CN111789109A - Amino acid cryopreservation liquid and preparation method thereof - Google Patents
Amino acid cryopreservation liquid and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an amino acid cryopreservation solution and a preparation method thereof, wherein each 100mL of the solution contains: 0.1-50g of amino acid freezing storage bionic ice control material, 5.0-45mL of polyalcohol, 0-15mL of DMSO, 0-30mL of serum and 0.1-1mol L of water-soluble sugar‑1And the balance of buffer solution. The cryopreservation liquid takes the amino acid bionic ice control material as a main component, has low DMSO content and low toxicity, and can achieve the same or even higher cell survival rate as a commercial cryopreservation liquid (containing DMSO 15%). 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 cells and tissues.
Description
The application requires that the patent application number of 201910282417.4, which is filed in 2019, 4, 9 and 9 to the intellectual property office of China, is the priority of the prior application with the name of 'amino acid frozen storage liquid and preparation method thereof'. The foregoing prior application is incorporated by reference into this application in its entirety.
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to an amino acid cryopreservation solution and a preparation method thereof.
Background
Cryopreservation refers to keeping the biological material at ultralow temperature to slow down or stop cell metabolism and division, and to continue to develop once normal physiological temperature is restored. Since the advent, this technique has become one of indispensable research methods in the field of natural science, and has been widely adopted. In recent years, with the increasing pressure of life, human fertility tends to decrease year by year, and fertility preservation is receiving more and more attention, and cryopreservation of human germ cells (sperm and oocyte), gonadal tissue, and the like is an important means for fertility preservation. In addition, as the world population ages, the need for cryopreservation of donated human cells, tissues or organs available for regenerative medicine and organ transplantation is also increasing dramatically. Therefore, how to efficiently store precious cells, tissues and organ resources in a freezing way becomes a scientific and technical problem to be solved urgently.
The most common cryopreservation method currently used is vitrification freezing. The vitrification freezing technology can 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. In addition, the organic solvents such as dimethyl sulfoxide (DMSO) or N, N-Dimethylformamide (DMF) with high concentration (not less than 15%) which are toxic to cells and are used in the existing vitrification freezing method cause toxic and side effects of the cryopreservation reagent to cells, and the survival rate of the frozen and preserved objects after recovery, even the safety and functional expression of filial generation are seriously influenced. 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
The invention provides an amino acid cryopreservation solution, wherein each 100mL of the solution contains: 0.1-50g of amino acid bionic ice control material, 5.0-45mL of polyalcohol, 0-15mL of DMSO, 0-30mL of serum and 0.1-1mol L of water-soluble sugar-1And the balance of buffer solution.
According to the invention, the amino acid bionic ice control material is selected from one or more than two of amino acid or polyamino acid.
As an embodiment of the invention, the amino acid bionic ice control material can be amino acid containing an ice-philic group and a hydrophilic group, or polyamino acid consisting of the amino acid containing the ice-philic group and the amino acid containing the hydrophilic group.
The hydrophilic group is a functional group capable of forming a non-covalent interaction with a water molecule, such as a hydrogen bond, van der waals interaction, electrostatic interaction, hydrophobic interaction, or pi-pi interaction with water; can be selected from hydroxyl (-OH), amino (-NH)2) Carboxylic acid group (-COOH), or amide group (-CONH)2) And the like;
the ice-philic group is a functional group that can form a non-covalent interaction with ice, for example, can form a hydrogen bond with ice, van der WaalsElectrostatic, hydrophobic or pi-pi interactions; illustratively, the oxophilic group may be selected from the group consisting of hydroxyl (-OH), amino (-NH)2) Phenyl (-C)6H5) Or pyrrolidinyl (-C)4H8N).
Illustratively, the amino acid bionic ice control material is selected from one or two of arginine, threonine, proline, lysine, histidine, glutamine, aspartic acid, glycine and the like, or polyamino acid consisting of the amino acids; for example, the amino acid bionic ice control material is a combination of arginine and threonine.
In one embodiment of the invention, the amino acid bionic ice control material is polyamino acid (the polymerization degree is not less than 2), preferably the polymerization degree is 2-40 (such as the polymerization degree is 6, 8, 15, 20 and the like), and the polyamino acid is a homopolymer of amino acid, such as one or a combination of more than two of poly-L-proline, poly-L-arginine and the like.
According to the invention, the content of the amino acid bionic ice control material in each 100mL of the cryopreservation solution is 0.5-50g, preferably 1.0-35g, for example, when the amino acid bionic ice control material is amino acid, the content of the amino acid bionic ice control material can be 5.0-35g, preferably 15-25 g; when the amino acid bionic ice control material is polyamino acid, the content of the amino acid bionic ice control material can be 0.5-9.0g, and preferably 1.0-5.0 g.
According to the invention, the polyol may be a polyol having from 2 to 5 carbon atoms, preferably a diol having from 2 to 3 carbon atoms, and/or a triol, such as any of ethylene glycol, propylene glycol, glycerol; ethylene glycol is preferred.
According to the present invention, the water-soluble sugar may be at least one of non-reducing disaccharide, water-soluble polysaccharide, and anhydrosugar, and is selected from sucrose, trehalose, water-soluble cellulose (e.g., hydroxypropylmethyl cellulose, etc.), polysucrose; sucrose is preferred. The water-soluble sugar can play a role in protecting cell membranes and avoiding cell sedimentation.
According to the present invention, the buffer may be selected from at least one of DPBS or hepes-buffered HTF buffer or other cell culture buffer.
According to the invention, the serum can be selected from human serum albumin or a substitute thereof for human-derived cryopreservation subjects, such as: the sodium dodecyl sulfate can be used for selecting fetal bovine serum or bovine serum albumin aiming at non-human-derived cryopreservation objects.
According to the invention, the DMSO content is between 0 and 10mL, preferably between 1.0 and 7.5mL, for example between 1.5 and 5mL, per 100mL of cryopreservation solution; as another embodiment of the present invention, the DMSO content is 0 per 100mL of the cryopreservation solution.
According to the invention, the serum content is 0.1-30mL, such as 5.0-20mL, 10-15mL, per 100mL of cryopreservation solution; as another embodiment of the present invention, the serum content is 0 per 100mL of the cryopreservation solution.
According to the invention, the content of the water-soluble sugar in each 100mL of 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。
According to the present invention, the polyol is contained in an amount of 5.0 to 40mL, for example, 6.0 to 20mL, 9 to 15mL, per 100mL of the cryopreservation solution.
According to the invention, the pH of the cryopreservation solution is from 6.5 to 7.6, for example from 6.9 to 7.2.
According to the present invention, the cryopreservation solution further contains 0.1 to 6.0g of polyvinyl alcohol (PVA) per 100mL of the cryopreservation solution.
According to the invention, the PVA is chosen from isotactic PVA, syndiotactic PVA and atactic PVA in one or a combination of two or more thereof, for example the PVA has a syndiotacticity of from 15% to 65%, in particular for example from 40% to 60%, from 53% to 55%. Atactic PVA is preferred, for example PVA with a syndiotacticity of 45% to 65%.
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%.
In one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL:
0.5-50g amino acid
Polyol 5.0-45mL
DMSO 0-10mL
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 each 100 mL:
L-Arg 2.0-20g
L-Thr 1.0-10g
ethylene glycol 5.0-15mL
DMSO 5.0-10mL
Serum 5.0-20mL
0.1-1.0mol L of cane sugar-1
DPBS margin.
As one embodiment of the present invention, the cryopreservation solution contains the following components per 100mL volume:
0.5-9.0g of polyamino acid
Polyol 5.0-45mL
DMSO 0-10mL
Serum 5.0-20mL
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:
poly-L-proline or poly-L-arginine 1.0-8.0g
Ethylene glycol 5-45mL
DMSO 0.1-10mL
Serum 5.0-20mL
0.1-1.0mol L of cane sugar-1
DPBS margin.
As one embodiment of the present invention, the cryopreservation solution contains the following components per 100mL volume:
0.5-9.0g of polyamino acid
Polyol 5.0-45mL
PVA 0.1-6g
0-20mL 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:
poly-L-proline or poly-L-arginine 1.0-8.0g
Ethylene glycol 5-45mL
PVA 0.1-6g
0-20mL of serum
0.1-1.0mol L of cane sugar-1
DPBS margin.
The invention also provides a preparation method of the cryopreservation liquid, which comprises the following steps:
(1) dissolving the amino acid bionic ice control material in partial buffer solution, and adjusting the pH value to form a solution 1; optionally, dissolving PVA in another part of buffer solution, and adjusting the pH to obtain a solution 2;
(2) dissolving water-soluble sugar in the buffer solution of the third part, and adding other components after the water-soluble sugar is completely dissolved to prepare a solution 3;
(3) and (3) cooling the solution 1, the optional solution 2 and the solution 3 to room temperature, mixing, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the cryopreservation solution.
According to the production method of the present invention, when the cryopreservation liquid contains serum, the serum is added at the time of use of the cryopreservation liquid.
According to the preparation method of the present invention, the PVA is dissolved by heating in a warm bath and stirring, for example, in a water bath or an oil bath; for example, the water bath temperature is 65-85 deg.C, 70-80 deg.C; the stirring is mechanical stirring such as magnetic stirring.
According to the preparation method of the present invention, in the step (2), the dissolution is ultrasonic-assisted dissolution.
The invention also provides a frozen equilibrium liquid, which contains 5.0-45mL of polyalcohol and the balance of buffer solution per 100 mL.
The frozen equilibrium liquid according to the invention, optionally also comprises DMSO 0-15mL, serum 0-30mL, and/or PVA 0-5.0 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 1.0-10mL, 5.0-7.5 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.
The frozen equilibrium liquid according to the invention has a PVA content of 0.1-5.0g, such as 0.1g, 0.5g, 1.0g, 2.0 g; as an embodiment of the present invention, the PVA content is 0.
In the frozen equilibrium liquid of the present invention, the polyhydric alcohol, the serum, and the buffer solution may be selected from the same types as those in the frozen stock solution.
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 100 mL.
In one embodiment of the present invention, the frozen equilibrium solution contains 7.5-15mL of polyhydric alcohol, 10-20mL of serum and the balance of buffer solution per 100 mL.
In one embodiment of the present invention, the frozen equilibrium solution comprises 1.0 to 5.0g of PVA, 7.5 to 15mL of polyol, and the balance of a buffer solution, in 100 mL.
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.
An amino acid reagent for cryopreservation, comprising the above equilibrium solution and the above cryopreservation solution, wherein the equilibrium solution and the cryopreservation solution are independent of each other.
According to the reagent for cryopreservation, the DMSO content in the cryopreservation solution is 0, and the frozen equilibrium solution contains 1.0-5.0g of PVA, 7.5-15mL of polyalcohol, 10-20mL of serum and the balance of buffer solution in each 100 mL; when the DMSO and the serum content in 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 100 mL.
Advantageous effects
The cryopreservation solution provided by the invention takes an amino acid bionic ice control material as a main ice control component, has wide source and good biocompatibility, and the prepared cryopreservation reagent can greatly reduce the using amount of DMSO (dimethyl sulfoxide) and even does not contain DMSO, has low toxicity and can achieve the same or even higher cell survival rate as the conventional commercialized cryopreservation solution containing more than 15% of DMSO. The cryopreservation liquid has simple composition, convenient raw material source and low cost, can be widely applied to cryopreservation of various cells and tissues, such as oocytes, embryos, stem cells, ovarian tissues, ovarian organs and the like, and can keep good biological activity.
Drawings
FIG. 1 is a photograph of a staining of a section of a fresh (unfrozen) ovarian organ;
FIG. 2 is a photograph of a stained section of a cryopreserved whole ovarian organ of comparative example 8 after thawing;
FIGS. 3-6 are photographs showing staining of frozen whole ovarian organs after thawing, in sequence, using examples 14-17;
FIG. 7 is a photograph of a staining of a section of fresh (unfrozen) ovarian tissue;
FIG. 8 is a photograph of a stained section of the cryopreserved ovarian tissue of comparative example 9 after thawing;
FIGS. 9-12 are photographs showing staining of thawed sections of frozen ovarian tissue of examples 18-21, in sequence.
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.
In the embodiment of the invention, poly-L-proline with the polymerization degree of 15 or 8 and the molecular weight of 1475 or 795 is adopted in the frozen preservation solution; the polymerization degree of the poly-L-arginine is 8, and the molecular weight of the poly-L-arginine is 1267. The polymerization degree of poly-L-proline in the thawing solution is 8, and the molecular weight is 795.
In the embodiment of the invention, the survival rate is the average survival rate of 3-12 repeated experiments.
EXAMPLE 1 oocyte and embryo cryopreservation
1. Preparing a cryopreservation solution: the following formula is adopted to prepare the cryopreservation liquid
The total volume of the cryopreservation liquid A is 100 mL: dissolving 16g of L-Arg and 8g of L-Thr in 25mL of DPBS, 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 a cryopreservation solution)-1) Ultrasonically dissolving the mixture in 25mL of DPBS, sequentially adding 10mL of glycol and 10mL of DMSO after all the sucrose is dissolved to obtain a solution 2, uniformly mixing the 2 solutions after the solution 1 and the solution 2 are restored to room temperature, adjusting the pH value to 6.9, fixing the volume by using the DPBS to make up the balance to 80% of the total volume, independently storing 20mL of fetal calf serum, and adding the fetal calf serum before the frozen preservation solution is used.
The total volume of the cryopreservation solution B is 100mL, 1.5g of poly-L-proline (with the polymerization degree of 15) is ultrasonically dissolved in 25mL of DPBS, and the pH is adjusted to 6.8 to obtain a solution 1; ultrasonically dissolving 17g (0.05mol) of sucrose in 25mL of DPBS, sequentially adding 10mL of ethylene glycol and 10mL of DMSO to obtain a solution 2 after the sucrose is completely dissolved, mixing the two solutions when the solution 1 and the solution 2 return to room temperature, adjusting the pH value to 7.0, adding DPBS to a constant volume to make up the balance to 80% of the total volume, storing 20mL of serum separately, and adding the serum before the frozen storage solution is used.
The total volume of the cryopreservation solution C is 100mL, 1.5g of poly-L-arginine (with the polymerization degree of 8) is ultrasonically dissolved in 25mL of DPBS, and the pH is adjusted to be 7.0 to obtain a solution 1; ultrasonically dissolving 17g (0.05mol) of sucrose in 20mL of DPBS, sequentially adding 10mL of ethylene glycol and 10mL of DMSO to obtain a solution 2 after the sucrose is completely dissolved, mixing the two solutions when the solution 1 and the solution 2 return to room temperature, adjusting the pH value to 7.0, adding DPBS to a constant volume to make up the balance to 80% of the total volume, storing 20mL of serum separately, and adding the serum before the frozen storage solution is used.
Cryopreservation liquid D: the total volume is 100mL, 1.5g of poly-L-proline is dissolved in another 20mL of DPBS by ultrasonic waves, and the pH is adjusted to 7.0 to obtain a solution 1; 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 2; 17g (0.05mol) of sucrose (sucrose in a final concentration of 0.5mol L in a cryopreservation solution)-1) Ultrasonically dissolving the mixture in 25mL of DPBS, sequentially adding 10mL of glycol to obtain a solution 3 after all the sucrose is dissolved, uniformly mixing the three solutions when the solutions 1, 2 and 3 are restored to room temperature, adjusting the pH value, and using the DPBS to perform constant volume to make up the balance to 100mL of the total volume for later use.
Cryopreservation liquid E: the total volume is 100mL, 1.5g of poly-L-arginine (with the polymerization degree of 8) is ultrasonically dissolved in another 20mL of DPBS, the pH is adjusted to be 7.0, and the solution is 1; 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 2; 17g (0.05mol) of sucrose (sucrose in a final concentration of 0.5mol L in a cryopreservation solution)-1) Ultrasonically dissolving the mixture in 25mL of DPBS, sequentially adding 10mL of glycol to obtain a solution 3 after all the sucrose is dissolved, uniformly mixing the three solutions when the solutions 1, 2 and 3 are restored to room temperature, adjusting the pH value, and using the DPBS to perform constant volume to make up the balance to 100mL of the total volume 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: heating 2.0g of PVA in a water bath at 80 ℃ and dissolving in 92.5 mL of DPBS by magnetic stirring, adjusting the pH to 7.0 when the PVA is completely dissolved, adding 7.5mL of ethylene glycol, and uniformly mixing 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, and 0.5mol L-1Sucrose 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 composition contains 10% (v/v) ethylene glycol, 20% (v/v) fetal bovine serum and 0.5mol L-1Sucrose 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)-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; when unfreezing, placing the frozen oocyte in the thawing solution I at 37 ℃ for balancing for 5 minutes, and then sequentially balancing in the thawing solutions II-IV for 3 minutes respectively; after culturing the thawed oocytes for 2 hours, the number of surviving cells was observed and the survival rate was calculated (see Table 1).
2. Embryo cryopreservation
The mouse embryo is firstly placed in the freezing balance liquid for balancing for 5 minutes, then placed in the freezing preservation liquid prepared according to the formula for 50 seconds, the embryo balanced in the freezing preservation liquid is placed on the freezing carrying rod, then quickly put into liquid nitrogen (-196 ℃) and continuously preserved after the carrying rod is sealed; when unfreezing, putting the frozen embryo into the unfreezing liquid I at 37 ℃ for balancing for 3 minutes, and then sequentially balancing in the unfreezing liquids II-IV for 3 minutes respectively; the thawed embryos were cultured for 2 hours, and the number of surviving embryos was observed to calculate the survival rate (see table 2).
TABLE 1 cryopreservation survival rates of oocytes from mice
TABLE 2 cryopreservation survival rates of mouse embryos
As can be seen from the data in tables 1 and 2, when the cryopreservation solution disclosed by the invention is used for cryopreservation of oocytes and embryos, the survival rate of the oocytes can reach more than 98%, and the survival rate of the embryos can reach more than 95%, and can reach or even be much higher than the cryopreservation recovery rate of the commercial cryopreservation solution (comparative examples 1-4) containing 15% DMSO and generally used clinically at present, and the cryopreservation effect of the amino acid bionic ice control material added is remarkably superior to that of the cryopreservation solution without the addition of the bionic ice control material in comparative example 2.
Example 2: cryopreservation of human umbilical cord mesenchymal stem cells
1. Preparing a cryopreservation solution: the following formula is adopted to prepare the cryopreservation liquid
Cryopreservation liquid F: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1)4.0g of poly-L-arginine (polymerization degree of 8), 1.0g of PVA, and the balance being DPBS.
Cryopreservation liquid G: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1)4.0g of poly-L-arginine (polymerization degree of 8), 7.5mL of DMSO, and the balance being DPBS.
Cryopreservation liquid H: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1)4.0g of poly-L-proline (polymerization degree of 8), 7.5mL of DMSO, and the balance of DPBS.
Cryopreservation liquid I: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1) 16g of L-Arg, 8g of L-Thr, 7.5mL of DMSO, and the balance being DPBS.
Cryopreservation liquid J: the total volume is 100mL, and the extract contains 25mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1) L-Arg 16g, L-Thr 8g, and the balance DPBS.
The preparation method of the cryopreservation solution was the same 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 4 #: each 1mL of the culture medium contained 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance a-MEM medium (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: after human umbilical cord mesenchymal stem cells on a culture dish are digested for 3 minutes by 25% pancreatin (the digestion time is controlled within 2-3 minutes), putting an isometric culture solution (10% FBS + a-MEM culture medium), gently blowing and beating until all the stem cells fall off, adding a 1.5ml centrifuge tube, centrifuging for 5 minutes at 1000rmp, discarding supernatant (separating cells from supernatant), adding 10 mu L of cryopreservation solution to the bottom of the centrifuge tube, gently blowing and beating to disperse stem cell clusters, placing 10 mu L of cryopreservation solution with stem cells on a cryopreservation slide, and placing the cryopreservation slide on liquid nitrogen (-196 ℃) for cryopreservation. When thawing, the frozen rod with cells and frozen preservation solution is directly placed into a culture medium at 37 ℃ for thawing. 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, even if no or only a small amount of DMSO (7.5%) is used, the survival rate of the stem cells can reach more than 80%, for example, the application examples 9 and 13, and when no DMSO is added at all, the survival rate can reach 92.4% and 71.0%, which shows that the reagent for freezing can not only reach the effectiveness of conventional cryopreservation liquid-cooled lyophilized cells, but also is much higher than the recovery rate of cryopreservation of a cryopreservation solution (comparative example 6) which is commonly used at present and contains 10% of DMSO, and the cryopreservation effect of the amino acid bionic ice control material is obviously better than that of the comparative examples 5 and 6 which do not contain the amino acid ice control material.
Example 3: cryopreservation of ovarian organ and ovarian tissue
1. Preparing a cryopreservation solution: the following formula is used for preparing the cryopreservation liquid and the frozen equilibrium liquid
Cryopreservation liquid K: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1)4.0g of poly-L-arginine (degree of polymerization: 8)1.0g of PVA and the balance of DPBS.
Cryopreservation liquid L: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1)4.0g of poly-L-arginine (polymerization degree of 8), 7.5mL of DMSO, and the balance being DPBS.
Cryopreservation liquid M: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1) 16g of L-Arg, 8g of L-Thr, 7.5mL of DMSO, and the balance being DPBS.
Cryopreservation liquid N: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5mol L) of sucrose-1)4.0g of poly-L-proline (polymerization degree of 8), 7.5mL of DMSO, and the balance of DPBS.
The preparation method of the cryopreservation solution was the same as in example 1.
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 added to 72.5mL of DPBS, mixed well, and used with 20m L serum.
2. Comparative example 3:
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).
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 whole ovarian organ of the mice and the ovarian tissue sections of the sexually mature mice 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 the cells are cultured for 2 hours in an incubator, the cells are fixed by 4% paraformaldehyde, embedded in paraffin, and stained by HE, and the morphology is observed under a microscope, and the results are shown in FIGS. 1 to 12, FIG. 1 is a photograph of staining a section of a fresh unfrozen ovarian organ, and FIG. 7 is a photograph of staining a section of a fresh unfrozen ovarian tissue.
TABLE 4 ovarian organ cryopreservation protocol
TABLE 5 ovarian tissue cryopreservation protocol
As can be seen from fig. 1-6, compared to comparative example 8 using the biomimetic ice-controlling material without amino acids and the fresh unfrozen ovarian organs, the original follicle structure in examples 14-17 is relatively intact, the mesenchymal structure is relatively intact, the cytoplasm of the cells is homogeneous, the light staining is relatively more, and the nucleus is shriveled and the deep staining is relatively less; the vessel wall structure is complete, the collapse of the vessel cavity is less, the cytoplasm of endothelial cells is homogeneous, the light staining is relatively more, and the nucleus is shrunk and the deep staining is relatively less. It can be seen that examples 14-17 are more effective in cryopreserving ovarian organs.
As can be seen from FIGS. 7-12, the protocol of examples 18-21 showed relatively intact follicular and antral follicular structures during the growth phase compared to comparative example 9 and fresh unfrozen ovarian tissue, and it can be seen that the cryopreservation solution of the present invention also has better effects than the prior art for cryopreservation of ovarian tissue.
Therefore, the cryopreservation liquid prepared by taking the amino acid 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 (10)
1. An amino acid-based cryopreservation solution, comprising per 100mL of the solution: 0.1-50g of amino acid bionic ice control material, 5.0-45mL of polyalcohol, 0-15mL of DMSO, 0-30mL of serum and 0.1-1mol L of water-soluble sugar-1And the balance of buffer solution.
2. The cryopreservation liquid of claim 1, wherein the amino acid bionic ice control material is selected from one or more of amino acid or polyamino acid;
preferably, the amino acid bionic ice control material is a polymer formed by amino acid containing an ice-philic group and a hydrophilic group, or the amino acid containing the ice-philic group and the amino acid containing the hydrophilic group; the hydrophilic group is a functional group which can form non-covalent interaction with water molecules, and the ice-philic group is a functional group which can form non-covalent interaction with ice;
preferably, the hydrophilic group can form a hydrogen bond, van der waals interaction, electrostatic interaction, hydrophobic interaction, or pi-pi interaction with water, and the ice-philic group can form a hydrogen bond, van der waals interaction, electrostatic interaction, hydrophobic interaction, or pi-pi interaction with ice;
preferably, theThe hydrophilic group is selected from hydroxyl (-OH), amino (-NH)2) Carboxylic acid group (-COOH), or amide group (-CONH)2) At least one of;
preferably, the oxophilic group is selected from the group consisting of hydroxyl (-OH), amino (-NH)2) Phenyl (-C)6H5) Or pyrrolidinyl (-C)4H8N).
3. The cryopreservation liquid of claim 1 or 2, wherein the amino acid bionic ice-controlling material is selected from one or more of arginine, threonine, proline, lysine, histidine, glutamine, aspartic acid, glycine or a polyamino acid consisting of the amino acids;
preferably, the amino acid bionic ice control material is polyamino acid with the polymerization degree being more than or equal to 2, such as one or a combination of more than two of poly-L-proline and poly-L-arginine.
4. The cryopreservation liquid according to any one of claims 1 to 3 wherein,
the polyhydric alcohol is a polyhydric alcohol with 2-5 carbon atoms, such as any one of ethylene glycol, propylene glycol and glycerol;
preferably, the water-soluble sugar is at least one of non-reducing disaccharide, water-soluble polysaccharide and anhydrosugar, and is selected from sucrose, trehalose, water-soluble cellulose (e.g., hydroxypropyl methylcellulose, etc.), polysucrose;
preferably, the buffer is selected from at least one of DPBS or hepes-buffered HTF buffer or other cell culture buffer.
Preferably, the serum is selected from human serum albumin or a substitute thereof for human-derived cryopreserved subjects, such as: the sodium dodecyl sulfate can be used for selecting fetal bovine serum or bovine serum albumin aiming at non-human-derived cryopreservation objects.
5. The cryopreservation solution of any one of claims 1 to 4 wherein the DMSO content is 1.0 to 10mL per 100mL of the cryopreservation solution;
preferably, the serum content is 10-15mL in each 100mL of the cryopreservation solution;
preferably, the content of the water-soluble sugar in each 100mL of the cryopreservation solution is 0.1-0.8mol L-1；
Preferably, the polyol content is 5.0-40mL per 100mL of the cryopreservation solution.
6. The cryopreservation liquid according to any one of claims 1 to 5, wherein the pH of the cryopreservation liquid is 6.5 to 7.6;
preferably, the cryopreservation solution further contains 0.1 to 6.0g of polyvinyl alcohol PVA per 100 mL.
7. The method for producing a cryopreservation liquid according to any one of claims 1 to 6, comprising the steps of:
(1) dissolving the amino acid bionic ice control material in partial buffer solution, and adjusting the pH value to form a solution 1; optionally, dissolving PVA in another part of buffer solution, and adjusting the pH to obtain a solution 2;
(2) dissolving water-soluble sugar in the buffer solution of the third part, and adding other components after the water-soluble sugar is completely dissolved to prepare a solution 3;
(3) and (3) cooling the solution 1, the optional solution 2 and the solution 3 to room temperature, mixing, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the cryopreservation solution.
Optionally, when the cryopreservation liquid contains serum, the serum is added at the time of use of the cryopreservation liquid.
8. A frozen equilibrium liquid is characterized by comprising 5.0-45mL of polyalcohol and the balance of buffer solution in volume of every 100 mL; optionally further comprising DMSO 0-15mL, serum 0-30mL, and/or PVA 0-5.0 g;
preferably, the DMSO content is 0, and the serum content is 0.1-30mL per 100mL volume;
preferably, the PVA content is 0.1 to 5.0 g.
9. The frozen equilibrium liquid according to claim 8, wherein the polyhydric alcohol is a polyhydric alcohol having 2 to 5 carbon atoms, such as any one of ethylene glycol, propylene glycol, and glycerin;
preferably, the buffer is selected from at least one of DPBS or hepes-buffered HTF buffer, or other cell culture buffer.
Preferably, the serum is selected from human serum albumin or a substitute thereof, such as Sodium Dodecyl Sulfate (SDS), for human cryopreservation subjects, and fetal bovine serum or bovine serum albumin for non-human cryopreservation subjects.
10. An agent for cryopreservation of amino acids, comprising the cryopreservation solution of any one of claims 1 to 6 and the frozen equilibrium solution of claims 8 to 9, the equilibrium solution and the preservation solution being present independently of each other;
preferably, the DMSO content in the cryopreservation solution is 0, and the frozen equilibrium solution contains 0-5.0g of PVA, 7.5-15mL of polyalcohol, 10-20mL of serum and the balance of buffer solution per 100 mL; preferably, when the serum content of the cryopreservation solution is also 0, the frozen equilibrium solution contains 1.0 to 5.0g of PVA, 7.5 to 15mL of polyol and the balance of buffer per 100 mL.
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