CN113383766A - Freezing storage liquid for stem cell freezing storage and preparation method thereof - Google Patents
Freezing storage liquid for stem cell freezing storage and preparation method thereof Download PDFInfo
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- CN113383766A CN113383766A CN202010171833.XA CN202010171833A CN113383766A CN 113383766 A CN113383766 A CN 113383766A CN 202010171833 A CN202010171833 A CN 202010171833A CN 113383766 A CN113383766 A CN 113383766A
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- 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
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- 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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The invention discloses a freezing storage solution for stem cell freezing storage and a preparation method thereof, wherein each 100mL of the solution contains: 0.1-50g of bionic ice control material, 0.1-15mL of DMSO, 0.1-30mL of serum and the balance of buffer solution. The cryopreservation liquid takes bionic ice control materials such as amino acids or polyvinyl alcohol and the like as main components, has low DMSO content, low toxicity and definite components, and can achieve the same or even higher cell survival rate as the existing cryopreservation liquid. The cryopreservation liquid has simple composition, convenient raw material source and low cost, and can be widely applied to cryopreservation of various cells, tissues or organs.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a freezing medium for stem cell freezing preservation 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. Stem cells are widely used in clinical treatment and regenerative medicine due to their functions of repairing tissue cell damage, replacing damaged cells, and stimulating the regeneration of body's own cells. For example, the human umbilical cord mesenchymal stem cells of the newborn fetus have wide clinical application prospect due to low immunogenicity, high cell content, strong proliferation capacity and the like; the cryopreservation of the human umbilical cord mesenchymal stem cells provides possibility for wide clinical application of the human umbilical cord mesenchymal stem cells. 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. Stem cells, such as mesenchymal stem cells, have the potential for self-renewal and multipotentiality and can be widely used in stem cell transplantation, autoimmune diseases or gene therapy vectors and the like. The prior cryopreservation reagent for stem cells generally comprises components such as a cryoprotectant, a stem cell growth medium and the like, and achieves the aims of supporting cells and reducing the formation of ice crystals. However, the growth medium easily causes the stem cells to be differentiated blindly, and the directional differentiation potential of the stem cells after recovery is influenced.
Disclosure of Invention
The present invention provides a cryopreservation solution, each 100mL of the solution containing: 0.1-50g of bionic ice control material, 0.1-15mL of DMSO, 0.1-30mL of serum and the balance of buffer solution.
According to the present invention, the cryopreservation liquid does not contain polyols and sugars.
According to the invention, the bionic ice control material is selected from at least one of polyvinyl alcohol or amino acid bionic ice control materials.
According to the present invention, the amount of polyvinyl alcohol contained in the cryopreservation solution per 100mL is preferably 0.1 to 6 g.
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%.
According to the invention, the amino acid bionic ice control material is selected from one or more than two of amino acid, polyamino acid or peptide compounds.
As an embodiment of the invention, the amino acid bionic ice control material can be an amino acid containing an ice-philic group and a hydrophilic group, or a polyamino acid or peptide compound 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 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 an amino acid homopolymer, such as one or a combination of more than two of poly-L-proline, poly-L-arginine and the like.
In an embodiment of the invention, the peptidic compound consists of an ice-philic amino acid, such as: threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc., with other hydrophilic amino acids selected from arginine, proline, alanine, etc., or Gluconolactone (GDL) or other sugars.
The peptide compound is a peptide formed by more than two amino acid units, such as: 2-8 amino acid units, specifically 2, 3, 4, 5 amino acid units; each amino acid unit is different. The arrangement of the hydrophilic amino acid and the other hydrophilic amino acid in the peptide compound is not particularly limited, and the peptide compound can be connected by amino acid connecting groups or chemical bonds known in the art, for example, the hydrophilic amino acid and the hydrophilic amino acid can be arranged in a single interval, or a plurality of hydrophilic amino acids can be connected to form a hydrophilic amino acid fragment or a hydrophilic amino acid fragment, and then the hydrophilic amino acid fragment or the hydrophilic amino acid fragment is respectively connected with the hydrophilic amino acid (or fragment) and the hydrophilic amino acid (or fragment).
In an embodiment of the invention, the peptidic compound is a polypeptide, such as at least one of L-Thr-L-Arg (TR), L-Thr-L-Pro (TP), L-Arg-L-Thr (RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), L-Ala-L-Ala-L-Thr (AAT), L-Thr-L-Cys-L-Thr (TCT). The polypeptides may be prepared by methods known in the art for polypeptide synthesis, for example by solid phase synthesis.
In another embodiment, the peptidic compound is a glycopeptide derivative, such as a gluconolactone or other glycopeptide derivative synthesized from carbohydrates and amino acids, such as GDL-L-Thr, GDL-L-Ser, and GDL-L-Val. The glycopeptide derivatives may be prepared according to art-known methods for the reaction of saccharides and amino acids, such as solid phase synthesis or by reacting saccharides and amino acids in an organic solvent.
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 present invention, the buffer may be selected from at least one of DPBS or hepes-buffered HTF buffers or other cell culture buffers known in the art.
According to the invention, the serum can be selected from human serum albumin or a substitute thereof, such as sodium dodecyl sulfate, for human cryopreservation subjects, and fetal bovine serum or bovine serum albumin for non-human cryopreservation subjects.
According to the invention, the DMSO content is between 0.1 and 15mL, preferably between 1.0 and 10mL, more preferably between 1 and 7.5mL, for example between 1.5 and 5mL, per 100mL of 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.
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.
In one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL:
0.5-50g amino acid
DMSO 0.1-10mL
0.1-30mL of serum
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
DMSO 0.1-10mL
serum 5.0-20mL
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
DMSO 0.1-10mL
Serum 5.0-20mL
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
DMSO 0.1-10mL
Serum 5.0-20mL
DPBS margin.
As one embodiment of the present invention, the cryopreservation liquid comprises the following components per 100mL volume:
PVA 1.0-5.0g
DMSO 0.1-10mL
0.1-20mL of serum
The balance of the buffer solution.
Preferably: the cryopreservation liquid comprises the following components in volume per 100 mL:
PVA 1.0-4.0g
DMSO 4-10mL
10-20mL of serum
DPBS margin.
The invention also provides a preparation method of the cryopreservation liquid, which comprises the following steps:
(1) dissolving the bionic ice control material in a part of buffer solution, and adjusting the pH value to form a solution 1;
(2) dissolving DMSO in a part of buffer solution to prepare a solution 2;
(3) and (3) mixing the solution 1 and the solution 2 after cooling to room temperature, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the frozen preservation solution.
According to the production method of the present invention, the serum is added when the cryopreservation solution is used.
According to the preparation method, when the bionic ice control material is polyvinyl alcohol, water bath heating is adopted and stirring is carried out; for example, the water bath temperature is 65-85 deg.C, 70-80 deg.C; the stirring is mechanical stirring such as magnetic stirring.
The invention also provides application of the cryopreservation liquid in stem cell cryopreservation. The stem cells can be cryopreserved using any stem cell freezing method known in the art, and in one embodiment, comprises adding a freezing fluid with stem cells into a cryopreservation tube, placing the cryopreservation tube in an isopropanol cryopreservation box, and freezing. In another embodiment, the cryopreservation solution described above is used for cryopreservation of stem cells using the microdroplet method.
Advantageous effects
The cryopreservation solution provided by the invention takes a polyvinyl alcohol or amino acid bionic ice control material as a main ice control component, the source is wide, the biocompatibility is good, the prepared cryopreservation reagent can be free of a cell culture medium, the components are determined, the composition is simple, and the higher cell survival rate can be maintained. The cryopreservation liquid provided by the invention is convenient in raw material source and low in cost, and can be widely applied to cryopreservation of various stem cells.
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 polymerization degree of the poly-L-proline adopted in the embodiment of the invention is 8 or 15, and the molecular weight is 795 or 1475; the polymerization degree of the poly-L-arginine is 8 or 15, and the molecular weight of the poly-L-arginine is 1267 or 2361.
In the embodiment of the invention, the survival rate is the average survival rate of 2-3 repeated experiments.
Examples
1. Preparing a cryopreservation solution: the following formula is adopted to prepare the cryopreservation liquid
The total volume of the cryopreservation solution A is 100mL, 2.0g of poly-L-arginine is ultrasonically dissolved in 25mL of DPBS, and the pH value is adjusted to 7.0 to obtain a solution 1; dissolving 10mL of DMSO in 20mL of DPBS to obtain solution 2, mixing the two solutions when the solution 1 and the solution 2 return to room temperature, adjusting the pH to 7.0, adding DPBS to a constant volume to make up the balance to 85% of the total volume, storing 15mL of serum separately, and adding the solution before use.
Cryopreservation liquid B: the total volume is 100mL, 2.0g of PVA is heated in a water bath at 80 ℃ and is dissolved in 25mL of DPBS by magnetic stirring, the pH value is adjusted to 7.0 after the PVA is completely dissolved and is cooled to the room temperature, 10mL of DMSO is dissolved in 25mL of DPBS to obtain a solution 2, the two solutions are mixed when the solution 1 and the solution 2 return to the room temperature, the pH value is adjusted, the constant volume is supplemented to 85% of the total volume, and 15mL of serum is separately stored and is added when a preservation solution is used.
Comparative example:
frozen preservation solution 1 #: each 1mL of the culture medium contained 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance of alpha-MEM.
Application example:
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 1.
And (3) freezing and storing the human umbilical cord mesenchymal stem cells in a freezing and storing tube of 300 mu L:
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 with 25% pancreatin for 2-3 minutes, then putting the cells into an equal volume of culture solution (10% FBS + a-MEM culture medium), gently blowing and beating until the stem cells are completely shed, centrifuging for 5 minutes at 1000r, discarding supernatant, adding 300 mu L of freezing solution into a centrifuge tube, gently blowing and beating to disperse stem cell clusters, adding the freezing solution with the stem cells into a 2ml cryopreservation tube, putting the cryopreservation tube into an isopropanol cryopreservation box, putting the cryopreservation tube into a refrigerator at-80 ℃, thawing after 24 hours, and staining trypan blue to check the survival rate (see table 1).
TABLE 1 human umbilical cord mesenchymal stem cell cryopreservation survival rate
| Numbering | Cryopreservation liquid | Survival rate |
| Application example 1 | A | 76.5％ |
| Application example 2 | B | 78.0％ |
| Comparative example 1 | Freezing fluid 1# | 76.4％ |
As can be seen from the data in Table 1, the cryopreservation liquid disclosed by the invention does not need to add a stem cell culture medium, so that the introduction of a culture medium with complex components is avoided, and only a bionic ice control material and a small amount of DMSO are added, so that the survival rate of stem cells can reach about 80% when the human umbilical cord mesenchymal stem cells are subjected to cryopreservation, which shows that the cryopreservation liquid has the effectiveness equivalent to that of the existing cryopreservation liquid with the culture medium added, and the components are simpler and more controllable, so that the cryopreservation liquid has a wide application prospect.
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. A cryopreservation solution comprising per 100mL of the solution: 0.1-50g of bionic ice control material, 0.1-15mL of DMSO, 0.1-30mL of serum and the balance of buffer solution.
2. The cryopreservation liquid according to claim 1, wherein the bionic ice control material is at least one selected from polyvinyl alcohol and amino acid bionic ice control materials.
3. The cryopreservation liquid according to claim 1 or 2, preferably containing polyvinyl alcohol in an amount of 0.1 to 6g per 100mL of the cryopreservation liquid;
preferably, the PVA is selected from one or a combination of two or more of isotactic PVA, syndiotactic PVA and atactic PVA, for example, the PVA has a syndiotacticity of 15% to 65%; preferably a random PVA, for example a PVA with a syndiotacticity of 45% to 65% of the PVA;
preferably, the PVA is selected from the group consisting of PVA having a molecular weight of 10-500kDa or higher, for example, a molecular weight of 10-30kDa, 30-50kDa, 80-90kDa, 200-500 kDa;
preferably, the PVA may be selected from PVAs having a degree of hydrolysis greater than 80%, for example from 80% to 99%.
4. The cryopreservation liquid according to claim 1 or 2, wherein the amino acid bionic ice control material is selected from one or more of amino acid, polyamino acid and peptide compound;
preferably, the amino acid bionic ice control material is 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;
preferably, the hydrophilic group is a functional group that can form a non-covalent interaction with a water molecule, such as a hydrogen bond with water, van der waals interaction, electrostatic interaction, hydrophobic interaction, or pi-pi interaction; can be selected from hydroxyl (-OH), amino (-NH)2) Carboxylic acid group (-COOH), or amide group (-CONH)2) And the like;
preferably, 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 oxophilic group may be selected from the group consisting of hydroxyl (-OH), amino (-NH)2) Phenyl (-C)6H5) Or pyrrolidinyl (-C)4H8N);
preferably, the amino acid bionic ice control material is selected from one or two of arginine, threonine, proline, lysine, histidine, glutamic acid, 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.
5. The cryopreservation liquid according to any one of claims 1 to 4, wherein the amino acid bionic ice control material is polyamino acid (polymerization degree is not less than 2), preferably the polymerization degree is 2 to 40 (such as polymerization degree is 6, 8, 15, 20 and the like), and is one or a combination of more than two of poly-L-proline, poly-L-arginine and the like;
preferably, the peptide compound is a peptide formed by more than two amino acid units, such as: 2-8 amino acid units, specifically 2, 3, 4, 5 amino acid units; each amino acid unit is different; for example, the peptidic compounds consist of ice-philic amino acids, such as: threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc. with other hydrophilic amino acids selected from arginine, proline, alanine, etc. or Gluconolactone (GDL) or other saccharides;
preferably, the peptidic compound is a polypeptide, such as at least one of L-Thr-L-Arg (TR), L-Thr-L-Pro (TP), L-Arg-L-Thr (RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), L-Ala-L-Ala-L-Thr (AAT), L-Thr-L-Cys-L-Thr (TCT);
preferably, the peptide compound is a glycopeptide derivative, such as a glycopeptide derivative synthesized from gluconolactone or other saccharides and amino acids, such as GDL-L-Thr, GDL-L-Ser, and GDL-L-Val.
6. The cryopreservation liquid according to any one of claims 1 to 5, wherein the content of the amino acid bionic ice-controlling material in each 100mL of the cryopreservation liquid is 0.5 to 50 g;
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 human serum albumin or a substitute thereof, such as sodium dodecyl sulfate, for human cryopreservation subjects, or fetal bovine serum or bovine serum albumin for non-human cryopreservation subjects;
preferably, the DMSO content is 0.1-15mL, and the DMSO content is 1.0-10mL in each 100mL of cryopreservation solution;
preferably, the serum content is 5.0-20mL in each 100mL of the cryopreservation solution;
preferably, the pH of the cryopreservation solution is 6.5 to 7.6.
7. The cryopreservation liquid according to any one of claims 1 to 6, which is composed of any one of: contains the following components in each 100 mL:
0.5-50g amino acid
DMSO 0.1-10mL
0.1-30mL of serum
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
DMSO 0.1-10mL
serum 5.0-20mL
DPBS margin;
preferably, the cryopreservation solution contains the following components per 100mL volume:
0.5-9.0g of polyamino acid
DMSO 0.1-10mL
Serum 5.0-20mL
The balance of 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
DMSO 0.1-10mL
Serum 5.0-20mL
DPBS margin;
preferably, the cryopreservation liquid comprises the following components in volume per 100 mL:
PVA 1.0-5.0g
DMSO 0.1-10mL
0.1-20mL of serum
The balance of buffer solution;
preferably: the cryopreservation liquid comprises the following components in volume per 100 mL:
PVA 1.0-4.0g
DMSO 4-10mL
10-20mL of serum
DPBS margin.
8. A method for producing a cryopreservation liquid according to any one of claims 1 to 7, comprising the steps of:
(1) dissolving the bionic ice control material in a part of buffer solution, and adjusting the pH value to form a solution 1;
(2) dissolving DMSO in a part of buffer solution to prepare a solution 2;
(3) and (3) mixing the solution 1 and the solution 2 after cooling to room temperature, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the frozen preservation solution.
9. The method for producing a cryopreservation liquid according to claim 8, wherein the serum is added at the time of use of the cryopreservation liquid;
when the bionic ice control material is polyvinyl alcohol, heating in a water bath and stirring; for example, the water bath temperature is 65-85 deg.C, 70-80 deg.C; the stirring is mechanical stirring such as magnetic stirring.
10. Use of the cryopreservation solution of any one of claims 1 to 7 in cryopreservation of stem cells, preferably comprising adding the cryopreservation solution with stem cells into a cryopreservation tube, placing the cryopreservation tube in an isopropanol cryopreservation box, and freezing.
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| CN113383767A (en) * | 2020-03-12 | 2021-09-14 | 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 |
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