We have successfully isolated cytotrophoblast cells from human term placentas [31 (link)]. Here, we isolated cytotrophoblast cells from
human first trimester placentas using similar methods with minor modifications. Briefly,
villous tissues were dissected free of membranes, rinsed, and minced in phosphate-buffered
saline (PBS) (Life Technologies). The villous samples were digested three times in a
digestion enzyme medium containing 1 mg/ml Dispase II (Life Technologies) and 0.1 mg/ml
DNase I (Roche) at 37°C for 15 min each cycle. Released cells were then purified on a
discontinuous Percoll gradient (GE Healthcare) and centrifuged at 730 × gfor 20 min at 4°C. The layer between the 45% and 35% Percoll aliquots containing
cytotrophoblast cells (density: 1.050–1.060 g/ml) were collected. Collected cells were
further immunopurified by eliminating CD45RB-positive cells of myeloid origin using a
phycoerythrin (PE)-conjugated anti-CD45RB antibody (Ab) (1:10; Miltenyi Biotec) and
anti-PE-microbeads (1:5; Miltenyi Biotec), and depleting fibroblasts using anti-fibroblast
microbeads (1:5; Miltenyi Biotec) according to the manufacturer's instructions.
human first trimester placentas using similar methods with minor modifications. Briefly,
villous tissues were dissected free of membranes, rinsed, and minced in phosphate-buffered
saline (PBS) (Life Technologies). The villous samples were digested three times in a
digestion enzyme medium containing 1 mg/ml Dispase II (Life Technologies) and 0.1 mg/ml
DNase I (Roche) at 37°C for 15 min each cycle. Released cells were then purified on a
discontinuous Percoll gradient (GE Healthcare) and centrifuged at 730 × gfor 20 min at 4°C. The layer between the 45% and 35% Percoll aliquots containing
cytotrophoblast cells (density: 1.050–1.060 g/ml) were collected. Collected cells were
further immunopurified by eliminating CD45RB-positive cells of myeloid origin using a
phycoerythrin (PE)-conjugated anti-CD45RB antibody (Ab) (1:10; Miltenyi Biotec) and
anti-PE-microbeads (1:5; Miltenyi Biotec), and depleting fibroblasts using anti-fibroblast
microbeads (1:5; Miltenyi Biotec) according to the manufacturer's instructions.
