Under sterile conditions, anaesthetized SD rats were placed in the supine position and their chests were opened. The thoracic aorta was removed and transferred to a culture dish with cold (4°C) DMEM (Figure 1A ). After removal of the fat tissue around the artery (Figure 1B ), the artery was longitudinally cut and placed in another cell culture dish containing DMEM (Figure 1C ). Then, we used a pair of ophthalmic curved tweezers to scrape the intima softly to get rid of endothelial cells (Figure 1D ). Later, we did not separate the adventitia or directly cut the artery into small tissue blocks. Two pairs of ophthalmic curved tweezers were used: one to press the artery to fix it and another to separate the media from the artery by pressing and pushing the artery with its blunt back side (Figure 2 ). After half of the media was removed, the same method was used to get the other half. Then, the media was cut into approximately 1-mm squares and transferred into cell culture plates. The plates were placed in a cell culture chamber for about 4 h to let the small tissue blocks adhere to the plates. DMEM containing 20% FBS was carefully added and the tissue blocks were incubated in the cell culture chamber without disturbance for the first 5 days.
In the traditional tissue explants method, all the steps were similar, and after the fat tissue was removed, the artery was directly cut into small tissue blocks and transferred to cell culture plates without removal of the adventitia.
In the enzyme digestion method, after the fat tissue was removed, the aorta was digested with 1 mg/mL collagenase II and 100 μg/mL elastase at 37°C for 1 h. Later, the cells were pelleted and plated in DMEM with 20% FBS. The next morning, the cells were washed with PBS 3 times and the media were refreshed every 48 h.
The A7r5 cell line has been widely used in vitro to study the physiology and pathophysiology of VSMCs [13 (link),14 (link)]. However, it has lost some VSMCs selectivity and has many differences from primary cultured VSMCs. Therefore, we chose the A7r5 cell line to compare its viability with primary cultured VSMCs.
The VSMCs obtained by the above methods (the new tissue explants method, the traditional tissue explants method, the enzyme digestion method, and A7r5 cell line) were identified through morphology and immunofluorescence detection of SM-actin. The purity of the VSMCs was tested through multiple fluorescent staining with DAPI and SM-actin antibody.
In the traditional tissue explants method, all the steps were similar, and after the fat tissue was removed, the artery was directly cut into small tissue blocks and transferred to cell culture plates without removal of the adventitia.
In the enzyme digestion method, after the fat tissue was removed, the aorta was digested with 1 mg/mL collagenase II and 100 μg/mL elastase at 37°C for 1 h. Later, the cells were pelleted and plated in DMEM with 20% FBS. The next morning, the cells were washed with PBS 3 times and the media were refreshed every 48 h.
The A7r5 cell line has been widely used in vitro to study the physiology and pathophysiology of VSMCs [13 (link),14 (link)]. However, it has lost some VSMCs selectivity and has many differences from primary cultured VSMCs. Therefore, we chose the A7r5 cell line to compare its viability with primary cultured VSMCs.
The VSMCs obtained by the above methods (the new tissue explants method, the traditional tissue explants method, the enzyme digestion method, and A7r5 cell line) were identified through morphology and immunofluorescence detection of SM-actin. The purity of the VSMCs was tested through multiple fluorescent staining with DAPI and SM-actin antibody.
