Isolation and Culture of Amniotic Fluid and Bone Marrow-Derived Mesenchymal Stem Cells

Approximately 5 ml of AF from healthy pregnant females (n=10) of gestation week 12~18 were collected during routine amniocentesis after informed consent. The AF cells obtained after centrifugation were resuspended in MSC growth medium (MSCGM) consisting of α-MEM supplemented with 16.5% fetal bovine serum, 1% glutamax and 1% bacteriostatic levels of penicillin-streptomycin (Gibco, MD, USA). The cells were seeded in T-25 cm2 flasks (BD Falcon) and incubated at 37°C under 5% CO₂. The medium containing non-adherent cells was changed on every 3rd day. The primary cultures of AF-MSC with semi-confluent growth were passaged by trypsinization and further expanded under the same culture conditions. The BM samples of patients with nutritional anemia or immune thrombocytopenia (ITP) (n=10) undergoing routine marrow examination were collected after informed consent and the BM-MSC were isolated and cultured using a protocol previously published by our laboratory (18 (link)). The cells of the 3^rd passage were used in the experiments.