Asunto(s)
Carcinoma de Células de Merkel/etiología , Poliomavirus de Células de Merkel/aislamiento & purificación , Infecciones por Polyomavirus/etiología , Neoplasias Cutáneas/etiología , Rayos Ultravioleta/efectos adversos , Anciano , Anciano de 80 o más Años , Carcinoma de Células de Merkel/epidemiología , Carcinoma de Células de Merkel/patología , Femenino , Humanos , Incidencia , Masculino , Nueva Zelanda/epidemiología , Infecciones por Polyomavirus/epidemiología , Infecciones por Polyomavirus/patología , Piel/patología , Piel/efectos de la radiación , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/patologíaRESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula , Separación Celular/métodos , Trofoblastos , Femenino , Humanos , EmbarazoRESUMEN
The mechanisms by which the fetus induces maternal physiological adaptations to pregnancy are unclear. Cellular debris, shed from the placental syncytiotrophoblast into the maternal blood and phagocytosed by maternal endothelial and immune cells, may be one of these mechanisms. Here we show that trophoblastic debris from normal first trimester placentae induces changes in the transcriptome and proteome of endothelial cells in vitro, which might contribute to the adaptation of the maternal cardiovascular system to pregnancy. Trophoblastic debris also induced endothelial cells to transcribe placenta-specific genes, including the vasodilator hormone CSH1, thereby expanding the effective functional size of the placenta. Our data suggest that the deportation of trophoblastic debris is an important part of the complex network of feto-maternal communication.