Isolation and primary culture of stem cells from the midgut epithelium of the silkworm <i>Bombyx mori</i>

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  • Fujiwara Nozomi
    The United Graduate School of Agricultural Sciences, Tottori University
  • Azuma Masaaki
    The United Graduate School of Agricultural Sciences, Tottori University
  • Itoh Masanobu
    Kyoto Institute of Technology
  • Kobayashi Jun
    Graduate School of Sciences and Technology for Innovation, Yamaguchi University

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  • Isolation and primary culture of stem cells from the midgut epithelium of the silkworm Bombyx mori

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Abstract

<p> The process of molting is essential for the growth of lepidopteran larvae and their metamorphosis into adults. However, the investigation of cellular renewal and regeneration is limited by the lack of efficient methods for the isolation and characterisation of midgut stem cells in vitro. Here, we demonstrate an easy and secure protocol for midgut stem cell isolation from molting larvae and pharate pupae of the silkworm, Bombyx mori. This meth- od is based on the physiology of stem cells, which lie loosely associated with the base of epithelial cells and multiply during the molting or metamorphosing phase. They finally intercalate or replace existing epithelial cells as they elongate and differentiate into mature forms. Using approximately 10 insects, it was possible to obtain >500,000 cells with high homogeneity and >80% viability. Stem cells prepared from the fourth instar molting larvae and pharate pupae were spherical and could be preserved at 4°C for 1-2 weeks with no viability loss. However, the number of living cells showed a steady decline when cultured in Grace’s insect culture medium for one week. Supplements such as fetal bovine serum, lipid-rich bovine serum albumin, or heat-treated native B. mori plasma did not support stem cell proliferation or suppress cell death. Although continuous stem cell culture is still difficult, the described method for midgut investigation could potentially utilized for biochemical studies of stem cells and pathological assays in vitro, which may lead to novel strategies for agricultural pest management.</p>

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