Isolation and Cultivation of Vascular Smooth Muscle Cells from the Mouse Circle of Willis

Cerebrovascular diseases are among the most prevalent diseases worldwide. Mechanistic research has demonstrated that cerebrovascular smooth muscle cells (CVSMCs) play an important role in various cerebrovascular diseases [1]. Pathophysiological alterations of cerebrovascular smooth muscle are associated with the development of several cerebrovascular disorders, such as stroke, cerebral edema, Alzheimer’s disease, and atherosclerosis [2]. Furthermore, CVSMCs are key for maintaining and regulating cerebral perfusion. In addition, gap junctions that connect smooth muscle cells are also involved in the transmission of vascular biochemical signals [3]. Smooth muscle cells are essential in regulating cerebrovascular tone, maintaining vascular integrity, and building brain homeostasis under physiological conditions [4‒6]. For example, cerebral atherosclerosis and vascular dysfunction in persistent hypertension lead to abnormal vascular congestion and obvious self-regulation functional abnormalities [7]. In stroke patients, alpha-smooth muscle actin (α-SMA) expression in vascular smooth muscle and the density of cerebral microvessels decrease, suggesting that the stroke process involves postischemic alterations in the cerebral vascular structure. Numerous studies have shown a cause-and-effect relationship between cerebrovascular inflammation and stroke [8, 9]. Specifically, lacunar strokes are thought to result from cerebral vascular lesions [10]. However, during phenotypic modulation, vascular smooth muscle cells (VSMCs) may cause a series of events that promote vascular inflammatory processes [9]. For instance, cerebrovascular injuries can induce severe vasogenic edema [11, 12], and cerebral herniation can be secondary to fluid-induced brain tissue compression [13]. VSMC hyperactivation can also accelerate vascular aging and atherosclerosis [14]. Furthermore, in patients with diabetes, glycosylation products are significantly upregulated in VSMCs, leading to increased microcirculation leakage in the patient’s brain [15].

In vitro culture of CVSMCs is a promising model for studying cerebrovascular disease mechanisms and selecting relevant therapeutic drugs. However, the cerebral vasculature of mice is more fragile than that of rats or their own aortas, making mouse CVSMC isolation very difficult. Therefore, in vitro studies of knockout mice are difficult owing to the technical challenges of primary cell isolation and cultivation. Moreover, most reports of isolated mouse CVSMCs have been limited to primary cultures, and few have reported passaging cells. Therefore, this study describes a simple and reproducible method to conveniently and efficiently obtain CVSMCs from mice and maintain their activity during the passaging process.

All experiments were performed using 5- to 6-week-old male C57BL/6J mice purchased from the animal experimental center of the Air Force Medical University, China. All animals with license lot number SCXK (2017-0021) were considered satisfactory for the experiment.

All animal feeding and experimental procedures followed the guidelines of the Institutional Ethics Review Board of the Air Force Medical University.

6-week-old healthy male C57BL/6J mice were euthanized using an overdose of 1.5% sodium pentobarbital administered via intraperitoneal injection, and the mouse bodies were immersed in 75% ethanol for 3–5 min to disinfect. The mice were sacrificed by cervical dislocation, and then the brains were quickly removed and placed in ice-cold Dulbecco’s phosphate-buffered saline (DPBS; Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) containing 1% penicillin-streptomycin (Gibco™; #15140163). Cerebral arteries (including the circle of Willis and its branches) were rapidly removed under a microscope (Olympus Corporation, Tokyo, Japan; #SZX7) and kept under sterile conditions (shown in Fig. 1). Isolated vessels were rinsed two to three times with DPBS. The vessels were then transferred into centrifuge tubes with collagenase type II solution (1 mg/mL dissolved in DPBS; Sigma-Aldrich, St. Louis, MO, USA) and mixed thoroughly. Next, the centrifuge tubes with the vessels were incubated in a 37°C shaker (80 times/min) for 15 min, after which the arteries were digested into flocculants. Dulbecco’s modified Eagle medium/F-12 (DMEM/F12; Gibco™; #A4192001) with 10% fetal bovine serum (FBS; Biological Industries, Cromwell, CT, USA; #04-001-1ACS) was added to the centrifuge tubes to terminate the enzymatic reaction, mixed, and then centrifuged (1,500 rpm, 5 min). After discarding the supernatant, 2 mL of DMEM/F12 containing 20% FBS and 1% penicillin-streptomycin was added to the centrifuge tube and mixed thoroughly. The above mixture was transferred to culture dishes (pretreated with 0.1% gelatin) and incubated at 37°C in a 5% carbon dioxide (CO₂) incubator. We verified whether the tissues and cells had adhered to the wall 48–72 h later. If we found a large amount of unadhered tissues and cells, the culture medium in the dishes were gently transferred to a centrifuge tube for centrifugation (1,000 rpm, 5 min), and the cell debris at the bottom of the dishes were gently washed twice with DPBS (shown in Fig. 2). After centrifugation, 3 mL of DMEM/F12 containing 20% FBS and 1% penicillin-streptomycin was added to the centrifuge tube to resuspend the tissues, which were then transferred to the original culture dishes to continue cultivation. After another 48 h, the culture was rechecked, and the above steps were repeated if there were still unattached cell tissues. Every 48 h after that, the cell growth was checked, and the medium was changed.

Previous studies showed that adding 20 ng/mL platelet-derived growth factor-BB (PDGF-BB; PeproTech; Rocky Hill, NJ, USA; #315-18) promotes VSMC proliferation [16]. Therefore, we also cultured cells with 20 ng/mL of PDGF-BB in the medium.

The cells were passaged when the cell growth density reached 80–90%. The cells were washed twice with DPBS and digested with 0.25% trypsin (Beyotime Biotechnology, Shanghai, China; #C0208-100 mL). The digestion was terminated immediately when a large part of the cells detached (observed under a microscope), which occurred after approximately 2 min at room temperature (RT). The collected cell suspension was centrifuged at 1,000 rpm for 5 min, and then the supernatant was discarded. The cells in the centrifuge tube were mixed with fresh medium and transferred to two new dishes for further culture.

The diameter of cerebral vessels in mice is small (0.20 ± 0.05 mm) and difficult to isolate; thus, the intima and fibrous outer layer are difficult to remove. Therefore, during cell passaging, the cells can be purified based on the digestion times of the fibroblasts and CVSMCs [17]. Fibroblasts detach from the wall before CVSMCs. Therefore, we microscopically observed the cells during the digestion step of passaging and discarded the cells digested after the first 20–30 s, which were mainly fibroblasts. Then, fresh trypsin was added to complete the passaging process.

There are more CVSMCs than fibroblasts and endothelial cells. Therefore, CVSMCs are proliferatively superior during culturing, but this can inhibit the proliferation of other cells. Moreover, CVSMC purification gradually improves as the number of cells increases. PDGF-BB stimulates VSMC proliferation; therefore, we added PDGF-BB to the cerebral artery CVSMC cultivation since it may enhance smooth muscle cell (SMC) proliferation (shown in Fig. 2) [18].

The cells’ shape, size, and growth pattern were observed under a microscope (Olympus Corporation; #CKX53).

The purified VSMCs (second passage after purification) were uniformly plated in laser confocal Petri dishes during the logarithmic growth phase. After 1 day, the medium was discarded. After fixation with 4% paraformaldehyde (diluted in PBS; Beyotime; #P0099-500 mL) for 20 min, the stimulated VSMCs were permeabilized with 0.1% Triton X-100 (Beyotime; #ST797-100 mL) in phosphate-buffered saline (PBS; Beyotime; #C0221A) for 10 min. Cells were blocked with 5% bovine serum albumin (#9048-46-8; Sigma-Aldrich) for 1 h at RT. Anti-α-SMA (Abcam; #ab5694), Anti-smooth muscle myosin heavy chain 11 (SMMHC, ProteinTech; #60222-1-Ig), and Anti-VE cadherin (Abcam; #ab205336) antibodies were added and incubated at 4°C overnight. On the second day, the cells were rewarmed for 30 min at RT and then washed 3 times (5 min each) with PBS. Goat anti-rabbit (ProteinTech; #SA00003-2) or goat anti-mouse (ProteinTech; #SA00013-1) secondary antibodies were added in a dark environment, and the cells were incubated at RT for 1 h. The cells were washed 3 times (5 min each) with PBS, and the nuclei were stained with 4′,6-Diamidino-2-phenylindole (Beyotime; #C1006) following the manufacturer’s instructions. The results were assessed by confocal microscopy (Zeiss, Oberkochen, Germany; #LSM 900).

When the second passage (P2) VSMCs were between 80 and 90% confluent, they were digested, counted, and then inoculated in 6-well plates at 5 × 10⁶ cells/ml and incubated in a 5% CO₂ incubator for 24 h. The first set of cells was digested with 0.25% trypsin (without ethylenediaminetetraacetic acid; Beyotime; #C0205) and collected. We used the Annexin V-FITC/Pi Cell Apoptosis Detection Kit (following the manufacturer’s instructions) and flow cytometry (BD FACSAria ™ III; Becton Dickinson, Franklin Lakes, NJ, USA) to analyze VSMC apoptosis (Servicebio, Wuhan, China; #G1511-50T).

The P2 primary CVSMCs were collected and washed twice with PBS, centrifuged at 1,000 rpm for 5 min, then resuspended in PBS. Anti-α-SMA (ProteinTech; #14395-1-AP), anti-smooth muscle protein 22 (SM22; ProteinTech; #60213-1-Ig), anti-calponin 1 (Bioss Antibodies Inc., Woburn, MA, USA; #bs-0095R), and anti-desmin (ProteinTech; #CL488-16520) antibodies were added and processed following the manufacturer’s instructions. Finally, the cells were examined using flow cytometry. Each experiment was repeated 3 times.

One thousand well-grown VSMCs were inoculated per well in 96-well plates and incubated at 37°C with 5% CO₂. The experimental group was incubated with 20 ng/mL PDGF-BB per well; the control group was incubated without PDGF-BB. At the same time, the same volume of PBS was used as the blank control group. All groups were subdivided based on the culture time into 1-, 2-, 3-, 4-, 5-, 6-, and 7-day groups. Each time group had six replicates. Two hours before the maximum incubation time (e.g., at hour 22 of 24), 10 μL of cell counting kit-8 solution (Dojindo, Kumamoto, Japan; #CK04) was added to each well, and the absorbance was measured at 450 nm using a multifunctional enzyme assay detector (BioTek, Winooski, VA, USA; #Synergy H1). In order to normalize the data, the final calculated data were obtained by subtracting the absorbance values of the blank control group from the absorbance values of the experimental group and the control group, respectively. Then, the cell proliferation rate was calculated and plotted using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA, www.graphpad.com).

Two passage of primary CVSMCs were collected for protein extraction, each sample was separated by electrophoresis, and then the proteins were transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA; #IPVH00010). The membranes were blocked for 1 h in tris-buffered saline with Tween (TBST) containing 50 g/L skim milk (pH 8.0), 10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.2% Tween-20. Then, various primary antibodies were added and incubated at 4°C overnight. The next day, the membranes were washed 3 times (10 min each) with TBST, and the secondary antibodies were added for 2 h at RT. After, the membranes were washed 3 times (10 min each) with TBST, and an enhanced chemiluminescence reagent (Beyotime; #P0018AM) was added to the membrane for visualization on X-ray film. The optical density of each target protein band was assessed using Fusion FX. EDGE (Vilber Bio Imaging, Paris, France, www.vilber.com) and normalized to the density of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) band in the same sample. The following primary antibodies were used: anti-α-SMA (ProteinTech; #14395-1-AP), anti-SM22 (ProteinTech; #60213-1-Ig), anti-osteopontin (OPN; Abcam, Cambridge, UK; #ab283656), anti-proliferating cell nuclear antigen (PCNA; ProteinTech; #60097-1-Ig), anti-cyclin D1 (ProteinTech; #60186-1-Ig), anti-p27 (ProteinTech; #67355-1-Ig), mouse anti-GAPDH (ProteinTech; #60004-1-Ig), and rabbit anti-GAPDH (ProteinTech; #10494-1-AP).

Every ten animals were used to isolate/culture primary CVSMCs, and all experiments were repeated 3 times. All data were normally distributed and presented as means ± standard errors. Between-group differences were assessed by one-way analysis of variance and t tests. A p value of <0.05 indicated statistical significance.

On the first day, only a few pieces of cerebrovascular fragments and cells were attached to the culture dish. After 3 days, a few cells had released from the tissue’s edge. After 7 days, the cells grew in parallel in the culture dish in several shapes and sizes, such as fusiform and ribbon. The cytoplasm and nuclei of the cells also varied. At this stage, the cells tended to be arranged in a net-like array. A typical peak-to-valley growth pattern was observed after approximately 14 days (shown in Fig. 2).

The second passage cells were cultured and immunofluorescently stained with VSMC-specific marker proteins (α-SMA and SMMHC) and endothelial cell-specific marker (VE-cadherin) proteins. In addition, the nuclei were counterstained with 4′, 6-Diamidino-2-phenylindole, and the images were merged.

Finally, the cells were identified by flow cytometry, and 97.4 ± 1.8% of VSMCs stained positively for α-SMA and SM22, 97.7 ± 2.0% of VSMCs stained positively for calponin and desmin. These are representative results to show the characterization of the VSMC culture and its purity (shown in Figure 3). It is worth noting that although the results in Figure 3 were obtained from P2 CVSMCs, the fluorescence results of P4 CVSMCs were almost the same as those of P2 (shown in online suppl. Data 3; for all online suppl. material, see https://doi.org/10.1159/000532033). This means that the cell phenotype of early passages (P2–P4) are stable.

PDGF-BB promotes VSMC proliferation; thus, we examined how adding PDGF-BB during cell culture affected VSMC dedifferentiation and the cell culture process. PDGF-BB increased VSMC proliferation and decreased the time required to reach the logarithmic growth phase. Also, OPN, α-SMA, and SM22 expression changed, indicating that PDGF-BB induced a phenotypic alteration of VSMCs from differentiation to dedifferentiation. Moreover, cell proliferation markers, such as PCNA, cyclin D1, and p27, are associated with VSMC proliferation and migration. We found markedly upregulated PCNA and cyclin D1 protein levels and decreased p27 protein expression in VSMCs incubated with PDGF-BB (shown in Fig. 4).

The VSMC apoptosis rate per group was detected by flow cytometry. The apoptosis rate of cells cultivated with and without PDGF-BB was 4.0 ± 0.2% and 3.7 ± 0.4%, respectively (shown in Fig. 5); the apoptosis rate did not differ between the groups (p > 0.05).

This study details the CVSMC isolation, primary cell cultivation, purification, and identification from the mouse circle of Willis. Previous reports have described CVSMC isolation, but they were limited to primary culture. Moreover, the reagents they used for cell isolation were complex and difficult to prepare. We compared the new method in this paper with several reported CVSMC isolation and culture methods. Obviously, our method is simpler and cheaper while ensuring cell purity, and the experimental steps in this article are described in detail (shown in Table 1). Therefore, to more conveniently and efficiently isolate and culture mouse CVSMCs, we improved the enzyme digestion time and conditions based on the traditional enzyme digestion method and isolated CVSMCs from the circle of Willis using only a single enzyme. This method is simple, reproducible, and can maintain cell activity during the passaging process. In addition, we increased the cell growth rate by adding PDGF-BB, a VSMC stimulator, to the culture environment, increasing the primary cell culture efficiency.

In our laboratory studies, SMCs were isolated from the explants’ edge after approximately 3 days and reached confluence after approximately 14 days. Furthermore, we obtained highly pure VSMCs during the second-generation purification process and identified a typical peak-to-valley growth pattern. Moreover, flow cytometry analyses demonstrated that >95% of cell-specific markers were positively expressed. Immunofluorescence results also confirmed that specific marker proteins were positively expressed. We also found that the phenotype of CVSMCs in the early passages (P2–P4) were stable and could be used for experiments, but the number of passage times and the stability of cell phenotype might be related to the conditions of different laboratories.

We attempted to shorten the cell culture cycle by adding 20 ng/mL of PDGF-BB to the cell culture medium, which greatly enhanced the cell growth rate. Cells cultivated with PDGF-BB reached confluence after 7–10 days and displayed a typical peak-to-valley growth pattern. Compared with the control group, contractile protein expression, such as α-SMA and SM22, decreased in VSMCs after adding PDGF-BB, but OPN expression (a synthetic protein) significantly increased [23]. In VSMCs, PCNA and cyclin D1 are pro-proliferation genes, whereas p27 has an anti-proliferation role [24‒26]. In this study, PCNA and cyclin D1 expression significantly increased in cells incubated with PDGF-BB, whereas p27 expression markedly decreased. These results suggest that PDGF-BB promoted cell proliferation and migration by altering the cytoarchitecture of the VSMCs. Notably, increased PDGF-BB expression indicates tumor angiogenesis in many tumors [27]. In addition, PDGF-BB is also a primary mediator for wound healing and tissue repair [28]. Therefore, if future studies incubate cells with PDGF-BB, its effect on cell structure should be considered.

Minimizing contamination with brain parenchyma and quickly isolating vessels during mouse cerebrovascular isolation is crucial to ensure cell viability. The efficiency of the vessel isolation primarily determines the number of CVSMCs; if it takes too long, fewer primary cells will be obtained in the subsequent cultivation. Therefore, we recommended no more than 10 min between sacrificing the mouse and isolating the blood vessels.

Finally, experimental conditions likely differ among laboratories. Therefore, the optimal enzymatic digestion time should be further explored based on various laboratory conditions. The digestion time is determined based on the degree to which the blood vessels are digested. In this study, blood vessel digestion was observed every 5 min, and the digestion was terminated when the blood vessels were flocculent or ruptured.

We obtained highly purified SMCs from the circle of Willis of mice and improved the experimental efficiency by including PDGF-BB, which shortened the cell cultivation period. These methods may provide ideal experimental material for studying underlying pathophysiological changes in neurovascular system diseases in vitro.

All animal feeding and experimental procedures followed the guidelines of the Institutional Ethics Review Board of the Air Force Medical University.

The authors have no conflicts of interest to declare.

This study was supported by the Shaanxi Provincial Key R & D Plan General Project (2021SF-146, 2022SF-246, 2021SF-253).

Junhui Xue, Wei Chang, Fengzhou Liu, and Yajuan Li were responsible for the conception and design of the research. Wei Chang, Yajuan Li, Peiran Zhang, and Shuai Qu performed the experiments. Wei Chang, Fengzhou Liu, Yajuan Li, Kehai Zang, and Shuai Qu analyzed the data and prepared the figures. Wei Chang and Jingyu Zhao drafted the manuscript. All authors listed contributed to the manuscript and approved the final version.

Wei Chang and Yajuan Li contributed equally to this work and should be considered co-first authors.

All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.