Amniotic fluid cell culture and karyotype analysis

Amniotic fluid (20 ml) was obtained (with the first 1–2 ml amniotic fluid discarded) and injected into two disposable sterile centrifuge tubes for the centrifugation at 190 × g for 10 min; the supernatant was subsequently discarded, and 2.5–3.0 ml cell suspension was inoculated into 5 ml of each of Gibco Amniomax-II (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and BIO-AMF-2 (Biological Industries, Kibbutz Beit-Haemek, Israel) amniotic fluid media, under sterile conditions, for 6~7-day static culture at 37°C and 5% CO₂. The cell growth was observed every day after the medium was changed. When the amniotic fluid cells adhered to the wall and grew vigorously, and the cells in metakinesis exhibited multiple clones under an inverted microscope, the amniotic fluid chromosomes in each culture bottle were collected separately and the slides produced as follows: Colcemid solution (Thermo Fisher Scientific, Inc.) was added to the culture bottle and incubated for a further 2 h at 37°C. The medium was removed from the culture bottle and saved in a prelabeled centrifuge tube. Trypsin-EDTA (1 ml) was added to the bottle and the cells washed by tilting the bottle from side to side. The solution was removed from the bottle and 1.5 ml of fresh trypsin-EDTA solution added and the cells bathed thoroughly with the solution by tilting the bottle. The cells were incubated at 37°C for 5 min and then added to the contents of the centrifuge tube and mixed prior to centrifugation at 190 × g for 10 min. The supernatant was removed and 5 ml of potassium chloride hypotonic solution 0.075 mol/l added to the cell, resuspended and incubated for 10–15 min in a water bath at 37°C. Freshly made fixative was added to each tube and mixed gently by inverting the tubes twice then centrifuged at 190 × g for 10 min and the supernatant discarded and the fixative step repeated an additional three times. Then the cells were suspended in a small volume of fixative to give a slightly opaque suspension and 3 to 4 drops were placed evenly on a cold wet slide an allow to dry. G-band staining (plus C-band staining when necessary) was used to prepare the chromosome specimens, and, in accordance with the International System for Human Cytogenomic Nomenclature (2013) (9), each specimen was analyzed 30 well-dispersed moderately-long metakinesis phases under a light microscope. When the chimera or abnormal karyotype was identified, the analysis was performed for a total of 100 mitotic phases.