James L. Vaughn

The establishment of two cell lines from the insectspodoptera frugiperda (lepidoptera; noctuidae)

SummaryThe history and characteristics of two cell lines developed from primary explants of pupal tissue from the insect,Spodoptera frugiperda (J. E. Smith), are described. One cell line, IPLB-SF-21, was developed with hemolymph-supplemented medium and has been maintained continuously on the medium. The second cell line, IPLB-SF-1254, was developed with a medium containing a combination of vertebrate sera plus hemolymph and was adapted to hemolymph-free medium at the 6th passage. The IPLB-SF-1254 line is 36 hr. The chromosomal morphology and distribution was typical of other lepidopteran cell lines. Serological studies showed that both cell lines have at least one antigen which also is common to tissue antigens from pupae ofSpodoptera frugiperda.

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

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.

The production of nuclear polyhedrosis viruses in large-volume cell cultures

Abstract Methods were tested for growing cell lines from the fall armyworm, Spodoptera frugiperda, in roller bottle cultures. The effects of inoculum size, medium volume, and serum level were tested for effect on the cell yield. A protocol is described which gives yields of 3–5 × 108 cells per bottle. Several protocols were then tested for producing the NPV of Autographa californica in this culture system and the results are described.

Improved Replication of Autographa californica Nuclear Polyhedrosis Virus in Roller Bottles: Characterization of the Progeny Virus

A reproducible growth curve was established for the propagation of Autographa californica (Speyer) nuclear polyhedrosis virus (ACMNPV) In a continuous insect line from Spodoptera frugiperda (J. E. Smith) during large-volume production. A newly developed method for quantitation of polyhedra inclusion bodies (PIB) by electron microscopy (EM) during the growth cycle was compared to hemocytometer counts. The virus particles (VP) ratio to PFU and VP per PIB were established by EM methods. Optimal yields of PIB and cell-free virus with biological activities were obtained in six consecutive production lots when the growth curve conditions were followed. The DNA of the viruses produced in the 1st and 8th passages in the S. frugiperda cell line was treated with restriction endonucleases and compared with that from the E-2 cloned variant of this virus. Data confirmed that the virus was the ACMNPV and also indicated that there were no detectable changes after 8 passages in insect cell culture.

Insect Cells for Insect Virus Production

Publisher Summary As more and more insects become resistant to chemical insecticides and as the pressures for a cleaner environment become greater, the demands for improved methods of controlling insect pests by biological means or through the combination of methods known as integrated pest management constantly increase. Cell culture systems, when perfected, would have several advantages for the production of viruses. It was obvious from the beginning of insect cell culture that for virus production, continuous insect cell lines would be required. Several insect cell lines are not substrate dependent and, therefore, can be grown in suspension systems. Although no insect cell lines have been shown to be substrate dependent, many will grow well in monolayers attached to either glass or plastic substrates. It is in the production of high virus yields that the most research is needed and where the benefits will be the greatest.

DIFFERENTIAL REQUIREMENTS OF TWO INSECT CELL LINES FOR GROWTH IN SERUM-FREE MEDIUM

SummaryThe development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 × 106 TCID50 extracellular virus and 4.4×108 polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects.

Insect cell nutrition: Emphasis on sterols and fatty acids

SummaryThe requirements for, or the utilization of, vitamins, sugars, amino acids, and lipids, as determined with established insect cell lines, are reviewed. Also discussed is the importance of inorganic salt balance and osmotic pressure. Nutrient requirements, as determined in experiments with established cells, are compared to media formulations based on the analysis of insect hemolymphs and to the dietary requirements of insects. It is suggested that culture media formulated in strict agreement with hemolymph analysis may be unnecessarily complex and do not ensure maximum growth.

A review of the use of insect tissue culture for the study of insect-associated viruses.

Viruses are associated with insects in a variety of host-pathogen relationships. The viruses may be specific pathogens of insects, as for example, are the polyhedrosis viruses; or they may be pathogens of higher animals or of plants, with the insects only the intermediate hosts and the vectors. Because of these diverse relationships, there is considerable interest among research workers in many areas of biology in the use of insect tissue culture as a tool for the study of viruses. Indeed the interest has often outdistanced progress in the field of insect tissue culture. Therefore, some discussion of several of the problems involved in the culture of insect tissue seems justified before reviewing the history and present status of the study of viruses.

The use of commercial serum replacements in media for the in vitro replication of nuclear polyhedrosis virus

Abstract Two commercially available fetal bovine serum replacements, previously used in media for the culture of vertebrate and insect cells, were evaluated in comparison with fetal bovine serum (FBS) for suitability as medium supplements for the production of the Autographa californica nuclear polyhedrosis virus in fall armyworm cell cultures. The overall patterns of viral protein synthesis were similar in infected cells in each of the media supplements, but some differences were noted in quantities of individual proteins produced and in the temporal regulation of the synthesis. The release of extracellular budded virions (ECV) in all media began at about the same time and the rate of release was similar. However, the final concentration of ECV was consistently lower in medium containing either CPSR-1 or CPSR-3 than that in FBS supplemented medium. The number of occlusion bodies (OB) per cell was also less in the CPSR-1 medium. In the CPSR-3 medium the number OBs per cell produced was not significantly different from those in the FBS medium. The LC 50 value of the OBs bioassayed in neonate Spodoptera frugiperda produced in the CPRS-3 medium was significantly lower than that of OBs from FBS medium but the LC 70 and LC 90 values were not. Abnormal OBs, that is OBs that contained few or no occluded virions, were observed in cells in media with all three supplements.

Use of commercial serum replacements for the culture of insect cells

SummaryTwo commercially available serum replacements developed for use in the culture of hybridoma and other mammalian cells were tested for their suitability as replacements for fetal bovine serum in insect cell culture medium. CPSR-1 and CPSR-3 both supported growth of the insect cell line IPLB-SF-21AE. CPSR-3 supported adequate growth, but cells in medium supplemented with CPSR-1 grew much slower and achieved only about half the final cell density of either FBS or CPSR-3 supplemented medium.