Smooth muscle cells were cultured from the medial layer of human aortic samples by enzymatic digestion. To prepare an isolated sample of the media, the endothelial layer of the aorta was removed by scraping the luminal surface gently with a scalpel. The tissue was washed in PBS, and then the adventitia and outer-most medial layers were peeled away with forceps. The remaining medial fragment was washed with PBS, and then used for the enzymatic digestion.
The enzymatic solution was a combination of 830 μl of M199 Media (ThermoFisher Scientific, 11150059; +0.5% FBS + 1% penicillin/streptomycin), 150 μl of Liberase™ (Roche, 05401020001) and 30 μl of DNAse. The prepared aortic media sample was cut into ∼1 × 1 cm fragments and added to a 1.5 ml Eppendorf tube containing the enzymatic solution. The sample was then placed in a SMC incubator (37°C, 5% CO2, 95% humidity) for 2 h. After the incubation, the digested supernatant was collected through a cell strainer and kept on ice until after the final incubation period. The undigested tissue was placed in a new Eppendorf tube with fresh enzymatic solution. The process was repeated for a second digestion, with an incubation period of 1.5 h. The supernatant from the second digestion was combined with that of the first, and the solution was then centrifuged at 750 RPM for 6 min at 4°C. The ensuing cell pellet was reconstituted in M199 media (+10% FBS + 1% penicillin/streptomycin), and the cells were plated on 0.4% gelatin-coated 60 mm cell culture dishes (Gelatin Type B Powder, Sigma-Aldrich, G9391; ThermoFisher Scientific Cell Culture Petri Dishes, 150340). The SMCs were maintained in the SMC incubator, as above.
The SMC media was changed 24 h after the isolation was completed, and then every 2 days until the cells reached confluence. When confluence was reached, the cells were trypsinized (Trypsin/EDTA solution, ThermoFisher Scientific, R001100) and plated on coverslips for immunocytochemistry (cell passage 1), or were plated on a fresh culture dish for continued growth and subsequent analysis of cellular senescence. For immunocytochemistry, serum was withdrawn from culture when the cells reached 80% confluence, and the cells were fixed with 4% paraformaldehyde after 72 h. For cellular senescence studies, the cells were re-platted each time confluence was reached until the cells stopped growing.
The enzymatic solution was a combination of 830 μl of M199 Media (ThermoFisher Scientific, 11150059; +0.5% FBS + 1% penicillin/streptomycin), 150 μl of Liberase™ (Roche, 05401020001) and 30 μl of DNAse. The prepared aortic media sample was cut into ∼1 × 1 cm fragments and added to a 1.5 ml Eppendorf tube containing the enzymatic solution. The sample was then placed in a SMC incubator (37°C, 5% CO2, 95% humidity) for 2 h. After the incubation, the digested supernatant was collected through a cell strainer and kept on ice until after the final incubation period. The undigested tissue was placed in a new Eppendorf tube with fresh enzymatic solution. The process was repeated for a second digestion, with an incubation period of 1.5 h. The supernatant from the second digestion was combined with that of the first, and the solution was then centrifuged at 750 RPM for 6 min at 4°C. The ensuing cell pellet was reconstituted in M199 media (+10% FBS + 1% penicillin/streptomycin), and the cells were plated on 0.4% gelatin-coated 60 mm cell culture dishes (Gelatin Type B Powder, Sigma-Aldrich, G9391; ThermoFisher Scientific Cell Culture Petri Dishes, 150340). The SMCs were maintained in the SMC incubator, as above.
The SMC media was changed 24 h after the isolation was completed, and then every 2 days until the cells reached confluence. When confluence was reached, the cells were trypsinized (Trypsin/EDTA solution, ThermoFisher Scientific, R001100) and plated on coverslips for immunocytochemistry (cell passage 1), or were plated on a fresh culture dish for continued growth and subsequent analysis of cellular senescence. For immunocytochemistry, serum was withdrawn from culture when the cells reached 80% confluence, and the cells were fixed with 4% paraformaldehyde after 72 h. For cellular senescence studies, the cells were re-platted each time confluence was reached until the cells stopped growing.
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