A method for the preparation of animal stem cells
By combining enzymatic hydrolysis, gradient centrifugation, and immunomagnetic bead purification with in vitro stable culture, the problems of large donor damage, low purity, and poor stability have been solved, achieving efficient, simple, and low-cost animal stem cell preparation, which is suitable for large-scale production of various animals.
Patent Information
- Application Number
- CN202610824852.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-09
- Publication Date
- 2026-08-25
AI Technical Summary
Existing methods for preparing animal stem cells suffer from problems such as significant donor damage, low purity, poor in vitro culture stability, and insufficient versatility, making it difficult to achieve efficient and large-scale preparation for different species.
A combined enzymatic hydrolysis, gradient centrifugation, and immunomagnetic bead purification system was adopted, along with an in vitro stable culture system. This included the recycling of the hydrolysate and optimization of the culture medium. Conventional reagents and energy-saving equipment were used, and the passage process was optimized to improve preparation efficiency and purity.
It achieves minimal donor damage, high stem cell purity, stable pluripotency, simple operation, and controllable cost, making it suitable for large-scale production and applicable to stem cell preparation in various animals, while reducing environmental burden and preparation costs.
Smart Images
Figure CN122628985A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a method for preparing animal stem cells. Background Technology
[0002] Stem cells are a type of cell with self-renewal capacity and multi-directional differentiation potential, playing a crucial role in the growth, development, tissue repair, and regeneration of animal organisms. Currently, methods for preparing animal stem cells mainly include bone marrow aspiration, blastocyst inner cell mass separation, and adipose tissue digestion. However, existing methods generally suffer from several technical bottlenecks: First, bone marrow aspiration and embryo separation are highly invasive, causing significant damage to donor animals, and the limited availability of donors makes large-scale acquisition difficult. Second, stem cells obtained using traditional separation methods have low purity and are easily contaminated with hematopoietic cells, fibroblasts, and other contaminating cells, leading to differentiation during subsequent culture and affecting the maintenance of stem cell pluripotency. Third, in vitro culture systems are imperfect, relying heavily on heterologous components such as serum, resulting in significant batch-to-batch variations, potential pathogen contamination risks, poor stem cell passage stability, and a tendency to lose pluripotency after long-term culture. Fourth, existing methods have poor adaptability to different species, failing to meet the needs of preparing stem cells from both mammals and birds, thus lacking versatility.
[0003] For example, existing methods for preparing rabbit bone marrow mesenchymal stem cells mostly use collagenase to digest bone marrow plugs. Although a certain amount of stem cells can be obtained, there are still problems such as incomplete removal of impurities and long culture cycles. On the other hand, the preparation of avian embryonic stem cells depends on special components such as egg yolk extracts, which is complicated and costly.
[0004] In addition, the stable in vitro culture of stem cells from livestock such as sheep has always been a technical challenge, as traditional culture systems are unable to maintain their long-term pluripotency.
[0005] Therefore, developing a method for preparing animal stem cells that is simple to operate, causes little damage, has high purity, is highly versatile, and can stably maintain the pluripotency of stem cells has become a technical problem that needs to be solved in the field of biotechnology. Summary of the Invention
[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing animal stem cells, which solves the problems of large donor damage, low stem cell purity, poor in vitro culture stability, and insufficient versatility of existing methods. This method enables efficient and large-scale preparation of animal stem cells from different species, while ensuring the pluripotency and activity of stem cells.
[0007] Technical solution
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] A method for preparing animal stem cells includes the following steps:
[0010] S1. Donor sample pretreatment: Select healthy, age-appropriate donor animals and select corresponding tissue samples according to the donor type—for mammals, select adipose tissue or bone marrow tissue, and for birds, select blastocyst tissue; perform aseptic treatment on the tissue samples, remove impurities such as connective tissue and blood vessels, rinse with physiological saline 3-5 times, cut into 1-2 mm³ tissue blocks, and set aside for later use.
[0011] S2. Compound Enzymatic Separation: The tissue blocks treated in step S1 are placed in the enzymatic hydrolysate and enzymatically hydrolyzed at a constant temperature of 37℃ and 5% CO2 for 1-2 hours with shaking. The enzymatic hydrolysate is a mixture of collagenase IV, hyaluronidase, and trypsin in a volume ratio of 2:1:1, with a concentration of 0.1-0.15%. The solvent is serum-free DMEM / F12 medium. Simultaneously, an enzymatic hydrolysate recycling device is used. The filtered hydrolysate is sterilely filtered, the concentration is replenished, and it is reused 1-2 times to reduce reagent waste and pollutant emissions. During enzymatic hydrolysis, the mixture is gently shaken every 15 minutes. After enzymatic hydrolysis, an equal volume of DMEM / F12 medium containing 10% fetal bovine serum is added to terminate the hydrolysis. The mixture is then filtered through a 200-mesh cell sieve, and the filtrate is collected. Optimized enzymatic hydrolysis parameters shorten the hydrolysis time by more than 30% compared to traditional methods, significantly improving cell separation efficiency and reducing the amount of enzymatic hydrolysis reagents used, thus reducing environmental pressure.
[0012] S3. Gradient centrifugation purification: Place the filtrate collected in step S2 into a centrifuge tube, centrifuge at 800 r / min for 5 min, discard the supernatant and retain the precipitate; resuspend the precipitate in physiological saline, centrifuge at 1500 r / min for 10 min, discard the supernatant; repeat the physiological saline resuspension-centrifugation operation twice to obtain a preliminarily purified stem cell precipitate; add DMEM / F12 medium containing 10% fetal bovine serum, 1 ng / mL basic fibroblast growth factor (bFGF), and 0.5 ng / mL leukemia inhibitory factor (LIF) to the precipitate, and resuspend to prepare a cell suspension;
[0013] S4. Secondary purification of immunomagnetic beads: Add CD44 antibody-modified immunomagnetic beads to the cell suspension from step S3, with an antibody concentration of 5 μg / mL, and incubate at 4°C in the dark for 30 min, gently inverting the centrifuge tube once every 5 min during incubation; after incubation, place the centrifuge tube in a magnetic field and let it stand for 5 min, discard the supernatant, and wash the magnetic beads 3 times with physiological saline containing 2% fetal bovine serum to remove unbound contaminating cells; then add stem cell culture medium to the magnetic field, gently pipette the magnetic beads, collect the cell suspension, and obtain high-purity stem cells;
[0014] S5. In vitro stabilization culture: The high-purity stem cell suspension obtained in step S4 was seeded into culture flasks coated with gelatin-coated solution at a seeding density of 5 × 10⁶. 5 Cells were cultured at a density of 1 / mL in an incubator at 37℃, 5% CO2, and 95% humidity. A new energy-saving incubator was used, which reduced energy consumption by 25% compared to traditional incubators. During the culture process, a half-volume medium replacement scheme was adopted to reduce medium loss by 40% and reduce waste liquid discharge. The first half-volume medium replacement was performed after 24 hours of culture to remove non-adherent cells, and then the medium was replaced every 48 hours. When the cell confluence reached 70-80%, the cells were digested and passaged with 0.25% trypsin at a passage ratio of 1:3. During the passage process, 1 ng / mL of vitamin C was added to maintain the pluripotency of stem cells. The passage process was optimized by using batch digestion and simultaneous passage to improve culture efficiency. Compared with traditional methods, the culture cycle of a single batch was shortened by 1-2 days, thus improving the preparation efficiency.
[0015] S6. Stem cell identification and preservation: Stem cells from passages 3-5 were cultured, and the positive expression rates of CD44 and CD90 were detected by flow cytometry. The expression of OCT4 and NANOG pluripotency markers was detected by immunofluorescence staining. Selected qualified stem cells were added to a cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) and 20% fetal bovine serum, and after gradient cooling, they were stored in liquid nitrogen to complete the preparation of animal stem cells.
[0016] As a further aspect of the present invention: in step S1, the donor animal is a mammal aged 2-20 weeks or an avian embryo that has been incubated for 3-5 days; the aseptic treatment is to soak the tissue sample in 75% alcohol for 3-5 minutes, and then rinse it with sterile physiological saline to remove alcohol residue.
[0017] As a further aspect of the present invention: in step S2, the pH value of the enzymatic hydrolysate is adjusted to 7.2-7.4, and the oscillation speed is 100-120 r / min; the filtration is carried out using a 200-mesh cell sieve, and the filtrate is gently blown away with a sterile pipette after filtration to avoid cell damage.
[0018] As a further aspect of the present invention: in step S3, the physiological saline is sterile phosphate buffer (PBS) with a pH of 7.2-7.4; 100 U / mL penicillin and 100 μg / mL streptomycin are also added to the stem cell culture medium to inhibit bacterial contamination.
[0019] As a further aspect of the present invention: in step S4, the particle size of the immunomagnetic beads is 1-3 μm, and the volume ratio of the immunomagnetic beads to the cell suspension is 1:10; during the rinsing process, the magnetic beads are gently blown to avoid the magnetic beads from aggregating and causing the loss of stem cells.
[0020] As a further aspect of the present invention: In step S5, the concentration of the gelatin coating solution is 0.1%, the coating conditions are incubation at 37°C for 30 minutes, the excess coating solution is discarded after coating, and the cells are air-dried before use; the passage digestion time is 1-2 minutes, the cell morphology is observed under a microscope during digestion, and when the cells become round and detach from the cell wall, culture medium is immediately added to terminate digestion.
[0021] As a further aspect of the present invention: in step S6, the positive expression rate of CD44 and CD90 in the flow cytometry detection needs to be ≥95%; in the immunofluorescence staining, OCT4 and NANOG markers need to be positively expressed; the gradient cooling step is as follows: place at 4℃ for 30 min, place at -20℃ for 1 h, place at -80℃ for 12 h, and then transfer to liquid nitrogen for storage.
[0022] The beneficial effects of this invention are:
[0023] Minimal donor damage and wide availability: This invention can select mammalian adipose tissue, bone marrow tissue or avian blastocyst tissue as samples. Adipose tissue and blastocyst tissue are easy to obtain, and the damage to the donor animal is far less than that of traditional bone marrow puncture and embryo separation methods. It is also compatible with mammals and birds, has strong versatility, and can achieve large-scale sample acquisition.
[0024] High stem cell purity: A combination of enzymatic hydrolysis, gradient centrifugation, and immunomagnetic bead secondary purification is adopted. Enzymatic hydrolysis can efficiently disperse tissue cells and reduce cell damage; gradient centrifugation can initially remove contaminating cells and cell debris; immunomagnetic beads target and bind to the CD44 marker on the surface of stem cells, which can specifically remove hematopoietic cells, fibroblasts and other contaminating cells. The final stem cell purity is ≥95%, effectively avoiding the impact of contaminating cells on subsequent culture.
[0025] High stability in in vitro culture: The optimized in vitro culture system, with the addition of bFGF, LIF and vitamin C, can effectively maintain the self-renewal capacity and pluripotency of stem cells and prevent differentiation during in vitro culture. At the same time, penicillin and streptomycin are added to inhibit contamination, and gelatin coating of culture flasks promotes stem cell adhesion. Stem cell passage can be stably maintained for more than 10 generations, still maintaining typical stem cell morphology and expression of pluripotency markers.
[0026] Simple to operate and cost-controllable: The steps of this invention are simple and do not require complicated experimental equipment. Operations such as compound enzymatic hydrolysis, centrifugation, and immunomagnetic bead purification are easy to standardize. The culture system uses conventional reagents and avoids the use of special and expensive ingredients such as egg yolk extract, thereby reducing the preparation cost and making it suitable for industrial-scale production.
[0027] Green and environmentally friendly with low energy consumption: The introduction of environmentally friendly technologies such as enzyme hydrolysate recycling and culture medium loss optimization reduces the waste of enzyme hydrolysate reagents and culture media, and reduces the discharge of waste liquid and waste reagents, which is in line with the development concept of green biotechnology. At the same time, energy-saving culture equipment is used to reduce energy consumption in the preparation process, reduce environmental burden, and achieve synergistic development of environmental protection and high efficiency.
[0028] Significantly improved efficiency: By optimizing enzymatic hydrolysis parameters and passage procedures, the cell separation and in vitro culture cycle is shortened, resulting in an overall preparation efficiency improvement of more than 30% compared to traditional methods. The design of enzymatic hydrolysate recycling and batch passage further improves operational efficiency, reduces time costs, and meets the needs of large-scale preparation.
[0029] Easy to popularize and promote: The reagents used in this invention are all conventional biological laboratory reagents, which are easy to purchase and inexpensive, and do not require special and expensive equipment; the operation steps are highly standardized, and no highly professional operators are required. They can be mastered with simple training and are suitable for laboratories, livestock breeding bases and biological enterprises of different sizes; at the same time, they are suitable for a variety of mammals and poultry, and are highly versatile. They can be quickly promoted and applied in different regions and fields, solving the problems of high difficulty and high threshold for popularization of existing technologies.
[0030] Broad application prospects: The stem cells prepared by this invention are highly pure, active, and stable in pluripotency. They can be widely used in animal husbandry breeding (such as the cultivation of superior gene-edited breeds), regenerative medicine (such as tissue repair), disease model construction, and biopharmaceuticals. This invention solves many pain points of existing stem cell preparation technologies and has important practical value and industrialization prospects.
[0031] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.
[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description
[0033] Figure 1 This is a schematic diagram of the structure of the present invention. Detailed Implementation
[0034] The present invention is illustrated below with specific embodiments, but these are not intended to limit the scope of the invention. Figure 1 As shown, a method for preparing animal stem cells,
[0035] Example 1: Preparation of sheep adipose-derived mesenchymal stem cells
[0036] This embodiment uses sheep as donors to prepare adipose-derived mesenchymal stem cells. The specific steps are as follows:
[0037] S1. Donor sample pretreatment: Select 8-week-old healthy sheep, and obtain 10g of adipose tissue from the groin under sterile conditions. Soak in 75% alcohol for 4 minutes, rinse 4 times with sterile physiological saline to remove connective tissue, blood vessels and other impurities, and cut into 1mm³ tissue blocks with sterile scissors for later use.
[0038] S2. Compound Enzymatic Separation: The tissue block was placed in 50 mL of enzymatic hydrolysate, which was a mixture of collagenase IV, hyaluronidase, and trypsin in a volume ratio of 2:1:1, with a concentration of 0.12%. The solvent was serum-free DMEM / F12 medium, and the pH was adjusted to 7.3. Under constant temperature conditions of 37℃ and 5% CO2, the enzymatic hydrolysis was carried out with shaking at 110 r / min for 1.5 h, with gentle shaking every 15 min. After the enzymatic hydrolysis was completed, 50 mL of DMEM / F12 medium containing 10% fetal bovine serum was added to terminate the enzymatic hydrolysis. The mixture was filtered through a 200-mesh cell sieve, and the filtrate was collected.
[0039] S3. Gradient centrifugation purification: Place the filtrate in a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, and retain the precipitate; add 20 mL of sterile PBS (pH 7.3) to the precipitate for resuspending, centrifuge at 1500 rpm for 10 min, and discard the supernatant; repeat the PBS resuspending-centrifugation operation twice to obtain the stem cell precipitate; add 30 mL of DMEM / F12 medium containing 10% fetal bovine serum, 1 ng / mL bFGF, 0.5 ng / mL LIF, 100 U / mL penicillin, and 100 μg / mL streptomycin to the precipitate, and resuspend to prepare a cell suspension;
[0040] S4. Secondary purification of immunomagnetic beads: Add 3m LCD44 antibody-modified immunomagnetic beads (particle size 2μm) to the cell suspension at an antibody concentration of 5μg / mL, and incubate at 4℃ in the dark for 30 min, gently inverting the centrifuge tube once every 5 min; after incubation, place the centrifuge tube in a magnetic field and let it stand for 5 min, discard the supernatant, wash the magnetic beads 3 times with PBS containing 2% fetal bovine serum, and gently pipette the magnetic beads after each wash; then add 20mL of stem cell culture medium to the magnetic field, gently pipette the magnetic beads, collect the cell suspension, and obtain high-purity adipose mesenchymal stem cells;
[0041] S5. In vitro stable culture: The cell suspension was seeded into culture flasks coated with 0.1% gelatin coating solution at a seeding density of 5 × 10⁶ cells / year. 5Cells were cultured at 37°C, 5% CO2, and 95% humidity in an incubator. After 24 hours of culture, the medium was changed halfway to remove non-adherent cells, and then the medium was changed every 48 hours. When the cell confluence reached 75%, the cells were digested with 0.25% trypsin for 1.5 minutes, and the digestion was stopped by adding culture medium. The cells were passaged at a ratio of 1:3, and 1 ng / mL of vitamin C was added during the passage.
[0042] S6. Stem cell identification and preservation: Flow cytometry analysis showed that the positive expression rate of CD44 was 96.2% and the positive expression rate of CD90 was 97.5%; immunofluorescence staining showed positive expression of OCT4 and NANOG, indicating that the stem cells were qualified. The qualified stem cells were added to a cryopreservation solution containing 10% DMSO and 20% fetal bovine serum, and incubated at 4℃ for 30 min, -20℃ for 1 h, and -80℃ for 12 h. They were then transferred to liquid nitrogen for preservation, thus completing the preparation of sheep adipose mesenchymal stem cells.
[0043] Example 2: Preparation of chicken embryonic stem cells
[0044] This embodiment uses chickens as donors to prepare embryonic stem cells. The specific steps are as follows:
[0045] S1. Donor sample pretreatment: Select healthy chicken embryos that have been incubated for 4 days, remove the blastocyst tissue under sterile conditions, soak in 75% alcohol for 3 minutes, rinse 3 times with sterile physiological saline to remove surface impurities, and cut into 1.5 mm³ tissue blocks for later use.
[0046] S2. Compound Enzymatic Separation: Place the tissue block into 30 mL of enzymatic hydrolysate, which is a mixture of collagenase IV, hyaluronidase, and trypsin in a volume ratio of 2:1:1, with a concentration of 0.1%. The solvent is serum-free DMEM / F12 medium, and the pH is adjusted to 7.2. Under constant temperature conditions of 37℃ and 5% CO2, the enzymatic hydrolysis is carried out at 100 rpm for 1 h, with gentle shaking every 15 min. After the enzymatic hydrolysis is completed, 30 mL of DMEM / F12 medium containing 10% fetal bovine serum is added to terminate the enzymatic hydrolysis. The solution is filtered through a 200-mesh cell sieve, and the filtrate is collected.
[0047] S3. Gradient centrifugation purification: Place the filtrate in a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, and retain the precipitate; add 15 mL of sterile PBS (pH 7.2) to the precipitate for resuspending, centrifuge at 1500 rpm for 10 min, and discard the supernatant; repeat the PBS resuspending-centrifugation operation twice to obtain the stem cell precipitate; add 20 mL of DMEM / F12 medium containing 10% fetal bovine serum, 1 ng / mL bFGF, 0.5 ng / mL LIF, 100 U / mL penicillin, and 100 μg / mL streptomycin to the precipitate, and resuspend to prepare a cell suspension;
[0048] S4. Secondary purification of immunomagnetic beads: Add 2m LCD44 antibody-modified immunomagnetic beads (1μm in diameter) to the cell suspension at an antibody concentration of 5μg / mL, and incubate at 4℃ in the dark for 30 min, gently inverting the centrifuge tube once every 5 min; after incubation, place the centrifuge tube in a magnetic field and let it stand for 5 min, discard the supernatant, and wash the magnetic beads 3 times with PBS containing 2% fetal bovine serum; then add 15mL of stem cell culture medium to the magnetic field, gently pipette the magnetic beads, collect the cell suspension, and obtain high-purity chicken embryonic stem cells;
[0049] S5. In vitro stable culture: The cell suspension was seeded into culture flasks coated with 0.1% gelatin coating solution at a seeding density of 5 × 10⁶ cells / year. 5 Cells were cultured at 37°C, 5% CO2, and 95% humidity in an incubator. After 24 hours of culture, the medium was changed halfway to remove non-adherent cells, and then the medium was changed every 48 hours. When the cell confluence reached 70%, the cells were digested with 0.25% trypsin for 1 minute, and the digestion was stopped by adding culture medium. The cells were passaged at a ratio of 1:3, and 1 ng / mL of vitamin C was added during the passage.
[0050] S6. Stem cell identification and preservation: Third-generation cultured stem cells were obtained, and flow cytometry analysis showed that the positive expression rate of CD44 was 95.8% and the positive expression rate of CD90 was 96.7%. Immunofluorescence staining showed that OCT4 and NANOG were positively expressed, and the identification was qualified. The qualified stem cells were added to a cryopreservation solution containing 10% DMSO and 20% fetal bovine serum, and after gradient cooling, they were transferred to liquid nitrogen for preservation, thus completing the preparation of chicken embryonic stem cells.
[0051] Example 3: Preparation of mouse bone marrow mesenchymal stem cells
[0052] In this embodiment, bone marrow mesenchymal stem cells were prepared using mice as donors. The specific steps are as follows:
[0053] S1. Donor sample pretreatment: Six-week-old healthy mice were selected, euthanized by cervical dislocation, soaked in 75% alcohol for 5 minutes, and the femur and tibia were separated under aseptic conditions. The surface tissue was removed, the two ends of the bones were cut off, bone marrow fluid was extracted with a sterile syringe, sterile physiological saline was injected, the bone marrow cavity was rinsed, and the bone marrow suspension was collected for later use.
[0054] S2. Compound Enzymatic Separation: The bone marrow suspension was placed in 40 mL of enzymatic hydrolysate, which was a mixture of collagenase IV, hyaluronidase, and trypsin in a volume ratio of 2:1:1, with a concentration of 0.15%. The solvent was serum-free DMEM / F12 medium, and the pH was adjusted to 7.4. Enzymatic hydrolysis was carried out at 37℃ and 5% CO2 for 2 h with shaking at 120 r / min, with gentle shaking every 15 min. After the enzymatic hydrolysis was completed, 40 mL of DMEM / F12 medium containing 10% fetal bovine serum was added to terminate the hydrolysis. The solution was filtered through a 200-mesh cell sieve, and the filtrate was collected.
[0055] S3. Gradient centrifugation purification: Place the filtrate in a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, and retain the precipitate; add 25 mL of sterile PBS (pH 7.4) to the precipitate for resuspending, centrifuge at 1500 rpm for 10 min, and discard the supernatant; repeat the PBS resuspending-centrifugation operation twice to obtain the stem cell precipitate; add 25 mL of DMEM / F12 medium containing 10% fetal bovine serum, 1 ng / mL bFGF, 0.5 ng / mL LIF, 100 U / mL penicillin, and 100 μg / mL streptomycin to the precipitate, and resuspend to prepare a cell suspension;
[0056] S4. Secondary purification of immunomagnetic beads: Add 2.5m of LCD44 antibody-modified immunomagnetic beads (3μm in diameter) to the cell suspension at an antibody concentration of 5μg / mL, and incubate at 4℃ in the dark for 30 min, gently inverting the centrifuge tube once every 5 min; after incubation, place the centrifuge tube in a magnetic field and let it stand for 5 min, discard the supernatant, and wash the magnetic beads 3 times with PBS containing 2% fetal bovine serum; then add 20mL of stem cell culture medium to the magnetic field, gently pipette the magnetic beads, collect the cell suspension, and obtain high-purity mouse bone marrow mesenchymal stem cells;
[0057] S5. In vitro stable culture: The cell suspension was seeded into culture flasks coated with 0.1% gelatin coating solution at a seeding density of 5 × 10⁶ cells / year. 5 Cells were cultured at 37°C, 5% CO2, and 95% humidity in an incubator. After 24 hours of culture, the medium was changed halfway to remove non-adherent cells, and then the medium was changed every 48 hours. When the cell confluence reached 80%, the cells were digested with 0.25% trypsin for 2 minutes, and the digestion was stopped by adding culture medium. The cells were passaged at a ratio of 1:3, and 1 ng / mL of vitamin C was added during the passage.
[0058] S6. Stem cell identification and preservation: 5th generation cultured stem cells were obtained. Flow cytometry showed that the positive expression rate of CD44 was 97.1% and the positive expression rate of CD90 was 98.3%. Immunofluorescence staining showed that OCT4 and NANOG were positive, and the cells were qualified. The qualified stem cells were added to a cryopreservation solution containing 10% DMSO and 20% fetal bovine serum, and after gradient cooling, they were transferred to liquid nitrogen for preservation, thus completing the preparation of mouse bone marrow mesenchymal stem cells.
[0059] Detailed Implementation Description
[0060] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the invention. In practical applications, parameters such as enzymatic hydrolysis time, centrifugation speed, and culture medium component ratio can be appropriately adjusted according to the donor animal species and tissue sample type to achieve the technical effects of the present invention. For example, for large mammals such as cattle and pigs, the amount of enzymatic hydrolysate and the enzymatic hydrolysis time can be appropriately increased; for poultry such as peacocks and ostriches, the acquisition time and culture conditions of blastocyst tissue can be adjusted, all without departing from the protection scope of the present invention.
[0061] The preparation method of this invention is simple to operate and highly standardized, enabling efficient preparation of animal stem cells from different species. It also has the advantages of being green and environmentally friendly, highly efficient, and easy to popularize and promote, solving many pain points of existing technologies. It not only reduces preparation costs and environmental burden, but also enables rapid application in various fields, and has important practical value, environmental value, and industrialization prospects.
[0062] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0063] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.
[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A method for preparing animal stem cells, characterized in that, Includes the following steps: S1. Donor sample pretreatment: Select healthy, age-appropriate donor animals and select corresponding tissue samples according to the donor type—for mammals, select adipose tissue or bone marrow tissue, and for birds, select blastocyst tissue; The tissue samples were aseptically processed to remove impurities such as connective tissue and blood vessels. They were rinsed 3-5 times with physiological saline and cut into 1-2 mm³ tissue blocks for later use. S2. Compound enzymatic hydrolysis and separation: The tissue blocks treated in step S1 are placed in the enzymatic hydrolysate and enzymatically hydrolyzed for 1-2 hours under constant temperature conditions of 37℃ and 5% CO2. The enzymatic hydrolysate is composed of collagenase IV, hyaluronidase and trypsin in a volume ratio of 2:1:1, with a concentration of 0.1-0.15% and the solvent is serum-free DMEM / F12 medium. After the enzymatic hydrolysis was completed, an equal volume of DMEM / F12 medium containing 10% fetal bovine serum was added to terminate the enzymatic hydrolysis. The solution was then filtered through a 200-mesh cell sieve, and the filtrate was collected. S3. Gradient centrifugation purification: Place the filtrate collected in step S2 into a centrifuge tube, centrifuge at 800 r / min for 5 min, discard the supernatant and retain the precipitate; Resuspend the precipitate in physiological saline, then centrifuge at 1500 r / min for 10 min and discard the supernatant. Repeat the saline resuspend-centrifugation process twice to obtain a preliminarily purified stem cell precipitate. Add DMEM / F12 medium containing 10% fetal bovine serum, 1 ng / mL basic fibroblast growth factor (bFGF), and 0.5 ng / mL leukemia inhibitory factor (LIF) to the precipitate, and resuspend to prepare a cell suspension; S4. Secondary purification of immunomagnetic beads: Add CD44 antibody-modified immunomagnetic beads to the cell suspension from step S3. The antibody concentration is 5 μg / mL. Incubate at 4°C in the dark for 30 min. After incubation, place the centrifuge tube in a magnetic field and let it stand for 5 minutes. Discard the supernatant and rinse the magnetic beads 3 times with physiological saline containing 2% fetal bovine serum. Then, stem cell culture medium is added to the magnetic field, and the cell suspension is collected to obtain high-purity stem cells. S5. In vitro stabilization culture: The high-purity stem cell suspension obtained in step S4 was seeded into culture flasks coated with gelatin-coated solution at a seeding density of 5 × 10⁶. 5 Cells / mL were incubated in an incubator at 37℃, 5% CO2, and 95% humidity. After 24 hours of incubation, perform the first half-volume medium change, and then change the medium every 48 hours thereafter. When the cell confluence reaches 70-80%, digest and passage the cells with 0.25% trypsin at a passage ratio of 1:3, and add 1 ng / mL of vitamin C during the passage process. S6. Stem cell identification and preservation: Stem cells cultured from the 3rd to 5th generation were collected, and the positive expression rates of CD44 and CD90 were detected by flow cytometry. The expression of OCT4 and NANOG pluripotency markers was detected by immunofluorescence staining. Select qualified stem cells, add them to cryopreservation solution for gradient cooling, and then store them in liquid nitrogen.
2. The method for preparing animal stem cells according to claim 1, characterized in that, In step S1, the donor animal is a mammal aged 2-20 weeks or a bird embryo that has been incubated for 3-5 days; The aseptic treatment involves soaking the tissue sample in 75% alcohol for 3-5 minutes, followed by rinsing with sterile saline to remove any alcohol residue.
3. The method for preparing animal stem cells according to claim 1, characterized in that, In step S2, the pH of the enzymatic hydrolysate is adjusted to 7.2-7.4, and the shaking speed is 100-120 r / min; After filtration, the filtrate is gently blown away with a sterile pipette.
4. The method for preparing animal stem cells according to claim 1, characterized in that, In step S3, the physiological saline is sterile phosphate buffer (PBS) with a pH of 7.2-7.4; The stem cell culture medium also contains 100 U / mL penicillin and 100 μg / mL streptomycin.
5. The method for preparing animal stem cells according to claim 1, characterized in that, In step S4, the immunomagnetic beads have a particle size of 1-3 μm and a volume ratio of 1:10 with the cell suspension. The magnetic beads are gently blown during the rinsing process.
6. The method for preparing animal stem cells according to claim 1, characterized in that, In step S5, the concentration of the gelatin coating solution is 0.1%, and the coating conditions are incubation at 37°C for 30 minutes. The passage digestion time is 1-2 minutes. When the cells become round and detach from the cell wall, culture medium is added to stop the digestion.
7. The method for preparing animal stem cells according to claim 1, characterized in that, In step S6, the positive expression rate of CD44 and CD90 in the flow cytometry detection is ≥95%. In the immunofluorescence staining, OCT4 and NANOG markers showed positive expression; The cryopreservation solution consists of 10% dimethyl sulfoxide (DMSO) and 20% fetal bovine serum; The gradient cooling process is as follows: place at 4℃ for 30 min, at -20℃ for 1 h, at -80℃ for 12 h, and then transfer to liquid nitrogen for storage. In step S2, an enzyme hydrolysate recycling device is used to reuse the filtered enzyme hydrolysate 1-2 times after sterile filtration and concentration replenishment. In step S5, an energy-saving incubator and a half-volume culture medium replacement optimization scheme are adopted to reduce energy consumption and reagent loss.
8. The method for preparing animal stem cells according to claim 1, characterized in that, The method is standardized in its operation steps, uses conventional biological reagents and conventional laboratory equipment, and does not require highly specialized operators. It can be mastered with simple training, making it easy to popularize and promote in different scales and fields.