Improved method for the production of insect cell cultures in large volume

Abstract

An improved semiautomated procedure for growing a cell line from the fall armyworm,Spodoptera frugiperda, under optimal conditions, in a stationary large scale culture, is described in detail. Complete supplementation of the medium, with final adjustment of osmolarity and pH prior to filtration, resulted in a 20-fold increase in cell yield and a reduced population doubling time with this insect cell line. This complete medium, when stored at 4°C for 6 months, supported cell growth equally well when compared to the yield of the cells grown in freshly prepared medium. Air used to pressurize the system was purifield to eliminate carbon monoxide and other contaminants. Antibiotic-free cell preparations were dispensed by a method applicable to commercial use. No bacterial, mycoplasmal, or viral contaminants were detected in the cell cultures grown in antibiotic-free medium for 20 months. Consistent reproducibility of cell yields per vessel was obtained. Cryopreserved insect cells were recovered and planted directly into roller bottles, which subsequently were scaled up to large volume production. Electron microscopic studies revealed that some cells were undergoing nuclear membrane invagination and some possessed lobed nuclei. These cells contained typical organelles, including abundant endoplasmic reticulum, mitochondria, polysomes, few microvilli, and dense bodies. These observations appeared to be normal properties of the continuous line, IPL-21 AE III.