A culture method for improving the viability and differentiation potential of fibroblast stem cells

Abstract

The present invention discloses a culture method for improving the viability and differentiation potential of fibroblast stem cells, and belongs to the field of cell culture technology. The present invention provides a cell culture dish, which is surface treated with graphene oxide, nano-zirconium boride, titanium dioxide and other reagents to improve the biocompatibility and cell adhesion of the cell culture dish; the method for culturing fibroblast stem cells using the above cell culture dish is to obtain fibroblast stem cells and transfer them to the cell culture dish for culture and adipogenic differentiation. During the culture process, the viability and differentiation potential of fibroblast stem cells are improved by optimizing the culture conditions to promote the growth and differentiation of fibroblast stem cells.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a culture method for improving the viability and differentiation potential of fibroblast stem cells. Background Art

[0002] The establishment of somatic cell lines and cryopreservation are fundamental and important methods for studying the characteristics of endangered species and preserving valuable resources. Based on this, research on fibroblasts has become a focus of attention. Fibroblasts, as the main cellular component of loose connective tissue, originate from mesenchymal cells during the embryonic period. They have distinct morphological characteristics, such as large cell volume, clear outer contour, and a spindle-shaped or star-shaped flattened structure. Furthermore, fibroblast nuclei are regularly oval, with large and prominent nucleoli. Fibroblasts exhibit highly active functional activities; their cytoplasm is weakly basic and exhibits significant protein synthesis and secretion activities. Under certain conditions, fibroblasts can interconvert with fibroblasts. This characteristic enables fibroblasts to play a crucial role in the body, playing a vital role in the repair of cell degeneration, necrosis, tissue defects, and bone trauma of varying degrees.

[0003] In recent years, the problem of insufficient viability and differentiation potential of fibroblast stem cells (FSCs) has become increasingly prominent. Firstly, the growth environment of FSCs has a significant impact on their viability and differentiation potential. Traditional culture methods often fail to fully simulate the natural in vivo growth environment, leading to limited cell activity and differentiation capacity. Secondly, factors such as the culture medium, additives, and culture conditions used during the culture process can also affect the viability and differentiation potential of FSCs. If these factors are not properly optimized, it may result in poor cell growth or a deviation from the expected differentiation direction. Furthermore, individual differences in cell source, age differences, and contamination during the process can also adversely affect the viability and differentiation potential of FSCs. Researchers are dedicated to studying culture methods that can improve the viability and differentiation potential of FSCs. This will be of great significance for saving endangered animals. Through the study of fibroblasts, we can gain a deeper understanding of the physiological characteristics and cellular mechanisms of endangered animals, providing a scientific basis for the protection of endangered species. Simultaneously, this will also bring new breakthroughs to the fields of regenerative medicine and disease treatment. Research on fibroblasts has significant value in endangered animal protection, regenerative medicine, and disease treatment. Establishing efficient methods and systems for isolating and culturing fibroblasts will help improve the protection level of endangered animals and provide new research directions and theoretical foundations for biomedical research on saving endangered species. Summary of the Invention

[0004] The purpose of this invention is to provide a culture method that improves the viability and differentiation potential of fibroblast stem cells, thereby solving the problem of insufficient viability and differentiation potential of fibroblast stem cells in the prior art.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A method for improving the viability and differentiation potential of fibroblast stem cells, comprising a culture dish, wherein the preparation method of the culture dish includes the following steps:

[0007] A1. Add boric acid and sucrose to acetic acid under stirring at room temperature, heat to 75-85℃ and continue stirring for 0.5-1h, cool to room temperature, add zirconium n-propoxide, stir at 55-65℃ for 3-4h, vacuum dry at 120℃ for 3-4h, grind to obtain material A, and calcine material A in an argon atmosphere to obtain nano-zirconium boride.

[0008] A2. Take the graphene oxide dispersion and glucose, mix them, stir for 20-30 min, add ammonia water, stir for 5-8 min, stir at 85-95℃ for 1-2 h, add the nano-zirconium boride and sodium dodecyl sulfonate, ultrasonically disperse for 30-40 min, stir in a water bath at 40-50℃ for 8-10 h, filter, take the solid phase, wash and dry to obtain material B;

[0009] A3. In a nitrogen atmosphere: Take ferrous chloride tetrahydrate and deionized water, add the material B, ultrasonically stir for 2-3 hours, stir in a water bath at 30-40℃ for 20-30 hours, and vacuum dry to obtain material C;

[0010] A4. Mix anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, and strontium nitrate. Add tetrabutyl titanate and stir for 2-3 hours. Heat in a hydrothermal reactor at 160°C for 2-3 hours. Vacuum dry and calcine at 450°C for 2-3 hours. Grind to obtain material D.

[0011] A5. Under light-protected conditions, bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) are placed in a high-speed mixer and mixed for 20-30 minutes to obtain a mixture. The mixture is then coated onto the surface of a circular glass dish with a radius of 5 cm, pressed evenly to a thickness of 2 mm, and then cured by irradiation with a UV lamp for 4-5 hours to obtain the culture dish.

[0012] As a further aspect of the present invention, a culture medium is also included, wherein the culture medium is evenly spread on the bottom surface of the culture dish with a thickness of 5 mm to obtain a cell culture dish; the culture medium is DMEM medium (purchased from Shandong Xiya Chemical Co., Ltd.).

[0013] As a further aspect of the present invention, the culture of fibroblast stem cells using the aforementioned cell culture dish includes the following steps:

[0014] S1. Obtain fibroblast stem cells;

[0015] S2. The fibroblast stem cells are transferred to the cell culture dish for culture.

[0016] S3. Select well-grown cultured fibroblast stem cells, freeze them, thaw them, and induce adipogenesis and differentiation in vitro.

[0017] As a further aspect of the present invention, in step A1, the ratio of boric acid, sucrose, acetic acid, and zirconium propoxide is 2-3g: 2-3g: 30-40mL: 5-6g.

[0018] As a further aspect of the present invention, in step A1, the firing process refers to heating from room temperature to 800°C at a rate of 5°C / min and holding for 10-20 min, then heating to 1200°C at a rate of 3°C / min and holding for 2-3 h, then heating to 1500-1600°C at a rate of 2°C / min and holding for 1-2 h, and then cooling to room temperature at a rate of 5°C / min.

[0019] As a further aspect of the present invention, in step A2, the ratio of the graphene oxide dispersion, glucose, ammonia, nano-zirconium boride, and sodium dodecyl sulfonate is 55-65 mL: 3.5-3.8 g: 70-80 μL: 6-8 mg: 1-1.2 mg.

[0020] As a further aspect of the present invention, in step A2, the concentration of the graphene oxide dispersion is 0.1 mmol / L; and the mass concentration of the ammonia water is 25%.

[0021] As a further embodiment of the present invention, in step A3, the ratio of ferrous chloride tetrahydrate, deionized water, and material B is 70-80g: 500mL: 4-6g.

[0022] As a further embodiment of the present invention, in step A4, the ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, strontium nitrate, and tetrabutyl titanate is 20-22 mL: 5-5.5 mL: 1-1.5 mL: 17-18 mL: 0.3-0.5 g: 4-6 mL; the concentration of the concentrated nitric acid is 16 mol / L; and the concentration of the ammonia is 14.8 mol / L.

[0023] As a further embodiment of the present invention, in step A5, the mass ratio of bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer is 40-50:40-50:2-3:1-2:1-1.2:1-1.2:0.5-0.8.

[0024] Beneficial effects of the present invention:

[0025] This invention discloses a method for improving the viability and differentiation potential of fibroblast stem cells. It provides a cell culture dish whose surface is treated with reagents such as graphene oxide, nano-zirconium boride, and titanium dioxide to improve biocompatibility and cell adhesion. The method utilizes this cell culture dish to culture fibroblast stem cells by obtaining them and transferring them to the dish for culture and adipogenic differentiation. During the culture process, optimized culture conditions promote the growth and differentiation of fibroblast stem cells, thereby improving their viability and differentiation potential. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings.

[0027] Figure 1 This is a magnified view of a portion of the surface of the culture dish prepared according to Example 1 of the culture method for improving the viability and differentiation potential of fibroblast stem cells proposed in this invention.

[0028] Figure 2 This is a fibroblast growth curve diagram of Example 4 of a culture method for improving the viability and differentiation potential of fibroblast stem cells proposed in this invention.

[0029] Figure 3 This is a diagram illustrating the in vitro differentiation of fibroblasts into adipocytes in Example 15 of a culture method for improving the viability and differentiation potential of fibroblast stem cells proposed in this invention.

[0030] Figure 4 These are comparative images of inducing fibroblasts to differentiate into adipocytes in Examples 15 and 16 of the culture method for improving the viability and differentiation potential of fibroblast stem cells proposed in this invention. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0032] Example 1

[0033] A method for preparing a cell culture dish includes the following steps:

[0034] A1. Boric acid and sucrose are added to acetic acid under stirring at room temperature. The mixture is heated to 75°C and stirred for 0.5 hours. After cooling to room temperature, zirconium propoxide is added. The mixture is stirred at 55°C for 3 hours and then vacuum dried at 120°C for 3 hours. The mixture is then ground to obtain material A. Material A is then calcined in an argon atmosphere to obtain nano-zirconium boride. The ratio of boric acid, sucrose, acetic acid, and zirconium propoxide is 2g:2g:30mL:5g. The calcination process involves heating from room temperature to 800°C at 5°C / min and holding for 10 minutes, then heating to 1200°C at 3°C / min and holding for 2 hours, then heating to 1500°C at 2°C / min and holding for 1 hour, and finally cooling to room temperature at 5°C / min.

[0035] A2. Take the graphene oxide dispersion and glucose, mix them, stir for 20 min, add ammonia water, stir for 5 min, stir at 85℃ for 1 h, add the nano-zirconium boride and sodium dodecyl sulfonate, ultrasonically disperse for 30 min, stir in a water bath at 40℃ for 8 h, filter, take the solid phase, wash, and dry to obtain material B; the ratio of the graphene oxide dispersion, glucose, ammonia water, nano-zirconium boride, and sodium dodecyl sulfonate is 55 mL: 3.5 g: 70 μL: 6 mg: 1 mg; the concentration of the graphene oxide dispersion is 0.1 mmol / L; the mass concentration of the ammonia water is 25%;

[0036] A3. In a nitrogen atmosphere: take ferrous chloride tetrahydrate and deionized water, add the material B, ultrasonically stir for 2 hours, stir in a water bath at 30°C for 20 hours, and vacuum dry to obtain material C; the ratio of ferrous chloride tetrahydrate, deionized water and material B is 70g:500mL:4g.

[0037] A4. Mix anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, and strontium nitrate. Add tetrabutyl titanate and stir for 2 hours. Heat in a hydrothermal reactor at 160°C for 2 hours, vacuum dry, calcine at 450°C for 2 hours, and grind to obtain material D. The ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, strontium nitrate, and tetrabutyl titanate is 20 mL: 5 mL: 1 mL: 17 mL: 0.3 g: 4 mL. The concentration of concentrated nitric acid is 16 mol / L. The concentration of ammonia is 14.8 mol / L.

[0038] A5. Under light-protected conditions, bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer were placed in a high-speed mixer and mixed for 20 minutes to obtain a mixture. The mass ratio of bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer was 40:40:2:1:1:1:0.5. The mixture was coated onto the surface of a circular glass dish with a radius of 5 cm, pressed evenly to a thickness of 2 mm, and then cured by irradiation with a UV lamp for 4-5 hours to obtain the culture dish. DMEM culture medium was evenly spread on the bottom surface of the culture dish to a thickness of 5 mm to obtain the cell culture dish.

[0039] Example 2

[0040] A method for preparing a cell culture dish includes the following steps:

[0041] A1. Boric acid and sucrose were added to acetic acid under stirring at room temperature. The mixture was heated to 80°C and stirred for 0.8 hours. After cooling to room temperature, zirconium propoxide was added. The mixture was stirred at 60°C for 3.5 hours and then vacuum dried at 120°C for 3.5 hours. The mixture was then ground to obtain material A. Material A was then calcined in an argon atmosphere to obtain nano-zirconium boride. The ratio of boric acid, sucrose, acetic acid, and zirconium propoxide was 2.5g:2.5g:35mL:5.5g. The calcination process involved heating the mixture from room temperature to 800°C at a rate of 5°C / min and holding for 15 minutes, then heating it to 1200°C at a rate of 3°C / min and holding for 2.5 hours, then heating it to 1540°C at a rate of 2°C / min and holding for 1.5 hours, and finally cooling it to room temperature at a rate of 5°C / min.

[0042] A2. Take the graphene oxide dispersion and glucose, mix them, stir for 20-30 min, add ammonia water, stir for 7 min, stir at 90℃ for 1.5 h, add the nano-zirconium boride and sodium dodecyl sulfonate, ultrasonically disperse for 35 min, stir in a water bath at 45℃ for 9 h, filter, take the solid phase, wash, and dry to obtain material B; the ratio of the graphene oxide dispersion, glucose, ammonia water, nano-zirconium boride, and sodium dodecyl sulfonate is 60 mL: 3.7 g: 75 μL: 7 mg: 1.1 mg; the concentration of the graphene oxide dispersion is 0.1 mmol / L; the mass concentration of the ammonia water is 25%;

[0043] A3. In a nitrogen atmosphere: ferrous chloride tetrahydrate and deionized water are mixed, and material B is added. The mixture is ultrasonically stirred for 2.5 h, stirred in a water bath at 35 °C for 25 h, and then vacuum dried to obtain material C. The ratio of ferrous chloride tetrahydrate, deionized water, and material B is 75 g: 500 mL: 5 g.

[0044] A4. Mix anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, and strontium nitrate. Add tetrabutyl titanate and stir for 2.5 hours. Heat in a hydrothermal reactor at 160°C for 2.5 hours, vacuum dry, calcine at 450°C for 2.5 hours, and grind to obtain material D. The ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, strontium nitrate, and tetrabutyl titanate is 21 mL: 5.3 mL: 1.3 mL: 17.5 mL: 0.4 g: 5 mL. The concentration of concentrated nitric acid is 16 mol / L, and the concentration of ammonia is 14.8 mol / L.

[0045] A5. Under light-protected conditions, bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer were placed in a high-speed mixer and mixed for 20-30 minutes to obtain a mixture. The mass ratio of bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer was 45:45:2.5:1.5:1.1:1.1:0.7. The mixture was coated onto the surface of a circular glass dish with a radius of 5 cm, pressed evenly to a thickness of 2 mm, and then cured by irradiation with a UV lamp for 4.5 h to obtain the culture dish. DMEM culture medium was evenly spread on the bottom surface of the culture dish to a thickness of 5 mm to obtain the cell culture dish.

[0046] Example 3

[0047] A method for preparing a cell culture dish includes the following steps:

[0048] A1. Boric acid and sucrose are added to acetic acid under stirring at room temperature. The mixture is heated to 85°C and stirred for 1 hour. After cooling to room temperature, zirconium propoxide is added. The mixture is stirred at 65°C for 4 hours and then vacuum dried at 120°C for 4 hours. The mixture is then ground to obtain material A. Material A is then calcined in an argon atmosphere to obtain nano-zirconium boride. The ratio of boric acid, sucrose, acetic acid, and zirconium propoxide is 3g:3g:40mL:6g. The calcination process involves heating from room temperature to 800°C at 5°C / min and holding for 20 minutes, then heating to 1200°C at 3°C / min and holding for 3 hours, then heating to 1600°C at 2°C / min and holding for 2 hours, and finally cooling to room temperature at 5°C / min.

[0049] A2. Take the graphene oxide dispersion and glucose, mix them, stir for 30 min, add ammonia water, stir for 8 min, stir at 95℃ for 2 h, add the nano-zirconium boride and sodium dodecyl sulfonate, ultrasonically disperse for 40 min, stir in a water bath at 50℃ for 10 h, filter, take the solid phase, wash, and dry to obtain material B; the ratio of the graphene oxide dispersion, glucose, ammonia water, nano-zirconium boride, and sodium dodecyl sulfonate is 65 mL: 3.8 g: 80 μL: 8 mg: 1.2 mg; the concentration of the graphene oxide dispersion is 0.1 mmol / L; the mass concentration of the ammonia water is 25%;

[0050] A3. In a nitrogen atmosphere: take ferrous chloride tetrahydrate and deionized water, add the material B, ultrasonically stir for 3 hours, stir in a water bath at 40°C for 30 hours, and vacuum dry to obtain material C; the ratio of ferrous chloride tetrahydrate, deionized water and material B is 80g:500mL:6g.

[0051] A4. Mix anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, and strontium nitrate. Add tetrabutyl titanate and stir for 3 hours. Heat in a hydrothermal reactor at 160°C for 3 hours, vacuum dry, calcine at 450°C for 3 hours, and grind to obtain material D. The ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, strontium nitrate, and tetrabutyl titanate is 22 mL: 5.5 mL: 1.5 mL: 18 mL: 0.5 g: 6 mL. The concentration of concentrated nitric acid is 16 mol / L. The concentration of ammonia is 14.8 mol / L.

[0052] A5. Under light-protected conditions, bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer were placed in a high-speed mixer and mixed for 30 minutes to obtain a mixture. The mass ratio of bisphenol A glycerol dimethacrylate, diethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer was 50:50:3:2:1.2:1.2:0.8. The mixture was coated onto the surface of a circular glass dish with a radius of 5 cm, pressed evenly to a thickness of 2 mm, and then cured by irradiation with a UV lamp for 5 hours to obtain the culture dish. DMEM culture medium was evenly spread on the bottom surface of the culture dish to a thickness of 5 mm to obtain the cell culture dish.

[0053] Comparative Example 1

[0054] The difference from Example 1 is that boric acid is not added.

[0055] Comparative Example 2

[0056] The difference from Example 1 is that no nano-zirconium boride is added.

[0057] Comparative Example 3

[0058] The difference from Example 1 is that no graphene oxide dispersion is added.

[0059] Comparative Example 4

[0060] The difference from Example 1 is that ferrous chloride tetrahydrate is not added.

[0061] Comparative Example 5

[0062] The difference from Example 1 is that the ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, strontium nitrate, and tetrabutyl titanate in step A4 is 20mL:22mL:1mL:0mL:0.3g:4mL.

[0063] Comparative Example 6

[0064] The difference from Example 1 is that the ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, strontium nitrate and tetrabutyl titanate in step A4 is 20mL:5.3mL:1mL:17mL:0g:4mL.

[0065] Comparative Example 7

[0066] The difference from Example 1 is that the ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia, strontium nitrate and tetrabutyl titanate in step A4 is 20mL:9mL:1mL:17mL:0.3g:0mL.

[0067] Comparative Example 8

[0068] The difference from Example 1 is that no polypropylene-graft-maleic anhydride compatibilizer is added.

[0069] Test Example 1

[0070] The surface roughness of the culture dishes prepared above was tested. The average surface roughness (Ra) of the culture dishes prepared in Examples 1-3 and Comparative Examples 1-8 was measured using a 2205 surface roughness meter. The test results are shown in Table 1 below.

[0071] Table 1

[0072]

[0073]

[0074] As shown in Table 1, the surfaces of the petri dishes prepared in Examples 1-3 of this application are rougher than those in Comparative Examples 1-8. (The rest of the text appears to be incomplete and requires further context.) Figure 1 This is a magnified view of a portion of the surface of the culture dish prepared in Example 1 of this application.

[0075] Test Example 2

[0076] The antibacterial properties of the culture dishes prepared in Examples 1-3 and Comparative Examples 5-7 were tested: 3 mL of pure BHI was added to a glass cuvette, and the "0" point was calibrated; 0.3 mL of fresh Streptococcus mutans bacterial culture 48 h after consolidation and transfer was added to the cuvette along with 2.7 mL of pure BHI, and the absorbance (A) value was measured to calculate the bacterial concentration. The bacterial culture was continuously diluted to adjust the density to 1 × 10⁻⁶. 7 CFU·mL -1 ; Take the culture dishes prepared in Examples 1-3 and Comparative Examples 5-7, add 2 mL of bacterial suspension to each culture dish as the experimental group; take glass dishes of the same size, add 2 mL of bacterial suspension as the control group; incubate at 37℃ with 5% CO2 for 24 h, remove, gently rinse with PBS buffer to remove non-adhesive bacteria, place in 1 mL of pure BHI, vortex and sonicate for 5 min to obtain bacterial suspension, take the bacterial suspension for serial serial dilution, take 100 μL to plate, incubate at 37℃ for 48 h, count colonies and calculate antibacterial rate. Antibacterial rate = (Ac - Ae) / Ac × 100%, where Ae is the average colony count of the test group and Ac is the average colony count of the control group. The test results are shown in Table 2 below.

[0077] Table 2

[0078]

[0079]

[0080] As shown in Table 2, the culture dishes prepared in Examples 1-3 of this application have excellent antibacterial properties.

[0081] Test Example 3

[0082] Biosafety tests were conducted on the cell culture dishes prepared in Examples 1-3 and Comparative Examples 1-8. Logarithmic growth phase mouse fibroblasts, after being resuscitated and passaged three times, were observed for cell morphology under a microscope, followed by digestion, centrifugation, resuspension, and counting to prepare a cell suspension. This cell suspension was added to the cell culture dishes prepared in Examples 1-3 and cultured in a constant-temperature cell culture incubator for 24 hours. The relative growth rate (RGR) was calculated, and the cell toxicity was evaluated according to the cytotoxicity grading criteria. According to the United States Pharmacopeia (USP), cytotoxicity was graded using a 5-point scale based on RGR: Grade 0, RGR ≥ 100%; Grade 1, RGR 75%-99%; Grade 2, RGR 50%-74%; Grade 3, RGR 25%-49%; Grade 4, RGR 1%-24%; Grade 5, RGR < 1%. Grades 0 and 1 were considered acceptable, Grade 2 should be evaluated in conjunction with cell morphology, and Grades 3-5 were considered unacceptable. The test results are shown in Table 3 below.

[0083] Table 3

[0084]

[0085]

[0086] As shown in Table 3, the cell culture dishes prepared in Examples 1-3 and Comparative Examples 1-8 of this application have good biocompatibility.

[0087] Test Example 4

[0088] The surface wettability of the culture dishes prepared in Examples 1-3 and Comparative Examples 1-4 were tested. The surface contact angle of the culture dishes prepared in Examples 1-3 and Comparative Examples 1-4 was measured using a contact angle meter (SL200, Kino Industry, USA). In the static contact angle measurement method, 1 μL of deionized water was dropped onto the surface of the culture dish. After waiting for 3 seconds for the droplet to stabilize, the contact angle was measured using the software of the contact angle meter. The average value of the left and right contact angles was used as the measurement result for each contact angle. Three points were randomly selected on the surface of each culture dish for measurement. Finally, the average contact angle of each group was calculated. The test results are shown in Table 4 below.

[0089] Table 4

[0090]

[0091]

[0092] As shown in Table 4, the culture dishes prepared in Examples 1-3 of this application have smaller contact angles and better interfacial wettability.

[0093] Example 4

[0094] The culturing of fibroblast stem cells using the cell culture dish prepared in Example 1 includes the following steps:

[0095] Obtaining fibroblast stem cells: Skin from the inner hind leg of a domestic goat was harvested. After shaving, the skin surface was disinfected with 20% iodine and 75% ethanol, and local anesthesia was administered. A 2cm × 2cm piece of full-thickness skin was then taken and immersed in sterile phosphate-buffered saline (PBS) containing 250 U / mL penicillin and 250 mg / L streptomycin. In a laminar flow hood, epidermal cells and subcutaneous tissue were gently scraped away, preserving the dermis. The skin was then cut into 1cm × 1cm pieces using ophthalmic scissors. The pieces were transferred to 50mL centrifuge tubes, and sterile antibiotic PBS solution was added. The tubes were washed three times, and the washings were discarded. The centrifuge tubes were then stored on ice.

[0096] Prepare 0.1% type I collagenase solutions using PBS, sterilize by filtration through a 100-mesh filter, and then transfer the solutions to 50 mL centrifuge tubes. Transfer the shredded skin tissue into the centrifuge tubes and digest in a 37°C water bath for 40 min, shaking every 10 min during digestion. Filter the digested cell suspensions through 40-mesh and 100-mesh filters, respectively. Centrifuge at 1200 rpm for 5 min, discard the supernatant, and then transfer the cells from the centrifuge tubes to the cell culture dishes prepared in Example 1. Incubate at 37°C and 5% CO2 for 1-2 days, monitoring every 2 days. Observe cell adhesion under a microscope. When the adherent cells reach 90%, purify the cells by washing them three times with PBS, adding 0.25% trypsin, and digesting the cells in a water bath at 37°C. Repeatedly pipette the cells until the rounded fibroblasts are completely and evenly mixed. Transfer the liquid to a 15mL centrifuge tube, centrifuge at 1200r / min for 2min, discard the supernatant, resuspend the cells, pipette them evenly, and passage them at a 1:2 ratio. Incubate the cells in a cell culture incubator at 37°C and 5% CO2. Observe cell growth every 2 days and change the medium and passage the cells in a timely manner according to the cell adhesion and growth.

[0097] Well-grown domestic goat cells were washed twice with PBS to remove any floating cells. Adhering cells were digested with pre-warmed 0.25% trypsin. The mixture was centrifuged at room temperature, and then 700 μL of complete culture medium was added. After thorough dissolution, the cells were transferred to cryovials, and 200 μL of fetal bovine serum and 100 μL of dimethyl sulfoxide were added and mixed thoroughly. Cells were cryopreserved in a programmed cryovial: Cells with cryopreservation solution were placed in a programmed cryovial and stored at -80°C for 24 hours before being stored in liquid nitrogen.

[0098] Resuscitation of goat fibroblasts: Preheat the water bath to 37°C. Remove the cryopreservation tubes from -80°C and place them in the water bath for 2-3 minutes to thaw the cryopreservation solution. Centrifuge at 1200 rpm for 3 minutes, retain the pellet, add it to the cell culture dish prepared in Example 1, resuspend it, and seed it into cell culture flasks. Culture in a cell culture incubator at 37°C and 5% CO2. Observe and record cell adhesion and growth on day 2 when changing the medium. Seed the resuscitated fibroblasts into 24-well plates (concentration 1×10⁻⁶). 4 Cells were added to each well (cells / mL) and 1 mL of cell suspension was added, followed by cell counting. Cells were cultured in a 37°C, 5% CO2 incubator. Cell counting was performed every 1 day, with 3 wells counted each time, for 9 consecutive days. The obtained data were used to plot a growth curve of goat fibroblasts (culture time on the x-axis and cell concentration on the y-axis), as shown in the attached figure. Figure 2 shown.

[0099] Example 5

[0100] The difference from Example 4 is that the cell culture dish prepared in Example 2 was used to culture fibroblast stem cells.

[0101] Example 6

[0102] The difference from Example 4 is that the cell culture dish prepared in Example 3 was used to culture fibroblast stem cells.

[0103] Example 7

[0104] The difference from Example 4 is that the cell culture dish prepared in Comparative Example 1 was used to culture fibroblast stem cells.

[0105] Example 8

[0106] The difference from Example 4 is that the cell culture dish prepared in Comparative Example 2 was used to culture fibroblast stem cells.

[0107] Example 9

[0108] The difference from Example 4 is that the cell culture dish prepared in Comparative Example 3 was used to culture fibroblast stem cells.

[0109] Example 10

[0110] The difference from Example 4 is that the cell culture dish prepared in Comparative Example 4 was used to culture fibroblast stem cells.

[0111] Example 11

[0112] The difference from Example 4 is that the cell culture dish prepared in Comparative Example 5 was used to culture fibroblast stem cells.

[0113] Example 12

[0114] The difference from Example 4 is that the cell culture dish prepared in Comparative Example 6 was used to culture fibroblast stem cells.

[0115] Example 13

[0116] The difference from Example 4 is that fibroblast stem cells were cultured using the cell culture dish prepared in Comparative Example 7.

[0117] Example 14

[0118] The difference from Example 4 is that the cell culture dish prepared in Comparative Example 8 was used to culture fibroblast stem cells.

[0119] The viability of fibroblasts from Examples 4-14 before cryopreservation and after thawing is recorded in Table 5 below.

[0120] Table 5

[0121]

[0122]

[0123] As shown in Table 5, the cell culture dish prepared in this application can effectively improve cell culture activity. This is because the culture dish prepared in this application improves the interfacial wettability, promotes the adhesion and proliferation of fibroblasts, and the higher surface roughness is conducive to the climbing and growth of fibroblasts, thereby improving the cell adhesion efficiency. At the same time, the introduction of antibacterial materials avoids the growth of microorganisms from disturbing the healthy growth of fibroblasts.

[0124] Example 15

[0125] Inducing fibroblasts to differentiate into adipocytes in vitro (e.g.) Figure 3 (As shown), including the following steps:

[0126] 1) First, prepare the differentiation culture medium, including the cell culture dish prepared in Example 1, and store it in a refrigerator at 4°C in the dark for later use.

[0127] 2) Inoculate fibroblasts cultured in Example 4 (5 × 10⁶ cells) into differentiation medium pretreated with 1% gelatin. 4 (cells / well);

[0128] 3) Incubate at 37℃ in a 5% CO2 incubator for 24 hours;

[0129] 4) After 24 hours, remove the culture medium and replace it with 3T3-L1 adipogenic differentiation induction medium (purchased from Shanghai Zeye Biotechnology Co., Ltd.) (1.5 mL / well);

[0130] 5) Change the lipid-inducing differentiation medium every 3 days, and induce differentiation for 2-3 weeks;

[0131] 6) After induction, remove the culture medium and add 1 mL of 4% paraformaldehyde to each well for fixation for 30 minutes;

[0132] 7) Wash twice with PBS;

[0133] 8) Add 1 mL of Alizarin Red S staining solution to each well and stain for 5-10 minutes;

[0134] 9) Wash three times with PBS, then observe and photograph under an inverted microscope, and count the percentage of adipocytes. The results are as follows: Figure 4 shown.

[0135] Example 16

[0136] Inducing fibroblasts to differentiate into adipocytes in vitro includes the following steps:

[0137] 1) First, prepare differentiation medium, including glass dishes containing only DMEM medium, and store them in a 4°C refrigerator away from light for later use;

[0138] 2) Inoculate fibroblasts cultured in Example 4 (5 × 10⁶ cells) into differentiation medium pretreated with 1% gelatin. 4 (cells / well);

[0139] 3) Incubate at 37℃ in a 5% CO2 incubator for 24 hours;

[0140] 4) After 24 hours, remove the culture medium and replace it with 3T3-L1 adipogenic differentiation induction medium (purchased from Shanghai Zeye Biotechnology Co., Ltd.) (1.5 mL / well);

[0141] 5) Change the lipid-inducing differentiation medium every 3 days, and induce differentiation for 2-3 weeks;

[0142] 6) After induction, remove the culture medium and add 1 mL of 4% paraformaldehyde to each well for fixation for 30 minutes;

[0143] 7) Wash twice with PBS;

[0144] 8) Add 1 mL of Alizarin Red S staining solution to each well and stain for 5-10 minutes;

[0145] 9) Wash three times with PBS, then observe and photograph under an inverted microscope, and count the percentage of adipocytes. The results are as follows: Figure 4 shown.

[0146] like Figure 4As shown in the comparison diagrams of induced fibroblast differentiation into adipocytes in Examples 15 and 16, the cell culture dishes prepared in this application can improve the differentiation potential of fibroblasts.

[0147] The foregoing has described some embodiments of the present invention in detail, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for increasing the viability and differentiation potential of fibroblast stem cells, characterized in that: The invention comprises a culture dish, wherein the preparation method of the culture dish comprises the following steps: A1. Add boric acid and sucrose to acetic acid under stirring at room temperature, heat to 75-85° C. and continue stirring for 0.5-1 h, cool, add zirconium n-propoxide, stir at 55-65° C. for 3-4 h, vacuum dry, grind to obtain material A, and sinter the material A in an argon atmosphere to obtain nano zirconium boride; A2, mixing graphene oxide dispersion and glucose, stirring, adding ammonia water, stirring, stirring at 85-95° C. for 1-2 h, adding the nano zirconium boride and sodium dodecyl sulfate, ultrasonically dispersing, stirring at 40-50° C. in a water bath for 8-10 h, filtering, washing the solid phase, and drying to obtain material B; A3. In a nitrogen atmosphere: mix ferrous chloride tetrahydrate and deionized water, add material B, stir with ultrasonic, stir in a water bath at 30-40° C. for 20-30 h, and dry in vacuum to obtain material C; A4, mix anhydrous ethanol, deionized water, concentrated nitric acid, ammonia water, and strontium nitrate, add butyl titanate, stir, heat in a hydrothermal kettle at 160°C for 2-3h, vacuum dry, calcine at 450°C for 2-3h, grind, and obtain material D; A5. Take bisphenol A propylene glycol dimethacrylate, triethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer in a high-speed mixer and mix for 20-30 minutes to obtain a mixed material. Apply the mixed material on the surface of a glass dish, press it evenly, and cure it under ultraviolet light to obtain the culture dish. It also includes a culture medium, which is evenly spread on the bottom surface of the culture dish to prepare a cell culture dish; the culture medium is a DMEM culture medium; The method of culturing fibroblast stem cells using the cell culture dish comprises the following steps: S1. Obtain fibroblast stem cells; S2, transferring the fibroblast stem cells to the cell culture dish for culture; S3, selecting the cultured fibroblast stem cells with good growth, freezing, thawing, and inducing adipogenesis differentiation in vitro; In step A1, the dosage ratio of boric acid, sucrose, acetic acid and zirconium n-propoxide is 2-3 g: 2-3 g: 30-40 mL: 5-6 g; In step A1, the firing refers to heating from room temperature to 800°C at 5°C / min and keeping the temperature for 10-20min, then heating to 1200°C at 3°C / min and keeping the temperature for 2-3h, then heating to 1500-1600°C at 2°C / min and keeping the temperature for 1-2h, and then cooling to room temperature at 5°C / min; In step A2, the ratio of the graphene oxide dispersion, glucose, ammonia water, nano zirconium boride, and sodium dodecyl sulfate is 55-65 mL: 3.5-3.8 g: 70-80 μL: 6-8 mg: 1-1.2 mg; In step A2, the concentration of the graphene oxide dispersion is 0.1 mmol / L; the mass concentration of the ammonia water is 25%; In step A3, the dosage ratio of ferrous chloride tetrahydrate, deionized water, and material B is 70-80 g: 500 mL: 4-6 g; In step A4, the ratio of anhydrous ethanol, deionized water, concentrated nitric acid, ammonia water, strontium nitrate and butyl titanate is 20-22 mL: 5-5.5 mL: 1-1.5 mL: 17-18 mL: 0.3-0.5 g: 4-6 mL; the concentration of concentrated nitric acid is 16 mol / L; the concentration of ammonia water is 14.8 mol / L; In step A5, the mass ratio of bisphenol A propylene glycol dimethacrylate, triethylene glycol dimethacrylate, dimethylaminoethyl methacrylate, camphorquinone, material C, material D, and polypropylene-graft-maleic anhydride compatibilizer is 40-50:40-50:2-3:1-2:1-1.2:1-1.2:0.5-0.8.

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