum; in another embodiment, the cryopreservation solution does not contain fetal bovine serum.

According to the invention, the PVA is selected from one or a combination of two or more of isotactic PVA, syndiotactic PVA and atactic PVA, for example the PVA has a syndiotacticity of from 15% to 60%, in particular for example from 50% to 60%, from 50% to 55%.

According to the invention, the PVA may be chosen from the group comprising PVA with a molecular weight of 10-500kDa, such as 10-30kDa, 30-50kDa, 80-90kDa, 200-500 kDa.

According to the present invention, the polyamino acid may be selected from at least one homopolymer of lysine, arginine, proline, threonine, histidine, and the like, or a copolymer of two or more amino acids.

According to the invention, the polyol may be a polyol having from 2 to 5 carbon atoms, preferably a diol having from 2 to 3 carbon atoms, and/or a triol, such as any of ethylene glycol, propylene glycol, glycerol; ethylene glycol is preferred.

According to the present invention, the water-soluble sugar may be at least one of non-reducing disaccharide, water-soluble polysaccharide, and anhydrosugar, and is selected from sucrose, trehalose, water-soluble cellulose (e.g., hydroxypropylmethyl cellulose, etc.), polysucrose; sucrose and hydroxypropyl methylcellulose are preferred. The water-soluble sugar can play a role in protecting cell membranes and avoiding cell sedimentation.

According to the present invention, the buffer may be selected from at least one of DPBS or hepes-buffered HTF buffer, or other cell culture medium buffer.

According to the invention, the serum may be selected from human serum albumin or analogues thereof, such as Sodium Dodecyl Sulphate (SDS); fetal bovine serum or bovine serum albumin can be selected for the non-human-derived cryopreservation object.

The preparation method of the cryopreservation liquid comprises the steps of dissolving the peptide compound in a buffer solution, cooling to room temperature, adjusting pH, dissolving other components in another buffer solution, cooling, mixing, adjusting pH, fixing the volume to a preset volume by using the buffer solution, and optionally adding serum when in use.

The preparation method comprises the following steps:

(1) dissolving a peptide compound in a part of buffer solution, cooling to room temperature, and adjusting the pH value to obtain a solution 1;

(2) dissolving water-soluble sugar in a part of buffer solution, and adding other components after the water-soluble sugar is completely dissolved to prepare a solution 2;

(3) optionally, dissolving PVA and/or polyamino acid in another part of buffer solution, cooling to room temperature, and adjusting pH to obtain solution 3;

(4) and (3) cooling the solution 1, the solution 2 and optionally the solution 3 to room temperature, mixing, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the cryopreservation solution.

According to the preparation method, in the step (3), the PVA is dissolved by heating in a warm bath; for example, the bath temperature is 60-85 deg.C, preferably 80 deg.C. In the step (3), the dissolving includes a stirring step.

According to the preparation method of the present invention, in the step (2), the dissolution is ultrasonic-assisted dissolution.

In the present invention, hydrophilic means capable of forming a non-covalent interaction with a water molecule, for example, capable of forming a hydrogen bond, van der waals interaction, electrostatic interaction, hydrophobic interaction or pi-pi interaction with water;

ice-philic refers to a non-covalent interaction with ice, such as hydrogen bonding, van der Waals interactions, electrostatic interactions, hydrophobic interactions, or pi-pi interactions with ice.

In the invention, when the cryopreservation solution is used, a cryopreservation equilibrium solution known in the field can be used, preferably, the DMSO content in the cryopreservation solution is 0, and the cryopreservation solution contains 7.5-15mL of polyalcohol, 10-20mL of serum and the balance of buffer solution per 100 mL; preferably, when the serum content of the cryopreservation solution is also 0, the frozen equilibrium solution contains 1.0 to 5.0g of PVA, 7.5 to 15mL of polyol and the balance of buffer per 100 mL. The components in the frozen equilibrium solution have the same meaning as in the frozen stock solution.

The invention also provides the application of the cryopreservation liquid, the frozen equilibrium liquid or the frozen preservation reagent, which is used for cryopreservation of cells, tissues or organs; for example for cryopreservation of oocytes, sperm or stem cells, ovarian tissue, embryos or ovarian organs.

Advantageous effects

The inventor of the invention finds that in the process of controlling the growth of ice crystals in an ice-water mixed phase, materials need to have good interaction with ice and water, so peptide compounds which are combined by ice-philic amino acids such as threonine and other hydrophilic amino acids or saccharides are synthesized according to an ice-philic-hydrophilic ice-controlling mechanism, the synthesized peptide compounds have good ice-controlling effect, can modify the appearance of the ice crystals, have no thermal hysteresis, and can keep excellent cell survival rate when being used for freezing and preserving fluid.

Drawings

FIG. 1: an electron micrograph of the ice crystal growth inhibition activity of GDL-L-Thr and a statistical plot of the size of ice crystals.

FIG. 2: the GDL-L-Thr modifies the appearance effect of ice crystals in pure water.

FIG. 3: an electron micrograph of the ice crystal growth inhibitory activity of the TR short chain peptide prepared in example 2 and a statistical plot of ice crystal size.

FIG. 4: the TR short-chain peptide prepared in example 2 modifies the morphological effect of ice crystals in pure water.

FIG. 5: example 7 peptoids R-COOH, R-CH₃And R-CH₂CH₃Inhibiting the growth activity of ice crystal.

FIG. 6: peptoids (A) R-COOH, (B) R-CH₃And (C) R-CH₂CH₃Modifying the appearance effect of the ice crystals in the pure water.

FIG. 7: photographs of sections of fresh, unfrozen ovarian organs from 3-day-old mice.

FIG. 8: the cryopreservation solution of comparative example 4 was used to cryopreserve a photograph of a thawed section of an intact ovarian organ.

FIG. 9: photographs of thawed sections of intact ovarian organs were cryopreserved using the cryopreservation solution of example 6.

FIG. 10: photographs of sections of fresh unfrozen ovarian tissue from sexually mature mice.

FIG. 11: photograph of frozen ovarian tissue section thawed by the cryopreservation solution of comparative example 5.

FIG. 12: photograph of thawed ovarian tissue section frozen using the cryopreservation solution of example 7.

Detailed Description

The preparation method of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 Synthesis of Compound of formula (1)

(1) 2-Chlorotrityl Chloride Resin (2-Chlorotrityl Chloride Resin) was placed in a reaction tube, and DCM (20mL g) was added^-1) And shaken for 30 minutes. And (3) filtering the sand core to remove the solvent, adding three times of molar excess Fmoc-L-Pro-OH, adding 8 times of molar excess DIEA, finally adding DMF to dissolve, and oscillating for 30 minutes. Methanol was capped for 30 minutes.

(2) The solvent DMF was removed and 20% piperidine/DMF solution (10mL g) was added^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) After 15 minutes the piperidine solution was removed. And (3) taking a small amount of resin, washing the resin with ethanol for three times, adding a ninhydrin reagent, heating the resin at 105-110 ℃ for 5 minutes, and taking a positive reaction when the resin turns dark blue.

(3) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice), adding Fmoc-L-Thr (tBu) -OH dissolved in DMF as little as possible into a reaction tube; double excess, HBTU double excess. Immediately thereafter, 8-fold excess of DIEA was added and the reaction was carried out for 30 minutes.

(4) After the solution is pumped out, a small amount of resin is taken out, washed with ethanol for three times, added with ninhydrin reagent, heated at 105-110 ℃ for 5 minutes, and colorless is a negative reaction, namely the reaction is complete.

(5) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Two times) washing followed by removal of the solvent and addition of 20% piperidine/DMF solution (10mL g)^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) Removing the piperidine solution after 15 minutes, taking a small amount of resin, washing the resin with ethanol, adding ninhydrin reagent, and heating at 105-110 ℃ for 5 minutesDark blue is a positive reaction.

(6) The product obtained by the above reaction was washed sequentially with DMF (15mL g-1, twice) and methanol (15mL g)^-1Twice) and DCM (15mL g)^-1Twice) after washing, the resin was drained.

(7) Using a cutting fluid (15mL g)^-1And TFA: water: EDT (electro-thermal transfer coating): tis 95:1:2:2, V/V) cut the product for 90 minutes. And blowing the cutting fluid by nitrogen, and then freeze-drying to obtain a crude polypeptide product.

(8) Purification of the polypeptide by HPLC and desalting, HPLC: tR 6.1mins (purification column model: Kromasil100-5C18,4.6mm 250 mm; gradient eluent: 0.1% TFA in acetonitrile and 0.1% aqueous TFA, 0mins-1:99, 20mins-1: 9). And (4) freeze-drying the purified solution to obtain a finished product L-Thr-L-Pro (marked as TP). The yield was about 80%. The mass spectrum identification 217.3 is [ M + H ] +.

EXAMPLE 2 Synthesis of Compound of formula (2)

(1) The 2-chlorotrityl chloride resin was placed in a reaction tube and DCM (20mL g) was added^-1) And shaken for 30 minutes. The sand core is filtered by suction to remove the solvent, three times molar excess of Fmoc-L-Thr (tBu) -OH is added, 8 times molar excess of DIEA is added, and finally DMF is added for dissolution and oscillation is carried out for 30 minutes. Methanol was capped for 30 minutes.

(2) The solvent DMF was removed and 20% piperidine/DMF solution (10mL g) was added^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) After 15 minutes the piperidine solution was removed. And (3) taking a small amount of resin, washing the resin with ethanol for three times, adding a ninhydrin reagent, heating the resin at 105-110 ℃ for 5 minutes, and taking a positive reaction when the resin turns dark blue.

(3) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice), adding Fmoc-Arg (Pbf) -OH dissolved by using DMF as little as possible into a reaction tube; double excess, HBTU double excess. Immediately thereafter, 8-fold excess of DIEA was added and the reaction was carried out for 30 minutes.

(4) After the solution is pumped out, a small amount of resin is taken out, washed with ethanol for three times, added with ninhydrin reagent, heated at 105-110 ℃ for 5 minutes, and colorless is a negative reaction, namely the reaction is complete.

(5) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Two times) washing followed by removal of the solvent and addition of 20% piperidine/DMF solution (10mL g)^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) And after 15 minutes, removing the piperidine solution, taking a small amount of resin, washing with ethanol, adding a ninhydrin reagent, heating at 105-110 ℃ for 5 minutes, and turning dark blue to be a positive reaction.

(6) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DCM (15mL g)^-1Twice) after washing, the resin was drained.

(7) Using a cutting fluid (15mL g)^-1And TFA: water: EDT (electro-thermal transfer coating): tis 95:1:2:2, V/V) cut the product for 90 minutes. And blowing the cutting fluid by nitrogen, and then freeze-drying to obtain a crude polypeptide product.

(8) Purification of the polypeptide by HPLC and desalting, HPLC: tR 4.8mins (purification column model: Kromasil100-5C18,4.6mm 250 mm; gradient eluent: 0.1% TFA in acetonitrile and 0.1% aqueous TFA, 0mins-1:99, 20mins-1: 4). And (4) freeze-drying the purified solution to obtain a finished product L-Thr-L-Arg (TR). The yield was about 80%. The mass spectrum identification 276.2 is [ M + H ] +.

EXAMPLE 3 Synthesis of Compound of formula (3)

(1) The 2-chlorotrityl chloride resin was placed in a reaction tube and DCM (20mL g) was added^-1) And shaken for 30 minutes. The sand core is filtered by suction to remove the solvent, three times molar excess of Fmoc-L-Thr (tBu) -OH is added, 8 times molar excess of DIEA is added, and finally DMF is added for dissolution and oscillation is carried out for 30 minutes. Methanol was capped for 30 minutes.

(2) The solvent DMF was removed and 20% piperidine/DMF solution (10mL g) was added^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) After 15 minutes the piperidine solution was removed. And (3) taking a small amount of resin, washing the resin with ethanol for three times, adding a ninhydrin reagent, heating the resin at 105-110 ℃ for 5 minutes, and taking a positive reaction when the resin turns dark blue.

(3) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice), adding Fmoc-Arg (Pbf) -OH dissolved by using DMF as little as possible into a reaction tube; double excess, HBTU double excess. Immediately thereafter, 8-fold excess of DIEA was added and the reaction was carried out for 30 minutes.

(4) After the solution is pumped out, a small amount of resin is taken out, washed with ethanol for three times, added with ninhydrin reagent, heated at 105-110 ℃ for 5 minutes, and colorless is a negative reaction, namely the reaction is complete.

(5) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Two times) washing followed by removal of the solvent and addition of 20% piperidine/DMF solution (10mL g)^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) And after 15 minutes, removing the piperidine solution, taking a small amount of resin, washing with ethanol, adding a ninhydrin reagent, heating at 105-110 ℃ for 5 minutes, and turning dark blue to be a positive reaction.

(6) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice) after washing, the resin was drained.

(7) Repeating the operations (3) to (5), and linking the amino acids Fmoc-L-Thr (tBu) -OH. The reaction product was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DCM (15mL g)^-1Twice) after washing, the resin was drained.

(8) Using a cutting fluid (15mL g)^-1And TFA: water: EDT (electro-thermal transfer coating): tis 95:1:2:2, V/V) cut the product for 90 minutes. And blowing the cutting fluid by nitrogen, and then freeze-drying to obtain a crude polypeptide product.

(9) Purification of the polypeptide by HPLC and desalting, HPLC: tR 3.9mins (purification column model: Kromasil100-5C18,4.6mm 250 mm; gradient eluent: 0.1% TFA in acetonitrile and 0.1% aqueous TFA, 0mins-1:99, 20mins-1: 4). Freeze-drying the purified solution to obtain the finished product L-Thr-L-Arg-L-Thr (TRT). The yield was about 75%. Mass spectrometry identification 377.4 is [ M + H ] +.

EXAMPLE 4 Synthesis of Compound of formula (4)

(1) The 2-chlorotrityl chloride resin was placed in a reaction tube and DCM (20mL g) was added^-1) And shaken for 30 minutes. The sand core is filtered by suction to remove the solvent, three times molar excess of Fmoc-L-Thr (tBu) -OH is added, 8 times molar excess of DIEA is added, and finally DMF is added for dissolution and oscillation is carried out for 30 minutes. Methanol was capped for 30 minutes.

(2) The solvent DMF was removed and 20% piperidine/DMF solution (10mL g) was added^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) After 15 minutes the piperidine solution was removed. And (3) taking a small amount of resin, washing the resin with ethanol for three times, adding a ninhydrin reagent, heating the resin at 105-110 ℃ for 5 minutes, and taking a positive reaction when the resin turns dark blue.

(3) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice), adding Fmoc-L-Pro-OH dissolved by using DMF as little as possible into a reaction tube; double excess, HBTU double excess. Immediately thereafter, 8-fold excess of DIEA was added and the reaction was carried out for 30 minutes.

(4) After the solution is pumped out, a small amount of resin is taken out, washed with ethanol for three times, added with ninhydrin reagent, heated at 105-110 ℃ for 5 minutes, and colorless is a negative reaction, namely the reaction is complete.

(5) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Two times) washing followed by removal of the solvent and addition of 20% piperidine/DMF solution (10mL g)^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) And after 15 minutes, removing the piperidine solution, taking a small amount of resin, washing with ethanol, adding a ninhydrin reagent, heating at 105-110 ℃ for 5 minutes, and turning dark blue to be a positive reaction.

(6) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice) after washing, the resin was drained.

(7) Repeating the operations (3) to (5),the linking amino acid Fmoc-L-Thr (tBu) -OH. The reaction product was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DCM (15mL g)^-1Twice) after washing, the resin was drained.

(8) The product was cleaved for 90 min using cleavage solution (15mL g-1, TFA: water: EDT: Tis ═ 95:1:2:2, V/V). And blowing the cutting fluid by nitrogen, and then freeze-drying to obtain a crude polypeptide product.

(9) Purification of the polypeptide by HPLC and desalting, HPLC: tR 7.6mins (purification column model: Kromasil100-5C18,4.6mm 250 mm; gradient eluent: 0.1% TFA in acetonitrile and 0.1% aqueous TFA, 0mins-1:99, 20mins-2: 8). And (4) freeze-drying the purified solution to obtain a finished product of L-Thr-L-Pro-L-Thr (TPT). The yield was about 70%. Mass spectrum identification 318.3 is [ M + H ] +

EXAMPLE 5 Synthesis of Compound of formula (5)

(1) The 2-chlorotrityl chloride resin was placed in a reaction tube and DCM (20mL g) was added^-1) And shaken for 30 minutes. The sand core is filtered by suction to remove the solvent, three times molar excess of Fmoc-L-Thr (tBu) -OH is added, 8 times molar excess of DIEA is added, and finally DMF is added for dissolution and oscillation is carried out for 30 minutes. Methanol was capped for 30 minutes.

(2) The solvent DMF was removed and 20% piperidine/DMF solution (10mL g) was added^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) After 15 minutes the piperidine solution was removed. And (3) taking a small amount of resin, washing the resin with ethanol for three times, adding a ninhydrin reagent, heating the resin at 105-110 ℃ for 5 minutes, and taking a positive reaction when the resin turns dark blue.

(3) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice), adding Fmoc-L-Ala-OH dissolved by using DMF as little as possible into a reaction tube; double excess, HBTU double excess. Immediately thereafter, 8-fold excess of DIEA was added and the reaction was carried out for 30 minutes.

(4) After the solution is pumped out, a small amount of resin is taken out, washed with ethanol for three times, added with ninhydrin reagent, heated at 105-110 ℃ for 5 minutes, and colorless is a negative reaction, namely the reaction is complete.

(5) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Two times) washing followed by removal of the solvent and addition of 20% piperidine/DMF solution (10mL g)^-1) After 5 minutes, the solvent was removed and a further 20% piperidine/DMF solution (10mL g)^-1) And after 15 minutes, removing the piperidine solution, taking a small amount of resin, washing with ethanol, adding a ninhydrin reagent, heating at 105-110 ℃ for 5 minutes, and turning dark blue to be a positive reaction.

(6) The product obtained in the above reaction was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DMF (15mL g)^-1Twice) after washing, the resin was drained.

(7) Repeating the operations (3) to (5) to link the amino acid Fmoc-L-Ala-OH. The reaction product was washed successively with DMF (15mL g)^-1Twice, methanol (15mL g)^-1Twice) and DCM (15mL g)^-1Twice) after washing, the resin was drained.

(8) Using a cutting fluid (15mL g)^-1And TFA: water: EDT (electro-thermal transfer coating): tis 95:1:2:2, V/V) cut the product for 90 minutes. And blowing the cutting fluid by nitrogen, and then freeze-drying to obtain a crude polypeptide product.

(9) Purification of the polypeptide by HPLC and desalting, HPLC: tR 7.9mins (purification column model: Kromasil100-5C18,4.6mm 250 mm; gradient eluent: 0.1% TFA in acetonitrile and 0.1% aqueous TFA in acetonitrile, 0mins-1:99, 20mins-1: 9). Lyophilizing the purified solution to obtain the final product L-Ala-L-Ala-L-Thr (AAT). The yield was about 70%. The mass spectrum identification 260.1 is [ M-8H ] +.

EXAMPLE 6 Synthesis of Compound of formula (6)

(1) 1.0g (0.56mol) of Gluconolactone (GDL) and 0.56mol of L-Thr were subjected to solid-phase synthesis to prepare GDL-L-Thr.

(2) HPLC purification, HPLC: tR 3.4mins (purification column model: Kromasil100-5C18 SHIMADDZUIntertsil ODS-SP (4.6 mm. times.250 mm. times.5 μ M), gradient eluent: 0.1% TFA acetonitrile solution and 0.1% TFA aqueous solution, 0.01-20mins-1:99, 20-30mins-21:79,30-40mins-100:0,40-50mins-1:99), yield about 50%, mass spectrometric identification 296.099 as [ M-H ] +.

The GDL-L-Thr prepared by the solid-phase synthesis method has higher purity and is more beneficial to product separation, and experimental results show that the GDL-L-Thr prepared by the solid-phase synthesis method has higher purity and keeps good capability of inhibiting ice crystal growth (figure 1).

EXAMPLE 7 Synthesis of Compound of formula (9)

(1) The DCM solution of dichlordimethyl silane was poured into the polypeptide synthesis tube, and after standing for 30 minutes the tube was air dried for use.

(2) 100mg of the resin was placed in a synthesis tube, 2mL of DMF was added, nitrogen was introduced, the resin was swollen for 10 minutes and filtered with suction.

(3) 1mL of 4-methylpiperidine/DMF solution was added for deprotection, and after 5 minutes, the solution was removed, and after 15 minutes, 1mL of 4-methylpiperidine/DMF solution was added, followed by bubbling and suction filtration.

(4) DMF rinse 5 times, bubble, suction filter.

(5) 2M, 0.5mL of bromoacetic acid/DMF solution, N, N-diisopropylcarbodiimide/DMF solution were added in this order, bubbled for 20 minutes, filtered with suction, and washed 3 times with DMF.

(6) 1M, 1mL of primary amine/DMF solution was added, bubbled for 30 minutes, rinsed with DMF, rinsed with dichloromethane (× 3).

(7) And (5) repeating the steps (5) and (6) until the molecular weight is required.

(8) Adding 4mL of lysate, fully shaking, introducing nitrogen to blow dry, finally freeze-drying and purifying to obtain the final finished product.

(9) R is-CH₃，-CH₂CH₃and-CH₂CH₂A peptoid of COOH. Mass spectrometric identification 444.6 for R as-CH₃Of [ M + H]⁺528.8 is R is-CH₂CH₃Of [ M + H]⁺792.1 is that R is-CH₂CH₂[ M + H ] of COOH]⁺。

Dissolving and dispersing a sample into a DPBS solution by adopting a sputtering freezing method for inhibiting the recrystallization of ice crystals (IRI), dripping 10-30 mu L of the solution onto the surface of a clean silicon wafer precooled at minus 60 ℃ at a height of more than 1.0m, heating to minus 6 ℃ at a speed of 20 ℃/min by using a cold-hot table, annealing for 30mins at the temperature, observing and recording the size of the ice crystals by using a polarizing microscope and a high-speed camera, and sealing the cold-hot table. Each sample was replicated at least three times, and the ice crystal size was counted using a Nano Measurer 1.2 with standard deviation as the error of the statistical result.

Ice crystal morphology (DIS) observation and Thermal Hysteresis (TH) measurement Using a nanoliter osmometer, a capillary was first melted with an alcohol burner external flame and simultaneously stretched to produce a capillary of very fine pore size, which was connected to a microsyringe. The immersion lens oil with higher viscosity is injected into the micron-aperture wafer, and the water solution dissolved with the material is injected into the micropores by using a microsyringe. The liquid drop is quickly frozen by controlling the temperature, and slowly heated to obtain the single crystal ice, the temperature is slowly reduced with the precision of 0.01 ℃, and the appearance of the ice crystal is observed by utilizing a microscope equipped with a high-speed camera and a TH test is carried out.

The IRI activity test was performed on 20. mu.L of the DPBS solution of TR prepared in example 2 above using the "sputter freezing method". The measured maximum ice crystal size (%) relative to DPBS is shown in fig. 3. The maximum ice crystal size of TR bound by chemical bonds is significantly smaller than the maximum ice crystal size of DPBS solutions of arginine and threonine at the same concentration.

The deionized water solution of TR prepared in the above example 2 is taken, and the observation of the ice crystal morphology by using nanoliter shows that TR has a weak effect of modifying the ice crystal morphology (supercooling degree-0.1 ℃, 0.4-0.01 ℃), as shown in FIG. 4. And no thermal hysteresis was measured.

IRI activity test was performed on 20. mu.L of the DPBS solution of GDL-L-Thr prepared in the above example 6 by the "sputter freezing method". The measured maximum ice crystal size (%) relative to DPBS is shown in fig. 1. The maximum ice crystal size of GDL-L-Thr bound by chemical bond is significantly smaller than that of DPBS solution of GDL and DPBS solution of L-Thr at the same concentration and smaller than that of DPBS solution mixed with GDL and L-Thr at the same concentration.

The deionized water solution of GDL-L-Thr prepared in the above example 6 was taken, and the observation of the morphology of ice crystals by nanoliter revealed that GDL-L-Thr had a weak effect of modifying the morphology of ice crystals (supercooling degree-0.1 ℃ C., 0.4-0.01 ℃ C.), as shown in FIG. 2. And no thermal hysteresis was measured.

The dpi activity test was performed using the "sputter freezing method" on 20 μ L of the DPBS solution of the compound prepared in the above example 7. The measured maximum ice crystal size (%) relative to DPBS is shown in fig. 5.

Taking deionized water solution of the three peptoids prepared in the above example 7, utilizing nanoliter to perform ice crystal morphology observation to find that R is-CH₃and-CH₂CH₃The peptoid has a more obvious effect of modifying the morphology of ice crystals, and R is-CH₂CH₂The morphology of COOH peptoids without modification of ice crystal morphology (supercooling degree-0.1 ℃, 0.4-0.01 ℃) is shown in FIG. 6, and no thermal hysteresis is detected for any of the three peptoids.

The results show that the prepared peptide compound has the effects of inhibiting the growth activity of ice crystals and modifying the appearance of the ice crystals, has no thermal hysteresis, can realize the effect of controlling the growth of the ice crystals, and can be used for freezing storage.

Example 8 oocyte and embryo cryopreservation experiments

(1) The TR synthesized in example 2 and the TPT synthesized in example 4 were used to prepare a cryopreservation solution as follows:

cryopreservation liquid 1: dissolving 28g of TR in 25mL of PBS by ultrasonic treatment in a total volume of 100mL, and adjusting the pH to 7.0 to obtain a solution 1; ultrasonically dissolving 0.05mol of sucrose in 25mL of DPBS, sequentially adding 10mL of ethylene glycol and 7.5mL of DMSO to obtain a solution 2 after the sucrose is completely dissolved, uniformly mixing the two solutions after the solution 1 and the solution 2 are returned to room temperature, adjusting the pH value, fixing the volume to 80% of the total volume by adopting the DPBS, and finally adding 20mL of serum before use.

Cryopreservation liquid 2: dissolving 28g of TPT in 25mL of PBS by ultrasonic treatment in a total volume of 100mL, and adjusting the pH value to 7.0 to obtain a solution 1; ultrasonically dissolving 0.05mol of sucrose in 25mLDPBS, sequentially adding 10mL of ethylene glycol and 7.5mLDMSO to obtain a solution 2 after the sucrose is completely dissolved, uniformly mixing the two solutions after the solution 1 and the solution 2 are restored to room temperature, adjusting the pH value, fixing the volume to 80% of the total volume by adopting DPBS, and finally adding 20mL of serum before use.

Comparative example 1:

cryopreservation liquid a: each 1mL of the mixture contained 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) serum, 0.5M sucrose, and the balance DPBS.

(2) Equilibrium solution and thawing solution:

freezing equilibrium liquid A: every 1mL of the reagent contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) serum and the balance of DPBS;

thawing solution B: thawing solution I (containing 1.0mol L)^-1Sucrose, 20% serum, balance DPBS); thawing solution II (containing 0.5mol L)^-1Sucrose, 20% serum, balance DPBS); thawing solution III (containing 0.25mol L)^-1Sucrose, 20% serum, balance DPBS); thawing solution IV (20% serum, balance DPBS).

(3) The cryopreservation liquid and the formula of the comparative example are used for preserving the oocyte of the mouse

Cryopreservation of mouse oocytes was performed using cryopreservation solutions 1 and 2 prepared in the above steps, and cryopreservation solution and cryoequilibrium solution of comparative example 1. The oocyte cryopreservation method comprises the steps of firstly placing oocytes in a cryopreservation liquid for balancing for 5 minutes, then placing the oocytes in the prepared cryopreservation liquid for 50 seconds, placing the oocytes which are balanced in the cryopreservation liquid on a freezing carrying rod, then quickly putting the oocytes into liquid nitrogen at (-196 ℃) and continuing to preserve the oocytes after the carrying rod is sealed; during thawing, placing the cryopreserved oocytes in a thawing solution I at 37 ℃ for 5 minutes, and sequentially balancing in the thawing solutions II-IV for 3 minutes respectively; the number of viable cells was observed after culturing the thawed oocytes for 2 hours. After thawing, the oocytes were cultured for 2 hours to calculate the survival rate of the oocytes (Table 1).

(4) Mouse embryo preservation by using the above cryopreservation liquid and comparative formula

The embryos of the mice are subjected to cryopreservation by using the cryopreservation liquids 1 and 2 prepared in the above steps, and the cryopreservation liquid and the frozen equilibrium liquid in the comparative example 1. The embryo cryopreservation method comprises the steps of firstly placing the embryo in a cryopreservation balance liquid for 5 minutes, then placing the embryo in the prepared cryopreservation liquid for 50 seconds, placing the embryo balanced in the cryopreservation liquid on a freezing carrying rod, then quickly putting the embryo into liquid nitrogen at (-196 ℃) and continuing to preserve after sealing the carrying rod; when unfreezing, placing the frozen embryo in the unfreezing liquid I at 37 ℃ for balancing for 5 minutes, and then balancing in the unfreezing liquids II-IV for 3 minutes respectively in sequence; the thawed embryos were cultured for 2 hours before observing the number of viable cells. After thawing, embryos were cultured for 2 hours to calculate survival (table 2).

In the examples, the survival rate is the average survival rate of 3-12 repeated experiments.

TABLE 1 cryopreservation survival rates of oocytes from mice

Numbering	Balancing liquid	Refrigerating fluid	Thawing solution	Total number of frozen eggs	Survival rate after 2 hours
						Comparative example 1	A	Cryopreservation liquid a	B	146	95％
Application example 1	A	Cryopreservation liquid 1	B	93	96.2％
						Application example 2	A	Cryopreservation liquid 2	B	48	90％

TABLE 2 cryopreservation survival rates of mouse embryos

Numbering	Balancing liquid	Refrigerating fluid	Thawing solution	Total number of embryos	Survival rate after 2 hours
						Comparative example 2	A	Cryopreservation liquid a	B	38	94.3％
Application example 3	A	Cryopreservation liquid 1	B	41	95.9％

The data in tables 1 and 2 show that the polypeptides of the present invention are used for cryopreservation of oocytes and embryos, and that the survival rate of oocytes and embryos of the existing commercial cryopreservation solution (DMSO content 15%) can be achieved by adding only a small amount of DMSO (7.5%), and the data of application example 1 and application example 3 show that TR polypeptides have more excellent effects for cryopreservation of oocytes and embryos.

Example 9: stem cell cryopreservation

Cryopreservation liquid 1: dissolving 28g of TR in 25mL of PBS by ultrasonic treatment in a total volume of 100mL, and adjusting the pH to 7.0 to obtain a solution 1; ultrasonically dissolving 0.05mol of sucrose in 25mL of DPBS, sequentially adding 10mL of ethylene glycol and 7.5mL of DMSO to obtain a solution 2 after the sucrose is completely dissolved, uniformly mixing the two solutions after the solution 1 and the solution 2 are returned to room temperature, adjusting the pH value, fixing the volume to 80% of the total volume by adopting the DPBS, and finally adding 20mL of serum before use.

Cryopreservation liquid 3: dissolving 28g of TR in 25mL of PBS by ultrasonic treatment in a total volume of 100mL, and adjusting the pH to 7.0 to obtain a solution 1; ultrasonically dissolving 0.05mol of sucrose in 25mL of DPBS, adding 10mL of ethylene glycol to obtain a solution 2 after the sucrose is completely dissolved, uniformly mixing the two solutions when the solution 1 and the solution 2 are restored to room temperature, adjusting the pH value, fixing the volume to 80% of the total volume by using the DPBS, and finally adding 20mL of serum before use.

Comparative example 2:

cryopreservation liquid b: each 1mL of the culture medium contained 10% (v/v) DMSO, 15% (v/v) serum, and the balance a-MEM (USA, Invitrogen, C12571500 BT).

The cryopreservation liquid is adopted to perform cryopreservation on the human umbilical cord mesenchymal stem cells according to the scheme in table 3. The cryopreservation method of the human umbilical cord stem cells is specifically a microdroplet method: digesting human umbilical cord mesenchymal stem cells on a culture dish for 2 minutes by using 25% pancreatin, putting the cells into an isometric culture solution (10% FBS + a-MEM culture medium), gently blowing and beating until the stem cells completely fall off, adding the cells into a 1.5ml centrifuge tube, centrifuging for 5 minutes at 1000rmp, discarding supernatant, separating the cells from the culture medium, adding 10uL of refrigerating fluid to the bottom of the centrifuge tube, gently blowing and beating to disperse stem cell clusters, placing the 10uL of refrigerating fluid with the stem cells on a freezing slide, and freezing and storing the freezing slide in liquid nitrogen (at the temperature of minus 196 ℃). When thawing, the frozen rods with cells and freezing fluid were placed directly in 37 ℃ a-MEM medium and thawed. After thawing, trypan blue staining was used to check for viability, and the number of cells was counted using the apparatus JIMBIO-FIL (see table 3).

TABLE 3 cryopreservation survival rate of human umbilical cord mesenchymal stem cells

Numbering	Cryopreservation liquid	Cryopreservation method	Survival rate
				Application example 4	Cryopreservation liquid 1	Micro-drop method	87.8％
Application example 5	Cryopreservation liquid 3	Micro-drop method	75.1％
				Comparative example 3	Cryopreservation solution b	Micro-drop method	76.6％

According to the results in table 3, it can be seen that when the cryopreservation solution of the present invention is added with no DMSO or only a small amount of DMSO (7.5%), the cell survival rate equivalent to that of the cryopreservation solution added with 10% DMSO in the prior art can be achieved, the amount of DMSO is greatly reduced, the damage and toxicity of DMSO to cells are reduced, and the passage stability and cell activity of the frozen stem cells can be greatly improved.

Example 10: cryopreservation of ovarian organs and ovarian tissues

Cryopreservation liquid 1: dissolving 28g of TR in 25mL of PBS by ultrasonic treatment in a total volume of 100mL, and adjusting the pH to 7.0 to obtain a solution 1; ultrasonically dissolving 0.05mol of sucrose in 25mL of DPBS, sequentially adding 10mL of ethylene glycol and 7.5mL of DMSO to obtain a solution 2 after the sucrose is completely dissolved, uniformly mixing the two solutions after the solution 1 and the solution 2 are returned to room temperature, adjusting the pH value, fixing the volume to 80% of the total volume by adopting the DPBS, and finally adding 20mL of serum before use.

Cryopreservation liquid a: each 1mL of the mixture contained 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) serum, 0.5M sucrose, and the balance DPBS.

Freezing equilibrium liquid A: every 1mL of the reagent contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) serum and the balance of DPBS;

thawing solution B: thawing solution I (containing 1.0mol L)^-1Sucrose, 20% serum, balance DPBS); thawing solution II (containing 0.5mol L)^-1Sucrose, 20% serum, balance DPBS); thawing solution III (containing 0.25mol L)^-1Sucrose, 20% serum, balance DPBS); thawing solution IV (20% serum, balance DPBS).

The complete ovarian organs of the mice and the ovarian tissue sections of the sexually mature mice within 3 days of the newborn period are respectively subjected to cryopreservation by using the cryopreservation liquid, the cryoequilibrium liquid of the comparative example and the cryopreservation liquid according to the schemes in tables 4 and 5.

And (3) balancing the whole ovarian organ or ovarian tissue section in a freezing balancing liquid at room temperature for 25 minutes, then placing the whole ovarian organ or ovarian tissue section in the prepared freezing preservation liquid for 15 minutes, then placing the whole ovarian organ or ovarian tissue section on a freezing carrying rod, and putting the whole ovarian organ or ovarian tissue section into liquid nitrogen for preservation. After thawing, the whole ovarian organ or tissue section is placed in culture medium (10% FBS + a-MEM) and then incubated at 37 deg.C with 5% CO₂After 2 hours of resuscitation in the incubator, morphology was observed by fixing with 4% paraformaldehyde, paraffin embedding, and HE staining, and the results are shown in fig. 7 to 12, fig. 7 is a photograph of a fresh unfrozen section of an ovarian organ, and fig. 10 is a photograph of a fresh section of an unfrozen ovarian tissue.

TABLE 4 ovarian organ cryopreservation protocol

Numbering	Balancing liquid	Cryopreservation liquid	Thawing solution	Form of the composition
					Application example 6	A	Cryopreservation liquid 1	B	FIG. 9
Comparative example 4	A	Cryopreservation liquid a	B	FIG. 8

TABLE 5 ovarian tissue cryopreservation protocol

Numbering	Balancing liquid	Cryopreservation liquid	Thawing solution	Form of the composition
					Application example 7	A	Cryopreservation liquid 1	B	FIG. 12
Comparative example 5	A	Cryopreservation liquid a	B	FIG. 11

According to fig. 7-9, it can be seen that, compared with comparative example 4 using the bionic ice-control material without adding peptides and fresh unfrozen ovarian organs, the slice photographs of the thawed ovarian organs preserved by freezing using the application example 6 show that the follicle structure is relatively complete, the mesenchymal structure is relatively complete, the cytoplasm is homogeneous, the light staining is relatively more, and the nucleus is shrunk and the deep staining is relatively less; the vessel wall structure is complete, the collapse of the vessel cavity is less, the cytoplasm of endothelial cells is homogeneous, the light staining is relatively more, and the nucleus is shrunk and the deep staining is relatively less. It can be seen that the application example 6 group had better cryopreservation effect on ovarian organs.

As can be seen from FIGS. 10 to 12, the protocol of example 7 was applied to the ovarian tissue of the adult mice which had not been frozen, and the follicles and antrum follicles in the growth phase were relatively intact as compared with the ovarian tissue of the fresh non-frozen adult mice, and it was found that the cryopreservation solution of the present invention was also effective in cryopreservation of the ovarian tissue as compared with the prior art.

Therefore, the cryopreservation liquid prepared by taking the peptide bionic ice control material as the main component can be simultaneously suitable for cryopreservation of oocytes, embryos, stem cells, reproductive organs and tissues, and can achieve good cell survival rate and biological activity.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A peptide compound is prepared by reacting an ice-philic amino acid with a hydrophilic amino acid, or Gluconolactone (GDL) or other saccharides.

2. The peptide compound according to claim 1, wherein said ice-philic amino acid is selected from at least one of threonine (L-Thr), glutamine (L-Gln), and aspartic acid (L-Asn), and said hydrophilic amino acid is selected from at least one of arginine, proline, and alanine;

preferably, the peptide compound is a polypeptide compound having two or more amino acid units, such as: 2-8 amino acid units, and the polypeptide compound contains more than two amino acid units.

3. The peptidic compound of any of claims 1 or 2, wherein the polypeptide compound is at least one of L-Thr-L-Arg (tr), L-Thr-L-Pro (tp), L-Arg-L-Thr (rt), L-Pro-L-Thr (pt), L-Thr-L-Arg-L-Thr (trt), L-Thr-L-Pro-L-Thr (tpt), L-Ala-L-Thr (aat), L-Thr-L-Cys-L-Thr (tct).

4. The peptide compound according to any one of claims 1 to 3, which has any one of the structures represented by formula (1) to formula (8):

5. a peptide compound represented by the structure:

wherein R is selected from substituted or unsubstituted alkyl, and the substituent can be selected from-OH, -NH₂、-COOH、-CONH₂Etc., e.g., R is substituted or unsubstituted C_1-6Alkyl, preferably R is-CH₃、-CH₂CH₃、-CH₂CH₂COOH; n is an integer of 1 or more and 1000 or less,

preferably, the compound represented by the formula (9) has a structure represented by any one of the following formulae:

6. use of a peptide compound according to any of claims 1 to 5 for controlling ice crystal growth in an aqueous solution.

7. Use of a peptide compound according to any of claims 1 to 5 for the preparation of a cryopreservation solution for cells or tissues, preferably in combination with other ice control materials selected from at least one of PVA, amino acids, polyamino acids and the like.

8. A cryopreservation solution comprising a peptide compound according to any one of 1 to 5; preferably, the peptide compound is contained in an amount of 0.1 to 50g per 100mL of the cryopreservation solution volume;

preferably, the adhesive further contains one or more of PVA, amino acid and polyamino acid;

preferably, it may also contain serum;

preferably, the peptide compound comprises 0.1-50g of peptide compound, 0-6.0g of PVA, 0-9.0g of polyamino acid, 0-15mL of DMSO, 5-45mL of polyalcohol and 0.1-1.0mol of L per 100mL^-10-30mL of serum, and the balance of buffer solution;

preferably, it contains 0.1-15mL of DMSO, e.g., 0.1-10mL of DMSO;

preferably, the cryopreservation solution contains 0.1 to 6.0g of PVA, for example 0.1 to 4.0g of PVA;

preferably, the cryopreservation solution contains 5.0 to 30mL of serum, for example 5.0 to 20mL of serum.

9. The cryopreservation fluid of claim 8, wherein the PVA is selected from one or a combination of two or more of isotactic PVA, syndiotactic PVA and atactic PVA, for example the PVA has a degree of syndiotacticity of 15% to 60%, particularly for example 50% to 60%, 50% to 55%;

preferably, the polyamino acid is selected from at least one homopolymer of lysine, arginine, proline, threonine, histidine, etc., or a copolymer of two or more amino acids;

preferably, the polyhydric alcohol is a polyhydric alcohol having 2 to 5 carbon atoms, such as any of ethylene glycol, propylene glycol, and glycerin;

preferably, the water-soluble sugar is at least one of non-reducing disaccharide, water-soluble polysaccharide and anhydrosugar, and is selected from sucrose, trehalose, water-soluble cellulose (e.g., hydroxypropyl methylcellulose, etc.), polysucrose;

preferably, the buffer is selected from at least one of DPBS or hepes-buffered HTF buffer or other cell culture medium buffer;

preferably, the serum is selected from human serum albumin or a substitute thereof for human-derived cryopreserved subjects, such as Sodium Dodecyl Sulfate (SDS); fetal bovine serum or bovine serum albumin can be selected for the non-human-derived cryopreservation object.

10. A method of preparing a cryopreservation solution according to claim 8 or 9 comprising dissolving the peptide compound in a buffer, adjusting the pH after cooling to room temperature, dissolving the other components in a further buffer, mixing after cooling, adjusting the pH, bringing to a predetermined volume with the buffer, optionally adding serum at the time of use,

preferably, the method comprises the following steps:

(1) dissolving a peptide compound in a part of buffer solution, cooling to room temperature, and adjusting the pH value to obtain a solution 1;

(2) dissolving water-soluble sugar in a part of buffer solution, and adding other components after the water-soluble sugar is completely dissolved to prepare a solution 2;

(3) optionally, dissolving PVA and/or polyamino acid in another part of buffer solution, cooling to room temperature, and adjusting pH to obtain solution 3;

(4) and (3) cooling the solution 1, the solution 2 and optionally the solution 3 to room temperature, mixing, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the cryopreservation solution.

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