Preparation of Cell Cultures and Vaccinia Virus Stocks

Patricia L. Earl1, Norman Cooper1, Linda S. Wyatt1, Bernard Moss1, Miles W. Carroll2

1 National Institutes of Allergy and Infectious Diseases, Bethesda, Maryland, 2 Oxford BioMedica, Oxford, null
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.12
DOI:  10.1002/0471140864.ps0512s13
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes the maintenance of cell lines used with vaccinia virus, both in monolayer cultures and in suspension. The suspended cell culture is then used in the preparation of vaccinia virus stocks. The preparation of chick embryo fibroblasts (CEF) is also presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Additionally, support protocols are presented for the titration of standard and MVA vaccinia virus stocks.

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

Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Culture of Monolayer Cells
  • Basic Protocol 2: Culture of Cells in Suspension
  • Basic Protocol 3: Preparation of a Vaccinia Virus Stock
  • Support Protocol 1: Titration of Vaccinia Virus Stocks by Plaque Assay
  • Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts
  • Basic Protocol 5: Preparation of an MVA Stock
  • Support Protocol 2: Titration of MVA Stocks by Immunostaining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Culture of Monolayer Cells

 Materials
  • Frozen ampule of cells (Table 5.12.2): S-C-1 (ATCC #CCL26), CV-1 (ATCC #CCL70), HuTK– 143B (ATCC #CRL8303), or BHK-21 (ATCC #CCL10) cells
  • 70% ethanol
  • Start-up medium (Table 5.12.2): complete MEM-20, complete MEM-20/BrdU (see recipes), or complete DMEM-20 (appendix 3C) 37°C
  • Maintenance medium (Table 5.12.2): complete MEM-10, complete MEM-10/BrdU (see recipes), orcomplete DMEM-10 (appendix 3C) 37°C
  • PBS (optional; appendix 2E)
  • Trypsin/EDTA: 0.25% (w/v) trypsin/0.02% (w/v) EDTA, 37°C
  • 25-cm2 and 150-cm2 tissue culture flasks
  • Humidified 37°C, 5% CO2 incubator

Basic Protocol 2: Culture of Cells in Suspension

 Materials
  • Frozen ampule of HeLa S3 cells (ATCC #CCL2.2)
  • 70% ethanol
  • Complete MEM-10 (see recipe), 37°C
  • Trypsin/EDTA: 0.25% (w/v) trypsin/0.02% (w/v) EDTA, 37°C
  • Complete spinner medium-5 (see recipe), 37°C
  • 25-cm2 tissue culture flask
  • Humidified 37°C, 5% CO2 incubator
  • Sorvall H-6000A rotor (or equivalent)
  • 100- or 200-ml vented spinner bottles and caps with filters (Bellco)
  • Additional reagents and equipment for counting cells with a hemacytometer (appendix 3C)

Basic Protocol 3: Preparation of a Vaccinia Virus Stock

 Materials
  • HeLa S3 cells from suspension culture (see Basic Protocol 2)
  • Complete MEM-10 and -2.5 (see recipe), 37°C
  • Vaccinia virus (ATCC #VR1354 or equivalent)
  • 0.25 mg/ml trypsin (2× crystallized and salt-free; Worthington; filter sterilize and store at –20°C)
  • Sorvall H-6000A rotor (or equivalent)
  • 150-cm2 tissue culture flask
  • Humidified 37°C, 5% CO2 incubator
  • Additional reagents and equipment for counting cells with a hemacytometer (appendix 3C)

Support Protocol 1: Titration of Vaccinia Virus Stocks by Plaque Assay

 Additional Materials (also see Basic Protocols 1 and 3)
  • BS-C-1 cells from confluent monolayer culture (see Basic Protocol 1)
  • Virus stock (see Basic Protocol 3)
  • 0.1% (w/v) crystal violet (Sigma) in 20% ethanol (store indefinitely at room temperature)
  • 6-well, 35-mm2 tissue culture dishes
  • Additional reagents and equipment for counting cells with a hemacytometer (appendix 2E)

Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts

 Materials
  • Nine-day-old embryonated eggs (Specific Pathogen Free Eggs, SPAFAS)
  • 70% ethanol
  • MEM with no additives, 37°C
  • Trypsin/EDTA: 0.25% (w/v) trypsin/0.02% (w/v) EDTA, 37°C
  • Complete MEM-10 (Table 5.12.2; see recipe), 37°C
  • Sterile dissecting scissors and forceps
  • 100-cm2 sterile petri dishes
  • 10-ml syringes
  • Sterile trypsinization flask with magnetic stir bar
  • Humidified 37° and 31°C, 5% CO2 incubators
  • 500-ml beakers with two layers of gauze taped over tops
  • Sorvall RC-3B centrifuge and 250-ml centrifuge bottles (or equivalent)
  • 150-cm2 tissue culture flasks

Basic Protocol 5: Preparation of an MVA Stock

 Materials
  • 150-cm2 tissue culture flasks of nearly confluent CEF (see Basic Protocol 4) or BHK-21 cells (see Basic Protocol 1)
  • Complete MEM-10 and -2.5 (see recipe), 37°C
  • Modified vaccinia virus Ankara (MVA; A. Mayr, Institut für Med. Mikrobiologie, Munich, Germany, or B. Moss, email bmoss@nih.gov)
  • 150-cm2 tissue culture flasks
  • Humidified 37°C, 5% CO2 incubator
  • Sorvall RC-3B centrifuge and sterile 250-ml centrifuge bottles (or equivalent)
  • Additional reagents and equipment for trypsinizing cells (see Basic Protocol 1)

Support Protocol 2: Titration of MVA Stocks by Immunostaining

 Materials
  • CEF (see Basic Protocol 4) or BHK-21 cells (see Basic Protocol 1) in a 150-cm2 tissue culture flask
  • Complete MEM-10 and -2.5 (see recipe), 37°C
  • MVA stock (see Basic Protocol 5)
  • 1:1 (v/v) acetone/methanol
  • PBS (appendix 2E), with and without 3% FBS
  • Rabbit anti-vaccinia antibody (e.g., Access BioMedica; or see Linscott, 1998)
  • Horseradish peroxidase–conjugated whole anti–rabbit Ig antibody (HRP-anti-rabbit; Amersham)
  • Dianisidine, or premade peroxidase substrate kit (Sigma)
  • PBS/H2O2 (add 10 µl 30% H2O2 to 10 ml PBS immediately before use)
  • 6-well, 35-mm2 tissue culture dishes
  • Humidified 37°C, 5% CO2 incubator
  • Additional reagents and equipment for trypsinizing cells (see Basic Protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
    Carroll, M.W. and Moss, B. 1997. Host range and cytopathogenicity of the highly attenuated MVA strain of vaccinia virus: Propagation and generation of recombinant viruses in a nonhuman mammalian cell line. Virology 238:198-211.
    Linscott, W.D. 1998. Linscott's Directory of Immunological and Biological Reagents, 10th ed. W.D. Linscott, Santa Rosa, CA.
    Mayr, A., Hochstein-Mintzel, V., and Stickl, H. 1975. Abstammung,eigenschaften and verwendung des attenuierten vaccinia-stammes MVA. Infection 3:6-14.
    Meyer, H., Sutter, G., and Mayer, A. 1991. Mapping of deletions in the genome of the highly attenuated vaccinia virus MVA and their influence on virulence. J. Gen. Virol. 72:1031-1038.
    Sutter, G. and Moss, B. 1992. Nonreplicating vaccinia virus efficiently expresses recombinant genes. Proc. Natl. Acad. Sci.U.S.A. 89:10847-10851.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library