Growth and Maintenance of Vero Cell Lines

Nicole C. Ammerman1, Magda Beier‐Sexton1, Abdu F. Azad1

1 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 4E
DOI:  10.1002/9780471729259.mca04es11
Online Posting Date:  November, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Vero cells are derived from the kidney of an African green monkey, and are one of the more commonly used mammalian continuous cell lines in microbiology and molecular and cell biology research. This unit includes protocols for the growth and maintenance of Vero cell lines in a research laboratory setting. Curr. Protoc. Microbiol. 11:A.4E.1‐A.4E.7. © 2008 by John Wiley & Sons, Inc.

Keywords: Vero cells; cell culture techniques; cell line

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation of Vero Cell Culture from Frozen Stocks
  • Basic Protocol 2: Maintenance of Vero Cell Culture
  • Basic Protocol 3: Preparation of Frozen Stocks of Vero Cell Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Propagation of Vero Cell Culture from Frozen Stocks

  Materials
  • Vero cell stock, frozen in liquid nitrogen or at −80°C
  • 70% ethanol solution (used for decontamination of laminar flow hood and objects brought into the hood)
  • Dulbecco's modification of Eagle medium (DMEM), supplemented with 10% heat‐inactivated fetal bovine serum (FBS), filter sterilized (see recipe)
  • 37°C water bath
  • 15‐ml conical tubes, sterile
  • 25‐cm2 or 50‐cm2 tissue culture flasks with vented caps, sterile

Basic Protocol 2: Maintenance of Vero Cell Culture

  Materials
  • Vero cells grown to a confluent monolayer in a 75‐cm2 flask with vented cap
  • DPBS without calcium or magnesium, filter‐sterilized (see appendix 2A)
  • 1× trypsin‐EDTA in DPBS without calcium or magnesium, filter‐sterilized (see recipe)
  • DMEM supplemented with 10% heat‐inactivated FBS, filter‐sterilized (see recipe)
  • Serological pipets, sterile
  • 15‐ml conical tubes, sterile
  • 75‐cm2 tissue culture flasks with vented caps, sterile

Basic Protocol 3: Preparation of Frozen Stocks of Vero Cell Culture

  Materials
  • Dimethyl sulfoxide (DMSO)
  • DMEM supplemented with 20% heat‐inactivated FBS, filter‐sterilized (see recipe)
  • Vero cells grown to a confluent monolayer in a 75‐cm2 flask with vented cap
  • DPBS without calcium or magnesium, filter‐sterilized (see appendix 2A)
  • 1× trypsin‐EDTA in DPBS without calcium or magnesium, filter‐sterilized (see recipe)
  • 15‐ml conical tubes, sterile
  • Serological pipets, sterile
  • Cryovials suitable for freezing at −80°C or liquid nitrogen, sterile
CAUTION: DMSO is hazardous; see unit 1.3 for guidelines on handling, storage, and disposal.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Chen, S.M., Popov, V.L., Feng, H.M., Wen, J., and Walker, D.H. 1995. Cultivation of Ehrlichia chaffeensis in mouse embryo, Vero, BGM, and L929 cells and study of Ehrlichia‐induced cytopathic effect and plaque formation. Infect. Immun. 63:647‐655.
   Hegde, R., Gomes, A.R., Byregowda, S.M., Hugar, P., Giridhar, P., and Renukaprasad, C. 2008. Standardization of large scale production of homologous live attenuated PPR vaccine in India. Trop. Anim. Health Prod. 40:11‐16.
   Mariotti, E., Mirabelli, P., Di Noto, R., Fortunato, G., and Salvatore, F. 2008. Rapid detection of Mycoplasma in continuous cell lines using a selective biochemical test. Leukemia Res. 32:323‐326.
   Nahapetian, A.T., Thomas, J.N., and Thilly, W.G. 1986. Optimization of environment for high density Vero cell culture: Effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites. J. Cell. Sci. 81:65‐103.
   Phelan, M.C. 2007. Basic techniques in mammalian cell tissue culture. Curr. Protoc.Cell Biol. 36:1.1.1‐1.1.18.
   Sato, J.D. and Kan, M. 2001. Media for culture of mammalian cells. Curr. Protoc. Cell Biol. 00:1.2.1‐1.2.15.
   Silva, A.C., Delgado, I., Sousa, M.F.Q., Carrondo, M.J.T., and Alves, P.M. 2008. Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine. Vaccine 26:3305‐3311.
   Uphoff, C.C. and Drexler, H.G. 2002. Comparative antibiotic eradication of Mycoplasma infections from continuous cell lines. In Vitro Cell. Biol. 38:86‐89.
   Zamboni, D.S., Mortara, R.A., and Rabinovitch, M. 2001. Infection of Vero cells with Coxiella burnetii phase II: Relative intracellular bacterial load and distribution estimated by confocal laser scanning microscopy and morphometry. J. Microbiol. Methods 42:223‐232.
Key References
   Coté, R.J. 2001. Aseptic technique for cell culture. Curr. Protoc. Cell Biol. 00:1.3.1‐1.3.10.
  This reference contains a protocol for proper sterile technique using a laminar flow hood.
   Simizu, E. and Terasima, T. (eds.) 1988. Vero Cells—Origin, Properties and Biomedical Applications. Department of Microbiology, Chiba University, Chiba, Japan.
  This reference describes the history of the Vero cell line, and it also includes an English language translation of the original article describing Vero cells (Yasumura and Kawakita, , below).
   Yasumura, Y. and Kawakita, Y. 1963. Studies on SV40 in tissue culture—Preliminary step for cancer research “in vitro.” Nihon Rinsho 21:1201‐1215. (article in Japanese)
  This is the original publication describing the isolation and initial propagation of the Vero cell line.
Internet Resources
  http://www.atcc.org
  The American Type Culture Collection (ATCC) Web site. The ATCC maintains a cell repository, which includes Vero cells. This site provides additional information regarding the growth and maintenance of Vero cells.
  http://www.ecacc.org.uk
  The European Collection of Animal Cell Cultures (ECACC) Web site. The ECACC maintains a cell repository, which includes Vero cells. This site provides additional information regarding the growth and maintenance of Vero cells.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library