CN111789106B - Application of cryopreservation liquid in organ and tissue cryopreservation - Google Patents
Application of cryopreservation liquid in organ and tissue cryopreservation Download PDFInfo
- Publication number
- CN111789106B CN111789106B CN202010171844.8A CN202010171844A CN111789106B CN 111789106 B CN111789106 B CN 111789106B CN 202010171844 A CN202010171844 A CN 202010171844A CN 111789106 B CN111789106 B CN 111789106B
- Authority
- CN
- China
- Prior art keywords
- solution
- cryopreservation
- pva
- use according
- 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.)
- Active
Links
Images
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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses application of a cryopreservation liquid in organ and tissue cryopreservation. The cryopreservation solution contains PVA, ethylene glycol and sucrose. The cryopreservation liquid takes PVA as a main cryoprotectant component, has low DMSO content and low toxicity, and has higher preservation efficiency when being used for ovarian tissues or organs. 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 application of a cryopreservation liquid in organ and tissue cryopreservation.
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 in the prior art, the invention provides a PVA-based cryopreservation liquid as well as a preparation method and application 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:
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 liquid comprises the following components in volume per 100 mL:
PVA 0.01-6.0g
ethylene glycol 5.0-30mL
10-20mL of serum
0.1-0.6mol L of cane sugar-1
DPBS margin.
As one embodiment of the present invention, the cryopreservation liquid comprises the following components per 100mL volume:
PVA 1.0-5.0g
5.0-20mL of polyol
DMSO 0.1-10mL
0.1-20mL of serum
0.2-0.8mol L of water-soluble sugar-1
The balance of the buffer solution.
Preferably: the cryopreservation liquid comprises the following components in volume per 100 mL:
PVA 1.0-4.0g
ethylene glycol 5.0-15mL
DMSO 4-10mL
10-20mL of serum
0.2-0.6mol L of cane sugar-1
DPBS margin.
As one embodiment of the present invention, the cryopreservation liquid is composed of, per 100mL volume:
PVA 0.1-6.0g
10-45mL of polyol
0.2-1.0mol 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:
PVA 1.0-5.0g
ethylene glycol 5.0-20mL
0.2-0.6mol L of cane sugar-1
DPBS margin.
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:
PVA 0-5.0g
polyol 5.0-45mL
DMSO 0-15mL
0-25mL of serum
The balance of the buffer solution.
The cryopreservation liquid comprises the following components in volume per 100 mL:
PVA 0.01-6.0g
0-9.0g of polyamino acid
Polyol 5.0-45mL
DMSO 0-15mL
0-30mL of serum
0.1-1.0mol L of water-soluble sugar-1
And (6) buffering the allowance.
The present invention also provides the use of the above-mentioned cryopreservation solution, cryoequilibrium solution or reagent for cryopreservation. Specifically, the method comprises the following steps:
the invention provides application of the cryopreservation liquid in organ and/or tissue cryopreservation,
the invention provides the application of the frozen equilibrium liquid in organ and/or tissue cryopreservation,
the invention provides an application of the reagent for cryopreservation in organ and/or tissue cryopreservation,
further, the use of the cryopreservation solution, the frozen equilibrium solution or the reagent for cryopreservation according to the present invention comprises: balancing the organ and/or tissue in the freezing balancing liquid, then putting the organ and/or tissue into the freezing preservation liquid, then putting the organ and/or tissue on a freezing slide and freezing and preserving by liquid nitrogen.
In one embodiment, the organ and/or tissue is ovarian tissue or an ovarian organ, for example, can be an ovarian tissue section or whole ovarian tissue.
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 the advantages of simple composition, convenient raw material source and low cost, and can be widely applied to cryopreservation of various 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:
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, 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:
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.
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; 1.5g of poly-L-proline(degree of polymerization 15) was sonicated in another 20mL of DPBS to adjust the pH to 7.0 to give 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
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# | 67 | 100.0% |
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 | c | D | Thawing solution 1# | 50 | 93.4% |
Application example 5 | c | D | Thawing solution 2# | 53 | 96.5% |
Application example 6 | c | E | Thawing solution 1# | 39 | 89.7% |
Application example 7 | c | E | Thawing solution 3# | 60 | 98.6% |
Comparative example 1 | a | Freezing fluid 1# | Thawing solution 1# | 146 | 95.0% |
Comparative example 2 | Equilibrium liquid 2# | Refrigerating fluid 2# | Thawing solution 1# | 96 | 81.9% |
Comparative example 3 | Equilibrium liquid 2# | Refrigerating fluid 2# | Thawing solution 2# | 44 | 94.7% |
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:
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 |
Serum (ml) | 20 |
DPBS(ml) | Balance of |
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
Numbering | Cryopreservation liquid | Cryopreservation method | Survival rate |
Application example 10 | C1 | Micro-drop method | 72.2% |
Application example 11 | D | Micro-drop method | 77.1% |
Application of the inventionExample 12 | F | Micro-drop method | 92.4% |
Comparative example 6 | Cryopreservation liquid 1# | Micro-drop method | 63.9% |
Comparative example 7 | Cryopreservation liquid 3# | Micro-drop method | 76.6% |
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 |
Ethylene glycol (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:
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 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.
The whole ovary organ or ovary tissue section is firstly put into the frozen equilibrium liquid to be balanced for 25 minutes at room temperature, then put into the prepared frozen preservation liquid for 15 minutes, and then the whole ovary is put intoOrgan or ovary tissue slices are placed on a freezing carrying rod and are put 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
Numbering | Balancing liquid | Cryopreservation liquid | Thawing solution | Form of the composition |
Application example 13 | c | D | Thawing solution 2# | FIG. 3 |
Application example 14 | b | C1 | Thawing solution 1# | FIG. 4 |
Application example 15 | b | F | Thawing solution 1# | FIG. 5 |
Comparative example 8 | a | Freezing fluid 1# | Thawing solution 1# | FIG. 2 |
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 | UnfreezingLiquid 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 (33)
1. The application of the cryopreservation liquid in organ and/or tissue cryopreservation is characterized in that the cryopreservation liquid contains 0.01-6.0g of PVA, 5.0-45mL of polyalcohol and 0.1-1.0mol L of water-soluble sugar in per 100mL of volume of the cryopreservation liquid-10-15mL of DMSO, 0-30mL of serum and the balance of buffer solution;
the PVA is random PVA, the syndiotactic degree is 45-65%, the molecular weight is 10-500kDa, and the degree of hydrolysis is more than 80%.
2. The use of claim 1, wherein the organ is an ovarian organ; the tissue is ovarian tissue section.
3. Use according to claim 1, wherein the DMSO content is 0.5-10 mL.
4. The use of claim 1, wherein the serum content is 10-20 mL.
5. Use according to claim 1, wherein the DMSO and/or serum content is 0.
6. Use according to any one of claims 1 to 5, wherein the polyol is a C2-5 polyol.
7. Use according to claim 6, wherein said polyol is any of ethylene glycol, propylene glycol, glycerol.
8. The use according to any one of claims 1 to 5, wherein the water-soluble saccharide is at least one of a non-reducing disaccharide, a water-soluble polysaccharide, a sugar anhydride.
9. Use according to claim 8, wherein the water-soluble sugar is selected from sucrose, trehalose, water-soluble cellulose or polysucrose.
10. The use according to any one of claims 1 to 5, wherein the buffer is at least one of DPBS, hepes-buffered HTF buffer or other cell culture buffer.
11. Use according to any one of claims 1 to 5, characterized in that the PVA content is between 0.1 and 5.5 g.
12. The use according to any one of claims 1 to 5, wherein the pH of the cryopreservation solution is from 6.5 to 7.6.
13. The use according to any one of claims 1 to 5, wherein the cryopreservation liquid consists 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.
14. The use according to any one of claims 1 to 5, wherein the cryopreservation liquid consists of, per 100mL volume:
PVA 1.0-5.0g
5.0-20mL of polyol
DMSO 0.1-10mL
0.1-20mL of serum
0.2-0.8mol L of water-soluble sugar-1
The balance of the buffer solution.
15. The use according to any one of claims 1 to 5, wherein the cryopreservation liquid consists of, per 100mL volume:
PVA 0.1-6.0g
10-45mL of polyol
0.2-1.0mol L of water-soluble sugar-1
The balance of the buffer solution.
16. The use according to claim 1, wherein the cryopreservation solution further comprises 0-9.0g of polyamino acid having a degree of polymerization of 2 or more per 100mL of volume.
17. The use according to claim 16, wherein the polyamino acid has a degree of polymerization of 2 to 40.
18. The use according to claim 1, wherein the cryopreservation solution further comprises 0-50g of amino acids per 100mL volume.
19. The use according to any one of claims 1 to 5, wherein the cryopreservation solution is prepared by a preparation method comprising the steps of:
(1) dissolving the PVA in a part of buffer solution, cooling to room temperature, and adjusting the pH value to obtain a solution 1;
(2) dissolving the water-soluble sugar in the rest buffer solution, and adding other components after the water-soluble sugar is completely dissolved to prepare a solution 3;
(3) and (3) cooling the solution 1 and the solution 3 to room temperature, and mixing to obtain the cryopreservation liquid.
20. The use according to any one of claims 16 to 18, wherein the cryopreservation solution is prepared by a preparation method comprising the following steps:
(1) dissolving the PVA in a part of buffer solution, cooling to room temperature, and adjusting the pH value to obtain a solution 1; dissolving polyamino acid or amino acid in a part of buffer solution, cooling to room temperature, and adjusting the pH value to form a solution 2;
(2) dissolving the water-soluble sugar in the rest buffer solution, and adding other components after the water-soluble sugar is completely dissolved to prepare a solution 3;
(3) and cooling the solution 1, the solution 2 and the solution 3 to room temperature, and mixing to obtain the cryopreservation liquid.
21. The use according to claim 19, wherein when the cryopreservation liquid contains serum, the serum is added at the time of use of the cryopreservation liquid.
22. The application of the frozen equilibrium liquid in the frozen preservation of organs and/or tissues is characterized in that the frozen equilibrium liquid contains 0.1 to 5.0g of PVA, 5.0 to 45mL of polyalcohol and the balance of buffer solution in per 100mL of volume;
the PVA is random PVA, the syndiotactic degree is 45-65%, the molecular weight is 10-500kDa, and the degree of hydrolysis is more than 80%.
23. The use of claim 22, wherein the frozen equilibration fluid further optionally comprises DMSO 0-15mL, serum 0-30 mL.
24. The use of claim 22, wherein the amount of PVA comprises 1.0-5.0g PVA per 100mL volume of frozen equilibrium solution.
25. The use according to claim 22, wherein the polyol is a C2-5 polyol.
26. The use of claim 25, wherein the polyol is a C2-C3 diol, triol.
27. The use according to claim 26, wherein the polyol is any one of ethylene glycol, propylene glycol and glycerol.
28. The use of claim 22, wherein the buffer is at least one of DPBS, hepes-buffered HTFD buffer, or other cell culture medium buffer.
29. The use of any one of claims 22-28, wherein the organ is an ovarian organ; the tissue is ovarian tissue section.
30. Use of a PVA-based agent for cryopreservation in organ and/or tissue cryopreservation, comprising the cryo-equilibration fluid according to any of claims 22 to 29 and the cryo-preservation fluid according to any of claims 1 to 21.
31. The use according to claim 30, wherein the cryopreservation solution contains 0 part of DMSO, and the frozen equilibrium solution contains 0.1-5.0g of PVA, 7.5-15mL of polyol, 10-20mL of serum, and the balance of buffer solution per 100mL of volume.
32. The use according to claim 30, wherein the cryopreservation solution contains 0% of DMSO and 0% of serum, and the frozen equilibrium solution contains 1.0-5.0g of PVA, 7.5-15mL of polyol and the balance of buffer solution per 100 mL.
33. The use according to any one of claims 30-32, the use comprising: balancing the organ and/or tissue in the freezing balancing liquid, then putting the organ and/or tissue into the freezing preservation liquid, then putting the organ and/or tissue on a freezing slide and freezing and preserving by liquid nitrogen.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910282416X | 2019-04-09 | ||
CN201910282416 | 2019-04-09 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111789106A CN111789106A (en) | 2020-10-20 |
CN111789106B true CN111789106B (en) | 2022-04-15 |
Family
ID=72806403
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010172544.1A Active CN111789108B (en) | 2019-04-09 | 2020-03-12 | Cryopreservation liquid and preparation method thereof |
CN202010171844.8A Active CN111789106B (en) | 2019-04-09 | 2020-03-12 | Application of cryopreservation liquid in organ and tissue cryopreservation |
CN202010171835.9A Active CN111789104B (en) | 2019-04-09 | 2020-03-12 | Application of cryopreservation liquid in stem cell cryopreservation |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010172544.1A Active CN111789108B (en) | 2019-04-09 | 2020-03-12 | Cryopreservation liquid and preparation method thereof |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010171835.9A Active CN111789104B (en) | 2019-04-09 | 2020-03-12 | Application of cryopreservation liquid in stem cell cryopreservation |
Country Status (1)
Country | Link |
---|---|
CN (3) | CN111789108B (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113383767B (en) * | 2020-03-12 | 2022-11-22 | 北京大学第三医院(北京大学第三临床医学院) | Application of frozen stock solution in stem cell freezing |
CN112514892B (en) * | 2020-12-25 | 2022-06-14 | 广州赛莱拉干细胞科技股份有限公司 | Exosome cryopreservation protection solution and preparation method thereof |
CN113229274A (en) * | 2021-05-20 | 2021-08-10 | 郑州优倍得生物科技有限公司 | Induced pluripotent stem cell cryopreservation liquid and cryopreservation method |
CN115399311A (en) * | 2021-05-26 | 2022-11-29 | 北京大学深圳医院 | Vitrification refrigerating fluid set and preparation method and application thereof |
CN114946836B (en) * | 2022-05-25 | 2024-06-04 | 成都诺医德医学检验实验室有限公司 | Micro-tissue serum-free frozen stock solution and application thereof |
CN116458497B (en) * | 2023-06-20 | 2023-09-01 | 细胞生态海河实验室 | Ovarian cell freezing solution and thawing solution and preparation method thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101491237A (en) * | 2009-03-03 | 2009-07-29 | 山东大学 | Use of tetrahydropyridines in cell, tissue, organ cryopreservation |
CN102124098A (en) * | 2008-06-27 | 2011-07-13 | 博傲沃德株式会社 | Cryopreservative composition for cell and tissue |
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 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2207787A1 (en) * | 1972-02-18 | 1973-08-23 | Guy Anders Heyden | Transporting- and storage medium for tissue biopsies - using aq chlorohexidene and buffer |
CN101250499A (en) * | 2008-04-11 | 2008-08-27 | 中山大学 | Stem cell nontoxic frozen stock solution |
CN102726367A (en) * | 2012-06-12 | 2012-10-17 | 宁波市畜牧工程技术研究服务中心 | Low-temperature protective agent of boar semen |
CN104839144A (en) * | 2015-04-30 | 2015-08-19 | 北京大学第三医院 | Vitrification freezing fluid of oocytes |
CN108207930B (en) * | 2016-12-15 | 2021-06-25 | 中国科学院理化技术研究所 | Cocktail type cryoprotectant and application thereof |
CN108235981B (en) * | 2016-12-23 | 2021-07-23 | 西比曼生物科技(香港)有限公司 | Cell cryopreservation liquid capable of being used clinically |
CN107183008A (en) * | 2017-05-27 | 2017-09-22 | 魏方萌 | A kind of placenta mesenchyma stem cell frozen stock solution and its cryopreservation methods |
CN109497044A (en) * | 2018-12-28 | 2019-03-22 | 深圳市昱杰生物科技有限公司 | A kind of Mammalian Embryo frozen stock solution and cryopreservation methods |
-
2020
- 2020-03-12 CN CN202010172544.1A patent/CN111789108B/en active Active
- 2020-03-12 CN CN202010171844.8A patent/CN111789106B/en active Active
- 2020-03-12 CN CN202010171835.9A patent/CN111789104B/en active Active
Patent Citations (3)
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 |
CN101491237A (en) * | 2009-03-03 | 2009-07-29 | 山东大学 | Use of tetrahydropyridines in cell, tissue, organ cryopreservation |
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 |
Non-Patent Citations (2)
Title |
---|
一种玻璃化冷冻保存对小鼠卵巢组织的影响;严晓南 等;《中国比较医学杂志》;20070531;第262-265页 * |
卵巢组织玻璃化冷冻的研究进展;王劲松 等;《中国比较医学杂志》;20141231;第71-74,81页 * |
Also Published As
Publication number | Publication date |
---|---|
CN111789106A (en) | 2020-10-20 |
CN111789104B (en) | 2022-06-17 |
CN111789104A (en) | 2020-10-20 |
CN111789108B (en) | 2022-03-25 |
CN111789108A (en) | 2020-10-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111789106B (en) | Application of cryopreservation liquid in organ and tissue cryopreservation | |
CN111789105B (en) | Application of amino acid cryopreservation liquid in stem cell cryopreservation | |
US10815456B2 (en) | Composition, kit and method for cryopreserving cells | |
CN111793110B (en) | Application of DMSO-free cryopreservation liquid in stem cell cryopreservation | |
CN113925049B (en) | Cell preservation solution for maintaining cell activity and preparation method and application thereof | |
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 | |
WO2020207152A1 (en) | Serum-free cryopreservation solution and preparation method and application thereof | |
CN110115265A (en) | A kind of embryo vitrifying freeze liquid | |
CN111789098B (en) | Application of amino acid freezing solution in cryopreservation of oocytes or embryos | |
CN111789103B (en) | Thawing solution for cryopreservation and thawing method | |
CN111789102B (en) | Application of thawing solution in thawing frozen and preserved oocyte or embryo | |
CN114557339A (en) | Embryonic stem cell serum-free preservation solution and preparation method and application thereof | |
JP7459130B2 (en) | Melt liquid and its preparation method and application | |
JP2023170612A (en) | Cryopreservation solution and cryopreservation method for porcine germ cell | |
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 |