Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera : Noctuidae)

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipunc ta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm. intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell type s were present in the cultures, including stem (regenerative), columnar, an d goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell densit y in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in sh ape and possess a brush border, while the goblet cells have a classic flask -like shape with a central cavity. Peritrophic membrane-like secretions wer e observed in all the culture flasks. Infection of these cells with multipl y embedded nucleopolyhedrovirus was confirmed, and we conclude that these m idgut cells can be used as an in vitro model system to study early events i n baculovirus infection.