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The remaining medial layer was cut into small pieces, placed in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, 10 mmol/L sodium pyruvate, and 20% FBS supplemented with 100 U/mL of penicillin and 100 g/mL of streptomycin, and incubated at 37 C in a humidified atmosphere containing 5% CO 2