The rabbit aortic vascular smooth muscle cells (RAVSMCs) were cultured with DMEM/F12 (SH30023.01; HyClone) supplemented with 10% fetal bovine serum (FBS; 1767839; Thermo Fisher Scientific), and 1% penicillin-streptomycin (15140–122; Thermo Fisher Scientific).
Human normal aorta tissues used for primary culture VSMCs (HAVSMCs) were obtained from recipients who underwent heart transplantation at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Aortic tissues were divided into pieces of approximately 1.5mm2 and stored in DMEM/F12 with 1% penicillin-streptomycin at 4 °C. Then, tissues were transferred into a new petri dish after removal of blood stains and connective tissue. The intima and media structures were identified under a stereo microscope and stripped of the intimal and residual adventitial tissues with forceps. The dissected media of the vessels were then cut into small pieces (1–2 mm) and transferred to cell culture flasks. The tissue blocks were spread evenly on the bottom of the flask with a control interval of approximately 2 mm. Five milliliters of DMEM/F12 medium supplemented with 10% FBS, 1% L-glutamine, and antibiotics was added to the flask, and the lid was loosely screwed on. The flask was placed in the incubator and stood upright for 30 min to allow explant attachment to the wall of the culture flask. After 30 min, the culture bottle was then lowered. The culture bottle was not moved for 5 days. A long spindle-shaped smooth muscle cell was observed around the tissue block in approximately one week. After the cells grew, the medium was renewed every 3 days, and the state of the cells was closely observed. The smooth muscle cells around the tissue block were evenly distributed, and the cells were routinely passaged when the degree of cell confluence was approximately 80%. After starvation for 12 h, RAVSMCs and HAVSMCs were treated with tubastatin A (S8049, Selleck) at different concentrations (0, 1, 5, 10, 15, 20 μM) for 48 h.
Human normal aorta tissues used for primary culture VSMCs (HAVSMCs) were obtained from recipients who underwent heart transplantation at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Aortic tissues were divided into pieces of approximately 1.5mm2 and stored in DMEM/F12 with 1% penicillin-streptomycin at 4 °C. Then, tissues were transferred into a new petri dish after removal of blood stains and connective tissue. The intima and media structures were identified under a stereo microscope and stripped of the intimal and residual adventitial tissues with forceps. The dissected media of the vessels were then cut into small pieces (1–2 mm) and transferred to cell culture flasks. The tissue blocks were spread evenly on the bottom of the flask with a control interval of approximately 2 mm. Five milliliters of DMEM/F12 medium supplemented with 10% FBS, 1% L-glutamine, and antibiotics was added to the flask, and the lid was loosely screwed on. The flask was placed in the incubator and stood upright for 30 min to allow explant attachment to the wall of the culture flask. After 30 min, the culture bottle was then lowered. The culture bottle was not moved for 5 days. A long spindle-shaped smooth muscle cell was observed around the tissue block in approximately one week. After the cells grew, the medium was renewed every 3 days, and the state of the cells was closely observed. The smooth muscle cells around the tissue block were evenly distributed, and the cells were routinely passaged when the degree of cell confluence was approximately 80%. After starvation for 12 h, RAVSMCs and HAVSMCs were treated with tubastatin A (S8049, Selleck) at different concentrations (0, 1, 5, 10, 15, 20 μM) for 48 h.
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