Development and Application of Insect Cell Culture Techniques

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Infection and development of microsporidians (Nosema bombycis NIS 001, N. sp. NIS M11, N. sp. F-30, N. mesnili AH, Thelohania sp. K-25) pathogenic to lepidopterous insects were studied in a cell line of Antheraea eucalypti. Microsporidian spores primed with 0.1 N potassium hydroxide solution were mixed with the A. eucalypti cell cultures and infected cultures were maintained for more than 18 months.Low cost insect cell culture media (MM) and its application to spinner culture of insect cells were investigated. Growth of two lepidopteran and one dipteran cell lines was compared under still and spinner culture conditions. Apparently, the insect cells cultured in plastic culture flasks showed higher growth rates when smaller culture vessels were employed.Also, a general purpose culture medium (MGM-450) for insect (Lepidopera, Diptera, Hemiptera and Coleoptera) cells was newly formulated.Cloning of insect cells using Bombyx mori cell line (SES-BoMo-15AII) was studied and establishment of the B. mori cell line resulted from a single cell was unsuccessful. It was suggested that a colonial coloning was practical method for cloning of insect cells at the present time.Spodoptera furgiperda Sf21AEII cells were highly sensitive to heat labile cytopathic factor (s) produced by several serovars of Bacillus thuringiensis. However, in vitro assay of the heat stable exotoxin (fly toxin) of B. thuringiensis using the similar system showed negative results.Addition of insect hemolymph factor (s) to Bombyx mori BM-N cells infected with a nuclear polyhedrosis virus, markedly enhanced the production of viral structural and inclusion proteins in vitro.Specific and common cell surface antigens of cultured lepidopteran cells were demonstrated and serological identification of insect cell lines was applicable for several insect cell lines.