Making Things Grow: Insect Cells, Stem Cells, and Primary Cell Lines All Pose Challenges for Cell Culturists

Date: June 22, 1998 Insect Cell Culture Media, Suppliers of Primary Cell Culture Media Advantages for Protein Expression Studies Since the mid-1950s cultures of insects--cockroaches, fruit flies, and leafhoppers, to name a few--have been the object of quiet study by physiologists and cell biologists. But along came genetic engineering and suddenly insect cultures have been put in the spotlight since they provide advantages over both bacterial and mammalian systems for recombinant protein production. While easy and inexpensive to use for large-scale production of proteins, bacterial systems often do not produce correctly modified and immunologically active proteins from eukaryotic sequences; mammalian cell cultures can be expensive to grow in bulk and require carefully controlled environments for optimum growth. In contrast to this, insect cells grow at room temperature and do not require CO2, which greatly simplifies the job of growing cells, while sporting a perfectly adequate transfection system for protein expression studies. Insect Cell Culture Media J.P. Burand, H. Lu, Replication of a Gonad-Specific Insect Virus in TN-368 Cells in Culture, Journal of Invertebrate Pathology, 70 (2):88-95,1997. R.R. Tonjes, et al., Characterization of Human Endogenous Retrovirus Type K Virus-Like Particles Generated from Recombinant Baculoviruses, Virology, 233 (2):280-91, 1997. X.Y. Cai et al., Expression, Purification, and Characterization of an Activated Cytokine-Suppressive Anti-Inflammatory Drug-Binding Protein 2 (CSBP2) Kinase from Baculovirus-Infected Insect Cells, Protein Expression and Purification, 10 (2):263-274, 1997. B. Yang et al., Very High Single Channel Water Permeability Of Aquaporin-4 in Baculovirus-Infected Insect Cells and Liposomes Reconstituted With Purified Aquaporin-4, Biochemistry, 36 (24):7625-32, 1997. M. Schmidt et al., Baculovirus-Mediated Large-Scale Expression And Purification Of A Polyhistidine-Tagged Rubella Virus Capsid Protein. Protein Expr Purif, 12 (3):323-30, 1998. X.G. Kong et al., Application Of Equine Infectious Anemia Virus Core Proteins Produced In A Baculovirus Expression System To Serological Diagnosis, Microbiological and Immunology, 41 (12):975-80, 1997. A.R. Karpf, D.T. Brown, Comparison Of Sindbis Virus-Induced Pathology In Mosquito And Vertebrate Cell Cultures, Virology, 240 (2):193-201, 1998. A. Roscoe et al., Cloning And Heterologous Expression Of NADPH-Cytochrome P450 Reductases From The Papaveraceae, Archives of Biochemistry and Biophysics, 348 (2):369-77, 1997. L.E. Pyle et al., Production Of Mature Human Apolipoprotein A-I In A Baculovirus-Insect Cell System: Propeptide Is Not Essential For Intracellular Processing But May Assist Rapid Secretion, Annals of Biochemistry, 253 (2):253-8, 1997 Z.X. Wang et al., Expression Of Chemokine Receptors In Insect Cells Using Baculovirus Vectors, Methods in Enzymology, 288:38-55, 1997. K. Cha et al., Rhodopsin Kinase: Expression In Baculovirus-Infected Insect Cells, And Characterization Of Post-Translational Modifications, Proceeding of the National Academy of Sciences, 94 (20):10577-82, 1997. T.P. Keeton and LA Bulla Jr., Ligand Specificity And Affinity Of BT-R1, The Bacillus Thuringiensis Cry1A Toxin Receptor From Manduca Sexta, Expressed In Mammalian And Insect Cell Cultures, Applied Environmental Microbiology, 63 (9):3419-25, 1997. Though insect cultures are robust and have been used extensively in protein expression studies for research purposes, their use in the commercial production of proteins appeared to be limited by differences in the glycosylation patterns in lepidopteran cells as compared to mammalian cells. Insect cells produce mannosylated glycoprotein structures and lack the terminal sialic acid or penultimate galactose saccharides found on native mammalian glycoproteins. This structure tends to decrease the effective half-life of proteins in vivo, making their use in therapeutic settings problematic. However, what might have been a weakness instead is a strength, as this difference in glycosylation patterns has been found to increase the immune response to insect cell-derived proteins. Hence, insect expression systems are increasingly finding a use in the production of commercial vaccines. A number of different insect cell lines are currently in use. Historically important but largely forgotten cell line is the S2 cell line, derived from a primary culture of late-stage Drosophila melanogaster embryos. Used for heterologous protein expression, this cell line is readily adaptable to growth in suspension, in addition to being easy to grow (I. Schneider, Journal of Embryology and Experimental Morphology, 27:363-5, 1972). The new juveniles on the block, receiving much of the attention from protein engineers, are Sf9 and Sf21 cell lines. Derived by scientists at the U.S. Department of Agriculture Insect Pathology Laboratory from the pupal ovarian tissue of the fall armyworm, Spondoptera frugiperda, these two lines are used extensively for the isolation and propagation of recombinant baculovirus stocks and recombinant proteins. The larger size of the cells in the Sf21 line makes it preferable for expression studies, in addition to the fact that it grows to a high density in suspension culture. The clonal isolate Sf9 cell line, on the other hand, is better designed for the formation of monolayers and plaques (D.R. O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, W.H. Freeman and Company, 1992). Finally, the High FiveTM cell line, BTI-TN-5BI-4, was developed at the Boyce Thompson Institute for Plant Research in Ithaca, N.Y., from embryonic ovarian cells of the cabbage looper, Trichoplusia ni (T.R. Davis et al., Bio/Technology, 10:1148-50, 1992). This cell line is easily grown in suspension and has increased protein production, reportedly as much as five- to ten-fold higher than Sf9 cells. (T.J. Wickham et al., Biotechnology Progress, 8:391-6, 1992, T.R. Davis et al., In Vitro Cell & Development, 29A:388-90, 1993) Products for Embryonic Stem Cell Culture Feeder Cells ATCC Cell Applications Genome Systems Lexicon Genetics LIF Becton Dickinson-human rec. Life Technologies Inc.-mouse Genzyme Corp-human rec. INTERGEN® PeproTech-mouse Roche Molecular Biochemicals-human Stem Cell Technologies Sigma-rec. Media Cell and Molecular Technologies INTERGEN® Life Technologies Lexicon Genetics Stem Cell Technologies Somewhat surprisingly, media that are supportive of insect cells differ only in a few parameters from other vertebrate media. The major points of departure are pH, with insect cells growing at lower pH ranges (6.0 to 6.9) than other vertebrate cells; osmolality, which is high due largely to the presence of amino acids; and ion ratios, which can vary from insect to insect. While attempts initially were made to simulate hemolymph, it was later learned that once the differences noted were taken into account, some standard mammalian media formulations could be adapted for growing insect cells. Nonetheless, a number of media specifically designed for insect cells, or for particular insect lines, have been devised. The basal media used for growth of many insect cells is a formulation devised by T.D.C. Grace in 1962 ( Nature, 195:788-9, 1962) -- still used despite the fact that it requires substantial amounts of serum or other additives. In addition several variations are available that have supplementation; these are called TNM-FH, or Hink's, when supplemented with yeastolate, lactalbumin hydrolysate, and glutamine, or IPL-41, when modified to include heavy metals. Among the media on the shelf are several designed for the growth of Drosophila cells (D-22, Schneider's, and Shields and Sang M3 media), as well as Mitsuhashi and Maramorosch, developed originally for leafhopper cells, and several formulations for Sf9 isolates (TC-100 and a variety of proprietary formulations). As interest mounted in the use of insect cells for protein production, companies responded by devising serum- and protein-free media designed for use with Sf9 and High Five cell lines in particular. In addition, media lacking certain amino acids are available for labeling studies. A listing of companies that offer insect cell culture media is found in an accompanying table. As the following recent references illustrate, insect cultures are finding a variety of uses. From the production of recombinant proteins and vaccines to the study of insect pathogens and disease states, insect cultures have proven their worth. The author can be reached at ldefrancesco@the-scientist.com. Insect Cell Culture Media, Suppliers of Primary Cell Culture Media

Making Things Grow: Insect Cells, Stem Cells, and Primary Cell Lines All Pose Challenges for Cell Culturists

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