CN111789105B - Application of amino acid cryopreservation liquid in stem cell cryopreservation - Google Patents
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Abstract
The invention discloses an application of amino acid cryopreservation liquid in stem cell cryopreservation, wherein each 100mL of the solution contains: amino acids0.1-50g of bionic ice control material for cryopreservation, 5.0-45mL of polyalcohol, 0-15mL of DMSO, 0-30mL of serum and 0.1-1mol L of water-soluble sugar ‑1 And 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 15 percent of DMSO). 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 filed by the intellectual property office of China in 2019, 4 and 9 is 201910282417.4, and the invention name is the priority of the prior application of 'an amino acid frozen preservative solution and a 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 application of an amino acid cryopreservation solution in stem cell cryopreservation.
Background
Cryopreservation is to store the biological material at ultralow temperature to slow down or stop cell metabolism and division, and to continue development once normal physiological temperature is recovered. Since its advent, this technology 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 ice crystal growth during rewarming, 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, even the safety of filial generation and the functional expression of the frozen preserved objects after recovery 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 liquid and provides application of the cryopreservation liquid in stem cell cryopreservation.
The amino acid-based cryopreservation solution of the present invention contains, 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 -1 And 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, such as a hydrogen bond with ice, van der waals interactions, electrostatic interactions, hydrophobic interactions, or pi-pi interactions; illustratively, the ice-philic group may be selected from the group consisting of hydroxyl (-OH), amino (-NH) 2 ) Phenyl (-C) 6 H 5 ) Or pyrrolidinyl (-C) 4 H 8 N).
Illustratively, the amino acid bionic ice-controlling 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 acid; for example, the amino acid bionic ice control material is a combination of arginine and threonine.
In one embodiment of the present invention, the amino acid-based biomimetic ice-controlling material is polyamino acid (polymerization degree is not less than 2), preferably polymerization degree is 2 to 40 (for example, polymerization degree is 6, 8, 15, 20, etc.), and the polyamino acid is a homopolymer of amino acid, for example, one or a combination of two or more of poly-L-proline, poly-L-arginine, etc.
According to the invention, the content of the amino acid bionic ice control material in every 100mL of the cryopreservation liquid is 0.5-50g, preferably 1.0-35g, for example, when the amino acid bionic ice control material is amino acid, the content can be 5.0-35g, preferably 15-25g; 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 1.0-5.0g.
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 selected from 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 cryopreservation solution.
According to the invention, the serum content is 0.1-30mL, such as 5.0-20mL, further such as 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 frozen product is frozen per 100mLThe content of the water-soluble sugar in the preservation solution is 0.1-1.0mol L -1 For example 0.1 to 0.8mol L -1 ，0.2-0.6mol L -1 (ii) a Specifically, for example, 0.25mol L -1 ，0.5mol L -1 ，1.0mol L -1 。
According to the invention, the polyol is present in an amount of 5.0 to 40mL, for example 6.0 to 20mL,9 to 15mL, per 100mL of 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%. Random PVA is preferred, for example PVA with a syndiotacticity of 45% to 65% of the PVA.
According to the invention, the PVA may be chosen from the group consisting of PVA with a molecular weight of 10-500kDa or higher, for example with a molecular weight of 10-30kDa, 30-50kDa, 80-90kDa, 200-500kDa.
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 100mL:
preferably: the cryopreservation liquid comprises the following components in each 100mL:
as one embodiment of the present invention, the cryopreservation solution contains the following components per 100mL volume:
preferably: the cryopreservation liquid comprises the following components in volume per 100mL:
as one embodiment of the present invention, the cryopreservation solution contains the following components per 100mL volume:
preferably: the cryopreservation liquid comprises the following components in volume per 100mL:
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 when the cryopreservation liquid is used.
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 in each 100 mL.
The frozen equilibration solutions according to the invention further optionally comprise DMSO 0-15mL, serum 0-30mL, and/or PVA 0-5.0g.
According to the frozen equilibrium liquid of the invention, the polyol content is 6.0-28mL, such as 7.0-20mL,10-15mL.
According to the frozen equilibrium solution of the invention, the DMSO content is 0.1-15mL, such as 1.0-10mL,5.0-7.5mL; as an embodiment of the invention, the DMSO content is 0.
The frozen equilibrium liquid according to the invention has a serum content of 0.1-30mL, such as 5.0-20mL,10-15mL; 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, 2.0g; 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.
In one embodiment of the present invention, the frozen equilibrium solution contains, per 100mL, 5.0-7.5mL of polyol, 5.0-7.5mL of DMSO, 10-20mL of serum, and the balance of buffer.
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 buffer solution, calculated as 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.
The invention provides application of the cryopreservation liquid in stem cell cryopreservation.
According to an embodiment of the present invention, the stem cell is a variety of stem cells known in the art to have a differentiation function, such as a pluripotent stem cell, a multipotent stem cell, or a multipotent stem cell, including but not limited to an embryonic stem cell, various types of mesenchymal stem cells (e.g., umbilical cord mesenchymal stem cell, adipose mesenchymal stem cell, bone marrow mesenchymal stem cell, etc.), a hematopoietic stem cell.
According to an embodiment of the invention, the stem cell cryopreservation method adopts a micro-drop method, for example, the application of the stem cell cryopreservation method comprises the following steps of adding a cryopreservation liquid into stem cells, dispersing by blowing to prepare a stem cell suspension, placing the stem cell suspension on a freezing slide and performing cryopreservation by liquid nitrogen (-196 ℃).
According to an embodiment of the present invention, thawing of the cryopreserved stem cells comprises placing the frozen slides with the stem cells in the a-MEM medium and thawing at 37 ℃.
In the present invention, "cryopreservation" and "cryopreservation" have the same meaning and are used interchangeably, and refer to preservation of a substance, or a cell, a tissue, or an organ at a low temperature so that the substance retains its original physicochemical and/or biological activity, physiological and biochemical functions.
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 sources and good biocompatibility, and the prepared cryopreservation reagent can greatly reduce the consumption of DMSO (dimethyl sulfoxide) or even does not add 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 disclosed by the invention is simple in composition, convenient in raw material source and low in cost, can be widely applied to cryopreservation of various cells and tissues, can be widely applied to cryopreservation of 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 the staining of thawed sections of frozen whole ovarian organs according to application examples 14-17;
FIG. 7 is a photograph of a fresh (unfrozen) ovarian tissue section stain;
FIG. 8 is a photograph of stained sections of cryopreserved ovarian tissue of comparative example 9 after thawing;
FIGS. 9-12 are photographs showing staining of thawed sections of frozen ovarian tissue according to examples 18-21, in sequence.
Detailed Description
The preparation process of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
In the embodiment of the invention, poly-L-proline with polymerization degree of 15 or polymerization degree of 8 and molecular weight of 1475 or 795 is adopted in the refrigerating fluid; 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.
The survival rate in the embodiment of the invention 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 100mL: 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 (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5mol L) -1 ) Ultrasonically dissolving the mixture in 25mL of DPBS, sequentially adding 10mL of glycol and 10mL of DMSO (dimethyl sulfoxide) to obtain a solution 2 after all sucrose is dissolved, uniformly mixing the 2 solutions when the solution 1 and the solution 2 are restored to room temperature, adjusting the pH value to 6.9, fixing the volume of 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 (0.05 mol) 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 (0.05 mol) of sucrose in 20mL of DPBS, sequentially adding 10mL of glycol and 10mL of DMSO to obtain a solution 2 after the sucrose is completely dissolved, mixing the two solutions uniformly after the solution 1 and the solution 2 are returned 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, independently storing 20mL of serum, and adding the serum before the frozen preservation 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 (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5mol L) -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: dissolving 1.5g of poly-L-arginine (with polymerization degree of 8) in another 20mL of DPBS by ultrasonic waves to obtain a solution 1, wherein the total volume is 100 mL; 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 (0.05 mol) of sucrose (the final concentration of sucrose in the cryopreservation solution is 0.5mol L) -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 20mL of serum was added at the time of use.
Frozen equilibrium liquid b: heating 2.0g of PVA in a water bath at 80 ℃, dissolving the PVA 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: every 1mL of the mixture contains 7.5% (v/v) of DMSO,7.5% (v/v) of glycol, 20% (v/v) of fetal calf serum and the balance of 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 -1 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 contains 10% (v/v) of ethylene glycol, 20% (v/v) of fetal bovine serum and 0.5mol L of -1 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) -1 Sucrose, 20% serum, balance DPBS); thawing solution II (containing 0.5mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution III (containing 0.25mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution IV (20% serum, balance DPBS).
Thawing solution 2#: thawing solution I (containing 1.0mol L) -1 20mg mL of sucrose (1) -1 10mg mL of PVA (1) -1 Polyproline of (a), the balance being DPBS); thawing solution II (containing 0.5mol L) -1 Sucrose, 20mg mL -1 5.0mg mL of PVA (1) -1 The balance being DPBS); thawing solution III (containing 0.25mol L) -1 Sucrose, 20mg mL -1 2.5mg mL of PVA (1) -1 Polyproline of (a), the balance being DPBS); thawing solution IV (20 mg mL) -1 With 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 equilibrated 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; the number of surviving cells was observed after culturing the thawed oocytes for 2 hours, 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
| Numbering | Balancing liquid | Cryopreservation liquid | Thawing solution | Total number of frozen eggs (grains) | Survival rate after 2 hours |
| Application example 1 | a | A | Thawing solution 1# | 67 | 98.5％ |
| Application example 2 | a | B | Thawing solution 1# | 109 | 96.3％ |
| Application example 3 | a | C | Thawing solution 1# | 67 | 95.5％ |
| Application example 4 | b | D | Thawing solution 1# | 39 | 89.7％ |
| Application example 5 | b | D | Thawing solution 2# | 60 | 98.6％ |
| Comparative example 1 | a | Refrigerating fluid 1# | Thawing solution 1# | 146 | 95.0％ |
| Comparative example 2 | Balance liquid 2# | Refrigerating fluid 2# | Thawing solution 1# | 96 | 81.9％ |
TABLE 2 survival rate of mouse embryos by cryopreservation
As can be seen from the data in tables 1 and 2, when the cryopreservation solution of the invention is used for cryopreservation of oocytes and embryos, the survival rate of the oocytes can reach more than 98%, the survival rate of the embryos can reach more than 95%, and the recovery rate of the oocytes can reach even far higher than that of the commercial cryopreservation solution (comparative examples 1-4) which is generally used clinically at present and contains 15% of DMSO, and the cryopreservation effect of the amino acid bionic ice control material added with the cryopreservation solution is obviously better than that of the cryopreservation solution without the bionic ice control material added with the comparative example 2.
Example 2: cryopreservation of human umbilical cord mesenchymal stem cells
1. Preparing a cryopreservation solution: the cryopreservation liquid is prepared according to the following formula
Cryopreservation liquid F: the total volume was 100mL, and the total volume contained 10mL of ethylene glycol, 20mL of serum, and 17g (0.5 mol L) of sucrose -1 ) 4.0g of poly-L-arginine (polymerization degree of 8), 1.0g of PVA and the balance of DPBS.
Cryopreservation liquid G: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5 mol 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: general assemblyThe volume of the product is 100mL, and the product contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5 mol 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 was 100mL, and the total volume contained 10mL of ethylene glycol, 20mL of serum, and 17g (0.5 mol L) of sucrose -1 ) 16g of L-Arg, 8g of L-Thr, 7.5mL of DMSO and the balance of DPBS.
Cryopreservation liquid J: the total volume is 100mL, and the extract contains 25mL of ethylene glycol, 20mL of serum and 17g (0.5 mol 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 -1 Sucrose and the balance DPBS.
Frozen stock solution 4#: each 1mL contained 10% (v/v) DMSO,15% (v/v) fetal bovine serum, and the balance a-MEM medium (USA, invitrogen, C12571500 BT).
Application example 2:
the cryopreservation liquids of the above examples and comparative examples were used to perform cryopreservation of human umbilical cord mesenchymal stem cells 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 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 10uL of refrigerating fluid to the bottom of the centrifuge tube, gently beating to disperse dry cell clusters, placing the 10uL of refrigerating fluid with the stem cells on a freezing slide, and placing the freezing slide on liquid nitrogen (-196 ℃) for freezing and preserving. 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 instrument JIMBIO-FIL, viability = number of viable cells/total number of cells (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 rates can reach 92.4% and 71.0%, which shows that the reagent for freezing can not only reach the effectiveness of conventional freezing liquid-cooled freeze-dried cells, but also is much higher than the recovery rate of the freezing preservation of a currently and commonly used cryopreservation solution (comparative example 6) containing 10% of DMSO, and the freezing preservation effect of the amino acid bionic ice control material added is obviously better than that of the comparative examples 5 and 6 without the amino acid ice control material added.
Example 3: cryopreservation of ovarian organ and ovarian tissue
1. Preparing a frozen preservation solution: the freezing preservation solution and the freezing equilibrium solution are prepared according to the following formula
Cryopreservation liquid K: the total volume was 100mL, and the total volume contained 10mL of ethylene glycol, 20mL of serum, and 17g (0.5 mol L) of sucrose -1 ) 4.0g of poly-L-arginine (polymerization degree of 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 (0.5 mol L) of sucrose -1 ) 4.0g of poly-L-arginine (polymerization degree of 8), 7.5mL of DMSO, and the balance of DPBS.
Cryopreservation liquid M: the total volume is 100mL, and the extract contains 10mL of ethylene glycol, 20mL of serum and 17g (0.5 mol 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 and serum20mL, 17g (0.5 mol L) sucrose -1 ) 4.0g of poly-L-proline (polymerization degree of 8), 7.5mL of DMSO, and the balance of DPBS.
The cryopreservation solution was prepared in the same manner 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 20mL of 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 20mL of serum was added at the time of use.
2. Comparative example 3:
frozen equilibrium liquid a: every 1mL of the mixture contains 7.5% (v/v) of DMSO,7.5% (v/v) of glycol, 20% (v/v) of fetal calf serum and the balance of 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 -1 Sucrose and the balance DPBS.
3. Thawing the liquid:
unfreezing liquid 1#: thawing solution I (containing 1.0mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution II (containing 0.5mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution III (containing 0.25mol L) -1 Sucrose, 20% serum, balance DPBS); thawing solution IV (20% 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, whole ovarian organs or ovarian tissue sections are placed in culture medium (10% FBS + a-MEM) and then incubated at 37 deg.C, 5% CO 2 Resuscitating and culturing in an incubator for 2 hours, fixing with 4% paraformaldehyde, embedding in paraffin, HE staining, and developingThe morphology was observed under the microscope and the results are shown in FIGS. 1-12, FIG. 1 is a photograph of a stained section of a fresh unfrozen ovarian organ, and FIG. 7 is a photograph of a stained section of a fresh unfrozen ovarian tissue.
TABLE 4 ovarian organ cryopreservation protocol
| Numbering | Balancing liquid | Cryopreservation liquid | Thawing solution | Form of |
| Application example 14 | b | K | Thawing solution 1# | FIG. 3 |
| Application example 15 | a | L | Thawing solution 1# | FIG. 4 |
| Application example 16 | a | M | Thawing solution 1# | FIG. 5 |
| Application example 17 | a | N | Thawing solution 1# | FIG. 6 |
| Comparative example 8 | a | Refrigerating fluid 1# | Thawing solution 1# | FIG. 2 |
TABLE 5 ovarian tissue cryopreservation protocol
| Numbering | Balancing liquid | Cryopreservation liquid | Thawing solution | Form of |
| Application example 18 | b | K | Thawing solution 1# | FIG. 9 |
| Application example 19 | a | L | Thawing solution 1# | FIG. 10 |
| Application example 20 | a | M | Thawing solution 1# | FIG. 11 |
| Application example 21 | a | N | Thawing solution 1# | FIG. 12 |
| Comparative example 9 | a | Freezing fluid 1# | Thawing solution 1# | FIG. 8 |
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 of the blood vessel is complete, the vessel cavity collapse is less, the cytoplasm of endothelial cells is homogeneous, the light staining is relatively more, and the nucleus is shriveled 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 follicles during the growth phase compared to comparative example 9 and fresh unfrozen ovarian tissue, and the use of the cryopreservation solution of the present invention for cryopreservation of ovarian tissue also showed a better effect than the prior art.
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 (17)
1. The application of the amino acid cryopreservation solution in stem cell cryopreservation is characterized in that the amino acid cryopreservation solution comprises the following components in each 100mL: 0.1-50g of amino acid bionic ice control material, 5.0-45mL of polyalcohol, 0-15mL of DMSO, 0-30mL of serum or human serum albumin or bovine serum albumin, 0.1-1mol L of water-soluble sugar -1 And the balance of buffer solution;
the amino acid bionic ice control material is selected from poly-L-arginine, and the polymerization degree of the poly-L-arginine is 8 to 15;
the pH value of the frozen preservation solution is 6.5-7.6.
2. The use of an amino acid-based cryopreservation solution for stem cell cryopreservation according to claim 1, wherein the polyol is a polyol having 2 to 5 carbon atoms.
3. The use of the cryopreservation solution of amino acids in stem cell cryopreservation according to claim 2, wherein the polyhydric alcohol is any one selected from ethylene glycol, propylene glycol and glycerol.
4. The use of an amino acid-based cryopreservation solution for cryopreservation of stem cells according to claim 1, wherein the water-soluble sugar is at least one of a non-reducing disaccharide, a water-soluble polysaccharide and a anhydrosugar.
5. The use of the cryopreservation solution of amino acids according to claim 4 in the cryopreservation of stem cells, wherein the water-soluble sugar is selected from sucrose, trehalose, water-soluble cellulose and polysucrose.
6. The use of the amino acid-based cryopreservation solution in stem cell cryopreservation according to claim 1, wherein the buffer is at least one selected from a DPBS buffer or a hepes-buffered HTF buffer or other cell culture buffers.
7. The use of an amino acid-based cryopreservation solution for cryopreservation of stem cells according to claim 1, wherein the DMSO content is 1.0-10mL per 100mL of the cryopreservation solution.
8. The use of an amino acid-based cryopreservation solution for stem cell cryopreservation according to claim 1, wherein the content of serum or human serum albumin or bovine serum albumin in each 100mL of the cryopreservation solution is 10 to 15mL.
9. The use of the amino acid-based cryopreservation solution for stem cell cryopreservation according to claim 1, wherein the content of the water-soluble sugar in 100mL of the cryopreservation solution is 0.1 to 0.8mol L -1 。
10. The use of an amino acid-based cryopreservation solution for cryopreservation of stem cells according to claim 1 characterised in that the polyol content per 100mL of cryopreservation solution is 5.0 to 40mL.
11. The use of the amino acid-based cryopreservation solution for cryopreservation of stem cells according to claim 1, wherein the cryopreservation solution further contains 0.1 to 6.0g of polyvinyl alcohol PVA per 100mL of the cryopreservation solution.
12. Use of an amino acid-based cryopreservation solution according to any one of claims 1 to 11 in the cryopreservation of stem cells, the amino acid-based cryopreservation solution being prepared by a method comprising the steps of:
(1) Dissolving the amino acid bionic ice control material in part of buffer solution, and adjusting the pH value to form a solution 1; optionally, dissolving PVA in a part of buffer solution, and adjusting the pH value to obtain a solution 2;
(2) Dissolving water-soluble sugar in part of buffer solution, and adding other components after the water-soluble sugar is completely dissolved to obtain solution 3;
(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 or human serum albumin or bovine serum albumin, the serum or human serum albumin or bovine serum albumin is added at the time of use of the cryopreservation liquid.
13. Use of the cryopreservation solution of amino acids according to claims 1 to 11 in cryopreservation of stem cells, wherein the stem cells are selected from any one of pluripotent, multipotent or multipotent stem cells.
14. The use of the cryopreservation solution of amino acids according to claim 13 in cryopreservation of stem cells selected from embryonic stem cells, various types of mesenchymal stem cells, hematopoietic stem cells.
15. The use of the cryopreservation solution of amino acids according to claim 14 in cryopreservation of stem cells, wherein the mesenchymal stem cells of each type are selected from umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and bone marrow mesenchymal stem cells.
16. Use of the amino acid-based cryopreservation solution according to claim 13 for cryopreservation of stem cells by the micro-drop method.
17. The use of the cryopreservation solution of amino acids according to claim 16 in the cryopreservation of stem cells, wherein the microdroplet method comprises the steps of adding the cryopreservation solution to stem cells, blowing and dispersing to prepare a stem cell suspension, placing the stem cell suspension on a cryoslide, and performing cryopreservation with liquid nitrogen.
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