A morphological and functional characterization of Bombyx mori larval midgut cells in culture

Abstract

Recent studies have shown that Bombyx mori larval midgut can transport proteins unaltered
following the transcellular pathway by transcytosis. The numerous steps involved in this complex
process are still unknown in the insect midgut, and a promising tool to elucidate this aspect is the
availability of single midgut cells in culture suitable for transport experiments. Mature midgut cells in
culture were obtained from stem cells isolated from B. mori larvae cultured in Grace’s medium
supplemented with 20-hydroxyecdysone (20-HE) and α-arylphorin. After three weeks, up to 60 % of
the cultured cells were differentiated into columnar and goblet cells, the two predominant cell types in the midgut epithelium. These cells presented in vitro the same shape, morphology and polarity
recorded in vivo, even if their dimensions were slightly reduced. Columnar cells displayed a well
developed cytoskeletal arrangement, with actin filaments highly organized within the thick brush
border and distributed in faint filaments in the cell cytoplasm. Microtubules formed a substantial net
just beneath the brush border and ran longitudinally from the apical to the basal pole of the cell.
Cultured cells homogenates displayed aminopeptidase N and alkaline phosphatase activity, proving
that these two enzymes, involved in vivo in the intermediate and final digestion, are expressed also in
vitro. The columnar cells differentiated in culture were able to internalize two model proteins with quite different transport rates.