CN111789100B - Application of DMSO-free cryopreservation solution in cryopreservation of oocytes or embryos - Google Patents
Application of DMSO-free cryopreservation solution in cryopreservation of oocytes or embryos Download PDFInfo
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- CN111789100B CN111789100B CN201910281982.9A CN201910281982A CN111789100B CN 111789100 B CN111789100 B CN 111789100B CN 201910281982 A CN201910281982 A CN 201910281982A CN 111789100 B CN111789100 B CN 111789100B
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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
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- 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
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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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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Abstract
The invention discloses a DMSO-free cryopreservation solution and application of a freezing equilibrium solution. The cryopreservation solution comprises 0.01-50.0g of bionic ice control material, 5.0-30mL of polyalcohol, 1-30g of water-soluble sugar, 0-30mL of serum and the balance of buffer solution in per 100mL of volume, wherein the bionic ice control material is selected from PVA and/or amino acid bionic ice control material. The cryopreservation solution and the cryopreservation equilibrium solution do not contain DMSO (dimethylsulfoxide), can achieve the same or even higher cell and tissue survival rate and function expression stability as commercial cryopreservation solution (containing 15 percent DMSO) when being used for cryopreservation of mouse oocytes and embryos, and have higher preservation efficiency. The cryopreservation solution is free of DMSO and serum, and further solves the problems that the commercial cryopreservation solution commonly used in clinical practice at present is poor in stability due to the fact that the commercial cryopreservation solution contains serum, and parasitic biological pollutants are easy to bring in.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to an application of oocyte or embryo cryopreservation.
Background
Since its advent, cryopreservation technology has become one of indispensable research methods in the field of natural science, and has been widely adopted. With the improvement of living standard and the development of medical technology, the cryopreservation of human germ cells (sperm and oocyte), gonadal tissues and the like becomes an important means for preserving fertility. 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 cryopreserve precious cell, tissue and organ resources has become an important issue in the field of life sciences.
The most common cryopreservation method currently used is vitrification freezing. Although the vitrification freezing technology can make liquid inside and outside cells directly become glass state in the rapid freezing process, the damage caused by the formation of ice crystal in the freezing process is avoided. However, prior art cryopreservation reagents are not effective in controlling ice crystal growth during rewarming, thereby damaging the cells. Dimethyl sulfoxide (DMSO) is a commonly used fluxing agent and a permeation type cell cryopreservation protective agent for in-vitro cell culture, but DMSO with different concentrations can trigger massive cell apoptosis after recovery of cryopreserved cells, a certain toxic effect is exerted on cells, and different types of cells have different sensitivities to DMSO concentrations, so that the application of a cryopreservation reagent taking DMSO as a main protective agent component is limited. At present, high-concentration (more than or equal to 15%) DMSO is generally used in a vitrification freezing method, but the high-concentration DMSO has high cytotoxicity, and the safety and functional expression of a preserved object (filial generation) are seriously influenced. In conclusion, the prior cryopreservation reagent has the problems of no capability of effectively controlling the growth of ice crystals in the rewarming process and high reagent toxicity.
Disclosure of Invention
To ameliorate the above-mentioned disadvantages of the prior art, the present invention provides the use of a DMSO-free cryopreservation solution for cryopreservation of oocytes or embryos.
The invention provides the following technical scheme:
a DMSO-free cryopreservation solution comprises 0.01-50.0g of bionic ice control material, 5.0-45mL of polyalcohol, and 0.1-1mol L of water soluble sugar per 100mL of volume -1 0-30mL of serum and the balance of buffer solution, wherein the bionic ice control material is selected from PVA and/or amino acid bionic ice control materials.
According to the invention, the amino acid bionic ice control material is selected from one or more of polyamino acid (the polymerization degree is more than or equal to 2, preferably 2-40, for example, the polymerization degree is 6, 8, 15, 20 and the like), amino acid and peptide compound.
According to the invention, the peptidic compounds are polypeptides (preferably peptides consisting of 2-8 amino acids, such as 2 peptides, 3 peptides, 4 peptides), glycopeptide derivatives, compounds of formula (I),
wherein R is selected from substituted or unsubstituted alkyl, and the substituent can be selected from-OH, -NH 2 、-COOH、-CONH 2 Etc., e.g., R is substituted or unsubstituted C 1-6 Alkyl, preferably R is-CH 3 、-CH 2 CH 3 、-CH 2 CH 2 COOH; n is an integer of 1 to 1000 inclusive, and 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.
According to the invention, the polyol may be any one of C2-5, preferably C2-C3, diols, triols, such as ethylene glycol, propylene glycol, glycerol.
According to the invention, the water-soluble sugar may be at least one of a non-reducing disaccharide, a water-soluble polysaccharide, a anhydrosugar, for example selected from sucrose, trehalose. 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 at least one of DPBS and hepes-buffered HTF buffer.
According to the invention, the serum can be selected from human serum albumin or Sodium Dodecyl Sulfate (SDS) as a substitute thereof aiming at human-derived freezing preservation objects, and can be selected from fetal bovine serum or bovine serum albumin aiming at non-human-derived freezing preservation objects.
According to the cryopreservation liquid of the invention, the bionic ice control material can be PVA, and the content of the PVA is 0.1-6.0g, such as 0.5-5.0 g; specifically, it may be 1.0g, 2.0g, 3.0g or 4.0 g.
According to the cryopreservation liquid, the bionic ice control material can be polyamino acid or amino acid, and the content of the polyamino acid or amino acid is 0.01-50g, such as 1.5-50 g; specifically, it may be 8.0g, 10g, 15g, 20g, 30g, or 40 g.
According to the cryopreservation liquid of the invention, the bionic ice control material can be a combination of PVA and polyamino acid, for example, the combination is composed of 0.1-5.0g of PVA and 1.0-9.0g of polyamino acid.
According to the cryopreservation liquid of the invention, the bionic ice control material can be a combination of PVA and amino acid, and consists of 0.1-5.0g of PVA and 8.0-35g of amino acid.
The cryopreservation solution according to the present invention has a polyol content of 6.0 to 28mL, for example, 7.0 to 20mL, 10 to 15mL, per 100 mL.
The cryopreservation solution according to the present invention has a serum content of 0.1 to 30mL, for example, 5.0 to 20mL, 10 to 15mL, per 100 mL.
The cryopreservation liquid according to the invention preferably has a serum content of 0 per 100 mL;
the cryopreservation solution according to the present invention contains 0.1 to 1.0mol L of the water-soluble sugar per 100mL of the cryopreservation solution -1 For example 0.1 to 0.8mol L -1 ，0.2-0.6mol L -1 (ii) a Specifically, for example, 0.25mol L -1 ，0.5mol L -1 ，1.0mol L -1 .
The pH of the cryopreservation solution according to the invention is 6.5 to 7.6, for example 6.9 to 7.2.
As one embodiment of the present invention, the cryopreservation liquid is composed of, per 100mL volume:
PVA 0.01-6.0g
polyol 5.0-45mL
0.1-30mL of serum
0.1-1.0mol L of water-soluble sugar -1
The balance of the buffer solution.
Preferably, the cryopreservation solution is administered per 100mL volume,
the composition consists of the following components:
PVA 1.0-6.0g
ethylene glycol 5-30mL
0.1-20mL of serum
0.2-0.8mol L of cane sugar -1
DPBS margin.
As one embodiment of the present invention, the cryopreservation liquid is composed of, per 100mL volume:
PVA 1.0-5.0g
10-45mL of polyol
0.1-1.0mol L of water-soluble sugar -1
The balance of the buffer solution.
Preferably, it consists of, per 100mL volume:
PVA 1.0-5.0g
10-30mL of ethylene glycol
0.2-0.8mol L of cane sugar -1
DPBS margin.
As one embodiment of the present invention, the cryopreservation liquid is composed of, per 100mL volume:
amino acid 2.0-50g
PVA 0.1-6g
10-30ml of polyol
0.1-1.0mol L of water-soluble sugar -1
10-20ml of serum
The balance of the buffer solution.
Preferably: the cryopreservation liquid is composed of the following components in volume per 100 mL:
L-Arg 5-18g
L-Thr 3-12g
PVA 1-6g
10-20ml of ethylene glycol
0.2-0.8mol L of cane sugar -1
10-20ml of serum
DPBS margin.
As one embodiment of the present invention, the cryopreservation liquid is composed of, per 100mL volume:
0.1-9.0g of polyamino acid
PVA 0.01-6.0g
10-30ml of polyol
0.1-1.0mol L of water-soluble sugar -1
The balance of the buffer solution.
Preferably, the following components: the cryopreservation liquid is composed of the following components in volume per 100 mL:
0.1-5g of polyproline
PVA 1-6g
10-20ml of ethylene glycol
0.2-0.8mol L of cane sugar -1
DPBS margin.
The invention also provides a preparation method of the cryopreservation liquid, which comprises the following steps: dissolving the bionic ice control material in a buffer solution, cooling to room temperature, adjusting the pH value, dissolving other components except the serum in the rest buffer solution, cooling, mixing, confirming or adjusting the pH value again, filling the rest buffer solution to a preset volume, and adding the serum when in use.
The preparation method comprises the following steps:
(1) dissolving PVA in a part of buffer solution, cooling to room temperature, and adjusting pH to obtain a solution 1;
(2) optionally, dissolving the polyamino acid or amino acid in a portion of the buffer, cooling to room temperature and adjusting the pH to form solution 2;
(3) dissolving water-soluble sugar in the other part of buffer solution, and adding other components except serum after the water-soluble sugar is completely dissolved to prepare a solution 3;
(4) and (3) cooling the solution 1, the optional solution 2 and the solution 3 to room temperature, mixing, adjusting the pH value, fixing the volume and complementing the balance of buffer solution to a preset volume to obtain the cryopreservation solution.
According to the production method of the present invention, when the cryopreservation liquid contains serum, the serum is added at the time of use of the cryopreservation liquid.
According to the preparation method of the present invention, in the step (1), PVA is dissolved in water bath heating; for example, the bath temperature is 60 to 95 ℃ and preferably 80 ℃. In the step (1), the dissolving includes a stirring step.
According to the preparation method of the present invention, in the step (2), the dissolution is ultrasound-assisted dissolution.
A frozen equilibrium liquid without DMSO contains 0-5.0g of PVA, 5.0-45mL of polyalcohol, 0-30mL of serum and the balance of buffer solution in every 100mL of volume.
According to the frozen equilibrium liquid of the invention, the PVA content is 0.1-5.0g, such as 0.1g, 0.5g, 1.0g, 2.0 g.
According to the frozen equilibrium liquid of the invention, the polyol content is 6.0-28mL, such as 7.0-20mL, 10-15 mL.
According to the frozen equilibrium liquid of the invention, the serum content is 0.1-30mL, such as 5.0-20mL, 10-15 mL. In one embodiment of the present invention, the serum is present in an amount of 0.
In one embodiment of the invention, the frozen equilibrium solution contains 7.5-15mL of polyalcohol, 10-20mL of serum and the balance of buffer solution based on the volume of 100 mL.
As an embodiment of the present invention, the frozen equilibrium solution contains 1.0 to 5.0g of PVA, 7.5 to 15mL of polyol and the balance of buffer solution per 100mL of volume.
In the frozen equilibrium liquid of the present invention, the PVA, the polyol and the serum may be selected from the group consisting of the corresponding components of the frozen stock solution.
The invention also provides a preparation method of the frozen equilibrium liquid, which comprises the steps of dissolving all the components in a buffer solution, storing serum separately and adding the serum when in use.
A reagent for cryopreservation without DMSO, comprising the above-mentioned equilibrium freezing solution and the above-mentioned cryopreservation solution, wherein the equilibrium freezing solution and the cryopreservation solution are present independently of each other.
According to the reagent for cryopreservation of the present invention, the serum content of the cryopreservation solution is 0, and the frozen equilibrium solution contains 1.0 to 5.0g of PVA, 7.5 to 15mL of a polyhydric alcohol, and the balance of a buffer solution per 100mL of volume.
According to the reagent for cryopreservation of the present invention, the frozen equilibrium solution comprises the following components per 100mL volume:
PVA 0-5.0g
0-15g of polyamino acid
Polyol 5.0-30mL
0-25mL of serum
The balance of buffer solution;
the cryopreservation liquid B comprises the following components in volume per 100 mL:
PVA 0.01-6.0g
0-50g of amino acid or polyamino acid
5.0-30mL of polyol
0-30mL of serum
0.1-1.0mol L of water-soluble sugar -1
The balance of the buffer solution.
According to the invention, the PVA is chosen from isotactic PVA, syndiotactic PVA and atactic PVA in one or a combination of two or more species, for example having a degree of syndiotacticity of from 15% to 60%, preferably from 45% to 60%, for example from 50% to 55%.
According to the invention, the PVA may be selected from the group consisting of PVAs having a molecular weight of 10-500kDa or higher, for example 10-30kDa, 30-50kDa, 80-90kDa, 200-500 kDa.
According to the invention, the PVA may be chosen from those having a degree of hydrolysis greater than 80%, for example a degree of hydrolysis of 80% to 99%, 82% to 87%, 87% to 89%, 89% to 99%, 98% to 99%.
According to the present invention, the polyamino acid may be selected from a homopolymer (degree of polymerization ≥ 2) of at least one of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine, and the like, or a copolymer of two or more amino acids.
According to the invention, the polypeptide may be selected from one or more 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).
According to the present invention, the glycopeptide derivative is a molecule comprising Gluconolactone (GDL) and an oxophilic amino acid by chemical bonding, for example: GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Thr, and the like.
According to the invention, the compound shown in the formula (I) has a structure shown in any one of the following formulas:
in the cryopreservation solution and the freezing equilibrium solution, the dosage of each component is based on the total volume of 100mL of the solution, and the balance is buffer solution.
The invention also provides application of the cryopreservation liquid and the frozen equilibrium liquid in cryopreservation of oocytes or embryos.
The application of the invention comprises that the oocyte or embryo to be preserved is firstly put into the frozen equilibrium liquid to be balanced for 2 to 5 minutes; then placing the oocyte or the embryo which is balanced in the cryopreservation liquid into liquid nitrogen for freezing after the oocyte or the embryo which is balanced in the cryopreservation liquid is balanced for 1 to 3 minutes.
The invention also provides application of the cryopreservation liquid in preparation of a freezing reagent used for cryopreservation of oocytes or embryos.
The invention also provides application of the frozen equilibrium liquid in preparation of a freezing reagent used in cryopreservation of oocytes or embryos.
Advantageous effects
The cryopreservation solution and the cryopreservation equilibrium solution provided by the invention do not contain DMSO (dimethyl sulfoxide), can achieve the same or even higher cell and tissue survival rate and functional expression stability as commercial cryopreservation solution (containing DMSO with the volume concentration of 15%) when being used for cryopreservation of mouse oocytes and embryos, and have higher preservation efficiency. The cryopreservation solution is free of DMSO and serum, and further solves the problems that the commercial cryopreservation solution commonly used in clinic at present is poor in stability due to the fact that the commercial cryopreservation solution contains serum, and parasitic biological pollutants are brought in. The cryopreservation liquid has simple composition, convenient raw material source and low cost.
Detailed Description
The preparation process 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.
The PVA adopted in the embodiment of the invention has the syndiotacticity of 50-55%, the molecular weight of 13-23kDa and the hydrolysis degree of 98%.
In the embodiment of the invention, the polymerization degree of poly-L-proline adopted in the refrigerating fluid is 15, and the molecular weight is 1475. The polymerization degree of poly-L-proline in the thawing solution is 8, and the molecular weight is 795.
In the embodiment of the invention, the survival rate is the average survival rate of 3-12 repeated experiments.
Examples
1. Preparing a cryopreservation solution: the cryopreservation liquid is prepared according to the following formula
The cryopreservation liquid A comprises the following components in every 100 ml:
| substance(s) | Dosage of |
| poly-L-proline (g) | 1.5 |
| PVA(g) | 2.0 |
| Ethylene glycol (mL) | 10 |
| Sucrose (mol L) -1 ) | 0.5 |
| DPBS(mL) | Balance of |
Heating 2.0g of PVA in a water bath at 80 ℃, dissolving in 25mL of DPBS (double layer barium sulfate) by magnetic stirring, and adjusting the pH to 7.0 to obtain a solution 1; ultrasonically dissolving 1.5g of poly-L-proline in another 20mL of DPBS, and adjusting the pH to 7.0 to obtain a solution 2; 17g (0.05mol) of sucrose (sucrose in a final concentration of 0.5mol L in a cryopreservation solution) -1 ) Ultrasonically dissolving the mixture in 25mL of DPBS, sequentially adding 10mL of glycol to obtain a solution 3 after all sucrose is dissolved, uniformly mixing the three solutions when the solutions 1, 2 and 3 are returned to room temperature, adjusting the pH value to 7.0, and adding the balance to the total volume of 100mL by using the DPBS for later use.
The cryopreservation liquid B comprises the following components in every 100 ml:
| substance(s) | Content (wt.) |
| L-Arg(g) | 8.0 |
| L-Thr(g) | 4.0 |
| PVA(g) | 2.0 |
| Ethylene glycol (mL) | 10 |
| Sucrose (mol L) -1 ) | 0.5 |
| Fetal bovine serum (mL) | 20 |
| DPBS(mL) | Balance of |
Preparing a frozen preservation solution: heating 2.0g of PVA in a water bath at 80 ℃, dissolving in 20mL of DPBS (double layer barium sulfate) by magnetic stirring, and adjusting the pH to 7.1 to obtain a solution 1; 8.0g of L-Arg and 4.0g of L-Thr were dissolved in 20mL of DPBS, and the pH was adjusted to 7.1 to obtain solution 2; 17g (0.05mol) of sucrose (sucrose in a final concentration of 0.5mol L in the cryopreservation solution) -1 ) Ultrasonically dissolving the mixture in 20mL of DPBS, and adding 10mL of glycol after all the sucrose is dissolved to obtain a solution 3; and after the solution 1, the solution 2 and the solution 3 are returned to the room temperature, uniformly mixing the three solutions, adjusting the pH value to 7.1, using DPBS to perform constant volume to make up the balance to 80% of the total volume, and adding 20mL of serum when in use.
Cryopreservation liquid C:
every 100ml contains the following components:
| substance(s) | Dosage of |
| PVA(g) | 2.0 |
| Ethylene glycol (mL) | 10 |
| Sucrose (mol L) -1 ) | 0.5 |
| Fetal bovine serum (mL) | 20 |
| DPBS(mL) | Balance of |
Heating 2.0g of PVA in a water bath at 80 ℃, dissolving in 25mL of DPBS by magnetic stirring, and adjusting the pH to 6.9 to obtain a solution 1; 17g (0.05mol) of sucrose (sucrose in a final concentration of 0.5mol L in the cryopreservation solution) -1 ) Ultrasonically dissolving the mixture in 25mL of DPBS, adding 10mL of ethylene glycol to obtain a solution 2 after all the sucrose is dissolved, mixing the two solutions when the solution 1 and the solution 2 are restored to room temperature, adjusting the pH value, fixing the volume and making up the balance to 80% of the total volume, and independently storing 20mL of serum to be added when a preservation solution is used.
Cryopreservation liquid D:
every 100ml contains the following components:
| substance(s) | Dosage of |
| PVA(g) | 2.0 |
| Ethylene glycol (mL) | 10 |
| Sucrose (mol L) -1 ) | 0.5 |
| DPBS(mL) | Balance of |
Heating 2.0g of PVA in a water bath at 80 ℃, dissolving in 30mL of DPBS by magnetic stirring, and adjusting the pH to 7.0 to obtain a solution 1; 17g (0.05mol) of sucrose (sucrose in a final concentration of 0.5mol L in the cryopreservation solution) -1 ) Ultrasonically dissolving the mixture in 25mL of DPBS, adding 10mL of ethylene glycol to obtain a solution 2 after all the sucrose is dissolved, mixing the two solutions when the solution 1 and the solution 2 return to room temperature, adjusting the pH value, and fixing the volume to make up the balance to 100mL for later use.
2. Preparing a frozen equilibrium solution: the freezing equilibrium liquid is prepared according to the following formula
Frozen equilibrium liquid a: heating 2.0g of PVA in a water bath at 80 ℃, dissolving in 50mL of DPBS by magnetic stirring, adjusting the pH to 7.0 when the PVA is completely dissolved, adding 7.5mL of ethylene glycol, uniformly mixing, and metering the volume to 100mL by using the DPBS for later use.
Frozen equilibrium liquid b: the total volume was 100mL, 7.5mL of ethylene glycol was dissolved in 72.5mL of DPBS and mixed well, and 20mL of serum was added at the time of use.
Comparative example:
frozen equilibrium liquid 1 #: each 1mL of the reagent contains 7.5% (v/v) DMSO, 7.5% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum and the balance DPBS;
frozen preservation solution 1 #: each 1mL of the suspension contains 15% (v/v) DMSO, 15% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, and the balance DPBS.
Frozen equilibrium liquid b: every 1mL of the mixture contains 7.5% (v/v) of glycol, 20% (v/v) of fetal calf serum and the balance of DPBS;
frozen preservation solution # 2: each 1mL of the mixture contained 10% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum, 0.5M sucrose, and the balance DPBS.
The recipes of the thawing solution adopted in the examples and the comparative examples of the present invention are three:
unfreezing liquid 1 #: thawing solution I (containing 1.0mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution II (containing 0.5mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution III (containing 0.25mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution IV (20% blood)Clear, balance DPBS).
Thawing solution 2 #: thawing solution I (containing 1.0mol L) -1 20mg mL of sucrose (1) -1 With the balance being DPBS); thawing solution II (containing 0.5mol L) -1 Sucrose, 20mg mL -1 The balance being DPBS); thawing solution III (containing 0.25mol L) -1 Sucrose, 20mg mL -1 With the balance being DPBS); thawing solution IV (20mg mL) -1 With the balance being DPBS).
Thawing solution # 3: thawing solution I (containing 1.0mol L) -1 20mg mL of sucrose (1) -1 10mg mL of PVA (A) -1 The balance being DPBS); thawing solution II (containing 0.5mol L) -1 Sucrose, 20mg mL -1 5.0mg mL of PVA (1) -1 The balance being DPBS); thawing solution III (containing 0.25mol L) -1 Sucrose, 20mg mL -1 2.5mg mL of PVA (1) -1 The balance being DPBS); thawing solution IV (20mg mL) -1 With the balance being DPBS).
Application example:
the cryopreservation of oocytes and embryos was performed using the cryo-equilibration fluid and the cryopreservation fluid of the above examples and comparative examples according to the protocols in tables 1 and 2, respectively.
1. Cryopreservation of oocytes
The mouse oocyte is firstly put into a frozen equilibrium liquid to be balanced for 5 minutes; then placing the oocytes in the prepared cryopreservation liquid for 1 minute, placing the oocytes which are balanced in the cryopreservation liquid on a freezing carrying rod, then quickly putting the oocytes into liquid nitrogen (-196 ℃) and continuing to store after sealing the carrying rod; placing the frozen oocytes in the thawing solution I at 37 ℃ for balancing for 5 minutes, and then sequentially balancing in the thawing solutions II-IV for 3 minutes respectively; after culturing the thawed oocytes for 2 hours, the number of surviving cells was observed and the survival rate was calculated (see Table 1).
2. Embryo cryopreservation
The mouse embryo is firstly placed in the freezing balance liquid to be balanced for 5 minutes, then placed in the freezing preservation liquid prepared according to the formula for 50 seconds, the embryo which is balanced in the freezing preservation liquid is placed on the freezing carrying rod, then quickly put into liquid nitrogen (-196 ℃) and continuously preserved after the carrying rod is sealed; placing the frozen embryos in the thawing solution I at 37 ℃ for balancing for 3 minutes, and then sequentially balancing in the thawing solutions II-IV for 3 minutes respectively; the thawed embryos were cultured for 2 hours, and the number of surviving embryos was observed to calculate the survival rate (see table 2).
TABLE 1 cryopreservation survival rates of oocytes from mice
| Number of | Balancing liquid | Cryopreservation liquid | Thawing solution | Total number of frozen eggs | Survival rate after 2 hours |
| Application example 1 | a | A | Thawing solution 1# | 39 | 89.7％ |
| Application example 1 | a | A | Thawing solution 2# | 60 | 98.6％ |
| Application example 2 | b | B | Thawing solution 1# | 109 | 94.8％ |
| Application example 3 | b | C | Thawing solution 1# | 90 | 97.7％ |
| Application example 4 | a | D | Thawing solution 1# | 50 | 93.4％ |
| Application example 5 | a | D | Thawing solution 3# | 53 | 96.5％ |
| Comparative example 1 | Equilibrium liquid 1# | Freezing fluid 1# | Thawing solution 1# | 96 | 81.9％ |
| Comparative example 2 | b | Refrigerating fluid 2# | Thawing solution 1# | 44 | 94.7％ |
TABLE 2 cryopreservation survival rates of mouse embryos
According to the data of the embodiment and the comparative example, the cryopreservation liquid and the freezing equilibrium liquid without DMSO have good preservation effect for cryopreservation of oocytes and embryos even without adding DMSO through the synergistic effect of the components, and the defect that the addition of DMSO with larger concentration in the existing cryopreservation liquid generates toxicity to the cells or embryos is overcome. Moreover, as can be seen from the application examples 5-8, even if serum is not added at the same time, under the combined action of the bionic ice control material and the osmotic protective agent ethylene glycol, the survival rate of the bionic ice control material can still be better than that of the existing commercial cryopreservation liquid, and the problems that the shelf life of the commercial cryopreservation liquid commonly used in clinic at present is short and parasitic biological pollutants can be brought in due to the serum are further solved.
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 without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (23)
1. The application of the cryopreservation liquid without DMSO (dimethyl sulfoxide) in cryopreservation of oocytes or embryos is characterized in that the cryopreservation liquid contains 0.01-50.0g of bionic ice control material, 5.0-45mL of polyalcohol and 0.1-1mol L of water-soluble sugar in volume of every 100mL -1 0-30mL of serum and the balanceThe bionic ice control material is a buffer solution, and the bionic ice control material is PVA or PVA and amino acid bionic ice control material; the PVA is selected from PVA with molecular weight more than 10kDa and less than or equal to 500 kDa;
the PVA is random PVA, and the syndiotacticity of the PVA is 15-60%;
the PVA is selected from PVA with a degree of hydrolysis greater than 80%;
the amino acid bionic ice control material is selected from one or more than two of polyamino acid, amino acid and peptide compounds with the polymerization degree of more than or equal to 2;
the peptide compound is polypeptide, glycopeptide derivative or a compound shown in a formula (I),
wherein R is selected from substituted or unsubstituted alkyl, and the substituent is selected from-OH, -NH 2 、-COOH、-CONH 2 N is an integer of 1 or more and 1000 or less;
the polypeptide is selected from one or more than two of L-Thr-L-Arg, L-Thr-L-Pro, L-Arg-L-Thr, L-Pro-L-Thr, L-Thr-L-Arg-L-Thr, L-Thr-L-Pro-L-Thr and L-Ala-L-Thr;
the glycopeptide derivative is at least one selected from GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr, GDL-L-Val and GDL-L-Ser.
2. The use according to claim 1, wherein R is a substituted or unsubstituted C1-6 alkyl group.
3. Use according to claim 2, characterized in that R is-CH 3 、-CH 2 CH 3 、-CH 2 CH 2 COOH。
4. The use of claim 1, wherein the ice control material is PVA, and the PVA content is 0.1-6.0 g.
5. Use according to any one of claims 1 to 4, wherein the polyol is a C2-5 polyol.
6. Use according to claim 5, wherein said polyol is any of ethylene glycol, propylene glycol, glycerol.
7. The use according to any one of claims 1 to 4, wherein the water-soluble saccharide is at least one of a non-reducing disaccharide, a water-soluble polysaccharide, a sugar anhydride.
8. Use according to claim 7, wherein said water-soluble sugar is selected from sucrose, trehalose.
9. The use according to any one of claims 1 to 4, wherein the buffer is at least one of DPBS and hepes-buffered HTF buffer.
10. Use according to any one of claims 1 to 4, wherein said ice control material is a combination of PVA with amino acids and/or polyamino acids, consisting of 0.1 to 5.0g of PVA and 8.0 to 35g of amino acids and/or 1.0 to 9.0g of polyamino acids.
11. Use according to any one of claims 1 to 4, characterized in that the PVA has a syndiotacticity of from 45% to 60%.
12. The use according to claim 1, wherein the polyamino acid is selected from the group consisting of a homopolymer of at least one of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine or a copolymer of two or more amino acids.
13. Use according to any one of claims 1 to 4, wherein the polyol content is from 6.0 to 28 mL.
14. The use according to any one of claims 1 to 4, wherein the serum level is 0.
15. Use according to any one of claims 1 to 4, wherein the water-soluble sugar is present in an amount of 0.1 to 1.0mol L -1 。
16. The use according to any one of claims 1 to 4, wherein the pH of the cryopreservation solution is from 6.5 to 7.6.
17. The use according to any one of claims 1 to 4, wherein the cryopreservation solution is prepared by: dissolving ice-controlling material in buffer solution, cooling to room temperature, adjusting pH, dissolving other components in the rest buffer solution, cooling, and mixing.
18. The use according to claim 17, wherein the cryopreservation fluid is prepared by a method comprising the steps of:
(1) dissolving PVA in a part of buffer solution, cooling to room temperature, and adjusting pH to obtain a solution 1;
(2) optionally, dissolving the polyamino acid or amino acid in a portion of the buffer, cooling to room temperature and adjusting the pH to form solution 2;
(3) dissolving water-soluble sugar in a part of buffer solution, adding other components except for serum after the water-soluble sugar is completely dissolved, and preparing a solution 3;
(4) cooling the solution 1, the optional solution 2 and the solution 3 to room temperature, mixing, adjusting the pH value, and fixing the volume to a preset volume by adopting a buffer solution to obtain the cryopreservation solution;
optionally, when the cryopreservation liquid contains serum, the serum is added at the time of use of the cryopreservation liquid.
19. The application of the freezing equilibrium liquid without DMSO in oocyte or embryo cryopreservation is characterized in that the freezing equilibrium liquid contains 0.1-4.0 g of PVA, 5.0-45mL of polyalcohol, 0-30mL of serum and the balance of buffer solution, wherein each 100mL of the freezing equilibrium liquid is counted; the PVA is selected from PVA with molecular weight more than 10kDa and less than or equal to 500 kDa; the PVA is random PVA, and the syndiotacticity of the PVA is 15-60%; the PVA is selected from the group consisting of PVA having a degree of hydrolysis greater than 80%.
20. Use according to claim 19, wherein the PVA has a syndiotacticity of 45% to 60%.
21. The use of claim 19 or 20, wherein the frozen equilibration fluid comprises 0.5-3.5 g PVA, 7.5-15mL polyol, and the balance buffer per 100 mL.
22. A method of cryopreservation using DMSO-free cryopreservation reagents, wherein an oocyte or embryo to be preserved is first placed in a cryo-equilibration solution according to any of claims 19 to 21 for 2 to 5 minutes; then, the oocyte or embryo which has been equilibrated in the cryopreservation solution of any one of claims 1 to 18 is placed in liquid nitrogen for freezing for 1 to 3 minutes.
23. The method of claim 22, wherein the serum content is 0 and the frozen equilibration fluid comprises 0.5-2.5g PVA, 7.5-15mL polyol, and the balance buffer, per 100 mL.
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| CN102124098A (en) * | 2008-06-27 | 2011-07-13 | 博傲沃德株式会社 | Cryopreservative composition for cell and tissue |
| CN104663649A (en) * | 2015-02-13 | 2015-06-03 | 安徽医科大学 | Human ovocyte cryoprotectant |
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| CN109221082A (en) * | 2018-09-14 | 2019-01-18 | 上海慧存医疗科技有限公司 | Cells frozen storing liquid, cryopreservation resuscitation method and its application |
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| CN102124098A (en) * | 2008-06-27 | 2011-07-13 | 博傲沃德株式会社 | Cryopreservative composition for cell and tissue |
| CN104663649A (en) * | 2015-02-13 | 2015-06-03 | 安徽医科大学 | Human ovocyte cryoprotectant |
| CN108207930A (en) * | 2016-12-15 | 2018-06-29 | 中国科学院理化技术研究所 | Cocktail type cryoprotectant and application thereof |
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