AMCs were isolated from amnion membranes as previously described by Jin et al. (28) and Sato et al. (55) with slight modifications. Primary AMCs were isolated from amnion membranes of women experiencing normal parturient at term (not in labor) and undergoing a repeat elective cesarean section. Reflected amnion (∼10 g) was peeled from the chorion layer and rinsed three or four times in sterile Hanks’ balanced salt solution (HBSS) (catalog no. 21-021-CV, Corning) to remove blood debris. The sample was then incubated with 0.05% trypsin/EDTA (catalog no. 25-053-CI, Corning) for 1 hour at 37°C (water bath) to disperse the cells and remove the epithelial cell layer. The amnion membrane pieces were then washed three to four times using cold HBSS to inactivate the enzyme. The washed amnion membrane was transferred into a second digestion buffer containing Minimum Essential Medium Eagle (catalog no. 10-010-CV, Corning), collagenase type IV (1 mg/ml), and deoxyribonuclease I (25 μg/ml) and incubated in a rotator at 37°C for 1 hour. The digested amnion membrane solution was neutralized using DMEM/F12 media (catalog no. 16-405-CV, Corning), filtered using a 70-μm cell strainer, and centrifuged at 3000 rpm for 10 min. The cell pellet was resuspended in complete DMEM/F12 media supplemented with 5% heat-inactivated FBS (catalog no. 35-010-CV, Corning), penicillin G (100 U/ml), and streptomycin (100 mg/ml) (catalog no. 30-001-CI, Corning); plated at 3 million to 5 million cells per T75; and incubated at 37°C with 5% CO2 until they were 80 to 90% confluent.

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