Co-culture method of SVGP12 and HMEC-1 cells

Abstract

The invention belongs to the technical field of nerve cell culture, and particularly relates to a co-culture method of SVGP12 and HMEC-1 cells. The method comprises the following steps: firstly, preparing a co-culture medium, on the basis of mixing DMEM and MCDB 131 according to a volume ratio, adding HEPES, Glutamax, N2, G-5, B-27, hEGF, penicillin-streptomycin and fetal calf serum, respectively adopting products with corresponding goods numbers for each component, and controlling specific final concentrations; and then respectively inoculating SVGP12 and HMEC-1 into culture flasks, culturing by using the co-culture medium, and carrying out passage. The method can realize indirect communication of two cells in a shared microenvironment, provides a reliable tool for researching a regulation mechanism of SVGP12 on HMEC-1, and also provides a reference for optimization design of a co-culture system.

Description

Technical Field

[0001] This invention belongs to the field of neural cell culture technology, specifically relating to a method for co-culturing SVGP12 and HMEC-1 cells. Background Technology

[0002] In in vitro cell culture research, culture medium and culture methods are core elements in constructing the cell growth microenvironment. Their rationality directly determines the stability of cell growth status and the authenticity of functional activity, and is of irreplaceable significance for in-depth exploration of cell-cell interaction mechanisms. Culture medium provides cells with the basic nutrients required for survival and metabolism, and also carries signaling molecules secreted by cells. The ratio of its components (such as serum concentration and types of growth factors) must be precisely matched to the physiological needs of cells to maintain typical cell morphology and functional characteristics. Culture methods, on the other hand, simulate the complex cell community environment in vivo by regulating cell growth patterns (such as adhesion and spatial distribution) and interaction forms (such as direct contact or indirect communication), and are key to achieving consistency between in vitro experimental results and in vivo physiological states.

[0003] For human vascular endothelial cells HMEC-1 and human astrocytes SVGP12, they work together to maintain vascular homeostasis and central nervous system function through close "cross-talk" in vivo. HMEC-1 relies on trophic factors secreted by astrocytes to maintain barrier function and substance transport activity, while SVGP12 adjusts its supportive role through feedback signals from endothelial cells. However, existing technologies have significant limitations in simulating this interaction: at the culture medium level, traditional single-cell culture often uses specialized culture media, which can meet the basic growth needs of a single cell, but cannot adapt to the synergistic nutritional needs of two cell types in a co-culture scenario. Simply mixing different specialized culture media often leads to nutritional imbalance or antagonism of signaling molecules, thereby inhibiting normal cell proliferation and functional expression. At the culture method level, single-cell culture often results in poor cell growth (such as decreased HMEC-1 adhesion ability and abnormal morphology) and degeneration of functional activity (such as downregulation of metabolic enzyme and transport protein expression) due to the lack of signal stimulation from xenogeneic cells. Existing co-culture methods either make it difficult to analyze the target cells individually due to cell mixing or interfere with the cell's own characteristics due to direct contact, and neither can accurately simulate the indirect communication between cells in vivo through paracrine signaling.

[0004] These issues make it difficult for in vitro cultured cells to reproduce the physiological state in vivo, severely hindering in-depth research on the interaction mechanism between HMEC-1 and SVGP12. Therefore, it is necessary to construct a co-culture system that balances compatible culture media and reasonable culture methods. Summary of the Invention

[0005] To address the aforementioned shortcomings, this invention provides a Transwell co-culture model of SVGP12 and HMEC-1. By customizing the co-culture medium and combining it with the Transwell culture method, an in vitro model that can simulate the "cross-dialogue" between cells in vivo is constructed. This not only improves the growth status and functional activity of HMEC-1, but also provides a reliable tool for revealing the regulatory mechanism of astrocytes on vascular endothelial cells, and provides an important reference for the optimized design of co-culture systems.

[0006] The technical solution of this invention is as follows: On one hand, the present invention provides a method for co-culturing SVGP12 and HMEC-1 cells, comprising the following steps: (1) SVGP12 and HMEC-1 were inoculated into culture flasks and cultured in co-culture medium; (2) After passage culture, SVGP12 and HMEC-1 cell suspensions were obtained by digestion, centrifugation and resuspending, respectively; (3) Inoculate SVGP12 and HMEC-1 cell suspensions into cell culture plates for culture; The co-culture medium includes DMEM, MCDB 131, Glutamax, N2, G-5, B-27, hEGF, penicillin-streptomycin, and fetal bovine serum.

[0007] In some embodiments, the co-culture medium is formulated as follows: DMEM and MCDB 131 are mixed at a volume ratio of 1:1, the final concentration of HEPES is 10 mM, the final concentration of Glutamax is 1% v / v, the final concentration of N2 is 1% v / v, the final concentration of G-5 is 1% v / v, the final concentration of B-27 is 1% v / v, the final concentration of hEGF is 10 ng / mL, the final concentration of penicillin-streptomycin is 1% v / v, and the final concentration of fetal bovine serum is 5% v / v.

[0008] In other embodiments, the co-culture medium is formulated as follows: DMEM and MCDB 131 are mixed at a volume ratio of 1:1, with a final concentration of HEPES of 5 mM, Glutamax of 0.1% v / v, N2 of 0.3% v / v, G-5 of 0.1% v / v, B-27 of 0.1% v / v, hEGF of 2 ng / mL, penicillin-streptomycin of 0.1% v / v, and fetal bovine serum of 1% v / v.

[0009] In some other embodiments, the co-culture medium is formulated as follows: DMEM and MCDB 131 are mixed at a volume ratio of 1:1, HEPES has a final concentration of 15 mM, Glutamax has a final concentration of 2% v / v, N2 has a final concentration of 2.5% v / v, G-5 has a final concentration of 2% v / v, B-27 has a final concentration of 2.5% v / v, hEGF has a final concentration of 18 ng / mL, penicillin-streptomycin has a final concentration of 2% v / v, and fetal bovine serum has a final concentration of 8% v / v.

[0010] Specifically, the digestion in step (2) is pancreatic enzyme digestion for 1-5 min; the centrifugation conditions are 500-700 rpm for 2-4 min.

[0011] Preferably, the digestion in step (2) is pancreatic enzyme digestion for 3 minutes; the centrifugation conditions are 600 rpm for 3 minutes.

[0012] Specifically, the cell culture plate in step (3) can be a Transwell cell culture plate.

[0013] Preferably, in step (3), SVGP12 is seeded into the lower chamber of the Transwell cell culture plate, and HMEC-1 is seeded into the upper chamber of the Transwell cell culture plate.

[0014] Preferably, the ratio of the number of SVGP12 to the number of HMEC-1 cells in step (3) can be 2:1.

[0015] Preferably, the inoculation density of the SVGP12 is 1.0 × 10⁻⁶. 5 -2.0×10 5 One per hole, specifically 1.1 × 10 5 1.2×10 5 1.3×10 5 1.4×10 5 1.5×10 5 1.6×10 5 1.7×10 5 1.8×10 5 1.9×10 5 2.0×10 5 The inoculation density of HMEC-1 is 5.0 × 10⁻⁶, including any point value within the range and any range consisting of two point values. 4 -1.0×10 5 One hole per hole, specifically 5.1 × 10 4 5.2×10 4 5.3×10 4 5.4×10 4 5.5×104 5.6×10 4 5.7×10 4 5.8×10 4 5.9×10 4 6.0×10 4 6.1×10 4 6.2×10 4 6.3×10 4 6.4×10 4 6.5×10 4 6.6×10 4 6.7×10 4 6.8×10 4 6.9×10 4 7.0×10 4 7.1×10 4 7.2×10 4 7.3×10 4 7.4×10 4 7.5×10 4 7.6×10 4 7.7×10 4 7.8×10 4 7.9×10 4 8.0×10 4 8.1×10 4 8.2×10 4 8.3×10 4 8.4×10 4 8.5×10 4 8.6×10 4 8.7×10 4 8.8×10 4 8.9×10 4 9.0×10 4 9.1×10 4 9.2×10 4 9.3×10 4 9.4×10 4 9.5×10 4 9.6×10 4 9.7×10 4 9.8×10 4 9.9×10 4 1.0×10 5 , and the range value formed by any point value within the range and any two point values.

[0016] Specifically, the cultivation conditions described in step (3) can be 5% CO2 and 37°C.

[0017] The beneficial effects of this invention are as follows: (1) This co-culture method achieves indirect communication between two cell types in a shared microenvironment through reasonable cell seeding density, Transwell physical separation design, and the use of matching culture medium. This design avoids the problem of cell separation in mixed culture and eliminates the interference of direct contact on the cell's own characteristics. It can be used to analyze HMEC-1 functional changes in a targeted manner, providing an ideal experimental system for precise research on the "cross-dialogue" mechanism between cells.

[0018] (2) This method can stably reproduce the specific functional regulatory effect of SVGP12 on HMEC-1. The experimental results showed that after culturing with this method, the transport function (ABCG2 upregulation) and metabolic activity (CYP family enzyme upregulation) of HMEC-1 were significantly enhanced, while the permeability (Integrin alpha 5) did not change significantly. This result clearly reveals the functional specificity of the interaction between the two cells, providing a reliable experimental basis for elucidating the division of labor and regulatory mechanism of astrocytes on vascular endothelial cells. Moreover, the results have high reproducibility, ensuring the credibility of the research conclusions. Attached Figure Description

[0019] Figure 1 To illustrate the effect of co-culture on the HMEC-1 transporter ABCG2, where A represents the expression level of the cellular transport marker gene protein, and B represents the Western blot quantification results.

[0020] Figure 2 To illustrate the effect of co-culture on the HMEC-1 transporter ABCB1, where A represents the expression level of the cellular transport marker gene protein, and B represents the Western blot quantification results.

[0021] Figure 3 To illustrate the effect of co-culture on the HMEC-1 transporter CYP2D6, A represents the expression level of the cellular transport marker gene protein; B represents the Western blot quantification results.

[0022] Figure 4 To illustrate the effect of co-culture on the HMEC-1 transporter CYP1B1, A represents the expression level of the cellular transport marker gene protein; B represents the quantification results from Western blot.

[0023] Figure 5 To illustrate the effect of co-culture on the HMEC-1 transporter CYP2E1, A represents the expression level of the cellular transport marker gene protein; B represents the Western blot quantification results.

[0024] Figure 6To investigate the effect of co-culture on the expression level of HMEC-1 permeability protein Integrin alpha 5.

[0025] Figure 7 The results show the quantitative results of the expression level of HMEC-1 permeability protein Integrin alpha 5 in co-culture, where A is 115 kDa and B is 19 kDa. Detailed Implementation

[0026] 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.

[0027] Example 1: Construction of a Transwell co-culture model of SVGP12 and HMEC-1 1. Transwell cell co-culture The specific steps are as follows: (1) HMEC-1 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNHu46) and SVGP12 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHU258) were seeded into 75 cm cells respectively. 2 Add co-culture medium (Table 1 ratio) to the culture flask and culture. Culture and passage, and observe the growth status of the cells.

[0028] (2) Place them at 75cm respectively 2 After the culture flasks were filled to 80-90% of SVGP12 and HMEC-1, the culture medium was discarded and the cells were washed three times with PBS.

[0029] (3) Add 2 mL of 0.25% trypsin to digest for 3 min, add culture medium to neutralize the trypsin (ratio 1:1), transfer the cell suspension to a centrifuge tube, centrifuge at 600 rpm / min for 3 min and discard the supernatant.

[0030] (4) Add culture medium to resuspend the cells, and take 20 μL of cell suspension to measure the cell concentration.

[0031] (5) The diluted cell suspension was seeded into 6-well Transwell cell culture plates at the following densities: SVGP12 in the lower chamber was 2.0 × 10⁻⁶. 5 / hole, upper chamber HMEC-1 is 1.0×10 5 / well, maintaining a cell ratio of 1:1.

[0032] (6) Place it in a 5% CO2, 37℃ incubator and observe its morphology regularly.

[0033] Table 1 Co-culture medium

[0034] 2. Extraction of total protein from HMEC-1 in Transwell chambers The specific steps are as follows: (1) Cells were cultured in 6-well Transwell plates to extract total protein from the cells (SVGP12 in the lower chamber of the 6-well plate was 2.0 × 10⁻⁶). 5 / hole, upper chamber HMEC-1 is 1.0×10 5 Seed the cells at a 1:1 ratio in each well. Culture for 2 days until cells reach 80-90% confluence (then extract the proteins from the upper cell layer). Mix the high-efficiency RIPA tissue / cell rapid lysis buffer with PMSF solution at a 100:1 volume ratio in the dark. Add 200 μL of this mixture to each well and lyse on ice for 10 min, then collect the mixture.

[0035] (2) After centrifuging at 4℃ and 12000 rpm / min for 10 min, the supernatant was collected and dispensed into 10 μL portions for BCA protein concentration determination.

[0036] (3) After determining the protein concentration using the BCA kit (Beyotime, catalog number: P0010), the protein was denatured according to the calculated data (protein denaturation method: the protein sample was mixed with SDS-PAGE protein loading buffer (5×) at a ratio of 4:1, and the mixture was placed in boiling water for 10 min. SDS-PAGE protein loading buffer (5×) manufacturer: Beyotime, catalog number: P0015-2mL). This was used for Western blot experiments.

[0037] 3. Western blot detection The specific steps are as follows: (1) Add the sample. According to the experimental design, the sample is added. The marker volume is 5 μL and the total protein volume of the sample is 10 μg.

[0038] (2) Electrophoresis, 200V for 30 min is sufficient.

[0039] (3) Electrospinning: Use a scraper to cut the target gel strip to a suitable size and retain it. Make the filter paper slightly larger than the lower gel layer containing the target strip, and the PVDF membrane slightly larger than the filter paper. Then prepare a "sandwich" in a white porcelain dish containing the electrospinning solution. Apply 300 mA voltage for 45 min.

[0040] (4) Sealing: Take out the membrane after electrotransfer, mark the front and back, and place it in a petri dish containing TBST with 5% (m / v) milk powder. Seal it on a shaker for 2 h.

[0041] (5) Incubating the primary antibody: Gently pour out the milk powder, and wash the PVDF membrane after sealing on a shaker with TBST 3 times for 10 min each time. Prepare a hybridization bag of appropriate size, seal the membrane and the primary antibody diluted in proportion, and incubate overnight in a refrigerator at 4°C.

[0042] (6) Incubating the secondary antibody: Take out the membrane and wash it on a shaker with TBST for 10 min, 3 times. Dilute the secondary antibody (rabbit anti-abcam / Abiocare, catalog number: ab171870; mouse anti-abcam / Abiocare, catalog number: ab6789), and incubate on a shaker for 1 h. Wash it 3 times in the same way.

[0043] (7) Exposure: Evenly drip the luminol solution prepared in a 1:1 ratio on the PVDF membrane, and expose and save the image.

[0044] Effect Example 1: Effect of SVGP12 on the intercellular transport function of HMEC-1 cells Taking the single culture group of HMEC-1 as the control, the changes in the expression levels of the transport marker proteins ABCG2 and ABCB1 in HMEC-1 after co-culture were detected by Western blot. Manufacturer of the ABCG2 antibody: abcam / Abiocare, catalog number: ab229193, manufacturer of the secondary antibody (rabbit IgG, polyclonal antibody - isotype control (ChIP Grade)): abcam / Abiocare, catalog number: ab171870; manufacturer of the ABCB1 antibody: CST / Cellsignal, catalog number: 13342S, manufacturer of the secondary antibody (rabbit IgG, polyclonal antibody - isotype control (ChIP Grade)): abcam / Abiocare, catalog number: ab171870.

[0045] Detection of the expression levels of ABCG2 and ABCB1: For the protein band diagram obtained through the Western blot experiment, use software to calculate the gray value of the experimental results, and then convert the protein bands into specific values for comparison.

[0046] The results showed that compared with single culture, the expression level of ABCG2 was significantly up-regulated after co-culture (P < 0.05) ( Figure 1 ), and the expression level of ABCB1 was not significant (P > 0.05) ( Figure 2 ).

[0047] Effect Example 2: Effect of SVGP12 on the metabolic function of HMEC-1 cells Using the HMEC-1 monoculture group as a control, the changes in the expression levels of the metabolic marker proteins CYP2D6, CYP1B1, and CYP2E1 in HMEC-1 after co-culture were detected by Western blot. The manufacturer of the CYP2D6 antibody: abcam / Abeam, product number: ab185625, and the manufacturer of the secondary antibody (rabbit IgG, polyclonal antibody - isotype control (ChIP Grade)) is abcam / Abeam, product number: ab171870; the manufacturer of the CYP1B1 antibody: abcam / Abeam, product number: ab185954, and the manufacturer of the secondary antibody (Goat Anti-MouseIgG H&L (HRP)) is abcam / Abeam, product number: ab6789; the manufacturer of the CYP2E1 antibody: abcam / Abeam, product number: ab28146, and the manufacturer of the secondary antibody (rabbit IgG, polyclonal antibody - isotype control (ChIP Grade)) is abcam / Abeam, product number: ab171870.

[0048] Detection of the expression levels of CYP2D6, CYP1B1, and CYP2E1:: The protein band images obtained through the western blot experiment were used, and software was used to calculate the gray values of the experimental results, and then the protein bands were converted into specific values for comparison.

[0049] The results showed that compared with monoculture, the expression levels of CYP2D6, CYP1B1, and CYP2E1 were significantly upregulated after co-culture (P < 0.05) ( Figures 3-5 )

[0050] Effect Example 3 Effect of SVGP12 on the permeability of HMEC-1 cells Using the HMEC-1 monoculture group as a control, the changes in the expression level of the permeability marker protein Integrin alpha 5 in HMEC-1 after co-culture were detected by Western blot. The manufacturer of the Integrin alpha 5 antibody: abcam / Abeam, product number: ab150361, and the manufacturer of the secondary antibody (rabbit IgG, polyclonal antibody - isotype control (ChIP Grade)) is abcam / Abeam, product number: ab171870.

[0051] Detection of the expression level of Integrin alpha 5: The protein band images obtained through the western blot experiment were used, and software was used to calculate the gray values of the experimental results, and then the protein bands were converted into specific values for comparison.

[0052] The results showed that compared with monoculture, the expression level of Integrin alpha 5 was not significant after co-culture (P > 0.05) ( Figures 6-7 ).

[0053] In summary, the experimental results indicate that SVGP12 has a certain promoting effect on the transport and metabolic functions of HMEC-1.

[0054] Example 2-Example 3 The difference from Example 1 lies in the co-culture medium for microvascular endothelial cells and astrocytes, as shown in Table 2: Table 2. Co-culture media for Examples 2-3

[0055] Comparative Example A comparative example was set up with reference to Example 1. The differences between the comparative example and Example 1 are shown in Table 3. The rest is the same as Example 1.

[0056] Table 3

[0057] SVGP12 and HMEC-1 were cultured using the culture media of Examples 1-3 and Comparative Examples 1-11, respectively. HMEC-1 was cultured at a culture medium of 1.0 × 10⁻⁶. 4 Cells were seeded into 96-well plates at 2.0 × 10⁶ wells, with 6 replicates per well. Cell morphology was observed at 24 h, 48 h, 72 h, and 96 h, and cell viability was assessed using the CCK8 assay. 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.

[0058] 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.

[0059] (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.

[0060] (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.

[0061] The absorbance was measured at 450 nm using an ELISA reader, and the OD values ​​for each group were recorded.

[0062] The detection results of HMEC-1 are shown in Table 4: Table 4. Results of HMEC-1 cell viability assay

[0063] The detection results of SVGP12 are shown in Table 5: Table 5. Cell viability assay results of SVGP12

[0064] As shown in Tables 4 and 5, the culture medium formulations in the examples were significantly superior to those in the comparative examples in maintaining the viability of HMEC-1 and SVGP12 cells, with Example 1 being the best. In terms of composition, replacing fetal bovine serum with horse serum or sheep serum, or altering their concentrations, significantly impacted cell viability in the comparative examples, indicating that fetal bovine serum and its appropriate concentration are crucial for cell viability. The absence of DMEM or MCDB-131 resulted in a significant decrease in cell viability, demonstrating that a 1:1 mixture of these two basal media is essential for maintaining cell viability. Inappropriate substitutions of the concentrations of added components such as N2, G-5, B-27, Glutamax, and hEGF also weakened support for cell viability, while the concentrations of each component in the examples better ensured cell viability.

[0065] 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 co-culturing SVGP12 and HMEC-1 cells, characterized in that, Includes the following steps: (1) SVGP12 and HMEC-1 were inoculated into culture flasks and cultured in co-culture medium; (2) After passage culture, SVGP12 and HMEC-1 cell suspensions were obtained by digestion, centrifugation and resuspending, respectively; (3) Inoculate SVGP12 and HMEC-1 cell suspensions into cell culture plates for culture; The co-culture medium includes DMEM, MCDB 131, HEPES, Glutamax, N2, G-5, B-27, hEGF, penicillin-streptomycin, and fetal bovine serum.

2. The co-cultivation method according to claim 1, characterized in that, The co-culture medium was formulated as follows: DMEM and MCDB 131 were mixed at a volume ratio of 1:1, with a final concentration of HEPES of 10 mM, Glutamax of 1% v / v, N2 of 1% v / v, G-5 of 1% v / v, B-27 of 1% v / v, hEGF of 10 ng / mL, penicillin-streptomycin of 1% v / v, and fetal bovine serum of 5% v / v.

3. The co-cultivation method according to claim 1, characterized in that, The co-culture medium was formulated as follows: DMEM and MCDB 131 were mixed at a volume ratio of 1:1, with a final concentration of 5 mM for HEPES, 0.1% v / v for Glutamax, 0.3% v / v for N2, 0.1% v / v for G-5, 0.1% v / v for B-27, 2 ng / mL for hEGF, 0.1% v / v for penicillin-streptomycin, and 1% v / v for fetal bovine serum.

4. The co-cultivation method according to claim 1, characterized in that, The co-culture medium was formulated as follows: DMEM and MCDB 131 were mixed at a volume ratio of 1:1, with a final concentration of HEPES of 15 mM, Glutamax of 2% v / v, N2 of 2.5% v / v, G-5 of 2% v / v, B-27 of 2.5% v / v, hEGF of 18 ng / mL, penicillin-streptomycin of 2% v / v, and fetal bovine serum of 8% v / v.

5. The co-cultivation method according to claim 1, characterized in that, The digestion in step (2) is pancreatic enzyme digestion for 1-5 min; the centrifugation conditions are 500-700 rpm for 2-4 min.

6. The co-cultivation method according to claim 1, characterized in that, The cell culture plate mentioned in step (3) is a Transwell cell culture plate.

7. The co-cultivation method according to claim 6, characterized in that, In step (3), SVGP12 is seeded into the lower chamber of the Transwell cell culture plate, and HMEC-1 is seeded into the upper chamber of the Transwell cell culture plate.

8. The co-culture method according to claim 1, characterized in that, In step (3), the ratio of the number of SVGP12 cells to the number of HMEC-1 cells in the suspension is 2:

1.

9. The co-cultivation method according to claim 8, characterized in that, The inoculation density of the SVGP12 was 1.0 × 10⁻⁶. 5 -2.0×10 5 The inoculum density of HMEC-1 was 5.0 × 10⁶ cells / well. 4 -1.0×10 5 One hole / hole.

10. The co-cultivation method according to claim 1, characterized in that, The cultivation conditions described in step (3) are 5% CO2 and 37°C.

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