CN111789100A - 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
- Publication number
- CN111789100A CN111789100A CN201910281982.9A CN201910281982A CN111789100A CN 111789100 A CN111789100 A CN 111789100A CN 201910281982 A CN201910281982 A CN 201910281982A CN 111789100 A CN111789100 A CN 111789100A
- Authority
- CN
- China
- Prior art keywords
- cryopreservation
- solution
- pva
- serum
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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 (dimethyl sulfoxide), can achieve the same or even higher cell and tissue survival rate and functional expression stability as the commercialized cryopreservation solution (containing 15% DMSO) when being used for cryopreservation of mouse oocytes and embryos, and have higher preservation efficiency. The cryopreservation liquid is free of DMSO and serum, and further solves the problems that the commercial cryopreservation liquid commonly used in clinic at present is poor in stability due to the fact that the commercial cryopreservation liquid contains serum, and parasitic biological pollutants are easy to bring.
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 the advent of the technology of cryopreservation, cryopreservation has become one of the 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. The vitrification freezing technology can make the liquid inside and outside the cell become glass state directly in the fast freezing process, so as to avoid the damage caused by the formation of ice crystal 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. Dimethyl sulfoxide (DMSO) is a fluxing agent and a permeable cell cryopreservation protective agent commonly used for in vitro cell culture, but DMSO with different concentrations can trigger massive cell apoptosis after the recovery of cryopreserved cells, so that the DMSO has a certain toxic effect on cells, and different types of cells have different sensitivities to DMSO concentrations, so that the application of the 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-10-30mL 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, -NH2、-COOH、-CONH2Etc., e.g., R is substituted or unsubstituted C1-6Alkyl, preferably R is-CH3、-CH2CH3、-CH2CH2COOH; 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 select human serum albumin or a substitute SDS (sodium dodecyl sulfate) thereof aiming at the human-derived cryopreservation object, and can select fetal bovine serum or bovine serum albumin aiming at the non-human-derived cryopreservation object.
According to the cryopreservation liquid disclosed by 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 is composed 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 solution according to the present invention, preferably having 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-1For 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 present 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 of 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 cryopreservation liquid is composed of the following components in volume per 100 mL:
polyproline 0.1-5g
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 for 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 invention, in the step (1), PVA is dissolved in water bath heating; for example, the temperature of the water bath 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 ultrasonic-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 each 100mL volume.
According to the frozen equilibrium liquid of the invention, the PVA content is 0.1-5.0g, for example 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.
The frozen equilibrium liquid according to the invention has a serum content of 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 present invention, the frozen equilibrium solution contains 7.5-15mL of polyol, 10-20mL of serum and the balance of buffer solution per 100mL of volume.
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
5.0-30mL of polyol
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 thereof, for example the PVA has a syndiotacticity of from 15% to 60%, preferably from 45% to 60%, for example from 50% to 55%.
According to the present invention, the PVA may be selected from the group consisting of PVA 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 at least one homopolymer (polymerization degree. gtoreq.2) of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine, etc., 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 liquid is free of DMSO and serum, and further solves the problems that the commercial cryopreservation liquid commonly used in clinic at present is poor in stability due to the fact that the commercial cryopreservation liquid contains serum, and parasitic biological pollutants are brought in. The cryopreservation liquid has the advantages of simple composition, convenient raw material source and low cost.
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.
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%.
The polymerization degree of poly-L-proline adopted in the refrigerating fluid in the embodiment of the invention 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 following formula is adopted to prepare the cryopreservation liquid
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 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 the sucrose is dissolved, uniformly mixing the three solutions when the solutions 1, 2 and 3 are restored to room temperature, adjusting the pH value to 7.0, and using the DPBS to perform constant volume to make up the balance to 100mL of the total volume 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 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 are dissolved in 20mL of DPBS, and the pH is adjusted to 7.1 to obtain a 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: a total volume of 100mL was prepared by dissolving 7.5mL of ethylene glycol in 72.5mL of DPBS, mixing, and adding 20mL of serum when used.
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 mixture contained 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: each 1mL of the mixture contains 7.5% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine 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:
thawing solution 1 #: 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).
Thawing solution 2 #: thawing solution I (containing 1.0mol L)-120mg mL of sucrose (1)-1With the balance being DPBS); thawing solution II (containing 0.5mol L)-1Sucrose, 20mg mL-1With the balance being DPBS); thawing solution III (containing 0.25mol L)-1Sucrose, 20mg mL-1With the balance being DPBS); thawing solution IV (20mg mL)-1With the balance being DPBS).
Thawing solution 3 #: thawing solution I (containing 1.0mol L)-120mg mL of sucrose (1)-110mg mL of PVA (A)-1Polyproline of (a), the balance being DPBS); thawing solution II (containing 0.5mol L)-1Sucrose, 20mg mL-15.0mg mL of PVA (1)-1Polyproline of (a), the balance being DPBS); thawing solution III (containing 0.25mol L)-1Sucrose, 20mg mL-12.5mg mL of PVA (1)-1Polyproline of (a), the balance being DPBS); thawing solution IV (20mg mL)-1With 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 oocyte in the prepared cryopreservation liquid for 1 minute, placing the oocyte on a freezing carrying rod after being balanced in the cryopreservation liquid, then quickly putting the oocyte into liquid nitrogen (minus 196 ℃) and continuing to preserve 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 for balancing for 5 minutes, then placed in the freezing preservation liquid prepared according to the formula for 50 seconds, the embryo balanced in the freezing preservation liquid is placed on the freezing carrying rod, then rapidly 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
| Numbering | 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 | UnfreezingLiquid 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 within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
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-10-30mL 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.
2. The application of claim 1, wherein the amino acid bionic ice control material is selected from one or more of polyamino acid, amino acid and peptide compound with the polymerization degree of more than or equal to 2;
preferably, the peptide compound is polypeptide, glycopeptide derivative or a compound shown in formula (I),
wherein R is selected from substituted or unsubstituted alkyl, and the substituent can be selected from-OH, -NH2、-COOH、-CONH2Etc., for example, R is a substituted or unsubstituted C1-6 alkyl group, preferably R is-CH3、-CH2CH3、-CH2CH2COOH; n is an integer of 1 or more and 1000 or less.
3. Use according to claim 1 or 2, the ice control material being PVA, the PVA being present in an amount of 0.1-6.0 g;
preferably, the polyol may be any of C2-5 polyols, such as ethylene glycol, propylene glycol, glycerol;
preferably, the water-soluble sugar may be at least one of non-reducing disaccharide, water-soluble polysaccharide and anhydrosugar, and is selected from sucrose and trehalose;
preferably, the buffer solution can be at least one of DPBS and hepes-buffered HTF buffer solution;
preferably, the serum can be human serum albumin or SDS as a substitute thereof for human-derived cryopreservation subjects, and can be fetal bovine serum or bovine serum albumin for non-human-derived cryopreservation subjects.
4. The use of any one of claims 1 to 3, wherein the biomimetic ice-controlling material is polyamino acid or amino acid, and the content of the ice-controlling material is 0.01 to 50 g;
preferably, the ice control material is a combination of PVA with amino acids and/or polyamino acids, for example consisting of 0.1-5.0g of PVA and 8.0-35g of amino acids and/or 1.0-9.0 polyamino acids.
5. Use according to any one of claims 1 to 4, wherein the PVA is selected from the group consisting of isotactic PVA, syndiotactic PVA and atactic PVA, in which case the PVA has a syndiotacticity of from 15% to 60%, preferably from 50% to 60%;
preferably, the PVA is selected from the group consisting of PVA having a molecular weight of 10-500kDa or higher;
preferably, the PVA is selected from PVAs having a degree of hydrolysis greater than 80%;
preferably, the polyamino acid is selected from at least one homopolymer (polymerization degree is not less than 2) of lysine, arginine, proline, threonine, histidine, glutamic acid, aspartic acid, glycine and the like or copolymer of more than two amino acids;
preferably, the glycopeptide derivative is a molecule comprising Gluconolactone (GDL) and an ice-philic amino acid through chemical bonding, for example: at least one of GDL-L-Thr, GDL-L-Gln, GDL-L-Asn, GDL-L-Phe, GDL-L-Tyr and GDL-L-Thr.
6. Use according to any one of claims 1 to 5, wherein the polyol content is from 6.0 to 28 mL;
preferably, the serum content is 0.
Preferably, the water-soluble sugar content is 0.1-1.0mol L-1；
Preferably, the pH of the cryopreservation solution is 6.5 to 7.6.
7. The use according to any one of claims 1 to 6, wherein the cryopreservation solution is prepared by: dissolving the ice control material in a buffer solution, cooling to room temperature, adjusting the pH value, dissolving other components in the rest buffer solution, cooling and mixing;
preferably, the 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 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.
8. The application of the freezing equilibrium liquid without DMSO in oocyte or embryo cryopreservation is characterized in that the freezing equilibrium liquid contains 0-5.0g of PVA, 5.0-45mL of polyalcohol, 0-30mL of serum and the balance of buffer solution in each 100 mL;
preferably, the PVA content is 0.1 to 4.0 g.
9. The use of claim 8, wherein the frozen equilibrium solution comprises 7.5-15mL of polyol, 10-20mL of serum, and the balance of buffer per 100 mL;
preferably, the frozen equilibrium solution contains 0.5-3.5g of PVA, 7.5-15mL of polyalcohol and the balance of buffer solution per 100 mL.
10. A method of cryopreservation using DMSO-free cryopreservation reagents, wherein the oocyte or embryo to be preserved is first equilibrated in a cryoequilibration solution according to any of claims 8 to 9 for 2 to 5 minutes; then placing the oocyte or embryo which is balanced in the cryopreservation solution of any one of claims 1 to 7 for 1 to 3 minutes, and freezing the oocyte or embryo which is balanced in the cryopreservation solution in liquid nitrogen;
preferably, the serum content is 0, and the frozen equilibrium solution contains 0.5-2.5g of PVA, 7.5-15mL of polyalcohol and the balance of buffer solution in each 100 mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910281982.9A CN111789100B (en) | 2019-04-09 | 2019-04-09 | Application of DMSO-free cryopreservation solution in cryopreservation of oocytes or embryos |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910281982.9A CN111789100B (en) | 2019-04-09 | 2019-04-09 | Application of DMSO-free cryopreservation solution in cryopreservation of oocytes or embryos |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111789100A true CN111789100A (en) | 2020-10-20 |
| CN111789100B CN111789100B (en) | 2022-09-06 |
Family
ID=72805718
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910281982.9A Active CN111789100B (en) | 2019-04-09 | 2019-04-09 | Application of DMSO-free cryopreservation solution in cryopreservation of oocytes or embryos |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111789100B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114451400A (en) * | 2022-01-14 | 2022-05-10 | 君创永晟(东莞)生物科技有限公司 | Application of cryopreservation liquid containing polypeptide in stem cell cryopreservation |
| CN114467917A (en) * | 2022-01-14 | 2022-05-13 | 君创永晟(东莞)生物科技有限公司 | Application of cryopreservation liquid containing polypeptide in cryopreservation of engineering cells and cell lines |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US20160369227A1 (en) * | 2013-12-19 | 2016-12-22 | Fertipro N.V | Methods for cryopreservation of stem cells via slow-freezing |
| CN108207930A (en) * | 2016-12-15 | 2018-06-29 | 中国科学院理化技术研究所 | A kind of cocktail type cryoprotector and its application |
| 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 |
-
2019
- 2019-04-09 CN CN201910281982.9A patent/CN111789100B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102124098A (en) * | 2008-06-27 | 2011-07-13 | 博傲沃德株式会社 | Cryopreservative composition for cell and tissue |
| US20160369227A1 (en) * | 2013-12-19 | 2016-12-22 | Fertipro N.V | Methods for cryopreservation of stem cells via slow-freezing |
| 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 | A kind of cocktail type cryoprotector and its application |
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114451400A (en) * | 2022-01-14 | 2022-05-10 | 君创永晟(东莞)生物科技有限公司 | Application of cryopreservation liquid containing polypeptide in stem cell cryopreservation |
| CN114467917A (en) * | 2022-01-14 | 2022-05-13 | 君创永晟(东莞)生物科技有限公司 | Application of cryopreservation liquid containing polypeptide in cryopreservation of engineering cells and cell lines |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111789100B (en) | 2022-09-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109221082B (en) | Cell cryopreservation liquid, cryopreservation recovery method and application thereof | |
| CN111789108B (en) | Cryopreservation liquid and preparation method thereof | |
| CN111789109B (en) | Amino acid cryopreservation liquid and preparation method thereof | |
| CN111793107B (en) | DMSO-free cryopreservation liquid and preparation method thereof | |
| CA3036391A1 (en) | Compositions and methods for cell cryopreservation | |
| JP4947948B2 (en) | Cell preservation solution | |
| CN111789100B (en) | Application of DMSO-free cryopreservation solution in cryopreservation of oocytes or embryos | |
| CN111789101B (en) | Application of PVA-based cryopreservation liquid in cryopreservation of oocytes or embryos | |
| CN111789098B (en) | Application of amino acid freezing solution in cryopreservation of oocytes or embryos | |
| EP3939428A1 (en) | Serum-free cryopreservation solution and preparation method and application thereof | |
| CN111789103B (en) | Thawing solution for cryopreservation and thawing method | |
| CN111789102B (en) | Application of thawing solution in thawing frozen and preserved oocyte or embryo | |
| AU2020256873B2 (en) | Thawing fluid, preparation method therefor and use thereof | |
| CN114557339A (en) | Embryonic stem cell serum-free preservation solution and preparation method and application thereof | |
| RU2791227C1 (en) | Defrosting liquid, its preparation method and its use | |
| WO2023064216A1 (en) | Composition comprising glass forming agent(s) for use as cryoprotectants and methods of making and using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |