Cornu cervi pantotrichum tissue cell culture method

Abstract

The invention relates to the technical field of tissue engineering, and particularly discloses a pilose antler tissue cell culture method which comprises the following steps: S1, cell in-vitro culture; s2, directional induced differentiation; step S3, ossification induction; the induction culture medium 1, the induction culture medium 2 and the ossification induction culture medium are added, amphotericin B capable of covering the fungal pollution risk is added into the induction culture medium 1, fetal calf serum contained in the induction culture medium 1 can neutralize trypsin, recombinant human HGF contained in the induction culture medium 2 can improve the migration ability of cells, and meanwhile, the ossification induction culture medium can be used for preparing the ossification culture medium. The recombinant human bFGF can synergistically promote proliferation, so that emigrated cells are quickly accumulated, and the situation that the cell density is too low due to insufficient proliferation after emigration is avoided; ascorbic acid-2-phosphate contained in the ossification induction culture medium can avoid oxidation failure of ascorbic acid, and the cell emigration efficiency is improved through the synergistic effect of the substances.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, and more specifically, to a method for culturing deer antler tissue cells. Background Technology

[0002] Deer antlers are bony appendages on the skull of deer with extremely high medicinal value. Their unique biological characteristic lies in their ability to shed and completely regenerate annually, with an extremely rapid regeneration process, growing up to 2 centimeters per day. The bone structure of deer antlers is highly differentiated, containing chondrocytes, osteoblasts, and pre-chondrocytes and pre-osteoblasts, thus providing an excellent model for research on limb regeneration and the ossification mechanism of long bones in the medical field.

[0003] Currently, deer antlers are mainly obtained through natural harvesting, but this method suffers from ecological damage and insufficient supply. Therefore, in vitro culture of deer antler tissue cells has great potential to meet market demand. Existing technologies for culturing deer antler tissue cells include tissue block culture and enzymatic digestion methods. Among these, tissue block culture has attracted widespread attention due to its simplicity and ability to preserve some of the extracellular matrix microenvironment; however, it suffers from low cell migration efficiency. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for culturing deer antler tissue cells.

[0005] The objective of this invention can be achieved through the following technical solutions: A method for culturing deer antler tissue cells includes the following steps: Step S1, In vitro cell culture: Take the antler stalk periosteum of a healthy sika deer, cut the antler stalk periosteum into tissue pieces, add collagenase I aqueous solution for one digestion treatment, centrifuge, wash, then inoculate and culture, add trypsin aqueous solution for a second digestion treatment, passage, and obtain passaged cells. Step S2, Directional Induction of Differentiation: Wash the passaged cells obtained in Step S1, add trypsin aqueous solution preheated to 37°C for digestion, add induction medium 1 preheated to 35-37°C, and agitate the culture flask wall to obtain a single-cell suspension; under sterile conditions, inoculate the single-cell suspension into complete culture medium; then incubate, add induction medium 2, and culture for 2-3 weeks to obtain deer antler proliferating chondrocytes; Step S3, Ossification Induction: Remove the old culture medium from the proliferating chondrocytes of deer antler, wash them, add ossification induction culture medium preheated to 37°C, and continue culturing to obtain deer antler tissue cells.

[0006] Furthermore, in step S1, the specific parameters for inoculation and culture are: RH 96-98%, temperature 35-37℃, CO2 volume concentration of 5-8%, and culture time of 5-7 days.

[0007] Furthermore, in step S1, during the first digestion process, the concentration of collagenase I aqueous solution is 0.6-1.2 mg / mL, and the amount of collagenase I aqueous solution added is 10-20 mL / g per gram of horn stalk periosteum tissue block.

[0008] Furthermore, in step S1, the tissue block inoculation density is controlled at 1-3 blocks / cm². 2 .

[0009] Furthermore, in step S1, during the secondary digestion process, the concentration of the trypsin aqueous solution is 0.6-1.2 mg / mL, and the amount of trypsin aqueous solution added is 14-18 mL / g per gram of horn stalk periosteum tissue block.

[0010] Furthermore, in step S1, when the cell fusion rate reaches 80-90%, passage is performed at a passage ratio of 1:3 to 1:5, for 1-3 generations.

[0011] Furthermore, in step S2, the culture plate used during the inoculation process is a 6-well cell culture plate, and the inoculation volume of the single-cell suspension is 2 × 10⁻⁶. 5 -5×10 5 Cells / well

[0012] Furthermore, in step S2, the amount of induction culture medium 1 added is 2-3 mL per well.

[0013] Furthermore, in step S2, the induction medium 2 is replaced with fresh medium every 2-3 days during the culture period.

[0014] Furthermore, in step S2, the specific parameters for the incubation treatment are: 37℃, 5% CO2, RH 96-98%, and the incubation time is 2.4-2.8h.

[0015] Furthermore, in step S2, the specific parameters for the digestion process are: RH 96-98%, temperature 35-37℃, CO2 volume concentration of 6-8%, and digestion time of 2-4 min.

[0016] Further, in step S2, the induction medium 1 is based on DMEM / F12, with the addition of 10-15% (v / v) fetal bovine serum, 1% (v / v) penicillin-antibody, 2.5 μg / mL amphotericin B, 5 ng / mL recombinant human IGF-1, 2 ng / mL recombinant human EGF, 10 mM HEPES buffer and 0.5 mM L-glutathione, and the pH is adjusted to 7.2-7.4.

[0017] Further, in step S2, the induction medium 2 is based on DMEM / F12, with the addition of 10-15% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 8 ng / mL recombinant human HGF, 4 ng / mL recombinant human bFGF, 10 µg / mL fibronectin, 5 µg / mL laminin, 50 µg / mL ascorbate-2-phosphate and 1 mM sodium pyruvate, and the pH is adjusted to 7.2-7.4.

[0018] Furthermore, in step S3, the specific parameters during the cultivation process are: RH 96-98%, temperature 35-37℃, and cultivation time of 5-7 weeks.

[0019] Further, in step S3, the osteolysis induction medium is based on DMEM / F12, with the addition of 10-12% (v / v) fetal bovine serum, 1% (v / v) penicillin antibiotics, 10 mM β-glycerophosphate sodium, 50 μg / mL ascorbic acid-2-phosphate, 0.1 µM dexamethasone, 10 ng / mL recombinant human BMP-2, 8 μg / mL hyaluronic acid, and 5 μg / mL chondroitin sulfate, and the pH is adjusted to 7.2-7.4.

[0020] Furthermore, in step S3, the ossification induction medium is replaced with fresh medium every 2-3 weeks during the culture period.

[0021] In summary, the present invention has the following beneficial effects: To improve the migration efficiency of deer antler tissue cells, this invention incorporates induction medium 1, induction medium 2, and osteolysis induction medium. Induction medium 1 not only adds amphotericin B to the traditional induction medium to cover the risk of fungal contamination, but also uses a very low concentration of amphotericin B to avoid affecting cell viability. Furthermore, the fetal bovine serum in induction medium 1 neutralizes trypsin, preventing residual trypsin damage to the cell membrane. The presence of recombinant human IGF-1 and recombinant human EGF repairs the decline in cell viability caused by digestion, increasing the adhesion rate after subsequent inoculation. HEPES buffer and antioxidants reduce pH fluctuations and oxidative stress damage to cells, preventing cells from entering a dormant state due to stress. Induction medium 2 contains recombinant human HGF, a core migration inducing factor that enhances cell migration ability. Simultaneously, recombinant human bFGF synergistically promotes proliferation, allowing the migrated cells to accumulate rapidly and preventing post-migration complications. The low cell density caused by "insufficient proliferation" can be addressed by adding fibronectin and laminin to mimic the microenvironment within deer antler, allowing cells to quickly adhere and form migration "anchor points." The addition of sodium pyruvate and high-glucose DMEM / F12 provides sufficient energy for migrating cells, preventing migration stagnation due to insufficient energy. The ascorbic acid-2-phosphate in the ossification induction medium prevents ascorbic acid from oxidizing and becoming ineffective, while reducing the damage to cells from oxidation products and lowering the risk of contamination from "dead cells feeding on bacteria" after cell death. Recombinant human BMP-2 is not only a key factor in ossification induction but also inhibits apoptosis during differentiation. The presence of hyaluronic acid and chondroitin sulfate can mimic the ossification microenvironment of deer antler, protecting the extracellular matrix secreted by migrating cells, preventing cell detachment due to matrix degradation, and providing support for the formation of ossification nodules, ensuring a sufficient quantity of functional deer antler tissue cells is ultimately obtained. Detailed Implementation

[0022] The main raw materials and their component contents used in the examples are shown below: The healthy sika deer were 4-year-old sika deer provided by the Zuojia Specialty Products Research Institute of the Chinese Academy of Agricultural Sciences; collagenase I aqueous solution was purchased from Merck Life Sciences, CAS No. 9001-12-1; trypsin was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., CAS No. 9002-07-7; sterile PBS buffer, specification 0.01M, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DMEM / F12 was purchased from Shanghai Shangbao Biotechnology Co., Ltd.; fetal bovine serum was purchased from Nanjing Senbeijia Biotechnology Co., Ltd., product number BC-SE-FB. S07; the bispecific antibody was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product code 60162ES76; amphotericin B was purchased from Beijing Solarbio Science & Technology Co., Ltd., product code IA0320; recombinant human IGF-1 was purchased from Shanghai Yuanye Biotechnology Co., Ltd., model S33080; recombinant human EGF was purchased from Wuhan Feien Biotechnology Co., Ltd.; HEPES buffer was purchased from Jiangsu Maige Biotechnology Co., Ltd., model BB0071; and the complete culture medium was purchased from Shanghai Yansheng Industrial Co., Ltd., a commercially available general-purpose complete culture medium for primary cells.

[0023] The present invention will be further described in detail below with reference to the embodiments.

[0024] Examples 1-3 and Comparative Examples 1-3 provide a method for culturing deer antler tissue cells. Example 1

[0025] This embodiment provides a method for culturing deer antler tissue cells, including the following steps: Step S1: In vitro cell culture: Take the antler stalk periosteum from healthy sika deer, cut it into 1mm×1mm×1mm tissue blocks, add 0.6mg / mL collagenase I aqueous solution for a first digestion treatment, centrifuge at 7000rpm for 10min, wash three times with sterile PBS buffer, and then inoculate and culture. Add trypsin aqueous solution for a second digestion treatment. When the cell confluence reaches 80%, passage is performed at a ratio of 1:3 to 1:5, for one passage to obtain passaged cells. During the first digestion treatment, the concentration of collagenase I aqueous solution is 0.6mg / mL, and the amount of collagenase I aqueous solution added is 10mL / g per gram of antler stalk periosteum tissue block. The specific parameters for inoculation and culture are: RH The culture time was 5 days, the temperature was 35℃, the volume concentration of CO2 was 5%, and the culture time was 5 days. During the secondary digestion process, the concentration of trypsin aqueous solution was 0.6 mg / mL. The amount of trypsin aqueous solution added was 14 mL / g per gram of horn stalk periosteum tissue block. Step S2, Directed Differentiation Induction: Wash the passaged cells obtained in Step S1 to remove residual serum, add trypsin aqueous solution preheated to 37℃, and digest them. The specific parameters for digestion are: RH 96%, temperature 35℃, CO2 volume concentration 6%, and digestion time 2 min. Add induction medium 1 preheated to 35℃, and agitate the culture flask wall 3 times to obtain a single-cell suspension. Under aseptic conditions, inoculate the single-cell suspension into complete culture medium. The culture plate used for inoculation is a 6-well cell culture plate, and the inoculation volume of the single-cell suspension is 2 × 10⁶ cells / well. 5 Cells / well; then incubated with the following parameters: 37℃, 5% CO2, RH 96%, for 2.4 hours. Induction medium 2 was added dropwise along the well wall, completed within 10 minutes at a rate of 3 drops / second. After addition, the cells were cultured for 2 weeks, with the induction medium 2 replaced every 2 days. This yielded proliferative chondrocytes of deer antler. Induction medium 1 was based on DMEM / F12, supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) penicillin-antibiotics, 2.5 μg / mL amphotericin B, 5 ng / mL recombinant human IGF-1, 2 ng / mL recombinant human EGF, and 10 mM... HEPES buffer and 0.5 mM L-glutathione were added, and the pH was adjusted to 7.2 with sterile 0.1 M hydrochloric acid aqueous solution. Induction medium 2 was based on DMEM / F12 medium, with the addition of 10% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 8 ng / mL recombinant human HGF, 4 ng / mL recombinant human bFGF, 10 µg / mL fibronectin, 5 µg / mL laminin, 50 µg / mL ascorbic acid and 1 mM sodium pyruvate, and the pH was adjusted to 7.2 with sterile 0.1 M sodium hydroxide aqueous solution. Step S3, Ossification Induction: Remove the old culture medium from the proliferating chondrocytes of deer antler, wash the cells three times with sterile PBS buffer to remove residual induction medium 2; add ossification induction medium preheated to 37℃, and continue culturing. The specific parameters during the culture process are: RH 96%, temperature 35℃, and culture time of 5 weeks, during which fresh ossification induction medium is replaced every 2 weeks to obtain deer antler tissue cells. The ossification induction medium is based on DMEM / F12, supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotic, 10mM β-glycerophosphate sodium, 50μg / mL ascorbic acid-2-phosphate, 0.1µM dexamethasone, 10ng / mL recombinant human BMP-2, 8μg / mL hyaluronic acid, and 5μg / mL chondroitin sulfate, and then adjust the pH to 7.2 with sterile 0.1M sodium hydroxide aqueous solution. Example 2

[0026] This embodiment provides a method for culturing deer antler tissue cells, including the following steps: Step S1: In vitro cell culture: Take the antler stalk periosteum from healthy sika deer, cut it into 2mm×2mm×2mm tissue blocks, add collagenase I aqueous solution for a first digestion treatment, control the centrifugation speed at 7500 rpm, centrifugation time at 12 min, wash 4 times with sterile PBS buffer, and then inoculate and culture. Add trypsin aqueous solution for a second digestion treatment. When the cell confluence reaches 85%, passage is performed at a passage ratio of 1:3 to 1:5, for 2 passages to obtain passaged cells. In the first digestion treatment, the concentration of collagenase I aqueous solution is 0.9 mg / mL, and the amount of collagenase I aqueous solution added is 15 mL / g per gram of antler stalk periosteum tissue block. In the second digestion treatment, the concentration of trypsin aqueous solution is 0.9 mg / mL, and the amount of trypsin aqueous solution added is 16 mL / g per gram of antler stalk periosteum tissue block. The specific parameters for inoculation and culture are: RH 97%, temperature 36℃, CO2 volume concentration 6.5%, incubation time 6 days; Step S2, Directed Differentiation Induction: The passaged cells obtained in Step S1 were washed to remove residual serum; trypsin aqueous solution preheated to 37°C was added for digestion, with the specific parameters controlled as follows: RH 97%, temperature 36°C, CO2 volume concentration 7%, and digestion time 3 min. Induction medium 1 preheated to 36°C was added, and the culture flask wall was agitated 4 times to obtain a single-cell suspension; under aseptic conditions, the single-cell suspension was inoculated into complete culture medium; the inoculation volume of the single-cell suspension was 3.5 × 10⁻⁶ cells / year. 5 Cells / well; then incubate, add induction medium 2 dropwise along the well wall, controlling the addition to be completed within 10 minutes, with a drop rate of 4 drops / second. After addition, culture for 2.5 weeks, changing the induction medium 2 fresh every 3 days during the culture period to obtain deer antler proliferative chondrocytes. Among them, induction medium 1 is based on DMEM / F12 medium, supplemented with 12% (v / v) fetal bovine serum, 1% (v / v) penicillin-antibiotics, 2.5 μg / mL amphotericin B, 5 ng / mL recombinant human IGF-1, 2 ng / mL recombinant human EGF, and 10 mM HEPES buffer and 0.5 mM L-glutathione were added, and the pH was adjusted to 7.3 with sterile 0.1 M hydrochloric acid aqueous solution. Induction medium 2 was based on DMEM / F12 medium, with the addition of 12% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 8 ng / mL recombinant human HGF, 4 ng / mL recombinant human bFGF, 10 µg / mL fibronectin, 5 µg / mL laminin, 50 µg / mL ascorbic acid, and 1 mM sodium pyruvate, and the pH was adjusted to 7.3 with sterile 0.1 M sodium hydroxide aqueous solution. Step S3, Ossification Induction: Remove the old culture medium from the proliferating chondrocytes of deer antler, wash the cells 4 times with sterile PBS buffer to remove residual induction medium 2; add ossification induction medium preheated to 37℃, and continue culturing. The specific parameters during the culture process are: RH 97%, temperature 36℃, and culture time of 6 weeks. Fresh ossification induction medium is replaced every 2 weeks during the period to obtain deer antler tissue cells. The ossification induction medium is based on DMEM / F12 medium, supplemented with 11% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 10mM β-glycerophosphate sodium, 50μg / mL ascorbic acid-2-phosphate, 0.1µM dexamethasone, 10ng / mL recombinant human BMP-2, 8μg / mL hyaluronic acid, and 5μg / mL chondroitin sulfate. The pH is then adjusted to 7.3 with sterile 0.1M sodium hydroxide aqueous solution. Example 3

[0027] This embodiment provides a method for culturing deer antler tissue cells, including the following steps: Step S1: In vitro cell culture: Take the antler stalk periosteum from healthy sika deer, cut it into 3mm×3mm×3mm tissue blocks, add collagenase I aqueous solution for a first digestion treatment, control the centrifugation speed at 8000rpm, centrifugation time at 14min, wash 5 times with sterile PBS buffer, and then inoculate and culture. Add trypsin aqueous solution for a second digestion treatment. When the cell confluence reaches 90%, passage is performed at a passage ratio of 1:3 to 1:5, for 3 passages to obtain passaged cells. In the first digestion treatment, the concentration of collagenase I aqueous solution is 1.2mg / mL, and the amount of collagenase I aqueous solution added is 20mL / g per gram of antler stalk periosteum tissue block. In the second digestion treatment, the concentration of trypsin aqueous solution is 1.2mg / mL, and the amount of trypsin aqueous solution added is 18mL / g per gram of antler stalk periosteum tissue block. The specific parameters for inoculation and culture are: RH 98%, temperature 37℃, CO2 volume concentration 8%, and culture time 7d. Step S2, Directed Differentiation Induction: The passaged cells obtained in Step S1 were washed to remove residual serum; trypsin aqueous solution preheated to 37°C was added for digestion, with the specific parameters controlled as follows: RH 98%, temperature 37°C, CO2 volume concentration 8%, and digestion time 4 min. Induction medium 1 preheated to 37°C was added, and the culture flask wall was agitated 5 times to obtain a single-cell suspension; under aseptic conditions, the single-cell suspension was inoculated into complete culture medium. The culture plate used for inoculation was a 6-well cell culture plate, and the inoculation volume of the single-cell suspension was 5 × 10⁶ cells / well. 5Cells / well; then incubated with the following parameters: 37℃, 5% CO2, RH 98%, for 2.8 hours. Induction medium 2 was added dropwise along the well wall, completed within 10 minutes at a rate of 5 drops / second. After addition, the cells were cultured for 3 weeks, with the induction medium 2 replaced every 3 days. This yielded proliferative chondrocytes of deer antler. Induction medium 1 was based on DMEM / F12, supplemented with 15% (v / v) fetal bovine serum, 1% (v / v) penicillin-antibiotics, 2.5 μg / mL amphotericin B, 5 ng / mL recombinant human IGF-1, 2 ng / mL recombinant human EGF, and 10 mM... HEPES buffer and 0.5 mM L-glutathione were added, and the pH was adjusted to 7.4 with sterile 0.1 M hydrochloric acid aqueous solution. Induction medium 2 was based on DMEM / F12 medium, with the addition of 15% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 8 ng / mL recombinant human HGF, 4 ng / mL recombinant human bFGF, 10 µg / mL fibronectin, 5 µg / mL laminin, 50 µg / mL ascorbic acid, 1 mM sodium pyruvate, and the pH was adjusted to 7.4 with sterile 0.1 M sodium hydroxide aqueous solution. Step S3, Ossification Induction: Remove the old culture medium from the proliferating chondrocytes of deer antler, wash the cells 5 times with sterile PBS buffer to remove residual induction medium 2; add ossification induction medium preheated to 37℃, and continue culturing. The specific parameters during the culture process are: RH 98%, temperature 37℃, culture time 7 weeks, during which fresh ossification induction medium is replaced every 3 weeks to obtain deer antler tissue cells. The ossification induction medium is based on DMEM / F12, supplemented with 12% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 10mM β-glycerophosphate sodium, 50μg / mL ascorbic acid-2-phosphate, 0.1µM dexamethasone, 10ng / mL recombinant human BMP-2, 8μg / mL hyaluronic acid, and 5μg / mL chondroitin sulfate. The pH is then adjusted to 7.4 with sterile 0.1M sodium hydroxide aqueous solution.

[0028] Comparative Example 1 Comparative Example 1 is the same as Example 1, except that in the preparation of induction medium 1, amphotericin B is replaced with an equal mass of bispecific antibiotics, while the other steps and raw materials are the same as in Example 1.

[0029] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that in the preparation of induction medium 2, recombinant human bFGF is replaced with an equal mass of recombinant human HGF, and the remaining steps and raw materials are the same as in Example 1.

[0030] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that in the preparation of the osteolysis induction medium, ascorbic acid-2-phosphate is replaced with an equal mass of ascorbic acid, while the other steps and raw materials are the same as in Example 1.

[0031] Performance testing Based on the scratch test, the migration efficiency of deer antler tissue cells obtained in Examples 1-3 and Comparative Examples 1-3 was determined. The specific detection process was as follows: (1) When the cell confluence reaches 90%, a standardized scratch is made using a 200 μL sterile pipette tip. After washing twice with sterile PBS buffer, three fields of view are selected under an inverted microscope, and the scratch width is measured using ImageJ software. The average value is taken as the initial scratch width (W0). (2) After 24 hours of culture, the scratch width was measured under the same field of view, and the average value was taken as the scratch width (W) after 24 hours of culture. t ); (3) Calculate the migration efficiency according to the following formula. Set up 3 replicate wells for each sample and take the average value as the final result; Migration efficiency (%) = (W0 - W) t ) / W0×100% The specific test results are shown in Table 1; Table 1. Results of migration efficiency test of deer antler tissue cells

[0032] As can be seen from the data in Table 1, compared with Comparative Examples 1-3, the deer antler tissue cells obtained by the culture methods in Examples 1-3 have a better migration efficiency.

[0033] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for culturing deer antler tissue cells, characterized in that, Includes the following steps: Step S1, In vitro cell culture: Take the antler stalk periosteum of a healthy sika deer, cut the antler stalk periosteum into tissue pieces, add collagenase I aqueous solution for a first digestion treatment, centrifuge, wash, then inoculate and culture, add trypsin aqueous solution for a second digestion treatment, passage, and obtain passaged cells. Step S2, Directional Induction of Differentiation: Wash the passaged cells obtained in step S1, add trypsin aqueous solution preheated to 37°C for digestion, add induction medium 1 preheated to 35-37°C, and agitate the culture flask wall to obtain a single-cell suspension; under sterile conditions, inoculate the single-cell suspension into complete culture medium, incubate, add induction medium 2, and culture for 2-3 weeks to obtain deer antler proliferative chondrocytes; Step S3, Ossification Induction: Remove the old culture medium from the proliferating chondrocytes of deer antler, wash them, add ossification induction culture medium preheated to 37°C, and continue culturing to obtain deer antler tissue cells.

2. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S1, the specific parameters for inoculation and culture are: RH 96-98%, temperature 35-37℃, CO2 volume concentration 5-8%, and culture time 5-7 days. During the first digestion process, the concentration of collagenase I aqueous solution is 0.6-1.2 mg / mL, and the amount of collagenase I aqueous solution added is 10-20 mL / g per gram of horn stalk periosteum tissue block. During the second digestion process, the concentration of trypsin aqueous solution is 0.6-1.2 mg / mL, and the amount of trypsin aqueous solution added is 14-18 mL / g per gram of horn stalk periosteum tissue block.

3. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S2, the culture plate used during the inoculation process is a 6-well cell culture plate, and the inoculation volume of single-cell suspension is 2 × 10⁻⁶. 5 -5×10 5 Cells / well 4. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S2, the amount of induction medium 1 added is 2-3 mL per well.

5. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S2, the induction medium 2 is replaced with fresh medium every 2-3 days during the culture period; the specific parameters for the incubation treatment are: 37℃, 5% CO2, RH 96-98%, and the incubation time is 2.4-2.8h; the specific parameters for the digestion treatment are: RH 96-98%, temperature 35-37℃, CO2 volume concentration of 6-8%, and digestion time of 2-4min.

6. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S3, the specific parameters during the cultivation process are: RH 96-98%, temperature 35-37℃, and cultivation time of 5-7 weeks.

7. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S2, the induction medium 1 is based on DMEM / F12, with the addition of 10-15% (v / v) fetal bovine serum, 1% (v / v) penicillin-antibody, 2.5 μg / mL amphotericin B, 5 ng / mL recombinant human IGF-1, 2 ng / mL recombinant human EGF, 10 mM HEPES buffer and 0.5 mM L-glutathione, and the pH is adjusted to 7.2-7.

4.

8. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S2, the induction medium 2 is based on DMEM / F12, with the addition of 10-15% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 8 ng / mL recombinant human HGF, 4 ng / mL recombinant human bFGF, 10 µg / mL fibronectin, 5 µg / mL laminin, 50 µg / mL ascorbic acid and 1 mM sodium pyruvate, and the pH is adjusted to 7.2-7.

4.

9. The method for culturing deer antler tissue cells according to claim 1, characterized in that, In step S3, the osteolysis induction medium is based on DMEM / F12, with the addition of 10-12% (v / v) fetal bovine serum, 1% (v / v) penicillin and antibiotics, 10 mM β-glycerophosphate sodium, 50 μg / mL ascorbic acid-2-phosphate, 0.1 µM dexamethasone, 10 ng / mL recombinant human BMP-2, 8 μg / mL hyaluronic acid and 5 μg / mL chondroitin sulfate, and the pH is adjusted to 7.2-7.4.

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