Primary single CTBs were isolated from human first-trimester villi as described previously with some modifications [27, 28]. First, following the same steps as those of primary hPDMSC isolation, the placental villi were minced into small pieces and rinsed extensively with PBS. Then, the digestion mixture containing 0.25% trypsin and 0.1 mg/ml DNAse I (Biodee, China) was added and incubated in a 37 °C shaking water bath for 30 min. The digested suspension was filtered through a nylon mesh (200 μm) and terminated with FBS. The remaining tissue was digested with the digestion mixture containing 1 mg/ml collagen I (Invitrogen, USA) and 0.1 mg/ml DNAse I in a 37 °C shaking water bath for 20 min. The digested suspension was filtered, and digestion was terminated again. The whole digestion procedure was repeated at least 3 times. Then, the whole-cell suspension was filtered through a nylon mesh (100 μm) and centrifuged at 1000 rpm for 10 min. The cell pellet was resuspended in 3 ml DMEM with 10% FBS, gently layered on the top of a preformed noncontinuous Percoll gradient (Phamaiva, USA) (75–25%), and centrifuged at 3000 rpm for 30 min. Single cells were collected between the 45 and 35% Percoll aliquots, resuspended, and counted. The freshly isolated cell suspension was seeded at a concentration of 1 × 108 cells/dish into 10-cm2 Petri dishes to collect CM or analyze by flow cytometry (or at a concentration of 1 × 105 cells/dish into 3-cm2 Petri dishes for identification). After being cultured for 1 h, the cell suspension was transferred to new Petri dishes and incubated overnight. The next day, the nonadherent cells were removed, and a new medium was added. Once the cells in the 10-cm2 Petri dishes were more than 90% confluent, the medium was replaced with serum-free DMEM, and the primary CTBs-CM was collected at different time points. The isolated cells in the 3-cm2 Petri dishes were passaged for identification.

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