Stem cell culture solution and culture method thereof
By preparing modified bFGF and EGF mixtures using MOFs embedding technology, the problem of unsatisfactory growth factor release in existing stem cell culture medium is solved, and a more stable stem cell culture environment is achieved.
Patent Information
- Application Number
- CN202510291655.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-06-13
AI Technical Summary
The existing method of embedding growth factors in stem cell culture medium has the problem of unsatisfactory release, which affects the culture effect of stem cells.
Modified bFGF and EGF mixtures were prepared by mixing 2-methylimidazole and zinc nitrate solutions, combined with ultrasonic irradiation and freeze-drying, and other steps to slow their release.
It effectively slows down the release of bFGF and EGF, extends its stability in stem cell culture medium, and improves the culture effect of stem cells.
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Figure SMS_1
Abstract
Description
Technical Field
[0001] The present invention relates to the field of stem cell culture, and specifically to a stem cell culture medium and a culture method thereof. Background Art
[0002] Stem cell culture medium is a culture medium specially designed to support the growth, proliferation and maintenance of undifferentiated state of stem cells. It simulates the in vivo microenvironment of stem cells by providing necessary nutrients, growth factors and signal molecules, ensuring that stem cells maintain their pluripotency or specific stemness characteristics in in vitro culture.
[0003] The main components of stem cell culture medium include basal culture medium, serum or serum substitutes, growth factors (such as bFGF, LIF, EGF), antibiotics, buffer systems (such as sodium bicarbonate, HEPES), nutritional supplements (such as glutamine, antioxidants) and matrix or adhesion proteins (such as Matrigel, polylysine), etc.
[0004] The existing method usually involves adding growth factors. In order to slow down the release of growth factors, the growth factors are usually encapsulated. However, experiments have found that although the existing encapsulation method can slow down the release, the release is basically completed after a certain period of time, resulting in unsatisfactory encapsulation release and affecting the culture effect of stem cells. Summary of the invention
[0005] The purpose of this application is to solve the problems in the prior art and disclose a method for preparing a stem cell culture medium, which is characterized by the following steps:
[0006] Take DMEM / F12 and Neurobasal Plus, mix them evenly, and filter the mixed basal culture medium through a filter. The volume of the culture medium is 500 copies.
[0007] Add B-27Supplement, L-glutamine, L-glutamic acid, modified bFGF and EGF mixture, 2-mercaptoethanol, penicillin-streptomycin, and HEPES buffer;
[0008] Mix thoroughly and evenly, place in a sterile centrifuge tube and store for later use.
[0009] In a preferred embodiment, the modified bFGF and EGF mixture is prepared as follows: first, a MOFs-encapsulated bFGF and EGF mixture is prepared, and an isolation agent is applied to the surface of the mixture to isolate the pores on the MOFs surface to slow down the overflow of bFGF and EGF.
[0010] In a preferred embodiment, the method for preparing the modified bFGF and EGF mixture is as follows: Dissolve 2.5 g of 2-methylimidazole in 20 mL of deionized water, stir until completely dissolved to obtain a first solution. Dissolve 0.75 g of zinc nitrate in 20 mL of deionized water, stir until completely dissolved to obtain a second solution. Dissolve 1 mg of bFGF and 1 mg of EGF in 5 mL of PBS buffer, gently mix to obtain a growth factor mixed solution C. Rapidly mix the first solution and the second solution, and immediately add the growth factor mixed solution C, gently stir evenly. Let it stand and react at room temperature for 1 hour, then centrifuge, wash, and dry to obtain the MOFs-embedded bFGF and EGF mixture. Add the mixture to 1 wt% aminomethylsilane by mass ratio and stir, while irradiating with ultrasonic waves for 1.5 h. Finally, separate the mixture from the solution by freeze-drying to obtain the modified bFGF and EGF mixture.
[0011] In an improved embodiment, the volume ratio of DMEM / F12 to Neurobasal Plus is 1:1.2.
[0012] In an improved embodiment, the filter is a 0.22 μm filter.
[0013] In an improved embodiment, the volumes of L-glutamine and L-glutamic acid are 5 and 0.25 respectively.
[0014] In an improved embodiment, the volumes of 2-mercaptoethanol, penicillin-streptomycin, and HEPES buffer are 0.5, 5, and 5 respectively.
[0015] This application also discloses a stem cell culture medium prepared according to the described method.
[0016] This application also discloses a method for culturing stem cells, and the specific steps are as follows:
[0017] S1. Obtain stem cells, resuspend the cells with the culture medium, adjust the cell density, and regularly change the culture medium to obtain a primary culture.
[0018] S2. Subculture the primary culture with the described culture medium for 3 - 7 generations to obtain stem cells.
[0019] The detailed steps of S2 are as follows: Take out the culture container from the incubator, aspirate the old culture medium; add an appropriate amount of PBS buffer, gently shake and then aspirate, repeat 2 times to remove residual serum and metabolic wastes; add 0.05% trypsin-EDTA solution to digest for 3 - 5 minutes, add the culture medium to terminate digestion and gently pipette to disperse the neurospheres into a single cell suspension; centrifuge the cell suspension (1000 rpm, 5 minutes), discard the supernatant, resuspend the cells with the stem cell culture medium, and adjust the cell density to 1 - 2×105 cells / mL, and evenly inoculate them into a new culture dish pre-coated with polylysine, then place it in an incubator at 37°C and 5% CO 2 and continue to culture. Replace the fresh culture medium every 2 - 3 days and observe the cell growth status. Specific implementation mode
[0020] The following is a detailed description of the present invention application.
[0021] I. Preparation of a mixture of bFGF and EGF encapsulated in MOFs.
[0022] Dissolve 2.5 g of 2-methylimidazole in 20 mL of deionized water and stir until completely dissolved to obtain a first solution. Dissolve 0.75 g of zinc nitrate in 20 mL of deionized water and stir until completely dissolved to obtain a second solution; dissolve 1 mg of bFGF and 1 mg of EGF in 5 mL of PBS buffer and gently mix well to obtain a third solution of growth factors; quickly mix the first solution and the second solution, and immediately add the third solution of growth factors and gently stir evenly. Let it stand and react at room temperature for 1 hour, then centrifuge, wash, and dry to obtain a mixture of bFGF and EGF encapsulated in MOFs, named mixture A.
[0023] II. Treatment of mixture A
[0024] Add mixture A to 1 wt% 3-aminopropyltriethoxysilane by mass and stir. The mass ratio of mixture A in the solution is 3.5%. At the same time, irradiate it under ultrasonic waves for 2 h. Finally, separate the mixture from the solution by freeze-drying method to obtain the treated bFGF and EGF mixture, named mixture B. In mixture B, due to the formation of a network-like 3-aminopropyltriethoxysilane polymer structure on the pore surface of MOFs, the pores are blocked, which can slow down the release of the encapsulated substances.
[0025] III. Preparation method of stem cell culture medium
[0026] Example 1: A preparation method of a stem cell culture medium, the steps are as follows:
[0027] Take DMEM / F12 and Neurobasal Plus media, mix them evenly at a volume ratio of 1:1.2, and filter the mixed basal medium using a 0.22 μm filter. Take 500 mL of the filtered medium. Add 10 mL of B-27 Supplement (50×), 5 mL of L-glutamine (200 mM), 0.25 mL of L-glutamic acid (200 mM), bFGF (10 μg / mL) 50 μL; EGF (10 μg / mL) 50 μL, 2-mercaptoethanol (55 mM) 0.5 mL, penicillin-streptomycin (100×) 5 mL, HEPES buffer (1 M) 5 mL; mix well, place in a sterile centrifuge tube, and store for later use.
[0028] Example 2: Take DMEM / F12 and Neurobasal Plus media, mix them evenly at a volume ratio of 1:1.2, and filter the mixed basal medium using a 0.22 μm filter. Take 500 mL of the filtered medium. Add 10 mL of B-27 Supplement (50×), 5 mL of L-glutamine (200 mM), 0.25 mL of L-glutamic acid (200 mM), Mixture A, 2-mercaptoethanol (55 mM) 0.5 mL, penicillin-streptomycin (100×) 5 mL, HEPES buffer (1 M) 5 mL; mix well, place in a sterile centrifuge tube, and store for later use, where the concentrations of bFGF and EGF are the same as in Example 1.
[0029] Example 3: Take DMEM / F12 and Neurobasal Plus media, mix them evenly at a volume ratio of 1:1.2, and filter the mixed basal medium using a 0.22 μm filter. Take 500 mL of the filtered medium. Add 10 mL of B-27 Supplement (50×), 5 mL of L-glutamine (200 mM), 0.25 mL of L-glutamic acid (200 mM), Mixture B, 2-mercaptoethanol (55 mM) 0.5 mL, penicillin-streptomycin (100×) 5 mL, HEPES buffer (1 M) 5 mL; mix well, place in a sterile centrifuge tube, and store for later use, where the concentrations of bFGF and EGF are the same as in Example 1.
[0030] IV. Stem cell culture method,
[0031] The specific steps are as follows: S1. Obtain neural stem cells, resuspend the cells with a culture medium, adjust the cell density, and regularly change the culture medium to obtain a primary culture. S2. Subculture the primary culture with the above-mentioned culture medium for 3 generations to obtain stem cells. The detailed steps of S2 are: Take out the culture container from the incubator, aspirate the old culture medium; add an appropriate amount of PBS buffer, gently shake and then aspirate, repeat 2 times to remove metabolic wastes; add 0.05% trypsin-EDTA solution to digest for 3 minutes, add the culture medium to terminate digestion and gently pipette to disperse the neurospheres into a single-cell suspension; centrifuge the cell suspension (1000 rpm, 5 minutes), discard the supernatant, resuspend the cells with the stem cell culture medium, and adjust the cell density to about 2×10 5 cells / mL, evenly inoculate into a new culture dish pre-coated with polylysine, and place it in an incubator at 37°C and 5% CO 2 2 incubator for continuous culture, change the fresh culture medium every 3 days, observe the cell growth status, and calculate the cell proliferation rate at different time periods. The results are as follows:
[0032]
[0033]
Claims
1. A method for preparing a stem cell culture medium, characterized in that: Here are the steps:
1. Take DMEM / F12 and Neurobasal Plus, mix them evenly, and filter the mixed basal culture medium through a filter. The volume of the culture medium is 500 copies.
2. Add B-27Supplement, L-glutamine, L-glutamic acid, treated bFGF and EGF mixture, 2-mercaptoethanol, penicillin-streptomycin, and HEPES buffer; 3. Mix thoroughly and evenly, place in a sterile centrifuge tube and store for later use.
2. The method for preparing stem cell culture medium according to claim 1, characterized in that: The method for preparing the modified bFGF and EGF mixed solution is as follows: first, a MOFs-embedded bFGF and EGF mixture is prepared, and an isolation agent is applied to the surface of the mixture to isolate the pores on the surface of the MOFs to slow down the overflow of bFGF and EGF.
3. The method for preparing a stem cell culture medium according to claim 2, characterized in that: The preparation method of the modified bFGF and EGF mixed solution is as follows: 2.5g 2-methylimidazole is dissolved in 20mL deionized water, stirred until completely dissolved, to obtain a first solution, 0.75g zinc nitrate is dissolved in 20mL deionized water, stirred until completely dissolved, to obtain a second solution; 1mg bFGF and 1mg EGF are dissolved in 5mL PBS buffer, gently mixed, to obtain a growth factor mixed solution C; the first solution and the second solution are quickly mixed, and the growth factor mixed solution C is immediately added, and gently stirred evenly. The reaction is allowed to stand at room temperature for 1 hour, and then centrifuged, washed, and dried to obtain a MOFs-encapsulated bFGF and EGF mixture, the mixture is added to 1wt% aminosilane by mass ratio and stirred, and ultrasonically irradiated for 1.5h, and finally the mixture is separated from the solution by freeze drying to obtain a modified bFGF and EGF mixture.
4. The method for preparing a stem cell culture medium according to claim 1, characterized in that: The volume ratio of DMEM / F12 and Neurobasal Plus is 1:1.
2.
5. The method for preparing a stem cell culture medium according to claim 2, characterized in that: The filter is a 0.22 μm filter.
6. The method for preparing a stem cell culture medium according to claim 2, characterized in that: The volumes of L-glutamine and L-glutamic acid are 5 and 0.25 respectively.
7. The method for preparing a stem cell culture medium according to claim 6, characterized in that: The volumes of 2-mercaptoethanol, penicillin-streptomycin, and HEPES buffer were 0.5, 5, and 5, respectively.
8. A stem cell culture medium, characterized in that: Prepared according to the method according to any one of claims 1 to 7.
9. A method for culturing stem cells, characterized in that: The specific steps are as follows: S1. Obtain stem cells, resuspend the cells in culture medium, adjust the cell density, and regularly replace the culture medium to obtain primary culture. S2. Subculture the primary culture with the culture medium described in claim 8 for 3 to 7 generations to obtain stem cells.
10. The stem cell culture method according to claim 9, characterized in that: The detailed steps of S2 are as follows: take the culture container out of the incubator and remove the old culture medium; add an appropriate amount of PBS buffer, shake gently and then remove, repeat twice to remove residual waste; add 0.05% trypsin-EDTA solution to digest for 3-5 minutes, add culture medium to terminate digestion and gently blow to disperse the neurospheres into a single cell suspension; centrifuge the cell suspension, discard the supernatant and resuspend the cells with stem cell culture medium to adjust the cell density to 1-2×10 5 cells / mL, and evenly inoculated into a new culture dish pre-coated with poly-lysine, and continued to culture in a 37°C, 5% CO2 incubator, replacing fresh culture medium every 2-3 days and observing the cell growth status.