Procedure for Harvesting Cells to Karyotype *
Amniotic fluids are centrifuged and cells suspended in medium, placed in 3 culture flasks. If initial amniotic fluid volume was low (8 ml or less), the cells would be split into 2 flasks and the supernatant amniotic fluid would be placed in a 3rd flask with an additional 1-2 ml of culture medium. This 3rd flask would be left undisturbed for 1 week. Three or more colonies are expected in each culture flask.
Cultures are fed twice a week. They are always fed the day before planned harvest. RPMI medium with 18-20% fetal bovine serum is used as culture medium.
On the morning of harvest Colcimid is added at a ratio of 0.1 ml Colcimid to 5 ml medium. The medium is not changed on the morning of harvest. Colcimid is left with the cells from 4 hours (active culture) to 7 hours (slow culture). The medium is poured off and saved, and 1 ml of trypsin-EDTA is added. That also is taken off and saved and then another ml of Trypsin EDTA is added to the cell layer. The flask is returned to the incubator for about 2 min. and when the cells come off the surface they and the fluid are added to the previous media and rinse. This mixture is centrifuged and the cell pellet is resuspended in hypo. The cells are fixed with two changes of fixative and then the cell suspension is dropped onto slides. If the original flask had two colonies, one should make two slides. For three or more colonies, one should make three or four slides.
Large epithelial cells have a low mitotic rate. For these cultures Colcimid is added the night before planned harvest and remains with the cells for up to 20 hours. Less than 10% of cultures are handled in this way.
The first flask of an amniotic fluid culture is expected to be harvested at 10-14 days and the second flask usually the day following the first flask. Essentially all cultures are completed with the harvest of two flasks and all work including karyotyping and report are completed on average in 18 days.
*Obtained from the laboratory of M.J. Mahoney and C. Smith, Yale University School of Medicine.