A simple method to isolate term trophoblasts and maintain them in extended culture

Abstract Introduction Primary trophoblast cultures obtained from term placentae are an important research tool. Term trophoblasts, while isolated as mononuclear cells, spontaneously fuse to form multinucleated syncytial clusters. Since term trophoblast cells do not replicate in vitro, contaminating cells can overgrow the culture limiting the lifespan of primary trophoblast cultures to about seven days. We aimed to develop a method that would allow the prolonged culture of term trophoblasts. Methods Trophoblasts were isolated from term placentae, following vaginal or cesarean section delivery, using either trypsin/DNase or dispase/DNase to digest the tissue. Purity of the trophoblasts was confirmed using flow cytometry prior to plating and during culture using immunocytochemistry. Cell death was examined with propidium iodide and trophoblast fusion monitored using PKH67 membrane stain. Results Digestion of term villous tissue with dispase/DNase resulted in the release of significantly more trophoblasts than digestion with trypsin/DNase (n = 8, p = 0.0051). Viability of the trophoblasts was unaffected by enzyme choice. The use of Advanced DMEM/F12 supplemented with 2% fetal bovine serum allowed culture of the trophoblasts with minimal cell death or contamination for 30 days. Despite prolonged culture over half of the trophoblasts remained mononuclear. Discussion We report a simple, optimized method to isolate and culture trophoblasts from term placentae for prolonged periods without substantial contamination with other cell types. Consistent with previous findings, trophoblasts cultured using our method were able to syncytialise, forming multi-nucleated syncytia. This extended growth time allows long term in vitro experimentation to further understand the nature of trophoblasts.