Microvascular endothelial cell culture method and application thereof

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a microvascular endothelial cell culture method and application thereof. A culture solution used in the culture method comprises a basic culture medium, fetal calf serum, HEPES, Glutamax, N2, G-5, B-27, hEGF and antibiotics, wherein the basic culture medium is formed by mixing DMEM (Dulbecco Modified Eagle Medium) and MCDB 131 in a ratio of 1: 1. According to the culture solution, by optimizing the components and the proportion thereof, the in-vitro proliferation rate of HMEC-1 cells can be remarkably promoted, and the high-activity state and normal functional characteristics of the cells in the long-term culture process are effectively maintained; the technical problems that the cell proliferation efficiency is low and the cell activity and function are difficult to maintain in a long-term culture state in an existing culture method are solved. The culture system provided by the invention is clear in component and simple and convenient to operate, and a stable and reliable technical platform is provided for related research of the microvascular endothelial cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell culture, and particularly relates to a microvascular endothelial cell culture method and application thereof. BACKGROUND

[0002] As an important component of the vascular system, microvascular endothelial cells are widely distributed in various tissues and organs throughout the body, not only constituting a physical barrier between blood and tissue, but also being the core unit involved in regulating key physiological and pathological processes such as vasomotion, inflammatory response, immune response, and neovascularization. Among numerous microvascular endothelial cell lines, HMEC-1 (human microvascular endothelial cell line 1) has become a classic model system for in vitro research on microvascular function due to its immortalization characteristics and retention of most of the biological characteristics of primary endothelial cells.

[0003] The HMEC-1 cell line is derived from human dermal microvascular endothelial cells and is immortalized by introducing SV40 large T antigen. It can stably express various endothelial cell markers such as CD31 and von Willebrand factor (vWF) in in vitro culture, and maintain the response ability to inflammatory stimuli and growth factors. These characteristics make it widely used in the fields of vascular biology research, inflammation mechanism exploration, pathogen-vascular interaction research, and drug screening.

[0004] However, the in vitro culture of HMEC-1 cells still faces significant challenges. The existing culture methods mostly use traditional endothelial cell culture medium formulations, but these formulations often cannot fully meet the specific growth needs of HMEC-1 cells. The main performance is that the cell proliferation rate is limited, it is difficult to obtain a sufficient number of cells for experiments in a short time; at the same time, in the long-term culture process, problems such as cell viability decline, morphological change and loss of functional characteristics easily occur. Especially after continuous passage, the cells are prone to premature aging, affecting the reliability and repeatability of experimental results.

[0005] In addition, the existing culture system is insufficient in maintaining the functional characteristics of HMEC-1 cells, resulting in unstable performance in research requiring highly functional cells such as angiogenesis assay and leukocyte adhesion experiment. This limitation seriously restricts the application value of the HMEC-1 cell model in fine research.

[0006] Therefore, developing a special culture method that can significantly improve the in vitro proliferation efficiency of HMEC-1 cells while effectively maintaining their cell viability, morphological characteristics and functional integrity has become a key technical problem urgently to be solved in the field. Such a method will greatly promote the in-depth development of microvascular-related research and provide a more reliable technical platform for the research fields of cardiovascular diseases, inflammatory diseases and tumor angiogenesis. SUMMARY

[0007] In view of the above, the present application provides a culture method for HMEC-1 cells and application thereof. The method establishes a stable and efficient culture system by optimizing the components and their proportions of the culture medium, which can significantly promote the in vitro proliferation of HMEC-1 cells, and effectively maintain the high activity state and normal functional characteristics of the cells during long-term culture.

[0008] The technical scheme of the present application is as follows: In one aspect, the present application provides a method for culturing microvascular endothelial cells, comprising the step of culturing microvascular endothelial cells using a culture medium, wherein the culture medium comprises the following components: a mixed basal medium of DMEM and MCDB 131; 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; hEGF with a final concentration of 2 ng / mL, 10 ng / mL or 18 ng / mL.

[0009] Specifically, the culture medium further comprises HEPES.

[0010] Preferably, the final concentration of HEPES can be 5-15 mM.

[0011] Preferably, the final concentration of HEPES can be 5 mM, 10 mM or 15 mM.

[0012] Specifically, the culture medium further comprises antibiotics.

[0013] Preferably, the antibiotics can be penicillin-streptomycin.

[0014] Preferably, the final concentration of penicillin-streptomycin can be 0.1%-2% v / v.

[0015] Preferably, the final concentration of penicillin-streptomycin can be 0.1% v / v, 1% v / v or 2% v / v.

[0016] Specifically, the microvascular endothelial cells can be HMEC-1.

[0017] In particular, in some embodiments, the culture medium consists of: DMEM mixed 1 : 1 with MCDB 131 ; 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 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; Penicillin-streptomycin at a final concentration of 1 %.

[0018] In particular, in some other embodiments, the culture medium consists of: DMEM mixed 1 : 1 with MCDB 131 ; 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; Penicillin-streptomycin at a final concentration of 0.1 %.

[0019] In particular, in yet other embodiments, the culture medium consists of: DMEM mixed 1 : 1 with MCDB 131 ; 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; Penicillin-streptomycin at a final concentration of 2%.

[0020] In another aspect, the present invention provides the application of the culture medium in the aforementioned method in the preparation of microvascular endothelial cell culture kits.

[0021] Specifically, the microvascular endothelial cells may be HMEC-1.

[0022] The beneficial effects of this invention are as follows: The microvascular endothelial cells cultured according to the formulation provided in this embodiment of the invention, The combination of the basic culture medium and the ratio of key additives provided by this invention form a complete and balanced culture system. Experiments have shown that it exhibits excellent proliferation capacity and cell viability throughout the entire culture cycle. Its proliferation rate is significantly better than conventional culture conditions and other comparative formulations, enabling the acquisition of a larger number of healthy cells in a shorter time, greatly improving experimental efficiency. The components in this formulation produce a crucial synergistic effect; any deviation from the type or concentration of a single component will lead to a significant decrease in culture results, thus establishing the uniqueness and irreplaceability of the formulation of this invention as an optimized whole. 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 HMEC-1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu46) 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 morphology was observed at 24h, 48h, 72h and 96h, and cell viability was detected by 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] As shown in Table 5, Example 1 exhibited the highest OD values ​​at all time points (24h, 48h, 72h, and 96h), indicating the strongest cell viability and fastest proliferation rate, especially reaching a peak of 1.962 at 72h, demonstrating that this culture medium formulation is most suitable for the rapid proliferation and maintenance of HMEC-1 cells. The OD values ​​of Example 2 were lower than those of Example 1 at all time points, but still better than most comparative examples, indicating that its culture conditions were acceptable. The OD value of Example 3 was the lowest among the examples, but still significantly higher than many comparative examples at 72h and 96h.

[0038] The OD values ​​of Comparative Example 1 (horse serum) and Comparative Example 2 (sheep serum) were both lower than those of Example 1, indicating that fetal bovine serum had the best effect on promoting the proliferation of HMEC-1 cells. The OD value of Comparative Example 5 (10% fetal bovine serum) was also lower than that of Example 1 (5%), indicating that excessively high serum concentration (10%) inhibited cell growth, and 5% may be a better choice.

[0039] The OD values ​​of Comparative Example 3 (without MCDB 131) and Comparative Example 4 (without DMEM) decreased significantly, indicating that a 1:1 mixture of DMEM and MCDB131 is necessary, and the absence of either will seriously affect cell growth.

[0040] In Comparative Examples 6–10, any deviation from the concentrations of components such as N2, G-5, B-27, Glutamax, and hEGF resulted in a decrease in OD values, indicating that the concentration ratio of these components in Example 1 was optimal, and any adjustment, whether single or combined, would affect cell viability.

[0041] 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 microvascular endothelial cells, characterized in that, The step includes culturing microvascular endothelial cells using a culture medium containing the following components: A mixed basal medium of DMEM and MCDB 131; Fetal bovine serum at final concentrations of 1% v / v, 5% v / v, or 8% v / v; Glutamax at final concentrations of 0.1% v / v, 1% v / v, or 2% v / v; N2 at final concentrations of 0.3% v / v, 1% v / v, or 2.5% v / v; G-5 at final concentrations of 0.1% v / v, 1% v / v, or 2% v / v; B-27 at final concentrations of 0.1% v / v, 1% v / v, or 2.5% v / v; hEGF at final concentrations of 2 ng / mL, 10 ng / mL, or 18 ng / mL.

2. The method according to claim 1, characterized in that, The culture medium also includes HEPES.

3. The method according to claim 2, characterized in that, The final concentration of HEPES is 5-15 mM.

4. The method according to claim 4, characterized in that, The culture medium also includes penicillin-streptomycin.

5. The method according to claim 4, characterized in that, The final concentration of the penicillin-streptomycin is 0.1%-2% v / v.

6. The method according to claim 1, characterized in that, The microvascular endothelial cells were HMEC-1.

7. The method according to any one of claims 1-6, characterized in that, The culture medium has the following components: A basal medium consisting of a 1:1 volume ratio of DMEM and MCDB 131; HEPES with a final concentration of 10 mM; Fetal bovine serum with a final concentration of 5% v / v; Glutamax at a final concentration of 1% v / v; N2 with 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; Penicillin-streptomycin with a final concentration of 1%.

8. The method according to any one of claims 1-6, characterized in that, The culture medium has the following components: A basal medium consisting of a 1:1 volume ratio of DMEM and MCDB 131; HEPES at 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; The final concentration of hEGF was 2 ng / mL; Penicillin-streptomycin with a final concentration of 0.1%.

9. The method according to any one of claims 1-6, characterized in that, The culture medium has the following components: A basal medium consisting of a 1:1 volume ratio of DMEM and MCDB 131; HEPES with a final concentration of 15 mM; Fetal bovine serum with a final concentration of 8% v / v; Glutamax at a final concentration of 2% v / v; N2 with 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; Penicillin-streptomycin with a final concentration of 2%.

10. The use of the culture medium in the method of any one of claims 1-9 in the preparation of a microvascular endothelial cell culture kit.

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