Culture method and application of human astrocytes

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a culture method and application of human astrocytes. According to the culture solution provided by the invention, DMEM (Dulbecco Modified Eagle Medium) and MCDB 131 are mixed according to a specific ratio to form a basic culture medium, and functional additives of HEPES, Glutamax, N2, G-5, B-27, hEGF (Human Epidermal Growth Factor) and the like with accurate concentration and fetal calf serum with proper concentration are matched. The culture solution can effectively improve the vitality and proliferation efficiency of SVGP12 cells, guarantees the growth stability and morphological consistency of the cells, can provide a high-quality and reliable cell source for the application of human astrocytes in the fields of neurobiology research, neurological disease mechanism exploration, drug screening and the like, and has important practical value.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for culturing human astrocytes and its application. Background Technology

[0002] As the most numerous glial cells in the central nervous system, astrocytes play an irreplaceable role in maintaining the homeostasis of the neural tissue microenvironment, providing nutritional support to neurons, regulating synapse formation and function, and participating in nerve injury repair and neuroinflammatory responses. Based on their important physiological functions, efficient in vitro culture techniques for human astrocytes (such as the commonly used SVGP12 cell line) have become a core technological support for basic neurobiological research, the exploration of the pathogenesis of neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), and the screening and safety evaluation of drugs for the nervous system, playing a crucial role in advancing research in these fields.

[0003] In the current technological field, the in vitro culture protocol for human astrocytes, represented by SVGP12, still faces many bottlenecks that urgently need to be overcome, making it difficult to meet the needs of scientific research and applications. These bottlenecks are mainly reflected in the following aspects: In existing culture protocols, SVGP12 cells often suffer from low adhesion rates and slow early growth, and are prone to proliferation arrest or even viability decline in the later stages of culture, making it difficult to obtain sufficient cell quantities in a short period, especially failing to meet the dual requirements of cell quantity and viability for large-scale experiments or long-term cell function studies; SVGP12 cells from different batches exhibit significant differences in morphological consistency and viability levels, the core reason being the existing… The existing protocols lack a unified and scientific standard for the selection and combination of key components in the culture system. This can easily lead to unstable cell growth due to component fluctuations, resulting in poor reproducibility of experimental results and affecting the reliability and comparability of research data. At the same time, existing protocols mostly use general cell culture systems and do not fully consider the characteristics of SVGP12 cells as a highly specialized nerve cell type. They lack targeted design for the nutritional environment required by SVGP12 cells (such as growth factors, serum types, osmotic pressure, pH buffering capacity, etc.), which can easily lead to an imbalance in nutrient supply (excess or deficiency). This can result in the inability to maintain normal cell physiological functions and make it difficult to use them to construct accurate in vitro cell models.

[0004] The aforementioned technical challenges severely limit the in-depth application of SVGP12 cells in related fields and hinder the development of neuroscience research and treatment technologies for nervous system diseases. Therefore, developing a new method that can overcome the limitations of existing technologies and achieve efficient and stable culture of SVGP12 cells is of significant practical importance and application value for improving the in vitro culture quality of this type of human astrocyte and advancing research in related fields. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a method for culturing human astrocytes and its application. The final concentrations of each component in the culture medium are precisely controlled to synergistically promote cell adhesion, proliferation, and viability maintenance. Fetal bovine serum is selected and controlled at an appropriate final concentration to avoid the inhibitory effects of xenogeneic serum or inappropriate concentrations on cell growth. This culture method effectively solves the pain points of existing technologies, achieving efficient and stable culture of human astrocytes (SVGP12), providing a high-viability, high-quality cell source for subsequent related research.

[0006] The technical solution of this invention is as follows: On one hand, the present invention provides a method for culturing human astrocytes, comprising seeding human astrocytes in a culture medium for culture, wherein the culture medium comprises a mixed basal medium of DMEM and MCDB 131, fetal bovine serum at a final concentration of 1% v / v, 5% v / v or 8% v / v, Glutamax at a final concentration of 0.1% v / v, 1% v / v or 2% v / v, N2 at a final concentration of 0.3% v / v, 1% v / v or 2.5% v / v, G-5 at a final concentration of 0.1% v / v, 1% v / v or 2% v / v, B-27 at a final concentration of 0.1% v / v, 1% v / v or 2.5% v / v, and hEGF at a final concentration of 2 ng / mL, 10 ng / mL or 18 ng / mL.

[0007] Specifically, the culture medium also includes buffer solution and antibiotics.

[0008] Preferably, the buffer solution is selected from HEPES, MOPS, TES, DIPSO, POPSO or HEPPSO; The antibiotic is selected from penicillin-streptomycin, gentamicin, amphotericin B, ampicillin, or tetracycline.

[0009] Preferably, the buffer solution may be HEPES; the antibiotic may be penicillin-streptomycin.

[0010] Preferably, the final concentration of HEPES can be 5-15 mM; the final concentration of penicillin-streptomycin can be 0.1%-2% v / v.

[0011] Preferably, the final concentration of HEPES can be 5mM, 10mM or 15mM; the final concentration of penicillin-streptomycin can be 0.1%v / v, 1%v / v or 2%v / v.

[0012] Specifically, the human astrocytes may be SVGP12, U-87MG, U-118MG, or U-251MG.

[0013] Preferably, the human astrocytes are SVGP12.

[0014] In some embodiments, the culture medium consists of a mixed basal medium of DMEM and MCDB 131 at a volume ratio of 1:1, HEPES at a final concentration of 10 mM, fetal bovine serum at a final concentration of 5% v / v, Glutamax at a final concentration of 1% v / v, N2 at a final concentration of 1% v / v, G-5 at a final concentration of 1% v / v, B-27 at a final concentration of 1% v / v, hEGF at a final concentration of 10 ng / mL, and penicillin-streptomycin at a final concentration of 1%.

[0015] In other embodiments, the culture medium consists of a mixed basal medium of DMEM and MCDB 131 at a volume ratio of 1:1, HEPES at a final concentration of 5 mM, fetal bovine serum at a final concentration of 1% v / v, Glutamax at a final concentration of 0.1% v / v, N2 at a final concentration of 0.3% v / v, G-5 at a final concentration of 0.1% v / v, B-27 at a final concentration of 0.1% v / v, hEGF at a final concentration of 2 ng / mL, and penicillin-streptomycin at a final concentration of 0.1%.

[0016] In some other embodiments, the culture medium consists of a mixed basal medium of DMEM and MCDB 131 at a volume ratio of 1:1, HEPES at a final concentration of 15 mM, fetal bovine serum at a final concentration of 8% v / v, Glutamax at a final concentration of 2% v / v, N2 at a final concentration of 2.5% v / v, G-5 at a final concentration of 2% v / v, B-27 at a final concentration of 2.5% v / v, hEGF at a final concentration of 18 ng / mL, and penicillin-streptomycin at a final concentration of 2%.

[0017] On the one hand, the present invention provides the application of the aforementioned culture method in neurobiological research, exploration of the mechanisms of neurodegenerative diseases, or screening of drugs for the nervous system.

[0018] Specifically, the human astrocytes may be SVGP12, U-87MG, U-118MG, or U-251MG.

[0019] Preferably, the human astrocytes are SVGP12.

[0020] The beneficial effects of this invention are as follows: (1) The culture medium formulations provided by the present invention can provide a suitable nutritional environment for cells, effectively promote cell proliferation, and meet the experimental requirements for cell quantity.

[0021] (2) The culture medium formula provided by the present invention makes the cell growth state more uniform and the vitality more stable, and can preserve the physiological functions of astrocytes to the greatest extent, meeting the experimental scenarios with high requirements for cell quality.

[0022] (3) The three formulations provided by this invention avoid the nutritional deficiencies of a single basic culture medium, and the choice of fetal bovine serum can effectively provide the growth factors and adhesion proteins required for cell growth, ensuring the basic growth needs of cells; on the other hand, the design of concentration gradients (medium concentration / low concentration / high concentration) provides flexible choices for different experimental needs: if the experiment needs to take into account both cell viability and cost, Example 2 (low concentration component) can be selected; if it is necessary to observe cell response in a specific high-nutrient environment, Example 3 (high concentration component) can be selected; if the best cell viability and stability are pursued, Example 1 is the first choice, which greatly improves the applicability and practicality of this culture method. Detailed Implementation

[0023] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0024] Example 1 SVGP12 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHU258) were seeded at a depth of 25 cm. 2 The culture medium in the cell culture flasks (Table 1) was placed in a 5% CO2, 37℃ incubator and cultured until the cell density reached 80-90%. Cell morphology was observed for subsequent passage. Cells cultured in the medium were then sputtered at 2.0 × 10⁶ cells / mL. 4 Cells were seeded into 96-well plates with 6 replicates per well. Cell adhesion and growth were observed after 4 hours. Cell morphology was observed in each culture medium at 24, 48, 72, and 96 hours, and cell viability was assessed using the CCK8 assay.

[0025] The CCK-8 experimental procedure was as follows: During passage, cells were seeded into four 96-well plates at 24 h, 48 h, 72 h, and 96 h, respectively. Six biological replicates were set up in each well.

[0026] (1) Discard the original culture medium in the cell culture plate, wash twice with sterile PBS, and replace the proliferation medium with 100uL per well.

[0027] (2) Add 10 μL of cck-8 solution to each well, avoid generating bubbles, and place in an incubator at 37°C for 1 hour.

[0028] The absorbance was measured at 450 nm using an ELISA reader, and the OD values ​​for each group were recorded.

[0029] Table 1. Culture medium of Example 1

[0030] Example 2 The difference between Example 2 and Example 1 is that the culture medium is different, as shown in Table 2. Everything else is the same as in Example 1.

[0031] Table 2. Culture medium of Example 2

[0032] Example 3 The difference between Example 3 and Example 1 is that the culture medium is different, as shown in Table 3. Everything else is the same as in Example 1.

[0033] Table 3. Culture medium of Example 3

[0034] Comparative Example A comparative example was set up with reference to Example 1. The difference between the comparative example and Example 1 is that the culture medium is different, as shown in Table 4. Everything else is the same as in Example 1.

[0035] Table 4 Comparative Culture Media

[0036] The test results of the examples and comparative examples are shown in Table 5: Table 5 Test Results

[0037] The results in Table 5 show that at the four detection time points of 24h, 48h, 72h, and 96h, the OD values ​​of Examples 1-3 were all higher than those of all comparative examples. Example 1 performed best, with an OD value of 1.154±0.047 at 96h; Examples 2 and 3 were slightly weaker, with OD values ​​of 0.914±0.075 and 0.932±0.031 at 96h, respectively. Comparative example 9, with the highest OD value among the comparative examples, had an OD value of only 0.729±0.022 at 96h, far lower than Example 1, and also lower than Examples 2 and 3, fully demonstrating the supporting advantage of the formulations in these examples for cell viability.

[0038] After replacing the fetal bovine serum in Example 1 with Comparative Example 1 (horse serum) and Comparative Example 2 (sheep serum), the 96h OD values ​​were 0.575±0.025 and 0.670±0.039, respectively, which were only 49.8% and 58.1% of those in Example 1. Moreover, the OD value of Comparative Example 1 stagnated from 72h to 96h, indicating that the fetal bovine serum selected in the examples was more suitable for cell growth.

[0039] Comparative Example 3 (without MCDB 131) and Comparative Example 4 (without DMEM) had their 96-hour OD values ​​removed, and were only 0.705±0.011 and 0.495±0.009, respectively, which were much lower than those in Example 1. This demonstrates that the 1:1 combination of DMEM and MCDB 131 in Example 1 is the basis for cell growth.

[0040] Component concentration: The OD values ​​of Comparative Examples 5 (10% fetal bovine serum) and 6-10 (adjusted additive concentration) were lower than those of Example 1. For example, the OD value of Comparative Example 10 at 24h was only 0.152±0.005, which was only 29.6% of that of Example 1. This shows that the component concentration ratio of Example 1 was accurate, and deviation from it would inhibit cell viability.

[0041] In summary, the examples demonstrated that by rationally selecting serum, combining it with basal culture medium, and precisely controlling the concentration of components, the high efficiency of human astrocytes SVGP12 was achieved, with results significantly superior to those of the comparative examples.

[0042] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A method for culturing human astrocytes, characterized by, The culture solution comprises DMEM and MCDB 131 mixed base medium, fetal bovine serum with a final concentration of 1% v / v, 5% v / v or 8% v / v, Glutamax with a final concentration of 0.1% v / v, 1% v / v or 2% v / v, N2 with a final concentration of 0.3% v / v, 1% v / v or 2.5% v / v, G-5 with a final concentration of 0.1% v / v, 1% v / v or 2% v / v, B-27 with a final concentration of 0.1% v / v, 1% v / v or 2.5% v / v, and hEGF with a final concentration of 2 ng / mL, 10 ng / mL or 18 ng / mL.

2. The culture method according to claim 1, characterized by, The culture solution further comprises a buffer and an antibiotic.

3. The culturing method according to claim 1, wherein, The buffer is selected from HEPES, MOPS, TES, DIPSO, POPSO or HEPPSO; The antibiotic is selected from penicillin-streptomycin, gentamicin, amphotericin B, ampicillin or tetracycline.

4. The culture method according to claim 3, characterized by, The buffer is HEPES; the antibiotic is penicillin-streptomycin.

5. The culture method according to claim 4, characterized by, The final concentration of the HEPES is 5-15 mM; the final concentration of the penicillin-streptomycin is 0.1%-2% v / v.

6. The culturing method according to claim 1, wherein The human astrocyte is SVGP12, U-87MG, U-118MG or U-251MG.

7. The culture method according to any one of claims 1 to 6, characterized by, The culture solution comprises DMEM and MCDB 131 mixed base medium with a volume ratio of 1:1, HEPES with a final concentration of 10 mM, fetal bovine serum with a final concentration of 5% v / v, Glutamax with a final concentration of 1% v / v, N2 with a final concentration of 1% v / v, G-5 with a final concentration of 1% v / v, B-27 with a final concentration of 1% v / v, hEGF with a final concentration of 10 ng / mL, and penicillin-streptomycin with a final concentration of 1%.

8. The culture method according to any one of claims 1 to 6, characterized by, The culture solution comprises DMEM and MCDB 131 mixed base medium with a volume ratio of 1:1, HEPES with a final concentration of 5 mM, fetal bovine serum with a final concentration of 1% v / v, Glutamax with a final concentration of 0.1% v / v, N2 with a final concentration of 0.3% v / v, G-5 with a final concentration of 0.1% v / v, B-27 with a final concentration of 0.1% v / v, hEGF with a final concentration of 2 ng / mL, and penicillin-streptomycin with a final concentration of 0.1%.

9. The culture method according to any one of claims 1 to 6, characterized by, The culture solution comprises DMEM and MCDB 131 mixed base medium with a volume ratio of 1:1, HEPES with a final concentration of 15 mM, fetal bovine serum with a final concentration of 8% v / v, Glutamax with a final concentration of 2% v / v, N2 with a final concentration of 2.5% v / v, G-5 with a final concentration of 2% v / v, B-27 with a final concentration of 2.5% v / v, hEGF with a final concentration of 18 ng / mL, and penicillin-streptomycin with a final concentration of 2%.

10. Use of the culture method according to any one of claims 1-9 in neurobiological research, mechanism exploration of neurodegenerative diseases or screening of nervous system drugs.

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