Growth of Insect Cells in Tissue Culture

Since Grace reported(1) the establishment in tissue culture of strains of cells from ovarian tissue of the saturniid moth Antheraea eu-calpti Scott, many unsuccessful attempts have been made to grow these cells in other laboratories. Since we have maintained these cells in monolayer culture for more than 2 years (with some 30 passages), a summary of our experiments may be helpful to others attempting to establish and maintain insect cells in tissue culture.

The cells initially obtained from Dr. Grace have been grown in monolayers in 1 × 6 inch glass tubes with teflon-lined screw-caps (Bell-co Glass Co.) and in plastic flasks (Falcon Plastics Division, Becton, Dickinson Co.). Seven ml of Grace's medium(l) supplemented with 3% (v/v) of Anther aea pernyi sterile heat-treated hemolymph+ was added to each 1×6 tube and 4 ml to each plastic flask (24 cm² surface). The initial cell count ranged from about 10³ to 10⁴ cells per ml. The tubes were placed on a slanting board (17° angle from horizontal) in an incubator at 28°-30°C. Medium changes have been routinely made on a weekly basis. Those cells found floating have been collected by centrifugation and used to start new tubes and flasks. Well developed monolayers covering the tube surface have usually been obtained within 3 weeks of starting the culture. As the monolayers are only loosely attached to the glass surfaces, care is needed in handling the tubes and flasks. The attached cells are aseptically scraped with a rubber policeman from the tubes once a month and these cell suspensions used to start new tubes and flasks. The glass tubes have not been specially washed or treated prior to use, and the sterile plastic flasks have been used as received from the manufacturer.