CELL CULTURES FROM DISPERSED EMBRYONIC HOUSE FLY TISSUES - TECHNIQUE MITOSIS AND CELL AGGREGATES

Six-hour old M. domestica house fly eggs were placed in porous, cellulose tissue embedding bags, sterilized, dechorionated, and crushed so that embryo cells were extruded through the bag. These cells and debris were filtered through a funnel with a coarse, fritted glass disk which passed only single cells. The cells were centrifuged at 810 rpm on a clinical centrifuge and the supernatant containing egg yolk, fat body, and debris was discarded. Cultures (2 × 106 cells/ml) were established with an empirically devised medium containing 20% fetal calf serum. Cell attachment was nearly complete in 4-6 h, and these cells assumed a normal spindle shaped, fibroblastic, or epithelioid appearance. Within 3-4 days, these attached cells aggregated into several types of clusters which appeared to consist of muscle cells, nerve cells, and unidentified spherical cells. The cultures were continuously maintained for 6 months to 1 year with mandatory subculturing. The division of nuclei and cells was demonstrated with tritiated thymidine, autoradiography, and time-lapse photomicrography. The cells were freeze-preserved by equilibration in 10% DMSO, 3% PVP, and 3% fetal calf serum and cooling at 2 °C/min to the temperature of liquid nitrogen. © 1969.