Canine ovary mesenchymal stem cells as well as isolated culture method and application thereof

Abstract

The invention discloses canine ovary mesenchymal stem cells as well as an isolated culture method and application thereof. According to the method, the platelet-rich plasma is used for coating the cell culture container, the first liquid changing time is shortened, and the culture temperature is set to be 38 DEG C of the normal body temperature of the dog for culture, so that the migration and adhesion rate of the mesenchymal stem cells of the ovary of the dog is increased, the purity of the P1-generation mesenchymal stem cells reaches 95% or above, and the yield of the P1-generation mesenchymal stem cells is increased. Compared with the traditional method, the similar purity can be achieved only by culturing more than P3 generation. According to the invention, the acquisition way of the canine mesenchymal stem cells is expanded, and the provided canine ovary mesenchymal stem cells can be used for treating and preventing various canine diseases, especially have a remarkable effect on treating canine atopic dermatitis, and obviously promote the regeneration of canine hair.

Description

Technical Field

[0001] This invention belongs to the field of stem cell and tissue engineering technology, and particularly relates to a method for isolating and culturing ovarian mesenchymal stem cells from canines, as well as the application of these mesenchymal stem cells in the preparation of drugs for treating skin defects, musculoskeletal injuries, and autoimmune diseases in canines. Background Technology

[0002] Mesenchymal stem cells (MSCs) are adult pluripotent stem cells widely found in various tissues, including adipose tissue, bone marrow, dental pulp, uterine decidua, and perinatal tissues (ovaries and umbilical cord). They are readily available and free from ethical controversy. MSCs are stable under in vitro culture conditions and effectively maintain their multipotent differentiation potential even after continuous passage. As stem cells originating from the mesoderm, MSCs can not only characteristically differentiate into mesodermal tissue cells such as adipocytes, chondrocytes, and osteoblasts, but under appropriate conditions, they can also differentiate across germ layers into various endoderm and ectoderm tissues and cells, such as nerve cells, hepatocytes, and β-islet cells. Extensive research has found no evidence that MSCs cause tumors; therefore, their safety is far superior to that of embryonic stem cells. MSCs have extremely low immunogenicity, allowing for allogeneic transplantation without immune rejection, and they also possess strong immunomodulatory capabilities. In view of this, MSCs are widely used in many fields such as anti-aging and health care, regenerative medicine, autoimmune diseases, metabolic diseases, and neurological and mental diseases, and are one of the most important research hotspots in the field of cell therapy.

[0003] As important companion animals to humans, dogs also suffer from many diseases similar to those in humans. Unfortunately, compared to the extensive and in-depth research on human MSCs, research on animal-related MSCs lags far behind. One of the main reasons limiting significant progress in animal-related MSC research is the lack of methods for obtaining, isolating, and preparing these tissues. Currently, the main sources of canine MSCs are adipose tissue, bone marrow, and ovaries and umbilical cords. The former two are invasive methods specifically designed for MSC acquisition, often unacceptable to pet owners; the latter two often require cesarean sections due to the habit of female dogs ingesting ovarian and umbilical cord tissues after giving birth. Therefore, selecting suitable tissues for isolating and preparing canine MSCs is a pressing issue. Furthermore, current mesenchymal stem cell preparation methods often require culturing to the 3rd generation (P3 generation) or higher to achieve sufficient purity; obtaining high-purity mesenchymal stem cells at lower passage numbers is also a problem that urgently needs to be solved.

[0004] Our research team discovered that canine ovarian tissue is rich in stromal vascular fraction (SVF), from which high-quality mesenchymal stem cells (MSCs) can be effectively isolated and cultured. Spaying / castration of female dogs is a routine procedure requested by pet owners, and the removed tissue is often discarded, wasting valuable animal tissue samples. Therefore, this invention provides a method for preparing MSCs using canine ovarian tissue, which not only makes full use of the tissue sample but also, since spaying / castration is mostly performed in puppyhood, yields young MSCs.

[0005] Therefore, there is an urgent need in the field for new methods to isolate and prepare mesenchymal stem cells from canine ovaries. Summary of the Invention

[0006] The purpose of this invention is to provide canine ovarian mesenchymal stem cells, their isolation and culture method, and their applications. This invention utilizes platelet-rich plasma-coated cell culture containers, shortening the initial medium change time, and sets the culture temperature at 38°C (normal canine body temperature), thereby promoting the migration and adhesion rates of canine ovarian mesenchymal stem cells. This results in P1 generation mesenchymal stem cells achieving a purity of over 95%, compared to traditional methods that often require culturing to P3 or higher to achieve similar purity. The canine ovarian mesenchymal stem cells provided by this invention can be used for the treatment and prevention of various canine diseases, particularly showing significant efficacy in treating canine atopic dermatitis and markedly promoting hair regeneration in dogs.

[0007] This invention is achieved through the following technical solution: The first aspect of this invention provides a method for isolating and culturing canine ovarian mesenchymal stem cells, comprising: (1) Pre-prepared platelet-rich plasma Dog blood was collected and mixed under aseptic conditions using sodium citrate solution as an anticoagulant. The plasma and red blood cells were separated by a first centrifugation at room temperature; the upper plasma layer was aspirated and a second centrifugation was performed, and the lower layer was resuspended to obtain PRP. (2) Tissue digestion Tissue was harvested from the female dog after castration surgery. After disinfection, the ovaries were removed, rinsed with DPBS (ThermoFisher, catalog number 14190), and then transferred to MEM-α medium (Vicente, catalog number 310-010-CL). Cut the ovary into 0.2~2 pieces. Tissue blocks of various sizes were centrifuged, the supernatant was discarded, and digestive enzyme solution was added for digestion. The digestion product was centrifuged again, and erythrocyte lysis buffer was added. The mixture was allowed to stand to obtain a suspension. The digestive enzyme solution was a mixture of type I collagenase (purchased from Worthington CLS-1, catalog number LS004196), type II collagenase (purchased from Worthington CLS-2, catalog number LS004176), and hyaluronidase (purchased from Worthington HSE, catalog number LS002594). Centrifuge the suspension and discard the supernatant to remove residual red blood cells. Wash the cell pellet with DPBS and then resuspend the pellet in mesenchymal stem cell culture medium to obtain the final cell suspension. (3) Cell culture The cell suspension obtained in step (2) was transferred to a PRP-coated culture container and then placed in a CO2 cell culture incubator to allow mesenchymal stem cells to migrate out and adhere to the container. Remove the culture container, aspirate the culture medium, wash twice with DPBS, add mesenchymal stem cell culture medium, and continue culturing in a CO2 cell incubator; thereafter, perform a complete medium change every 3 days and continue culturing until the confluence rate of adherent cells in the culture container reaches 40%-70%; (4) Cell passage Utilizing digestive enzymes ( The cells (purchased from Thermo Fisher, catalog number 12605010) were dissociated, centrifuged, and then resuspended in mesenchymal stem cell culture medium. The cells were then seeded into a culture vessel for culture. The medium was changed every 1 to 3 days until the adherent cell fusion rate reached 70-90%, thus obtaining P1 generation ovarian mesenchymal stem cells. (5) Cell cryopreservation The obtained ovarian mesenchymal stem cells were added to a cryopreservation solution, aliquoted into cryovials, transferred to a programmed cooling box, and slowly cooled in a -80°C freezer. The cells were then transferred to liquid nitrogen for cryopreservation the following day. The cryopreservation solution contained... Culture medium, dimethyl sulfoxide, and canine serum.

[0008] Specifically, the method for isolating and culturing canine ovarian mesenchymal stem cells includes the following steps: (1) Pre-prepared platelet-rich plasma Using a 3.0%-4.0% (W / V) sodium citrate solution as an anticoagulant, under aseptic conditions, collect 10 mL of canine blood using a blood collection tube containing 1 mL of sodium citrate solution, and gently invert and mix 5-8 times. Perform the first centrifugation at room temperature at a speed of 1500-2000 rpm for 8-15 minutes to separate plasma and red blood cells; carefully aspirate the plasma portion above the leukocyte layer (buffy coat) and transfer it to a sterile conical centrifuge tube, then perform the second centrifugation at room temperature at a speed of 2500-3500 rpm for 8-15 minutes. The lower layer is collected and resuspended to obtain PRP. After being added to a cell culture container and spread evenly, it is placed in a cell culture incubator and incubated for 1-2 hours for later use. (2) Tissue digestion Tissue was harvested from the castrated female dog and then soaked in 75% alcohol for 10-60 seconds and rinsed repeatedly with saline solution to disinfect the tissue. The ovary was dissected, blood vessels and fascia were removed, and the ovary was washed with DPBS (Thermo Fisher, catalog number 14190, containing 200 mg / L KCl, 200 mg / L KH2PO4, 8 g / L NaCl, 2.16 g / L Na2HPO4·7H2O) and then transferred to MEM-α medium (Vicente, catalog number 310-010-CL). The ovary was minced into tissue pieces of 0.2-2 mm³, centrifuged to remove the supernatant, and a digestive enzyme solution was added. The mixture was then incubated on a constant-temperature shaking incubator at 34-40°C and 30-100 rpm for 0.5-3 hours. The incompletely digested tissue pieces were then removed by filtering through a 40-150 μm cell filter. The digestive enzyme solution contained a mixture of type I collagenase (purchased from Worthington CLS-1, catalog number LS004196), type II collagenase (purchased from Worthington CLS-2, catalog number LS004176), and hyaluronidase (purchased from Worthington HSE, catalog number LS002594). Centrifuge the digestion product at 1000 rpm for 10 minutes, resuspend the precipitate in DPBS, add red blood cell lysis buffer (purchased from eBioscience, catalog number 00-4300-54, containing 8.29 g / L ammonium chloride, 1 g / L potassium bicarbonate, and 37 mg / L EDTA) and mix thoroughly. Incubate at room temperature for 10 minutes to obtain a suspension. Centrifuge the suspension at 800-2000 rpm for 5-15 minutes, discard the supernatant, and gently tap the bottom of the tube to loosen the precipitate. If there are still many red blood cells remaining, repeat 2-3 times. The cell pellet was washed twice with DPBS and then resuspended in mesenchymal stem cell culture medium to obtain the final cell suspension. (3) Cell culture The cell suspension obtained in step (2) was transferred to a PRP-coated culture container, and then the culture container was placed in a CO2 cell culture incubator and cultured for 0.5 to 4 hours to allow mesenchymal stem cells to migrate out and adhere to the wall. Remove the culture container, aspirate the culture medium, wash twice with PBS, add mesenchymal stem cell culture medium, and continue culturing in a CO2 cell incubator; thereafter, perform a complete medium change every 3 days and continue culturing until the adherent cell confluence rate in the culture container reaches 40%-70%; (4) Cell passage Cells were dissociated using a digestive enzyme (TrypLE™ Express, Thermo Fisher, catalog number 12605010), centrifuged, and then resuspended in mesenchymal stem cell culture medium. The cells were then seeded into culture containers and cultured. The medium was changed every 1-3 days until the adherent cell confluence rate reached 70-90%, yielding P1 generation ovarian mesenchymal stem cells. The cells were then passaged as described above. (5) Cell cryopreservation The obtained ovarian mesenchymal stem cells were added to a cryopreservation solution, aliquoted into cryovials, transferred to a programmed cooling box, and slowly cooled in a -80°C freezer. The cells were then transferred to liquid nitrogen for cryopreservation the following day. The cryopreservation solution contained... Culture medium, dimethyl sulfoxide, and canine serum.

[0009] Preferably, the concentration of the sodium citrate solution in step (1) is 3.2% or 3.8% (W / V); more preferably, the concentration of the sodium citrate solution is 3.2% (W / V).

[0010] Preferably, in step (1), the first centrifugation operation is performed at a speed of 2000 rpm for 10 minutes; the second centrifugation operation is performed at a speed of 3000 rpm for 10 minutes.

[0011] Preferably, the cell culture container in step (1) is a TC-treated 10 cm cell culture dish or a T75 cell culture dish.

[0012] Preferably, the mother dog in step (2) is under 1 year old.

[0013] Preferably, the soaking time of the 75% alcohol soak in step (2) is 30 seconds.

[0014] Preferably, the digestive enzyme solution in step (2) is prepared by dissolving a mixture of 0.05-0.5g type I collagenase, 0.05-0.5g type II collagenase, and 0.01-0.2g hyaluronidase in 100mL of MEM-α medium, then filtering it through a 0.22μm sterile PES filter for sterilization. More preferably, the digestive enzyme solution is prepared by dissolving a mixture of 0.08-0.25g type I collagenase, 0.01-0.2g type II collagenase, and 0.02-0.1g hyaluronidase in 100mL of MEM-α medium, then filtering it through a 0.22μm sterile PES filter for sterilization. More preferably, the digestive enzyme solution is prepared by dissolving a mixture of 0.15g type I collagenase, 0.1g type II collagenase, and 0.05g hyaluronidase in 100mL of MEM-α medium. Dissolve the MEM-α medium, filter it through a 0.22μm sterile PES filter for sterilization, and then use it for later use.

[0015] Preferably, in step (2), the constant temperature shaking incubator is set at 36-38°C and 80 rpm for 1-2 hours for digestion.

[0016] Preferably, the cell filter in step (2) is a 100 μm filter.

[0017] Preferably, the mesenchymal stem cell culture medium in step (2) is based on MEM-α medium (Vicente, catalog number: 310-010-CL), supplemented with: 10~20% (V / V) canine serum, 2.0~10.0 mg / L transferrin, 3.0~8.0 μg / L sodium selenite, 8.0~32.0 mg / L putrescine, 2.1~12.6 μg / L progesterone, 2.0~10.0 μg / L biotin, 0.5~2.0 μg / mL L-carnitine, 5.0~10.0 μg / mL D(+)-galactose, 15.0~40.0 mg / L reduced glutathione, 20.0~60.0 mg / L ascorbic acid; 5.0~10.0 mg / L glycine, 3.0~17.8 mg / L glutathione, and 3.0~17.8 mg / L glutathione. mg / L alanine, 4.4~26.4 mg / L asparagine, 4.4~26.6 mg / L aspartic acid, 4.9~29.4 mg / L glutamate, 20.0~60.0 mg / L proline, 3.5~21.0 mg / L serine; 0.5~2.5 mg / L choline chloride, 0.5~2.5 mg / L D-calcium pantothenate, 0.5~2.5 mg / L folic acid, 0.6~2.4 mg / L nicotinamide, 0.5~2.5 mg / L pyridoxal hydrochloride, 0.05~0.25 mg / L riboflavin, 0.5~2.5 mg / L thiamine hydrochloride, 1.0~5.0 mg / L inositol.

[0018] Preferably, in step (3), the cells are cultured in a CO2 cell culture incubator with a CO2 concentration of 5% and culture conditions of 38°C for 2 hours.

[0019] Preferably, the cell cryopreservation solution in step (5) consists of 50% (V / V) MEM-α medium, 10% (V / V) dimethyl sulfoxide and 40% (V / V) canine serum.

[0020] A second aspect of the present invention provides canine ovarian mesenchymal stem cells obtained by the above-described isolation and culture method.

[0021] A third aspect of this invention provides a culture medium for isolating and culturing canine ovarian mesenchymal stem cells, comprising: MEM-α medium (Vicente, catalog number: 310-010-CL) as the basal medium, supplemented with: 10-20% (V / V) canine serum, 2.0-10.0 mg / L transferrin, 3.0-8.0 μg / L sodium selenite, 8.0-32.0 mg / L putrescine, 2.1-12.6 μg / L progesterone, 2.0-10.0 μg / L biotin, 0.5-2.0 μg / mL L-carnitine, 5.0-10.0 μg / mL D(+)-galactose, 15.0-40.0 mg / L reduced glutathione, 20.0-60.0 mg / L ascorbic acid; 5.0-10.0 mg / L glycine, 3.0-17.8 mg / L... mg / L alanine, 4.4~26.4 mg / L asparagine, 4.4~26.6 mg / L aspartic acid, 4.9~29.4 mg / L glutamate, 20.0~60.0 mg / L proline, 3.5~21.0 mg / L serine; 0.5~2.5 mg / L choline chloride, 0.5~2.5 mg / L D-calcium pantothenate, 0.5~2.5 mg / L folic acid, 0.6~2.4 mg / L nicotinamide, 0.5~2.5 mg / L pyridoxal hydrochloride, 0.05~0.25 mg / L riboflavin, 0.5~2.5 mg / L thiamine hydrochloride, 1.0~5.0 mg / L inositol.

[0022] Preferably, the culture medium comprises: MEM-α medium (Vicente, catalog number: 310-010-CL) as the basal medium, supplemented with: 15% (V / V) canine serum, 5.5 mg / L transferrin, 5.0 μg / L sodium selenite, 16.1 mg / L putrescine, 6.3 μg / L progesterone, 5 μg / L biotin, 1.0 μg / mL L-carnitine, 7.5 μg / mL D(+)-galactose, 30.73 mg / L reduced glutathione, 50 mg / L ascorbic acid; 7.5 mg / L glycine, 8.9 mg / L alanine, 13.2 mg / L asparagine, 13.3 mg / L aspartic acid, 14.7 mg / L glutamate, 40 mg / L proline, 10.5 mg / L serine; 1 mg / L choline chloride, 1 mg / L... D-Calcium Pantothenate, 1 mg / L Folic Acid, 1.2 mg / L Nicotinamide, 1 mg / L Pyridoxal Hydrochloride, 0.1 mg / L Riboflavin, 1 mg / L Thiamine Hydrochloride, 2 mg / L Inositol.

[0023] The fourth aspect of this invention provides the application of the aforementioned canine ovarian mesenchymal stem cells in the preparation of drugs for treating or preventing canine diseases; the canine diseases are selected from skin defects, musculoskeletal system injuries, autoimmune diseases, diabetes, liver / kidney failure, and cognitive impairment; for example: skin and mucous membrane tissue defects; musculoskeletal system injuries such as bones, muscles, joints, and ligaments; autoimmune diseases such as atopic dermatitis, inflammatory bowel disease, and aplastic anemia; diabetes; liver and kidney failure; and cognitive impairment.

[0024] Preferably, the canine disease is atopic dermatitis.

[0025] This invention provides canine ovarian mesenchymal stem cells, their isolation and culture methods, and their applications, expanding the tissue sources of mesenchymal stem cells and avoiding the waste of ovarian tissue after castration in female dogs. By utilizing platelet-rich plasma-coated cell culture containers, this invention shortens the initial medium change time and sets the culture temperature at 38°C (normal canine body temperature), thereby promoting the migration and adhesion rates of canine ovarian mesenchymal stem cells and achieving a purity of over 95% for P1 generation mesenchymal stem cells. In contrast, traditional methods often require culturing to P3 or higher to achieve similar purity.

[0026] The method of this invention is simple to operate, convenient and practical, and yields high-purity, abundant, and rapidly proliferating mesenchymal stem cells with the potential to differentiate into osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, and glandular cells. Since castration in female dogs is mostly performed in puppyhood, the stem cells in their ovaries are relatively immature and have good viability, showing broad clinical application prospects. The obtained mesenchymal stem cells can be cryopreserved long-term in liquid nitrogen at -196°C using conventional cell cryopreservation methods, establishing an ovarian stem cell bank and laying the foundation for future in-depth stem cell research and clinical treatment.

[0027] This invention utilizes a specialized culture medium to culture canine ovarian mesenchymal stem cells, which can maintain cell stemness while promoting rapid stem cell proliferation. The canine ovarian mesenchymal stem cells provided by this invention can be used for the treatment and prevention of various canine diseases, especially showing significant efficacy in treating canine atopic dermatitis and markedly promoting hair regeneration in dogs. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 To separate canine ovarian tissue; where A: tissue from castration of the female dog; B: bilateral ovarian tissue removed.

[0030] Figure 2 To prepare canine ovarian mesenchymal stem cells; in A: digested canine ovarian tissue was seeded into a 10cm cell culture dish (comparative example) for 48 hours. After washing with DPBS to remove excess tissue and cells, a large number of long spindle-shaped adherent MSCs were observed; in B: digested canine ovarian tissue was seeded into a PRP-coated 10cm cell culture dish (example) for 2 hours. After washing with DPBS to remove excess tissue and cells, long spindle-shaped adherent MSCs were observed.

[0031] Figure 3 Primary canine ovarian mesenchymal stem cells (P0) were cultured for 7 days; A: After canine ovarian tissue was seeded into a 10cm cell culture dish (comparative example) and cultured for 7 days, a large number of MSCs growing in a vortex pattern were observed; B: After canine ovarian tissue was seeded into a 10cm cell culture dish coated with PRP (example) and cultured for 7 days, a large number of MSCs growing in a vortex pattern were observed.

[0032] Figure 4Flow cytometry analysis was performed on canine ovarian mesenchymal stem cells (D-MSCs / O-P1) P1 generation. A: Canine ovarian mesenchymal stem cells (comparative example) P1 generation: 72.15%–81.37% of cells were positive for characteristic markers of mesenchymal stem cells (CD29, CD73, CD90, CD105), 23.87%–36.48% were negative for characteristic markers (CD14, CD19, CD34, CD45), and 0.46% were HLA-DR. B: Canine ovarian mesenchymal stem cells (example) P1 generation: 95.51%–99.75% of cells were positive for characteristic markers of mesenchymal stem cells (CD29, CD73, CD90, CD105), and 0.26%–0.48% were negative for characteristic markers (CD14, CD19, CD34, CD45, HLA-DR).

[0033] Figure 5 Flow cytometry analysis was performed on canine ovarian mesenchymal stem cells (D-MSCs / O-P3) P3 generation. A: Canine ovarian mesenchymal stem cells (comparative example) P3 generation: 98.28%–99.97% of cells were positive for characteristic markers of mesenchymal stem cells (CD29, CD73, CD90, CD105), and 0.04%–0.79% were negative for characteristic markers (CD14, CD19, CD34, CD45, HLA-DR). B: Canine ovarian mesenchymal stem cells (example) P3 generation: 95.51%–99.75% of cells were positive for characteristic markers of mesenchymal stem cells (CD29, CD73, CD90, CD105), and 0.26%–0.48% were negative for characteristic markers (CD14, CD19, CD34, CD45, HLA-DR).

[0034] Figure 6 Osteogenic and adipogenic differentiation were induced in canine ovarian mesenchymal stem cells. Osteogenic induction: After 2 weeks of induction culture, canine ovarian mesenchymal stem cells from the examples (P1 generation) and the comparative example (P3 generation) showed a strong positive reaction to Alizarin Red staining, with over 94% of the cells showing orange-red calcification nodules, while the uninduced control group was mostly negative. Adipogenic induction: After 2 weeks of induction culture, canine ovarian mesenchymal stem cells from the examples (P1 generation) and the comparative example (P3 generation) showed that intracellular lipid droplets were specifically stained red by Oil Red O staining, indicating that the cells had transformed into adipocytes, while the uninduced control group was negative.

[0035] Figure 7Case studies of canine ovarian mesenchymal stem cell therapy for atopic dermatitis include: a 13-year-old Border Collie receiving 3×10⁷ ovarian mesenchymal stem cells (P3 generation in the comparative example) before and after treatment (red box indicates before treatment); B1: a 12-year-old Teddy dog ​​before treatment; B2: the same dog after receiving 1×10⁷ ovarian mesenchymal stem cells (P1 generation in the example); C1: a 14-year-old Chinese Rural Dog before treatment; C2: the same dog after receiving 2×10⁷ ovarian mesenchymal stem cells (P1 generation in the example). Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example: Preparation of mesenchymal stem cells from canine ovaries (1) Pre-prepared platelet-rich plasma Platelet-rich plasma (PRP) coated into cell culture dishes: Take 10 mL of anticoagulated whole blood from castrated female dogs (under 1 year old) (1 mL of 3.2% (w / v) sodium citrate solution added to 9 mL of canine blood); centrifuge at 2000 rpm for 10 minutes at room temperature; carefully aspirate the plasma portion of the upper buffy coat and transfer it to a sterile 15 mL conical centrifuge tube; then centrifuge at 3000 rpm for 10 minutes at room temperature; collect the lower 2 mL of plasma and add it to a TC-treated 10 cm cell culture dish, spread it evenly, and incubate in a cell culture incubator for 1-2 hours for later use.

[0038] (2) Tissue digestion Ovarian tissue digestion: In a biosafety cabinet, the tissue excised during castration in the female dog was removed from the sample collection bottle and placed in a stainless steel cup. It was then disinfected by immersion in 75% alcohol for 30 seconds, followed by repeated rinsing with physiological saline to disinfect the tissue. The tissue was then placed in a stainless steel dish containing an appropriate amount of DPBS. The ovary was dissected from the adipose tissue using surgical forceps, removing blood vessels and fascia. After rinsing again with DPBS, the tissue was transferred to a 6cm glass culture dish containing 5mL of MEM-α culture medium. The ovary was minced into tissue pieces of approximately 0.5-1mm³ using a scalpel and ophthalmic scissors. The tissue pieces were collected and transferred to 15mL conical centrifuge tubes, centrifuged at 2000 rpm for 10 minutes, and the supernatant was discarded. A digestive enzyme solution was added (final concentration: 0.15% (w / v) type I collagenase, 0.1% (w / v) collagenase). Type II collagenase and 0.05% (w / v) hyaluronidase in MEM-α solution (filtered through a 0.22 μm sterile PES filter for sterilization) were placed on a constant temperature shaking water bath at 37°C and 80 rpm for 1 hour for digestion. A 100 μm cell filter was placed on a 50 mL conical centrifuge tube to collect the digestion product. The filter was washed with twice the volume of DPBS, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was gently tapped at the bottom of the tube to loosen it. The precipitate was resuspended in 2 mL of DPBS, and 20 mL of erythrocyte lysis buffer was added and mixed thoroughly. The mixture was incubated at room temperature for 10 minutes to lyse the erythrocytes in the suspension. The suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The precipitate was gently tapped at the bottom of the tube to loosen it (this step was repeated once). The cell pellet was washed twice with DPBS and then resuspended in mesenchymal stem cell culture medium to obtain the final cell suspension.

[0039] In this embodiment, the mesenchymal stem cell culture medium was based on MEM-α, supplemented with: 15% (V / V) canine serum; 5.5 mg / L transferrin, 5.0 μg / L sodium selenite, 16.1 mg / L putrescine, 6.3 μg / L progesterone, 5 μg / L biotin, 1.0 μg / mL L-carnitine, 7.5 μg / mL D(+)-galactose, 30.73 mg / L reduced glutathione, 50 mg / L ascorbic acid; 7.5 mg / L glycine, 8.9 mg / L alanine, 13.2 mg / L asparagine, 13.3 mg / L aspartic acid, 14.7 mg / L glutamate, 40 mg / L proline, 10.5 mg / L serine; 1 mg / L choline chloride, 1 mg / L D-Calcium Pantothenate, 1 mg / L Folic Acid, 1.2 mg / L Nicotinamide, 1 mg / L Pyridoxal Hydrochloride, 0.1 mg / L Riboflavin, 1 mg / L Thiamine Hydrochloride, 2 mg / L Inositol.

[0040] (3) Cell culture Add the cell suspension obtained in step (2) to a 10cm cell culture dish coated with PRP, place it in a 5% CO2 cell culture incubator, and culture at 38°C for 2 hours. Remove the culture container from the incubator, discard the culture medium, wash twice with DPBS (spindle-shaped adherent cells can be clearly seen under a microscope), add 10mL of mesenchymal stem cell culture medium, and place it in a 38°C 5% CO2 cell culture incubator for continued culture. Perform the first complete medium change after 3 days (at this time, cell clones growing in a turbine shape can be seen), and continue culture. Perform a complete medium change every 3 days thereafter.

[0041] (4) Cell passage Under a microscope, when the confluence rate of adherent cells in the culture vessel reached 60%, the culture supernatant was discarded. After washing twice with DPBS, 2 mL of digestion solution (TrypLE™ Express, a commercial product of Thermo Fisher, diluted 4 times with DPBS) was added to cover the culture dish. Digestion was carried out for 5 minutes, and 2 mL of culture medium was added to stop the digestion. The cells were repeatedly pipetted several times to dissociate them, and the cell suspension was collected and transferred to a 15 mL conical centrifuge tube. The culture dish was washed with 4 mL of DPBS and transferred to the centrifuge tube. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cell pellet was collected, and the cells were resuspended in mesenchymal stem cell culture medium. After counting, the cells were seeded at 1 × 10⁶ in TC-treated T75 culture flasks and placed in a 38°C 5% CO₂ cell culture incubator for further culture. The medium was changed every 3 days until the confluence rate reached more than 80%, thus obtaining P1 generation canine ovarian mesenchymal stem cells. Subsequent passages were then carried out using the above culture method. During the process, the cell growth status was dynamically observed, and cells from passages P1, P3, P5, P7, and P9 were collected for flow cytometry analysis of cell surface markers.

[0042] (5) Cell cryopreservation Take a portion of the canine ovarian mesenchymal stem cells obtained in step (4), and obtain cell pellet through digestion, centrifugation, recovery, and counting. Resuspend the cells in an appropriate amount of cell cryopreservation solution (50% (V / V) MEM-α, 10% (V / V) dimethyl sulfoxide, 40% (V / V) canine serum), aliquot them into cell cryopreservation tubes at 1×10⁶ / mL, transfer them to a cell programmed cooling box, and place them in a -80℃ freezer for slow cooling. After 6-8 hours, transfer them to liquid nitrogen for cryopreservation. Before cryopreservation, input the detailed information of the mesenchymal stem cells (dog age, breed, genetic information, vaccination information, virus detection information, etc.) into a computer database to establish a data archive for future reference.

[0043] In this embodiment, the obtained canine ovarian mesenchymal stem cells exhibited a relatively uniform spindle-shaped and whorled growth pattern, with rapid adhesion, rapid proliferation, and easy digestion by trypsin. Phenotypic analysis of the obtained cells showed that in passage P1, the percentage of cells with characteristic positive markers such as CD29, CD73, CD90, and CD105 was 95.51%–99.75%, while the percentage of cells with characteristic negative markers such as CD14, CD19, CD34, CD45, and HLA-DR was 0.26%–0.48%. By passage 9 (P9), the morphology and growth characteristics of the cells showed no significant changes, with 98.58%–99.75% of cells showing positive markers and 0.04%–0.52% showing negative markers. Tests for endotoxin, mycoplasma, and chlamydia were all negative; bacterial and fungal cultures were also negative. In step (1) of this embodiment, the culture vessel is coated with canine blood PRP to promote the migration, adhesion, and growth of MSCs by utilizing the abundant chemokines and growth factors in platelets. Simultaneously, the culture temperature is set at 38°C, the normal body temperature of dogs, which further promotes rapid migration and adhesion of MSCs. Based on this, a mesenchymal stem cell-specific culture medium supplemented with various amino acids, vitamins, and antioxidants is used to promote rapid proliferation of MSCs and maintain their stemness. Through the method of this embodiment, high-purity P1 generation canine ovarian mesenchymal stem cells were obtained, which is not only significantly superior to the comparative embodiment but also superior to most current methods for preparing mesenchymal stem cells (most of which require up to P3 generation).

[0044] Comparative Example: (1) Digestion and processing of ovarian tissue Inside the biosafety cabinet, tissue excised from the castration procedure in the female dog was removed from the sample collection bottle and placed in a stainless steel cup. It was then disinfected by immersion in 75% alcohol for 30 seconds, followed by repeated rinsing with physiological saline to sterilize the tissue. The tissue was then placed in a stainless steel dish containing an appropriate amount of DPBS. The ovary was dissected from the adipose tissue using surgical forceps, and blood vessels and fascia were removed. After rinsing again with DPBS, the tissue was transferred to a 6cm glass culture dish containing 5mL of MEM-α culture medium. The ovary was then minced into tissue pieces of approximately 0.5–1 mm³ using a scalpel and ophthalmic scissors. The tissue pieces were collected and transferred to 15mL conical centrifuge tubes, centrifuged at 2000 rpm for 10 minutes, and the supernatant was discarded. A digestive enzyme solution was added (final concentration: 0.15% (w / v) type I collagenase, 0.1% (w / v) collagenase). Type II collagenase and 0.05% (w / v) hyaluronidase in MEM-α solution (filtered through a 0.22 μm sterile PES filter for sterilization) were placed on a constant temperature shaking water bath at 37°C and 80 rpm for 1 hour for digestion. A 100 μm cell filter was placed on a 50 mL conical centrifuge tube to collect the digestion product. The filter was washed with twice the volume of DPBS, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The precipitate was gently tapped at the bottom of the tube to loosen it. The precipitate was resuspended in 2 mL of DPBS, and 20 mL of erythrocyte lysis buffer was added and mixed thoroughly. The mixture was incubated at room temperature for 10 minutes to lyse the erythrocytes in the suspension. The suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The precipitate was gently tapped at the bottom of the tube to loosen it (this step was repeated once). The cell pellet was washed twice with DPBS and then resuspended in mesenchymal stem cell culture medium (MEM-α medium containing 15% (v / v) canine serum in this comparative example) to obtain the final cell suspension.

[0045] (2) Cell culture The cell suspension (4 mL) obtained in step (1) was seeded into a 10 cm cell culture dish and placed in a 5% CO2 cell culture incubator and cultured at 37°C for 8 hours. The culture dish was then removed from the incubator, and 5 mL of mesenchymal stem cell culture medium was added. The dish was then cultured at 37°C in a 5% CO2 incubator. After 48 hours of culture, the culture medium was discarded, and the dish was washed twice with DPBS and the medium was completely replaced. The medium was then completely replaced every 3 days thereafter.

[0046] (3) Cell passage Under a microscope, when the confluence of adherent cells in the culture vessel reached 60%, the culture supernatant was discarded. After washing twice with DPBS, 2 mL of digestion solution (TrypLE™ Express, a commercial product of Thermo Fisher, diluted 4 times with DPBS) was added to cover the culture dish. Digestion was carried out for 5 minutes, and 2 mL of culture medium was added to stop the digestion. The cells were dissociated by repeatedly pipetting with a Pasteur pipette, and the cell suspension was collected and transferred to a 15 mL conical centrifuge tube. The culture dish was washed with 4 mL of DPBS and transferred to the centrifuge tube. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cell pellet was collected, and the cells were resuspended in mesenchymal stem cell culture medium. After counting, the cells were seeded at 1 × 10⁶ in TC-treated T75 culture flasks and placed in a 37°C 5% CO₂ cell culture incubator for further culture. The medium was changed every 3 days until the confluence reached more than 80%, thus obtaining P1 generation canine ovarian mesenchymal stem cells. Subsequent passages were then carried out using the above culture method. During the process, the cell growth status was dynamically observed, and cells from passages P1, P3, P5, P7, and P9 were taken for flow cytometry analysis to detect cell surface markers (high expression of CD29, CD73, CD90, and CD105, and low / no expression of CD14, CD19, CD34, CD45, and HLA-DR).

[0047] (4) Cell cryopreservation Take a portion of the canine ovarian mesenchymal stem cells obtained in step (3), and after digestion, counting, and centrifugation, obtain cell pellets. Add an appropriate amount of cell cryopreservation solution (50% (V / V) MEM-α, 10% (V / V) dimethyl sulfoxide, 40% (V / V) canine serum) to resuspend the cells. Aliquot the cells into cryopreservation tubes at 1×10⁶ / mL, transfer them to a programmed cooling box, and place them in a -80℃ freezer for slow cooling. After 6-8 hours, transfer them to liquid nitrogen for cryopreservation. Before cryopreservation, input the detailed information of the mesenchymal stem cells (dog age, breed, genetic information, vaccination information, virus detection information, etc.) into a computer database to establish a data archive for future reference.

[0048] In the comparative example, steps (1) to (4) adopted the currently common method of preparing mesenchymal stem cells from tissues, namely: after digesting the tissue block with digestive enzymes, the mesenchymal stem cells were separated by utilizing their adherent growth characteristics.

[0049] Cellular analysis results in this comparative study showed that spindle-shaped adherent cells were clearly visible under a microscope after 48 hours of culture of canine ovarian mesenchymal stem cells, and turbine-shaped cell clones formed in about 8 days. During the culture process, these cells were found to have relatively uniform morphology, rapid proliferation rate, fast adhesion rate, and were easily digested by trypsin. Even after passage to P9, their morphology and growth characteristics remained largely unchanged. Flow cytometry results showed that in the P1 generation, the percentage of cells with characteristic positive markers for mesenchymal stem cells (CD29, CD73, CD90, CD105, etc.) was 72.15%–81.37%, while the percentage of cells with characteristic negative markers (CD14, CD19, CD34, CD45, etc.) was 23.87%–36.48%, and HLA-DR was less than 1%. In the P3 generation, the percentage of cells with characteristic positive markers (CD29, CD73, CD90, CD105, etc.) was 98.28%–99.97%, while the percentage of cells with characteristic negative markers (CD14, CD19, CD34, CD45, HLA-DR, etc.) was 0.04%–0.79%. Detection of endotoxin, mycoplasma, and chlamydia was negative. Bacterial and fungal cultures were also negative.

[0050] Application Example 1: Identification of the multi-directional differentiation potential of ovarian MSCs The canine ovarian mesenchymal stem cells obtained from the examples (P1 generation) and the comparative example (P3 generation) were subjected to the following experiments to detect their differentiation potential. The results showed that both possessed osteogenic and adipogenic differentiation potential. Figure 6 As shown.

[0051] Osteogenic induction: Canine ovarian MSCs in logarithmic growth phase were harvested, and the cell density was adjusted to 1×10⁵ / well. Cells were seeded and cultured in 6-well cell culture plates. When confluence reached approximately 60%, the culture medium was replaced with osteogenic induction medium, consisting of: DMEM / F12 medium, 15% (v / v) canine serum, 1 μM dexamethasone, 50 mg / L sodium ascorbate phosphate, and 10 mM β-glycerophosphate. The medium was changed every 3–4 days. After continuous induction, alizarin red staining was performed for identification. Detection method: The induction medium was discarded, and the cells were washed twice with DPBS, excess water was aspirated, and the cells were fixed with 4% paraformaldehyde for 30 min, followed by washing three times with DPBS. 1 mL of 40 mM alizarin red staining solution (pH=4.1) was added to the wells, and the cells were gently incubated at room temperature for 5 minutes. Unadulterated dye was aspirated, and the cells were washed three times with DPBS until no red color was observed in the washings. The cells were then observed under a light microscope. After one week of culture, the cell morphology underwent significant changes, with the appearance of small black lumps and a cloudy, hazy appearance on the cell surface. The spindle-shaped, fibroblast-like structure transformed into a polygonal shape, resembling neuronal cells, with long filamentous protrusions appearing around the cell periphery and extending outwards. At two weeks of culture, Alizarin Red staining showed a strong positive reaction, with over 94% of the cells stained orange-red. In contrast, the uninduced control group was mostly negative, with only about 5% showing a weak positive result, indicating that the cells had transformed into osteoblasts.

[0052] Adipogenic induction: Canine ovarian MSCs in logarithmic growth phase were harvested, and the cell density was adjusted to 1×10⁵ / well. Cells were seeded and cultured in 6-well cell culture plates. When confluence reached approximately 80%, the culture medium was replaced with adipogenic induction medium, consisting of: DMEM / F12 medium, 15% (v / v) canine serum, 1 μM dexamethasone, 10 μM insulin, 0.5 mM IBMX, and 200 μM indomethacin. The medium was changed every 3–4 days. After continuous induction, Oil Red O staining was performed for identification. Oil Red O staining method: The induction medium was discarded, the cells were rinsed twice with DPBS, excess water was aspirated, and the cells were fixed with 4% paraformaldehyde for 30 min. Then, the cells were stained with diluted Oil Red O staining solution in the dark for 10 min, rinsed twice with DPBS, and observed and photographed. After 3 days of culture, the cells underwent morphological changes, gradually shrinking and shortening from a spindle-shaped fibroblast-like structure, with over 90% of the cells becoming cuboidal or polygonal. After 7 days of continuous culture, tiny lipid droplets were visible within the cells under a microscope. As the culture time increased, the lipid droplets gradually enlarged and merged. By 2 weeks of culture, fused lipid droplets filled the entire cell. Oil Red O staining showed that the intracellular lipids were specifically stained red, indicating that the cells had transformed into adipocytes.

[0053] Application Example 2: Application of canine ovarian mesenchymal stem cells in the treatment of canine atopic dermatitis The canine ovarian mesenchymal stem cells obtained from the examples (P1 generation) and the comparative example (P3 generation) were used in a treatment experiment for canine atopic dermatitis. The corresponding canine ovarian mesenchymal stem cells were taken from liquid nitrogen, rapidly thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, mesenchymal stem cell culture medium was added, and the cells were seeded into T75 culture flasks. The mesenchymal stem cell culture medium was MEM-α based, supplemented with: 15% (V / V) canine serum; 5.5 mg / L transferrin, 5.0 μg / L sodium selenite, 16.1 mg / L putrescine, 6.3 μg / L progesterone, 5 μg / L biotin, 1.0 μg / mL L-carnitine, 7.5 μg / mL D(+)-galactose, 30.73 mg / L reduced glutathione, 50 mg / L ascorbic acid; 7.5 mg / L glycine, 8.9 mg / L alanine, 13.2 mg / L asparagine, 13.3 mg / L aspartic acid, 14.7 mg / L glutamate, 40 mg / L proline, 10.5 mg / L serine; 1 mg / L choline chloride, 1 mg / L D-Calcium Pantothenate, 1 mg / L Folic Acid, 1.2 mg / L Nicotinamide, 1 mg / L Pyridoxal Hydrochloride, 0.1 mg / L Riboflavin, 1 mg / L Thiamine Hydrochloride, 2 mg / L Inositol. When the cell confluence reaches 90%, discard the culture supernatant; wash twice with DPBS, then add 2 mL of digestion solution (in this example, Thermo Fisher's commercially available TrypLE™ Express, diluted 4 times with DPBS) to cover the culture dish, digest for 5 minutes, then add 2 mL of culture medium to stop digestion; repeatedly pipette several times to dissociate the cells, collect the cell suspension, and transfer it to a 15 mL conical centrifuge tube; wash the culture dish with 4 mL of DPBS, transfer the cell suspension to the centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and collect the cell pellet; wash the cells twice with physiological saline, and count them; weigh the dog to be treated, adjust the total cell count to 1 million to 2 million cells / kg body weight, and dissolve the cell suspension in 100 mL of sodium chloride injection solution to prepare the canine ovarian mesenchymal stem cell intravenous injection preparation. Transport the canine ovarian mesenchymal stem cell intravenous injection preparation via cold chain to the veterinary hospital for stem cell therapy.

[0054] The canine ovarian mesenchymal stem cell (P1 generation) intravenous injection formulation from the embodiment was used to treat canine atopic dermatitis.

[0055] Case 1: A 12-year-old Teddy dog ​​weighing 5 kg presented with hair loss, an unpleasant odor, and significantly reduced mobility, almost unable to jump. After 4 months of treatment with 1×10⁷ mesenchymal stem cells, the dog's hair grew back noticeably, the odor disappeared, and its mobility significantly improved, allowing it to jump onto a 50cm seat. Follow-up for six months showed the condition remained stable without recurrence.

[0056] Case 2: A 14-year-old Chinese rural dog, weighing 13 kg, with hair loss (almost complete baldness) accompanied by subcutaneous masses and lethargy; after 4 months of treatment with 2×107 mesenchymal stem cells, the dog's hair grew significantly (covering the entire body surface), the subcutaneous masses disappeared, and its vitality was significantly enhanced.

[0057] The canine atopic dermatitis was treated with an intravenous injection of canine ovarian mesenchymal stem cells (P3 generation) from a comparative example.

[0058] Dog 3: A 13-year-old Border Collie weighing 25 kg, with hair loss that did not grow back after shaving and a significant decrease in activity level; after 4 months of treatment with 3×10⁷ mesenchymal stem cells, the dog's hair grew back significantly, its activity level and mental state improved significantly, and it interacted more actively with its owner.

[0059] The above data indicators show that the canine mesenchymal stem cells isolated by the method of this invention have the ability to differentiate into osteoblasts, adipocytes, etc., confirming that the canine mesenchymal stem cells obtained by the method of this invention have stem cell characteristics.

[0060] Atopic dermatitis (AD) is defined as "a genetically predisposed inflammatory and pruritic allergic skin disease with characteristic clinical features." Currently recommended treatments for AD include oral systemic immunosuppressants such as corticosteroids, cyclosporine, and oclacitinib. However, these immunosuppressive drugs have limited efficacy, and long-term use can lead to serious side effects. In this invention, we utilize the powerful immunomodulatory and tissue-repairing capabilities of canine ovarian mesenchymal stem cells (P1 generation) to treat canine AD via intravenous infusion. The condition of the affected dogs was effectively controlled long-term and completely, with no observed side effects. Simultaneously, the mental and motor abilities of the treated dogs showed significant improvement. The canine ovarian mesenchymal stem cells (P1 generation) used in our embodiment achieved the same therapeutic effect as the comparative canine ovarian mesenchymal stem cells (P3 generation), and no significant difference was observed in safety and efficacy between the two.

[0061] This invention discloses a method for isolating, preparing, and culturing mesenchymal stem cells from canine ovarian tissue, expanding the methods for obtaining canine mesenchymal stem cells. The method is simple, convenient, practical, and highly efficient. PRP-coated culture dishes promote chemotaxis, migration, and adhesion of mesenchymal stem cells, increasing the yield of primary cells. Optimized culture media and conditions help maintain cell stemness and promote rapid cell proliferation. This invention can obtain ovarian mesenchymal stem cells with over 95% purity at P1 generation, enabling the treatment of canine diseases, including: skin and mucous membrane defects; injuries to the musculoskeletal system such as bones, muscles, joints, and ligaments; autoimmune diseases such as atopic dermatitis, inflammatory bowel disease, and aplastic anemia; diabetes; liver and kidney failure; and cognitive impairment.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for isolating and culturing canine ovarian mesenchymal stem cells, comprising: (1) Pre-prepared platelet-rich plasma Dog blood was collected and mixed under aseptic conditions using sodium citrate solution as an anticoagulant. The plasma and red blood cells were separated by a first centrifugation at room temperature; the upper plasma layer was aspirated and a second centrifugation was performed, and the lower layer was resuspended to obtain PRP. (2) Tissue digestion Tissue was harvested from the female dog after castration surgery. After disinfection, the ovaries were removed, rinsed with DPBS (Thermo Fisher, catalog number 14190), and then transferred to MEM-α medium (Vicente, catalog number 310-010-CL). The ovary was minced into tissue pieces of 0.2-2 mm3, centrifuged and the supernatant was discarded. A digestive enzyme solution was added for digestion. The digestion product was centrifuged and then red blood cell lysis buffer was added. The mixture was allowed to stand to obtain a suspension. The digestive enzyme solution contained a mixture of type I collagenase (purchased from Worthington CLS-1, catalog number LS004196), type II collagenase (purchased from Worthington CLS-2, catalog number LS004176), and hyaluronidase (purchased from Worthington HSE, catalog number LS002594). Centrifuge the suspension and discard the supernatant to remove residual red blood cells. Wash the cell pellet with DPBS and then resuspend the pellet in mesenchymal stem cell culture medium to obtain the final cell suspension. (3) Cell culture The cell suspension obtained in step (2) was transferred to a PRP-coated culture container and then placed in a CO2 cell culture incubator to allow mesenchymal stem cells to migrate out and adhere to the container. Remove the culture container, aspirate the culture medium, wash twice with PBS, add mesenchymal stem cell culture medium, and continue culturing in a CO2 cell incubator; thereafter, perform a complete medium change every 3 days and continue culturing until the adherent cell confluence rate in the culture container reaches 40%-70%; (4) Cell passage Cells were dissociated using digestive enzymes (TrypLE™ Express, purchased from Thermo Fisher, catalog number 12605010), centrifuged, and then resuspended in mesenchymal stem cell culture medium. The cells were then seeded into culture containers for culture. The medium was changed every 1 to 3 days until the adherent cell confluence rate reached 70-90%, thus obtaining P1 generation canine ovarian mesenchymal stem cells. (5) Cell cryopreservation The obtained ovarian mesenchymal stem cells were added to a cryopreservation solution, aliquoted into cryovials, transferred to a programmed cooling box, and slowly cooled in a -80°C freezer. The cells were then transferred to liquid nitrogen for cryopreservation the following day. The cryopreservation solution contained... Culture medium, dimethyl sulfoxide, and canine serum.

2. The method for isolating and culturing canine ovarian mesenchymal stem cells according to claim 1, characterized in that: The concentration of the sodium citrate solution in step (1) is 3.0%-4.0% (W / V).

3. The method for isolating and culturing canine ovarian mesenchymal stem cells according to claim 1, characterized in that: In step (1), the first centrifugation operation is performed at a speed of 1500-2000 rpm for 8-15 minutes; the second centrifugation operation is performed at a speed of 2500-3500 rpm for 8-15 minutes.

4. The method for isolating and culturing canine ovarian mesenchymal stem cells according to claim 1, characterized in that: The digestive enzyme solution in step (2) is a mixture containing 0.05~0.5% (W / V) type I collagenase, 0.05~0.5% (W / V) type II collagenase, and 0.01~0.2% (W / V) hyaluronidase.

5. The method for isolating and culturing canine ovarian mesenchymal stem cells according to claim 1, characterized in that: The digestion process in step (2) involves adding a digestive enzyme solution, digesting on a constant-temperature shaking incubator at 34-40°C and 30-100 rpm for 0.5-3 hours, followed by... Cellular filters remove undigested tissue fragments.

6. The method for isolating and culturing canine ovarian mesenchymal stem cells according to claim 1, characterized in that: The mesenchymal stem cell culture medium in step (2) is based on MEM-α medium (Vicente, catalog number: 310-010-CL), supplemented with: 10~20% (V / V) canine serum, 2.0~10.0 mg / L transferrin, 3.0~8.0 μg / L sodium selenite, 8.0~32.0 mg / L putrescine, 2.1~12.6 μg / L progesterone, 2.0~10.0 μg / L biotin, 0.5~2.0 μg / mL L-carnitine, 5.0~10.0 μg / mL D(+)-galactose, 15.0~40.0 mg / L reduced glutathione, and 20.0~60.0 mg / L ascorbic acid; 5.0~10.0 mg / L glycine, 3.0~17.8 mg / L alanine, 4.4~26.4 mg / L asparagine, 4.4~26.6 mg / L aspartic acid, 4.9~29.4 mg / L glutamic acid, 20.0~60.0 mg / L proline, 3.5~21.0 mg / L serine; 0.5~2.5 mg / L choline chloride, 0.5~2.5 mg / L D-calcium pantothenate, 0.5~2.5 mg / L folic acid, 0.6~2.4 mg / L nicotinamide, 0.5~2.5 mg / L pyridoxal hydrochloride, 0.05~0.25 mg / L riboflavin, 0.5~2.5 mg / L thiamine hydrochloride, 1.0~5.0 mg / L inositol.

7. The method for isolating and culturing canine ovarian mesenchymal stem cells according to claim 1, characterized in that: In step (3), the cells are cultured in a CO2 cell culture incubator with a CO2 concentration of 5% and culture conditions of 38°C for 2 hours.

8. The method for isolating and culturing canine ovarian mesenchymal stem cells according to claim 1, characterized in that: The cell cryopreservation solution in step (5) is 50% (V / V). The culture medium consisted of 10% (v / v) dimethyl sulfoxide and 40% (v / v) canine serum.

9. A canine ovarian mesenchymal stem cell obtained by the isolation and culture method according to any one of claims 1-8.

10. A culture medium for isolating and culturing canine ovarian mesenchymal stem cells, comprising: Using MEM-α medium (Vicente, catalog number: 310-010-CL) as the basal medium, the following were added: 10-20% (V / V) canine serum, 2.0-10.0 mg / L transferrin, 3.0-8.0 μg / L sodium selenite, 8.0-32.0 mg / L putrescine, 2.1-12.6 μg / L progesterone, 2.0-10.0 μg / L biotin, 0.5-2.0 μg / mL L-carnitine, 5.0-10.0 μg / mL D(+)-galactose, 15.0-40.0 mg / L reduced glutathione, and 20.0-60.0 mg / L ascorbic acid; 5.0~10.0 mg / L glycine, 3.0~17.8 mg / L alanine, 4.4~26.4 mg / L asparagine, 4.4~26.6 mg / L aspartic acid, 4.9~29.4 mg / L glutamic acid, 20.0~60.0 mg / L proline, 3.5~21.0 mg / L serine; 0.5~2.5 mg / L choline chloride, 0.5~2.5 mg / L D-calcium pantothenate, 0.5~2.5 mg / L folic acid, 0.6~2.4 mg / L nicotinamide, 0.5~2.5 mg / L pyridoxal hydrochloride, 0.05~0.25 mg / L riboflavin, 0.5~2.5 mg / L thiamine hydrochloride, 1.0~5.0 mg / L inositol.

11. The use of canine ovarian mesenchymal stem cells according to claim 9 in the preparation of drugs for treating or preventing canine diseases.

12. The application according to claim 11, characterized in that: The canine diseases mentioned include skin defects, musculoskeletal injuries, autoimmune diseases, diabetes, liver / kidney failure, or cognitive impairment.

13. The application according to claim 11, characterized in that: The canine disease described is atopic dermatitis.

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