2.1. Amniotic-Fluid-Derived MSCs Isolation, Culture, and Expansion

AF samples (about five milliliters) were obtained by biopsy (amniocentesis) from mid-second-trimester (16–24 weeks) or third-trimester (28–34 weeks) pregnant woman who needed prenatal diagnosis, and no abnormalities were revealed by genetic analysis using protocols approved by the Ethics Committee of Biomedical Researches of Vilnius District, number 158200-123-428-122. Samples were maintained at room temperature for about 4 hours prior to isolation of amniotic cells using two-stage protocol [23]. The sample was centrifuged at 1,800 rpm for 20 min, the supernatant was removed, and the cell pellet was washed once in DMEM medium (Sigma-Aldrich Ltd.) without serum to remove blood and cell debris. After centrifugation, the cell pellet was resuspended in 5 mL of growth medium (AmnioMAX™-C100 basal medium with AmnioMAX™-C100 supplement (Gibco, Life Technologies, Grand Island, NY, USA)), containing 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Grand Island, NY, USA) and plated in a 25 cm² culture flask (TPP, Switzerland). Amniocytes were incubated for 10–15 days at 37°C in 5% CO₂, when first colonies appeared (first stage). For culturing MSC (second stage), nonadhering AF cells were collected from primary culture and further expanded in a new 25 cm² culture flask at 37°C in 5% CO₂. After the appearance of the cell colonies, the growing medium was changed every 3 days. When cells reached confluence at 80%, subculturing into higher passages was performed by trypsinization with 0.05% trypsin-EDTA (Gibco, Life Technologies, Grand Island, NY, USA) for 3 min. A morphologically homogeneous population of fibroblast-like cells was obtained after two rounds of subculture. Cell morphology was observed by phase-contrast microscope (Nicon Eclipse TS100). Cell population doubling levels are presented as the average values calculated as follows: cell culture time (days)/number of passages.