Growth by Cell Division in Insect Tissue Culture

Abstract

Abstract 1. 1. Extensive cellular outgrowths were produced in insect tissue culture; the ovarian tissue of diapause silkmoth pupae was used and a medium consisting essentially of salts, sugars, organic acids, lactalbumin hydrolysate, and haemolymph. The presence of TC yeastolate improved the appearance of the cultures. 2. 2. Cellular outgrowth was not increased by partially substituting relatively large amounts of lysine, histidine, and serine for the lactalbumin, nor by including the vitamin B complex, cholesterol, or glutathione in the medium. It was considerably reduced by the omission of haemolymph, which also affected the ability of the cells to adhere to the surface of the coverglass. 3. 3. A simple technique was devised for permanently staining the cultures in situ to permit an evaluation of cellular migration and mitosis in the outgrowths. 4. 4. The outgrowths were composed of several kinds of cells. Prominent were those of the ovariole sheath and the intermediate layer. Cells of the inner follicle layer and the extremely large nurse cells were also capable of wandering into the cellular field. Epithelial sheets and brush-like extensions were formed by the cells of the ovariole sheath and connective tissue cap respectively. 5. 5. Mitosis in the outgrowths seemed to be mainly confined to the cells of the ovariole sheath and intermediate layer. In the stained preparations these cells displayed mitosis at all stages. It is likely that the cells of the inner follicle layer were also capable of mitosis when present in the outgrowths. 6. 6. In 5 to 6 day old cultures the mean mitotic index was 1.0 per cent, in those of 8 to 9 days 0.5 per cent, in 12 day cultures 0.3 per cent, and in 21 day less than 0.3 per cent. Since cells in culture took about 30 minutes to complete mitosis, it is estimated from the mitotic indices that several generations of cells were produced. This compares favourably with growth by cell division in early vertebrate tissue culture.