Synchronization culture of amniotic fluid cells using excess thymidine block followed by deoxycytidine release and its application to high-resolution banding analysis of chromosomes

We describe a simple synchronization culture technique of amniotic fluid (AF) cells to yield many earlier mitotic divisions with extended chromosomes. AF cell samples obtained by amniocentesis were cultured in the usual manner. Thirty hours after the first subculture, they were exposed to excess thymidine (0.5 mM). This cell cycle block was released by adding deoxycytidine (10 microM) 18 hr after synchronization. At exactly 7.5 hr after the release, the cultures were treated with Colcemid (0.02 microgram/ml) for 20 min then harvested. The mitotic index and the ratio of cells in the earlier mitotic stages were much higher in the synchronized cultures than in the control cultures. The same favorable effects were obtained also in AF cell cultures by combining this technique with ethidium bromide or actinomycin D treatment. The technique was less toxic to the cells, and was simple and reproducible. It was successfully applied to prenatal cytogenetic diagnosis of 2 families with a subtle inherited chromosome abnormality, so it is recommended for high-resolution banding analysis of AF cells and possibly chorionic villus samples.