Growth and Maintenance of Mosquito Cell Lines

Raquel Hernandez1, Dennis T. Brown1

1 North Carolina State University, Raleigh, North Carolina
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 4J
DOI:  10.1002/9780471729259.mca04js17
Online Posting Date:  May, 2010
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Mosquito cells (Aedes albopictus) are among the most common insect cells emerging as new sources of cell cultures to use in basic research and in the pharmaceutical industry. They adapt well to growth in suspension; can be used in bioreactors for the production of expressed proteins, virus, and virus‐like particles; can be used in studies requiring lower growth temperatures than mammalian cells (28°C or below); and (because they are cholesterol auxotrophs) can be adapted to grow in dilipidated or serum‐free medium for experiments requiring these conditions. Procedures applicable to the laboratory maintenance of mosquito cell lines are described. Curr. Protoc. Microbiol. 17:A.4J.1‐A.4J.8. © 2010 by John Wiley & Sons, Inc.

Keywords: mosquito cell culture; semisuspension cells; cholesterol auxotrophs

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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation of Mosquito Cells
  • Basic Protocol 2: Freezing and Thawing Mosquito Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Propagation of Mosquito Cells

  • Mosquito cells, growing in semisuspension or frozen stock (if using frozen stock, see protocol 2), e.g., C6/36 cells (ATCC #CRL‐1660)
  • 1× complete Mitsuhashi and Maramorosch (M&M) medium (see recipe) or 1× complete EMEM medium (see recipe)
  • Tissue culture flasks or other suitable vessel
  • 5‐ to 10‐ml pipets

Basic Protocol 2: Freezing and Thawing Mosquito Cells

  • Mosquito cells grown to log phase in tissue culture flask: U4.4, C7‐10 (contact authors at or C6/36 cells (ATCC #CRL‐1660)
  • Lifting buffer (see recipe), optional
  • Freezing medium (see recipe) made with M&M or EMEM (and containing DMSO), depending on optimal growth medium
  • 1× complete EMEM medium, 37°C
  • 10‐ to 50‐ml centrifuge tubes
  • 2‐ml cryotubes
  • Nalgene Cryo 1°C freezing container
  • Liquid nitrogen freezer
  • 37°C water bath
  • 25‐cm2 flasks
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Literature Cited

Literature Cited
   Bowers, D.F., Coleman, C.G., and Brown, D.T. 2003. Sindbis virus‐associated pathology in Aedes albopictus (Diptera: Culicidae). J. Med. Entomol. 40:698‐705.
   Brown, D.T., Smith, J.F., Gliedman, J.B., Riedel, B., Filtzer, D., and Renz, D. 1976. Morphogenesis of Sindbis virus in cultured mosquito cells. In Invertebrate Tissue Culture, Applications in Medicine, Biology, and Agriculture (E. Kurstak and K. Maramorosch, eds.) pp. 35‐48. Academic Press, New York.
   Cox, M.M., Patriarca, P.A., and Treanor, J. 2008. FluBlok, a recombinant hemagglutinin influenza vaccine. Influenza Other Resp. Viruses 2:211‐219.
   Hafer, A., Whittlesey, R., Brown, D.T., and Hernandez, R. 2009. Differential incorporation of cholesterol by Sindbis virus grown in mammalian or insect cells. J. Virol. 83:9113‐9121.
   Igarashi, A. 1978. Isolation of a Singh Aedes albopictus cell clone sensitive to Dengue and Chickungunya viruses. J. Gen. Virol. 40:531‐544.
   Karpf, A.R. and Brown, D.T. 1998. Comparison of Sindbis virus‐induced pathology in mosquito and vertebrate cell cultures. Virology 240:193‐201.
   Karpf, A.R., Blake, J.M., and Brown, D.T. 1997. Characterization of the infection of Aedes albopictus cell clones by Sindbis virus. Virus Res. 50:1‐13.
   Krammer, F., Nakowitsch, S., Messner, P., Palmberger, D., Ferko, B., and Grabherr, R. 2010. Swine‐origin pandemic H1N1 influenza virus‐like particles produced in insect cells induce hemagglutination inhibiting antibodies in BALB/c mice. Biotechnol. J. 5:17‐23.
   Krebs, K.C. and Lan, Q. 2003. Isolation and expression of a sterol carrier protein‐2 gene from the yellow fever mosquito, Aedes aegypti. Insect Mol. Biol. 12:51‐60.
   Mudiganti, U., Hernandez, R., Ferreira, D., and Brown, D.T. 2006. Sindbis virus infection of two model insect cell systems—A comparative study. Virus Res. 122:28‐34.
   Sarver, N. and Stollar, V. 1977. Sindbis virus‐induced cytopathic effect in clones of Aedes albopictus (Singh) cells. Virology 80:390‐400.
   Scheefers‐Borchel, U., Scheefers, H., Edwards, J., and Brown, D.T. 1981. Sindbis virus maturation in cultured mosquito cells is sensitive to actinomycin D. Virology 110:292‐301.
   Singh, K.R.P. 1967. Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Curr. Sci. India 36:506‐508.
   Stollar, V. 1993. Insect‐transmitted vertebrate viruses: Alphatogaviruses. In Vitro Cell. Dev. Biol. Anim. 29A:289‐295.
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