Establishment of primary and continuous cultures of epithelial cells from larval lepidopteran midguts

Abstract

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°C or 18†C for 15 to 20 h. Dissociated cells were resuspended in culture medium at 105 cells per 25 cm2-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 μg/ml gentamicin. Dissociated cells resuspended in fetal bovine serum attached to petri dishes after 72 to 96 h.