CN111789108B - Cryopreservation liquid and preparation method thereof - Google Patents
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Abstract
The invention discloses a cryopreservation liquid and a preparation method thereof. The cryopreservation liquid takes PVA as a main cryoprotectant component, has low DMSO content and low toxicity, can achieve the same or even higher cell survival rate and functional expression stability as commercial cryopreservation liquid (containing DMSO 15%) when being used for cryopreservation of cells or tissues, particularly oocytes, and has higher preservation efficiency. The cryopreservation liquid has simple composition, convenient raw material source and low cost, and can be widely applied to cryopreservation of various cells, tissues and organs.
Description
The application requires that the patent application number filed in 2019, 4, 9 and 9 to the intellectual property office of China is 201910282416.X, and the invention name is the priority of the prior application of 'PVA-based cryopreservation liquid and a preparation method thereof'. 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 PVA (polyvinyl alcohol) -based cryopreservation liquid and a preparation method thereof.
Background
Cryopreservation refers to keeping cells, tissues or organs at an ultralow temperature to slow down or stop the metabolism and division of cells, and to continue their development once the 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 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. The prior vitrification freezing method uses high-concentration (more than or equal to 15 percent) organic solvents, 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 high reagent toxicity.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a PVA-based cryopreservation liquid and a preparation method thereof.
The invention provides the following technical scheme:
a cryopreservation solution comprises PVA 0.01-6.0g, polyalcohol 5.0-45mL, and water soluble sugar 0.1-1.0mol L per 100mL volume-10-15mL of DMSO, 0-30mL of serum and the balance of buffer.
According to the invention, the PVA is chosen from atactic PVA, for example having a degree of syndiotacticity of 45% to 65%, 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 invention, the polyol may be a polyol having 2 to 5 carbon atoms, preferably a diol or triol having 2 to 3 carbon atoms, such as any of ethylene glycol, propylene glycol and glycerol.
According to the invention, the water-soluble sugar may be at least one of a non-reducing disaccharide, a water-soluble polysaccharide, a sugar anhydride, for example selected from sucrose, trehalose, water-soluble cellulose, polysucrose; sucrose, hydroxypropylmethylcellulose and the like are preferable. The water-soluble sugar can play a role in protecting cell membranes and avoiding cell sedimentation.
According to the invention, the serum can be selected from human serum albumin or an alternative, such as Sodium Dodecyl Sulfate (SDS), for human cryopreservation subjects and fetal bovine serum or bovine serum albumin for non-human cryopreservation subjects.
According to the present invention, the buffer may be at least one of DPBS, hepes-buffered HTF buffer, and other known cell culture buffers.
According to the cryopreservation solution of the present invention, the PVA content is 0.1 to 5.5g, 0.5 to 5.0g, for example, 1.0g, 2.0g, 3.0g, 4.0 g.
According to the cryopreservation solution of the present invention, the polyol content is 6.0 to 40mL, for example, 7.0 to 20mL, 10 to 15 mL.
The cryopreservation solution according to the invention, the DMSO content is 0.1-15mL, such as 1.0-10mL, preferably 1.5-7.5 mL; as another embodiment of the invention, the DMSO content is 0.
The cryopreservation solution according to the invention has a serum content of 0.1-30mL, such as 5.0-20mL, 10-15 mL; as another embodiment of the present invention, the serum level is 0.
The content of the water-soluble sugar in the cryopreservation solution is 0.1-1.0mol L-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 comprises the following components per 100mL volume:
preferably, the cryopreservation liquid comprises the following components in volume per 100 mL:
as one embodiment of the present invention, the cryopreservation liquid comprises the following components per 100mL volume:
preferably: the cryopreservation liquid comprises the following components in volume per 100 mL:
as one embodiment of the present invention, the cryopreservation liquid is composed of, per 100mL volume:
preferably: the cryopreservation liquid is composed of the following components in volume per 100 mL:
according to the present invention, the cryopreservation solution may further comprise 0 to 9.0g of polyamino acid (having a degree of polymerization of 2 or more, e.g., 6, 8, 15, 20, etc.) per 100mL of volume, for example, 1.0 to 6.0g, 2.0 to 5.0g, 3.0g, or 4.0g of polyamino acid. Preferably, the polymerization degree of the polyamino acid is 6 to 25.
According to the present invention, the polyamino acid may be selected from at least one or a polymer of two or more amino acids among lysine, arginine, proline, threonine, and the like.
According to the present invention, the cryopreservation solution may further comprise 0 to 50g of an amino acid per 100mL of volume, for example, 1.0 to 25g, 4.0 to 20g, or 8.0 to 10g of at least one or a combination of two or more amino acids selected from lysine, arginine, proline, threonine histidine, glutamine, aspartic acid, glycine and the like.
The invention also provides a preparation method of the cryopreservation liquid, which 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 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, confirming or adjusting the pH again, and filling the rest 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 production method of the present invention, in the step (1), the PVA is dissolved by heating in a warm bath, such as a water bath or an oil bath; for example, the temperature of the 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 contains PVA 0-5.0g, polyalcohol 5.0-45mL, and buffer solution in balance by volume of 100 mL.
The frozen equilibrium liquid according to the invention also optionally comprises 0-15mL of DMSO and 0-30mL of serum.
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.
According to the frozen equilibrium solution of the invention, the DMSO content is 0.1-15mL, such as 1-10mL, 1.5-7.5 mL; as an embodiment of the invention, the DMSO content is 0.
The frozen equilibrium liquid has the serum content of 0.1-30mL, such as 5.0-20mL and 10-15 mL; in one embodiment of the present invention, the serum is present in an amount of 0.
As an embodiment of the invention, the frozen equilibrium solution contains 5.0-7.5mL of polyalcohol, 5.0-7.5mL of DMSO, 10-20mL of serum and the balance of buffer solution in each volume of 100 mL.
As an embodiment of the invention, the frozen equilibrium liquid comprises 7.5-15mL of polyalcohol, 10-20mL of serum and the balance of buffer solution in each 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.
The PVA, polyol, serum and buffer in the frozen equilibrium solution have the same meanings as in 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 the buffer liquid, storing the serum separately and adding the serum when in use.
A reagent for cryopreservation based on PVA, 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.
In one embodiment of the present invention, the reagent for cryopreservation has a DMSO content of 0, and the frozen equilibrium solution contains 0 to 5.0g of PVA, 7.5 to 15mL of a polyol, 10 to 20mL of serum, and the balance of a buffer solution per 100mL of volume. When the DMSO and the serum content of the cryopreservation solution are both 0, the frozen equilibrium solution contains 1.0-5.0g of PVA, 7.5-15mL of polyalcohol and the balance of buffer solution in each 100mL volume.
According to the reagent for cryopreservation of the present invention, the frozen equilibrium solution comprises the following components per 100mL volume:
the cryopreservation liquid comprises the following components in volume per 100 mL:
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
(1) The cryopreservation liquid provided by the invention takes PVA as a main component, and has the advantages of low DMSO content, low toxicity and good safety.
(2) The random PVA with a specific structure is selected as the main ice control material, and the random PVA has more excellent spreading performance on an ice-water interface, so that the growth of ice crystals can be effectively inhibited, the damage of the ice crystals to cells is reduced, and when the random PVA is used for freezing preservation solution, a good freezing preservation effect can be realized by using a small amount of PVA.
(3) The cryopreservation liquid disclosed by the invention is used for cryopreservation of cells or tissues, particularly oocytes and embryos, and can achieve the same or even higher cell survival rate and functional expression stability as commercial cryopreservation liquid (containing 15% of DMSO) under the combined action of special PVA and other permeability protective agents and non-permeability protective agents in specific proportions, and has higher preservation efficiency.
(4) The cryopreservation liquid has simple composition, convenient raw material source and low cost, and can be widely applied to cryopreservation of various cells (oocytes, embryos, stem cells and the like), tissues and organs.
Drawings
FIG. 1 is a photograph of a fresh ovarian organ section stained;
FIG. 2 is a photograph showing staining of a frozen whole ovarian organ of comparative example 8 after thawing;
FIGS. 3-5 are sequential photographs showing staining of frozen whole ovarian organs after thawing, using examples 13-15;
FIG. 6 is a photograph of a staining of a fresh ovarian tissue section;
FIG. 7 is a photograph showing staining of a thawed section of cryopreserved ovarian tissue of comparative example 9;
FIGS. 8-10 are sequential photographs showing staining of thawed sections of frozen ovarian tissue using examples 16-18.
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%.
In the embodiment of the invention, poly-L-proline with polymerization degree of 8 or 15 and molecular weight of 795 or 1475 is adopted in the cryopreservation liquid; the polymerization degree of poly-L-arginine is 8, and the molecular weight is 1267. The polymerization degree of poly-L-proline in the thawing solution is 8, and the molecular weight is 795.
The survival rate in this example is the average of 3-12 replicates per group.
Example 1: cryopreservation of oocytes and embryos
1. Preparing a cryopreservation solution: the following formula is adopted to prepare the cryopreservation liquid
Cryopreservation liquid a:
each 100mL of the composition contains the following components:
| substance(s) | Content (wt.) |
| PVA(g) | 2.0 |
| Ethylene glycol (mL) | 10 |
| DMSO(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 value to 7.0 to obtain a solution 1 after the PVA is completely dissolved and cooled to room temperature; 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 glycol and 10mL of DMSO 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, fixing the volume to make 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 B:
every 100ml contains the following components:
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, keeping constant volume and supplementing 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:
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.
Cryopreservation liquid E:
every 100ml contains the following components:
| 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 |
Liquid preparation: 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 (with the polymerization degree of 15) 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 the cryopreservation solution)-1) Dissolving the mixture in 25mL of DPBS by ultrasonic, adding 10mL of glycol in sequence to obtain a solution 3 after all the sucrose is dissolved, mixing the 3 solutions uniformly after the solutions 1, 2 and 3 are returned 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: 7.5mL of ethylene glycol and 7.5mL of DMSO were added to 65mL of DPBS, mixed well, and 20m L serum was added at the time of use.
Frozen equilibrium liquid b: 7.5mL of ethylene glycol was dissolved in 72.5mL of DPBS, mixed well and added with 20mL of serum when used.
And (3) freezing the equilibrium solution c, namely heating 2.0g of PVA in a water bath at 80 ℃, dissolving in 50mL of DPBS by magnetic stirring, adjusting the pH value to 7.0 when the PVA is completely dissolved, adding 7.5mL of ethylene glycol, uniformly mixing, adjusting the pH value, and fixing the volume to make up the balance to 100mL for later use.
Comparative example 1:
frozen equilibrium liquid a: 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 2 #: 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 formulations of the thawing solutions used in example 1 and comparative example 1 were as follows:
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 1:
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; 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; 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 5 minutes, then placed in the freezing preservation liquid prepared by the formula in the embodiment or the comparative example for 50 seconds, the embryo which is balanced in the freezing preservation liquid is placed on the freezing carrying rod, then the embryo is quickly put into liquid nitrogen (-196 ℃) and is continuously preserved after the carrying rod is sealed; when unfreezing, the embryo is placed in the unfreezing liquid I at 37 ℃ for balancing for 3 minutes, and then the embryos are respectively balanced in the unfreezing liquids II to IV for 3 minutes in sequence; 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
TABLE 2 cryopreservation survival rates of mouse embryos
| Numbering | Balancing liquid | Cryopreservation liquid | Thawing solution | Total number of frozen embryos | Survival rate after 2 hours* |
| Application example 8 | c | D | Thawing solution 1# | 41 | 95.8％ |
| Application example 9 | c | E | Thawing solution 1# | 42 | 95.2％ |
| Comparative example 4 | a | Freezing fluid 1# | Thawing solution 1# | 38 | 94.3％ |
| Comparative example 5 | Equilibrium liquid 2# | Refrigerating fluid 2# | Thawing solution 1# | 39 | 82.4％ |
The data show that the cryopreservation solution can achieve the survival rate of more than 90% and even 100%, and can achieve or far exceed the recovery rate of cryopreservation of the commercial cryopreservation solution containing 15% DMSO and commonly used in clinical practice at present, and the comparison between the application example 1 (containing 10% DMSO) and the comparison example 1, namely the commercial oocyte cryopreservation solution (containing 15% DMSO) shows that the survival rate of oocytes is remarkably improved by adding PVA; application examples 2-3 also show that the PVA-added cryopreservation solution completely free of DMSO can achieve higher oocyte or embryo survival rate, and solves the problems that the commercial cryopreservation solution commonly used in clinic at present is high in DMSO concentration and causes great damage to cells; moreover, application examples 5 and 7-9 show that higher survival rate of oocytes or embryos can be realized under the condition that DMSO and serum are not added in refrigerating fluid, equilibrium fluid and thawing fluid. Solves the problems of short quality guarantee period, parasitic biological pollutant brought by the commercial cryopreservation liquid containing serum which is generally used in clinic at present.
Example 2: stem cell cryopreservation
Cryopreservation liquid C1:
every 100ml contains the following components:
| substance(s) | Dosage of |
| PVA(g) | 1.0 |
| Ethylene glycol (mL) | 10 |
| Sucrose (mol L)-1) | 0.5 |
| Fetal bovine serum (mL) | 20 |
| DPBS(ml) | Balance of |
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 |
Cryopreservation liquid F:
every 100ml contains the following components:
the cryopreservation solution was prepared in the same manner as in example 1.
2. Comparative example 2:
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, and 0.5mol L-1Sucrose and the balance DPBS.
Frozen preservation solution 3 #: each 1mL of the culture medium contained 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance a-MEM (USA, Invitrogen, C12571500 BT).
Application example 2:
cryopreservation of human umbilical cord mesenchymal stem cells was performed using the cryopreservation liquids of the above examples and comparative examples according to the protocol in table 3.
1. Cryopreservation of human umbilical cord mesenchymal stem cells by microdroplet method
The cryopreservation method of the human umbilical cord stem cells used by the invention specifically comprises the following steps: 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 a freezing solution to the bottom of the centrifuge tube, gently blowing and beating to disperse stem cell clusters, placing the 10uL of the freezing solution with the stem cells on a freezing slide, and freezing and storing the freezing slide at (-196 ℃) by using liquid nitrogen. When unfreezing, the freezing carrying rod with the cells and the freezing liquid is directly put into a culture medium at 37 ℃ for unfreezing. 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
When the cryopreservation solution disclosed by the invention is used for cryopreservation of human umbilical cord mesenchymal stem cells, the survival rate of the stem cells can reach 92.4% and 72.2% (application examples 12 and 10) even without DMSO, and the survival rate can reach 77.1% even without DMSO and serum, so that the survival rate of the conventional cryopreservation reagent is reached, which shows that the reagent for freezing can reach the effectiveness of conventional cryopreservation solution for freezing and freeze-drying cells, and even reach or be much higher than the recovery rate of cryopreservation solution (comparative example 7) containing 10% DMSO and commonly used at present, and the cryopreservation effect based on PVA is obviously better than that of comparative example 6 without PVA.
Example 3: freezing and storing complete ovary organ and ovary tissue section
1. Preparing a cryopreservation liquid
Cryopreservation liquid D:
every 100ml contains the following components:
| substance(s) | Dosage of |
| PVA(g) | 2.0 |
| Second twoAlcohol (mL) | 10 |
| Sucrose (mol L)-1) | 0.5 |
| DPBS(mL) | Balance of |
The frozen preservation solution C1 contains the following components in each 100 ml:
cryopreservation liquid F:
every 100ml contains the following components:
| substance(s) | Dosage of |
| poly-L-arginine (g, degree of polymerization 8) | 4.0 |
| PVA(g) | 1.0 |
| Ethylene glycol (ml) | 10 |
| Sucrose (mol L)-1) | 0.5 |
| Fetal bovine serum (mL) | 20 |
| DPBS(ml) | Balance of |
The cryopreservation solution was prepared in the same manner as in example 1.
Preparing a frozen equilibrium solution: the freezing equilibrium liquid is prepared according to the following formula
Frozen equilibrium liquid b: 7.5mL of ethylene glycol was dissolved in 72.5mL of DPBS, mixed well and added with 20mL of serum when used.
And (3) freezing the equilibrium solution c, namely heating 2.0g of PVA in a water bath at 80 ℃, dissolving in 50mL of DPBS by magnetic stirring, adjusting the pH value to 7.0 when the PVA is completely dissolved, adding 7.5mL of ethylene glycol, uniformly mixing, adjusting the pH value, and fixing the volume to make up the balance to 100mL for later use.
2. Comparative example:
frozen equilibrium liquid a: 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, and 0.5mol L-1Sucrose and the balance DPBS.
3. Thawing the liquid:
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).
Application example 3:
the frozen equilibrium solution and the frozen preservation solution of the examples and the comparative examples were used to perform the frozen preservation of the ovary organ of the mouse and the ovary tissue section of the sexually mature mouse within 3 days of the newborn period according to the protocols in tables 4 and 5, respectively.
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 ovarian tissue section was placed in culture medium (10% FBS + a-MEM) and then incubated at 37 ℃ with 5% CO2After 2 hours of recovery culture in the incubator, the morphology was observed by fixing with 4% paraformaldehyde, paraffin embedding and HE staining, and the results are shown in FIGS. 1-10, in which FIG. 1 is a photograph of a section of a fresh unfrozen ovarian organ and FIG. 6 is a photograph of a section of a fresh unfrozen ovarian tissue.
TABLE 4 ovarian organ cryopreservation protocol
TABLE 5 ovarian tissue section cryopreservation protocol
| Numbering | Balancing liquid | Cryopreservation liquid | Thawing solution | Form of the composition |
| Application example 16 | c | D | Thawing solution 2# | FIG. 8 |
| Application example 17 | b | C1 | Thawing solution 1# | FIG. 9 |
| Application example 18 | b | F | Thawing solution 1# | FIG. 10 shows a schematic view of a |
| Comparative example 9 | a | Freezing fluid 1# | Thawing solution 1# | FIG. 7 |
As can be seen from fig. 1 to 5, in the application examples 13 to 15, the original follicle structure was relatively intact, the matrix structure was relatively intact, the cytoplasm was homogeneous, the light staining was relatively more, and the nucleus was shrunken and the deep staining was relatively less, compared to the application example 8 in which polyvinyl alcohol was not added and the application example of the fresh unfrozen ovarian organ; 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 examples 13-15 group had better cryopreservation effect on ovarian organs.
As can be seen from FIGS. 6-10, the protocol of examples 16-18 was compared with that of comparative example 9 and fresh unfrozen adult mouse ovarian tissue, and the follicle and antrum follicle structure in the growth phase were relatively intact, so that the cryopreservation solution of the present invention also has better effects than the prior art in cryopreservation of ovarian tissue.
Therefore, the cryopreservation liquid prepared by taking the PVA bionic ice control material as the main component has a good effect of inhibiting the growth of ice crystals, can reduce the using amount of DMSO in a preservation system, even does not add DMSO, can keep good biocompatibility, 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 (30)
1. A cryopreservation solution characterized by comprising 0.01-6.0g of PVA, 5.0-45mL of a polyhydric alcohol, and 0.1-1.0mol L of a water-soluble sugar per 100mL of the solution-1Comprises 0-15mL of DMSO, 0-30mL of serum and the balance of buffer solution;
the PVA is random PVA, the syndiotactic degree of the PVA is 45-65%, the molecular weight of the PVA is 10-500kDa, and the degree of hydrolysis is more than 80%.
2. The cryopreservation liquid according to claim 1, wherein the DMSO content is 0.5-10 mL.
3. The cryopreservation liquid according to claim 1, wherein the serum content is 10-20 mL.
4. The cryopreservation solution of claim 1 wherein the DMSO and/or serum content is 0.
5. The cryopreservation liquid according to any one of claims 1 to 4, wherein the polyol is a C2-5 polyol.
6. The cryopreservation liquid according to claim 5, wherein the polyhydric alcohol is any one of ethylene glycol, propylene glycol and glycerin.
7. The cryopreservation liquid according to any one of claims 1 to 4, wherein the water-soluble sugar is at least one of a non-reducing disaccharide, a water-soluble polysaccharide and a sugar anhydride.
8. The cryopreservation liquid according to claim 7, wherein the water-soluble sugar is selected from sucrose, trehalose, water-soluble cellulose and polysucrose.
9. The cryopreservation liquid of any one of claims 1 to 4, wherein the buffer is at least one of DPBS, hepes-buffered HTF buffer or other cell culture buffer.
10. The cryopreservation liquid of any one of claims 1 to 4 wherein the PVA content is 0.1 to 5.5 g.
11. The cryopreservation liquid according to any one of claims 1 to 4, wherein the pH of the cryopreservation liquid is 6.5 to 7.6.
12. The cryopreservation liquid according to any one of claims 1 to 4, which is composed of, per 100mL of 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.
13. The cryopreservation liquid according to any one of claims 1 to 4, which is composed of, per 100mL of volume:
PVA 1.0-5.0g
5.0-20mL of polyol
DMSO 0.1-10mL
0.1-20mL of serum
0.2-0.8 mol L of water-soluble sugar-1
The balance of the buffer solution.
14. The cryopreservation liquid according to any one of claims 1 to 4, which is composed of, per 100mL of volume:
PVA 0.1-6.0g
10-45 mL of polyol
0.2-1.0 mol L of water-soluble sugar-1
The balance of the buffer solution.
15. The cryopreservation liquid according to any one of claims 1 to 4, further comprising 0 to 9.0g of polyamino acid having a degree of polymerization of 2 or more per 100mL of volume.
16. The cryopreservation liquid of claim 15 wherein the polymerization degree of the polyamino acid is 2 to 40.
17. The cryopreservation liquid according to any one of claims 1 to 4, further comprising 0 to 50g of an amino acid per 100mL of volume.
18. The method for producing a cryopreservation liquid according to any one of claims 1 to 17, 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 the rest part of buffer solution, and adding other components after the water-soluble sugar is completely dissolved to obtain a solution 3;
(4) and (3) cooling the solution 1, the optional solution 2 and the solution 3 to room temperature, and then mixing to obtain the cryopreservation solution.
19. The method for producing a cryopreservation liquid according to claim 18, wherein when the cryopreservation liquid contains serum, the serum is added at the time of use of the cryopreservation liquid.
20. The frozen equilibrium liquid is characterized by comprising 0.1-5.5g of PVA, 5.0-45mL of polyalcohol and the balance of buffer solution in per 100mL of volume, wherein the PVA is atactic PVA, the syndiotactic degree of the PVA is 45-65%, the hydrolysis degree is more than 80%, and the molecular weight is 10-500 kDa.
21. The frozen equilibrium liquid according to claim 20, further comprising DMSO 0-15 mL.
22. The frozen equilibrium liquid according to claim 20, further comprising 0-30mL of serum.
23. A frozen equilibrium liquid according to any one of claims 20 to 22, characterized in that the PVA content contains 1.0-5.0g PVA per 100mL volume of frozen equilibrium liquid.
24. A frozen equilibrium liquid according to any one of claims 20 to 22, characterized in that the polyol is a C2-5 polyol.
25. The frozen equilibrium liquid according to claim 24, wherein the polyhydric alcohol is a dihydric or trihydric alcohol having the formula C2-C3.
26. The frozen equilibrium liquid according to claim 24, wherein said polyhydric alcohol is any one of ethylene glycol, propylene glycol and glycerol.
27. The frozen equilibrium liquid according to any one of claims 20 to 22, wherein said buffer is at least one of DPBS, hepes-buffered HTF buffer, or other cell culture buffer.
28. A reagent for cryopreservation based on PVA, comprising the frozen equilibrium solution of any one of claims 20 to 27 and the frozen stock solution of any one of claims 1 to 17.
29. The reagent for cryopreservation on based on PVA according to claim 28, wherein when the DMSO content in the cryopreservation solution is 0, the cryoequilibrium solution contains 0.1 to 5.5g of PVA, 7.5 to 15mL of polyol, 10 to 20mL of serum, and the balance of buffer per 100mL of volume.
30. The reagent for cryopreservation on based on PVA according to claim 28 or 29, wherein the cryopreservation solution contains DMSO and serum in both amounts of 0, and the cryoequilibrium solution contains 1.0 to 5.0g of PVA, 7.5 to 15mL of polyol, and the balance of buffer solution per 100 mL.
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