Chang basal medium

AFSC were isolated based on previously published methods from our group[8 (link), 13 (link)]. Primary human amniotic fluid was obtained from patients in their second trimester undergoing planned amnioreduction as part of a therapeutic treatment for twin-twin transfusion syndrome (TTTS). Amniotic fluid was centrifuged at 1200 rpm for 10 min, and collected cells were plated at 2500 cells/cm2 on standard plastic Petri dishes and cultured in a modified α-Minimum Essential Media: 63% αMEM (Invitrogen, Carlsbad, CA), 18% Chang Basal Medium (Irvine Scientific, Santa Ana, CA), 2% Chang C supplement (Irvine Scientific), 15% fetal bovine serum (PAA Laboratories, Dartmouth, MA), and GlutaMAX (Invitrogen) at 37°C and 5% CO2 in a humidified environment. Media was changed every two to three days, and cells were passaged at 60–70% confluence. At the first passage, a subpopulation of progenitor cells was isolated through fluorescence-activated cell sorting for expression of the membrane receptor CD117/c-kit (BD Biosciences, Bedford, MA). Cell colonies were detached into single cells (Accutase; Sigma-Aldrich, St. Louis, MO; 37°C, 10 min), and c-kit+ cells were collected using a Dako MoFlo sterile cell sorter. All studies of primary human cells were approved by the Institutional Review Boards of both Baylor College of Medicine and Rice University, and subjects gave informed consent.