ESTABLISHMENT OF PRIMARY AND CONTINUOUS CULTURES OF EPITHELIAL-CELLS FROM LARVAL LEPIDOPTERAN MIDGUTS

Midgut epithelial cells from larvae of Bombyx mori, Choristoneura fumiferana and Lymantria dispar were isolated by incubating everted guts in 200 IU/ml collagenase-Type XI at 13 degrees C or 18 degrees C for 15 to 20 h. Dissociated cells were resuspended in culture medium at 10(5) cells per 25 cm(2)-vented flask and cell survival was assessed over a 31-day incubation period. Cell survival was 98% on day 1, decreased to about 75% by day 3 and equilibrated at this level for the remainder of the incubation period. Columnar and goblet cells were isolated from the midgut at a ratio of 4:1. During a 31-day incubation period, primary cultures of Lymantria and Bombyx cells retained a polarized morphology; columnar cells are rectangular with microvilli on an apical border while goblet cells are goblet shaped with a deep invagination of the membrane that is observed as a central channel. Choristoneura cells had similar characteristics but, both cell types were more oval-shaped. The cultured cells began to divide after 2 months, and currently we have cells in their 18th passage. The culture medium contained Mitsuhashi and Maramorosh medium, 10% Fetal bovine serum and 50 mu g/ml gentamicin. Dissociated cells resuspended in fetal bovine serum attached to petri dishes after 72 to 96 h.