CN115590018B - Clinical mesenchymal stem cell cold storage preservation solution for dogs and application method thereof - Google Patents
Clinical mesenchymal stem cell cold storage preservation solution for dogs and application method thereof Download PDFInfo
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Classifications
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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
-
- 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
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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Abstract
The invention provides a clinical mesenchymal stem cell cold storage preservation solution for dogs, which comprises the following raw materials: water for injection, canine albumin, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate, dextran 40 and water-soluble vitamin E; according to the weight ratio, the feed ratio of the water for injection, the canine albumin, the glucose, the sodium citrate, the citric acid, the sodium chloride, the sodium bicarbonate, the glucan 40 and the water-soluble vitamin E is 100:10-20:0.9-1.2:0.25-0.6:0.015-0.04:0.6-0.7:0.035-0.04:1-2:0.00002-0.00008. The canine mesenchymal stem cell cold storage preservation solution prepared by the invention can be directly used for clinic, can maintain the activity and stability of canine mesenchymal stem cells within 7 days under cold storage condition, has no toxic or side effect, does not need ultralow temperature storage, and is suitable for wide popularization and application.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a clinical mesenchymal stem cell cold storage preservation solution for dogs and an application method thereof.
Background
Mesenchymal stem cells (mesenchymal stem cells, MSCs) are cells derived from mesoderm, have the potential to self-renew and differentiate in multiple directions, and can be isolated from various tissues and organs (such as bone marrow, fat, placenta and umbilical cord). MSCs have effects of promoting tissue repair, inhibiting inflammation, and reconstructing organism immunity. With respect to these characteristics, veterinary students put their eyes on clinical therapeutic applications in dogs, and have achieved satisfactory results in the treatment of skin wounds, liver and kidney injuries, arthritis, bone injuries, myocarditis, and other diseases.
MSC is different from common biological products and medicines, is the most basic unit of life with life activity, and is a difficult problem in clinical popularization and application. The existing preservation modes mainly comprise freezing preservation and refrigerating preservation. The freeze preservation, namely the preservation at the liquid nitrogen temperature (-196 ℃) shows that the stem cells can be preserved for a plurality of decades, the good activity state, the characteristics of the stem cells and the functions thereof can be still maintained after recovery, the main components of the preservation solution are serum and dimethyl sulfoxide (DMSO), however, the DMSO has cytotoxicity, vascular toxicity and hepatorenal toxicity, for example, the cells stored in jelly are directly used after recovery, and the excessive DMSO can damage the bodies of dogs. And the cryopreservation mode needs ultralow temperature equipment and needs recovery and other processes before transplantation, which is a challenge for the used pet hospital equipment. In the aspect of cold storage, the current research shows that the stem cells can be stored for 72 hours at the temperature of 4-8 ℃ under the environment of the cold storage liquid, the activity is kept to be more than 80%, and the storage time is shorter, so that the current cold storage is only suitable for short-distance storage application, cannot meet the conditions of long-distance transportation and advanced storage of pet hospitals, and limits the application of stem cell therapy in a wider range. Therefore, a preservation solution is urgently needed to be researched, and the stem cells can be preserved for a long time under the condition of refrigeration, so that the preservation solution is suitable for long-distance transportation.
Disclosure of Invention
In order to solve the defects and problems in the prior art, the invention provides the cold preserving solution for the mesenchymal stem cells for dogs and the application method thereof, so that the mesenchymal stem cells for dogs can be preserved for a long time under the cold preserving condition, the cold preserving solution can be suitable for long-distance transportation, and the cold preserving solution can be directly applied to clinic and has no side effect on dogs.
According to a first aspect of the invention, the invention provides a clinical mesenchymal stem cell cryopreservation solution for dogs, which comprises the following raw materials: water for injection, canine albumin, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate, dextran 40 and water-soluble vitamin E; according to the weight ratio, the feed ratio of the water for injection, the canine albumin, the glucose, the sodium citrate, the citric acid, the sodium chloride, the sodium bicarbonate, the glucan 40 and the water-soluble vitamin E is 100:10-20:0.9-1.2:0.25-0.6:0.015-0.04:0.6-0.7:0.035-0.04:1-2:0.00002-0.00008.
The cold storage preservation solution prepared from the raw material components is matched with each other in a certain proportion, so that the long-time preservation of the canine mesenchymal stem cells can be realized under the cold storage condition, namely, the higher activity of the canine mesenchymal stem cells can be maintained for a long time by the cell preservation solution under the cold storage condition, and the long-distance transportation of the canine mesenchymal stem cells can be realized under the cold storage condition. Long-term experiments show that when the mesenchymal stem cell cryopreservation solution prepared by the invention is used for preserving cells under the cryopreservation condition, the activity (cell survival rate) of the cells after 7 days can still be maintained to be more than 70%.
In terms of biological cell preservation, the most widely used cryopreservation methods in the market at present usually adopt a plurality of small molecular substances as cryopreservants, wherein the small molecular substances comprise organic solvents such as dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, methanol, ethanol, propanol and formamide, and the small molecular substances have a certain toxicity to cells when the content of the small molecular substances is too high. In particular, the most commonly used dimethyl sulfoxide has a high-efficiency protective effect on cells, but the too high content of dimethyl sulfoxide has larger toxicity on cells. When the cells are frozen and preserved by the refrigerant, the refrigerant can avoid forming ice crystals in the cells under the condition of ultralow temperature, and protect the cells from being damaged by the ice crystals, so that the cells can be preserved for a long time under the ultralow temperature. However, the frozen and preserved cells need to be recovered when in use, and the freezing and recovery both need specific experimental instruments and strict experimental conditions, which increases the experimental difficulty and the experimental cost.
In order to reduce the experiment difficulty and cost, the refrigerated preservation solution of the cells has been developed to a certain extent in recent years, and compared with the refrigerated preservation solution, the refrigerated preservation solution has a simpler preservation process of the cells, does not need to be frozen or resuscitated, and can avoid some damage to the cells possibly caused by the freezing and resuscitated process, so that a specific experimental instrument is not needed, and the preservation cost is lower. And the cells preserved by the cold storage preservation solution do not need to be resuscitated, so that the method can be directly applied to clinic. However, the conventional refrigerating preservation solution can be preserved for about 72 hours under the refrigerating condition (such as 4-8 ℃) and the activity is kept at about 80%, so that the conventional refrigerating preservation solution is only suitable for short-distance transportation. The long-term experiment shows that the cell cold preservation solution prepared by adopting the specific raw material components can preserve cells for a long time under the cold preservation condition, and the cells can keep good activity, so that the practical transportation time and the activity use requirements are met. In addition, the solvent of the cold storage preservation solution in the scheme is water, and is nontoxic to dogs.
In addition, at present, a normal temperature preservation method is rarely adopted for preserving cells in the market, but the preservation time of the cells is extremely short by adopting the normal temperature preservation method, and the cells lose activity in 1-2 days generally, even cannot meet the short-distance transportation, and cannot meet the requirement for preserving the cells in most cases.
Preferably, the feed ratio of the water for injection, the canine albumin, the glucose, the sodium citrate, the citric acid, the sodium chloride, the sodium bicarbonate, the glucan 40 and the water-soluble vitamin E is 100:10:1.03:0.4:0.028:0.66:0.034:1:0.00005 according to the weight ratio. Experiments show that when the obtained mesenchymal stem cell cryopreservation liquid is used for preserving cells, the cell activity of the mesenchymal stem cell cryopreservation liquid after the mesenchymal stem cell cryopreservation liquid is preserved for 7 days under the cryopreservation condition can still reach more than 80%, and the mesenchymal stem cell cryopreservation liquid has an excellent cell preservation effect.
Preferably, the clinical mesenchymal stem cell cold storage preservation solution for dogs is prepared by uniformly mixing water for injection, albumin for dogs, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate, dextran 40 and water-soluble vitamin E, filtering and sterilizing.
Preferably, in the filter sterilization operation, a filter membrane having a pore size of 0.22 μm is used.
According to another aspect of the invention, an application method of a clinical mesenchymal stem cell cryopreservation solution for dogs is provided, and the clinical mesenchymal stem cell cryopreservation solution for dogs is used for preserving the mesenchymal stem cells of dogs.
Preferably, the application method of the clinical mesenchymal stem cell cryopreservation solution for dogs comprises the following specific steps: and (3) pre-cooling the clinical mesenchymal stem cell cold preservation solution for dogs at the temperature of 4-8 ℃, adding the canine mesenchymal stem cells into the cold preservation solution, and continuously preserving the mesenchymal stem cells at the temperature of 4-8 ℃.
When the mesenchymal stem cell cold preserving fluid prepared by the method is used for preserving the canine mesenchymal stem cells, the mesenchymal stem cell cold preserving fluid is required to be precooled so as to ensure that the canine mesenchymal stem cells can keep the same cold preserving condition in the preservation process, and adverse effects on the preservation of the cells due to the preservation temperature difference are avoided.
Preferably, the canine mesenchymal stem cells are subcultured P3-P5 passage cells (passage 3 to passage 5).
The canine mesenchymal stem cells adopted by the invention are passage cells of P3-P5 after passage culture of canine tissues, and the passage cells of P3-P5 have higher bioactivity and higher cell safety, can reach a certain cell content after in vitro culture and amplification, and meet clinical use standards.
Preferably, the concentration of the canine mesenchymal stem cells is 1 '10' after the canine mesenchymal stem cells are added to the canine clinical mesenchymal stem cell cryopreservation solution 6 ~5´10 6 /ml. The concentration of the preserved canine mesenchymal stem cells is controlled within a certain range, so that the best protection effect on the cells can be ensured.
The formula and the proportion of the cell preservation solution are regulated, so that the prepared cell preservation solution can have an excellent preservation effect on canine mesenchymal stem cells under the condition of refrigeration, the activity of the cells can reach more than 70% under the long-time refrigeration preservation for 7 days, the existing refrigeration preservation solution can be preserved for about 72 hours only under the refrigeration temperature, and compared with the existing refrigeration preservation solution, the cold preservation solution can be preserved for a long time under the refrigeration condition, and the long-distance transportation requirement is met.
Drawings
FIG. 1 is a bar graph showing cell viability at different storage times in examples 1 to 3 and comparative examples 1 to 5 according to the present invention.
Fig. 2 is a microscopic image of canine mesenchymal stem cells of example 1 of the present invention after being stored for 168 hours and stained with trypan blue.
FIG. 3 is a microscopic image showing the adherent growth of cells obtained by re-seeding canine mesenchymal stem cells of example 1 of the present invention after 168 hours of storage in a petri dish for 12 hours of culture.
FIG. 4 is a microscopic image (200') of the adherent growth of cells obtained by re-seeding canine mesenchymal stem cells of example 1 of the present invention after 168h of storage in a petri dish for 12h of culture.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. The reagents referred to below are all commercially available, unless otherwise indicated. For simplicity, some of the operations do not detail the parameters, steps, and instrumentation used for operation, which are well known and reproducible by those skilled in the art.
The canine mesenchymal stem cells used in the following examples and comparative examples were prepared by the following methods (canine adipose mesenchymal stem cells are taken as an example):
(1) Taking the inguinal fat of a healthy dog in a 50ml centrifuge tube containing 20% of double-antibody PBS in a sterile operating room, cleaning blood on adipose tissue with normal saline, and then placing the cleaned adipose tissue in the centrifuge tube to cut the adipose tissue with an ophthalmic scissors;
(2) Adding 5ml of collagenase with concentration of 1mg/ml into the adipose tissue treated in the step (1), placing into a water bath kettle at 37 ℃ for digestion for 1h, taking out, adding a proper amount of stop solution for stopping digestion, filtering by using a 200-mesh screen, centrifuging the filtered solution for 5min at a rotating speed of 1200rpm, and discarding the supernatant;
(3) Step (d)(2) Adding DMEM culture solution containing 10% foetal calf serum, 1% diabody and 1% L-glutamine into the residue after discarding supernatant, re-suspending cells, inoculating into culture dish, placing at 37deg.C, 5% CO 2 Culturing in a constant temperature incubator. The cell state is observed every day, after the cell wall is grown to 80% -90%, the cell is passaged, P3 generation cells are taken for adipogenic osteogenic differentiation, and the expression of the surface proteins CD34, CD44 and CD90 is identified by immunofluorescence.
(4) And (3) taking the identified P3-P5 generation cells in the step (3) for use in preservation experiments of the following examples and comparative examples.
Example 1
1. Preparing cold storage preserving solution for canine mesenchymal stem cells
Uniformly mixing water for injection, canine albumin, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate, glucan 40 and water-soluble vitamin E (Trolox) in a weight ratio of 100:10:1.03:0.4:0.028:0.66:0.034:1:0.00005, and filtering and sterilizing the obtained mixed solution by using a filter membrane with a pore diameter of 0.22 mu m to obtain the canine mesenchymal stem cell cold storage preservation solution.
2. Treatment before transportation of canine mesenchymal stem cells
(1) Taking P3-P5 generation canine mesenchymal stem cells which are adhered to 80% -90%, stopping digestion by using 0.25% (mass concentration) pancreatin digestion by using a DMEM culture solution containing 10% (mass concentration) fetal bovine serum, centrifuging at 1200rpm for 5min, discarding the supernatant, re-suspending the cells by using normal saline, and centrifuging at 1200rpm for 5min again, discarding the supernatant;
(2) Adding the prepared canine mesenchymal stem cell cold storage preservation solution into the residues after the supernatant is discarded in the step (1) (the prepared canine mesenchymal stem cell cold storage preservation solution needs to be pre-cooled in an environment of 4-8 ℃ in advance), and adjusting the cell density to 1X 10 6 ~5×10 6 Obtaining a canine mesenchymal stem cell suspension by using/ml; taking 1ml of canine mesenchymal stem cell suspension into a 2ml EP tube, sealing and placing in an environment of 4-8 ℃.
And (3) preserving and transporting the prepared canine mesenchymal stem cell suspension in a light-shielding environment at 4-8 ℃ and packaging and transporting the suspension by using an ice box in the transportation process. In addition, the prepared canine mesenchymal stem cell suspension can also be directly injected into the body of a canine directly or intravenously.
Example 2
The present example is different from example 1 in that the formulation ratio adopted for preparing the canine mesenchymal stem cell cryopreservation solution is adjusted as follows: the feed ratio of the water for injection, the canine albumin, the glucose, the sodium citrate, the citric acid, the sodium chloride, the sodium bicarbonate, the glucan 40 and the water-soluble vitamin E (Trolox) is 100:15:0.9:0.25:0.014:0.7:0.035:1.5:0.00002; the remainder was identical to example 1.
Example 3
The present example is different from example 1 in that the formulation ratio adopted for preparing the canine mesenchymal stem cell cryopreservation solution is adjusted as follows: the feed ratio of the water for injection, the canine albumin, the glucose, the sodium citrate, the citric acid, the sodium chloride, the sodium bicarbonate, the glucan 40 and the water-soluble vitamin E (Trolox) is 100:20:1.2:0.6:0.04:0.6:0.033:2:0.00007; the remainder was identical to example 1.
Comparative example 1
This comparative example is different from example 1 in that dextran 40 and water-soluble vitamin E (Trolox) were not added when preparing a canine mesenchymal stem cell cryopreservation solution; the remainder was identical to example 1.
Comparative example 2
This comparative example is different from example 1 in that water-soluble vitamin E (Trolox) was not added when preparing a canine mesenchymal stem cell cryopreservation solution; the remainder was identical to example 1.
Comparative example 3
This comparative example differs from example 1 in that dextran 40 was not added when preparing a canine mesenchymal stem cell cryopreservation solution; the remainder was identical to example 1.
Comparative example 4
The comparative example is different from example 1 in that dextran 40 is not added when preparing the cold storage preservation solution of canine mesenchymal stem cells, and the feeding ratio of water for injection, canine albumin, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate and water-soluble vitamin E (Trolox) is 100:10:1.03:0.4:0.028:0.66:0.034:1.00005; the remainder was identical to example 1.
Comparative example 5
The comparative example is different from example 1 in that water-soluble vitamin E (Trolox) is not added when preparing the cold storage preservation solution of the canine mesenchymal stem cells, and the feeding ratio of water for injection, canine albumin, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate and dextran 40 is 100:10:1.03:0.4:0.028:0.66:0.034:1.00005; the remainder was identical to example 1.
Test case
1. Experimental construction mode
The canine mesenchymal stem cell suspensions obtained in examples 1-3 and comparative examples 1-5 were stored at 4-8 ℃ in a dark environment, were sampled after 24h, 48h, 72h, 96h, 120h, 144h and 168h, respectively, and after staining with 0.4% trypan blue, the cell viability was detected with a fully automatic cell counting instrument.
2. Experimental results
The canine mesenchymal stem cell suspensions obtained in examples 1-3 and comparative examples 1-5 were stored at 4-8 ℃ in a dark environment, and the cell survival rates at different times are shown in the following table 1.
Table 1 results of cell viability calculations (n=3, results averaged) at different times in a dark storage environment at 4-8 °c
The data in table 1 above were rearranged and analyzed to obtain the bar graph results in fig. 1. As can be seen from Table 1 and FIG. 1, the cell viability tended to decrease with the increase in shelf life, and as can be seen from the bar chart in FIG. 1, the cell viability decrease in examples 1 to 3 was significantly smaller than that in comparative examples 1 to 5. Further, as can be seen from Table 1, the cell viability was higher in examples 1 to 3 than in comparative examples 1 to 5 at each sampling time point. The cell survival rate of the cells in the canine mesenchymal stem cell preservation solution in examples 1-3 after preservation for 72 hours at 4-8 ℃ is above 90%, and the cell survival rate after preservation for 96 hours is above 84%, so that the clinical use requirements are fully met. And the canine mesenchymal stem cell cryopreservation solution prepared by the formula adopted in the example 1 has the best preservation effect on canine mesenchymal cells, and the cell survival rate after 168 hours can still reach 80.2%. In comparative examples 1 to 5, one or more of dextran 40 and water-soluble vitamin E (Trolox) were omitted, and the cell survival rate was lower than that of examples 1 to 3 regardless of whether the amounts of the remaining components were adjusted, which indicates that the activity of canine mesenchymal stem cells in the preservation process and the preservation time of the cells could be effectively maintained by the mutual cooperation of the components in the formulation of the canine mesenchymal stem cell cryopreservation solution of the present invention. In comparative example 1, the canine mesenchymal stem cell cryopreservation solution was prepared without adding dextran 40 and water-soluble vitamin E (Trolox), and the cell viability was only 62.8% after 96 hours of preservation, and the decrease was significant. In comparative examples 2 and 3, dextran 40 and water-soluble vitamin E (Trolox) were omitted from the cold storage solutions of canine mesenchymal stem cells, the cell survival rates of the cells after 72 hours of storage were 75.1% and 66.4%, respectively, and the cell survival rate after 96 hours of storage was only 68.5%, less than 60%, which is far inferior to examples 1 to 3. In comparative examples 4 and 5, after omitting dextran 40 and water-soluble vitamin E (Trolox), the amounts of the rest components are increased, the survival rates of cells after 72 hours of storage are 62% and 77%, and the survival rates of cells after 96 hours of storage are less than 60% and 70.1%, respectively, so that the requirements of clinical cell activity cannot be met.
In addition, the present invention also reseedes the cells stored for 168 hours in example 1 to a petri dish, and observes the cell adhesion after 12 hours. Fig. 2 is a microscopic image of the canine mesenchymal stem cells of example 1 after being stained with trypan blue for 168 hours, and after the cells of example 1 after being stored for 168 hours are re-inoculated into a petri dish for 12 hours, the cell attachment conditions are shown in fig. 3 and fig. 4, and the cell attachment conditions under different magnifications are shown in fig. 3 and fig. 4, respectively. From fig. 3 to 4, it can be seen that the cells after 168 hours of preservation grow well after re-inoculation, so that the preservation solution can realize that the canine mesenchymal stem cells are preserved for a certain time at 4 ℃ to 8 ℃, the cell survival rate and the cell proliferation activity are not affected basically in the preservation process, and the preservation solution is suitable for long-time preservation and long-time transportation application of the canine mesenchymal stem cells.
The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention, but these modifications or substitutions are all within the scope of the present invention.
Claims (7)
1. The clinical mesenchymal stem cell cold storage preservation solution for dogs is characterized by comprising the following raw materials: water for injection, canine albumin, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate, dextran 40 and water-soluble vitamin E;
according to the weight ratio, the feed ratio of the water for injection, the canine albumin, the glucose, the sodium citrate, the citric acid, the sodium chloride, the sodium bicarbonate, the glucan 40 and the water-soluble vitamin E is 100:10-20:0.9-1.2:0.25-0.6:0.015-0.04:0.6-0.7:0.035-0.04:1-2:0.00002-0.00008;
the mesenchymal stem cells are canine adipose mesenchymal stem cells;
the refrigerating condition is 4-8 ℃;
the clinical mesenchymal stem cell cold storage preservation solution for dogs is prepared by uniformly mixing water for injection, albumin for dogs, glucose, sodium citrate, citric acid, sodium chloride, sodium bicarbonate, dextran 40 and water-soluble vitamin E, filtering and sterilizing.
2. The canine mesenchymal stem cell cryopreservation solution for clinical use according to claim 1, wherein: according to the weight ratio, the feed ratio of the water for injection, the canine albumin, the glucose, the sodium citrate, the citric acid, the sodium chloride, the sodium bicarbonate, the glucan 40 and the water-soluble vitamin E is 100:10:1.03:0.4:0.028:0.66:0.034:1:0.00005.
3. The canine mesenchymal stem cell cryopreservation solution for clinical use according to claim 1, wherein: in the filter sterilization operation, a filter membrane having a pore size of 0.22 μm was used.
4. A method for applying the clinical mesenchymal stem cell cryopreservation solution for dogs, which is characterized in that the clinical mesenchymal stem cell cryopreservation solution for dogs according to any one of claims 1 to 3 is adopted to preserve the mesenchymal stem cells of dogs.
5. The method for applying the canine mesenchymal stem cell cryopreservation solution according to claim 4, comprising the following specific steps: and (3) pre-cooling the clinical mesenchymal stem cell cold preserving fluid of the dogs at the temperature of 4-8 ℃, adding the canine mesenchymal stem cells into the pre-cooling preserving fluid, and continuously preserving the canine mesenchymal stem cells at the temperature of 4-8 ℃.
6. The method for applying the clinical mesenchymal stem cell cryopreservation solution for dogs according to claim 5, wherein the method comprises the following steps: the canine mesenchymal stem cells are subcultured P3-P5 passage cells.
7. The method for applying the clinical mesenchymal stem cell cryopreservation solution for dogs according to claim 6, wherein the method comprises the following steps: after the canine mesenchymal stem cells are added into the clinical mesenchymal stem cell cryopreservation solution for dogs, the concentration of the canine mesenchymal stem cells is 1×10 6 ～5×10 6 /mL。
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