Summary
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.
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Vaughn, J. L.; Goodwin, R. H.; Tompkins, G. J.; McCawley, P. The establishment of two cell lines from the insect,Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13: 213–217; 1977.
Lynn, D. E.; Hink, W. F. Infection of synchronized TN-368 cell cultures with alfalfa, looper nuclear polyhedrosis virus. J. Invertebr. Pathol. 32: 1–5; 1978.
Granados, R. R. Infection and replication of insect pathogenic viruses in tissue culture. Adv. Virus Res. 20: 189–236; 1976.
Hink, W. F. Cell lines from invertebrates. Methods Enzymol. 58: 450–466; 1979.
Kurtti, T. J.; Brooks, M. A. The effect of osmotic pressure and pH on the growth ofHeliothis zea cells in vitro. Rodriguez, J. G. ed. International conference on the significance of insect and mite nutrition, University of Kentucky, 1972. Insect and mite nutrition; significance and implications in ecology and pest management. Amsterdam: North Holland Publishing Co.; 1972: 387–395.
Kurtti, T. J.; Chaudhary, S. P. S.; Brooks, M. A. Influence of physical factors on the growth of insect cells in vitro. II. Sodium and potassium as osmotic pressure regulators of moth cell growth. In Vitro 11: 274–285; 1975.
Vaughn, J. L. The production of nuclear polyhedrosis viruses in large-volume cell cultures. J. Invertebr. Pathol. 28: 233–237; 1976.
Patterson, M. K., Jr. Measurement of growth and viability of cells in culture. Methods Enzymol. 58: 141–152; 1979.
Perry, V. P.; Kroener, C. A.; Martin, J. L. Protected freezing of cell suspensions. Procedure 76161. TCA Manual 1: 119–120; 1975.
Weiss, S. A.; Lester, T. L.; Kalter, S. S.; Heberling, R. L. Chemically defined serum-free media for the cultivation of primary cells and their susceptibility to viruses. In Vitro 16: 616–628; 1980.
Lewis, K. Self contained breathing apparatus requires clean air. Fire Chief Magazine, October; 1971.
Weiss, S. A. Concentration of baboon endogenous virus in large-scale production by use of hollowfiber ultrafiltration technology. Biotechnol. Bioeng. 22: 19–31; 1980.
McGarrity, G. J.; Sarama, J.; Vanaman, V. Factors influencing microbiological assay of cell-culture mycoplasmas. In Vitro 15: 73–81; 1979.
Russell, W. C.; Newman, C.; Williamson, D. H. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 253: 461–462; 1975.
Schaeffer, W. I. Proposed usage of animal tissue culture terms. Usage of vertebrate cell, tissue, and organ tissue culture terminology. In Vitro 15: 649–653; 1979.
Honn, K. V.; Singley, J. A.; Chavin, W. Fetal bovine serum: a multivariate standard. Proc. Soc. Exp. Biol. Med. 149: 344–347; 1975.
McLimans, W. F. Mass culture of mammalian cells. Methods Enzymol. 58: 194–211; 1979.
Perlman, D.; Rahman S. B.; Semar, J. B. Antibiotic control ofMycoplasma in tissue culture. Appl. Microbiol. 15: 82–85; 1967.
Faulkner, P.; Brown, M.; Potter, K. N. Utilization of tissue culture techniques to clone an insect cell line and to characterize strains of baculovirus. Kurstak, E.; Maramorosch, K. eds. International conference on invertebrate tissue culture, 4th, Mont Gabriel, Quebec, 1975; Invertebrate tissue culture. Applications in medicine, biology, and agriculture. New York: Academic Press; 1976: 347–360.
Nicklin, P. M.; House, W. Large-scale production of virus. Biotechnol. Bioeng. 18: 723–737; 1976.
Corbeil, M.; Trudel, M.; Payment, P. Production of cells and viruses in a new multiple tube tissue culture propagator. J. Clin. Microbiol. 10: 91–95; 1979.
Harms, E.; Wendenburg, J. Large-scale perfusion culture of cells growing on surfaces with automatic gas and medium control. Cytobiologie 18: 67–75; 1978.
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The majority of this work was done under contract No. 58-3204-8-5, U.S. Department of Agriculture, Science and Education Administration, Beltsville Agriculture Research Center, Beltsville, MD.
Mention of a copy or proprietary product does not necessarily constitute an endorsement by the U.S. Department of Agriculture.
An erratum to this article is available at http://dx.doi.org/10.1007/BF02628413.
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Weiss, S.A., Smith, G.C., Kalter, S.S. et al. Improved method for the production of insect cell cultures in large volume. In Vitro 17, 495–502 (1981). https://doi.org/10.1007/BF02633510
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DOI: https://doi.org/10.1007/BF02633510