Preparation of Cell Cultures and Vaccinia Virus Stocks

Catherine A. Cotter1, Patricia L. Earl1, Linda S. Wyatt1, Bernard Moss1

1 Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.16
DOI:  10.1002/cpmb.33
Online Posting Date:  January, 2017
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Abstract

The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range‐restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.

Keywords: cell culture; vaccinia virus; modified vaccinia virus Ankara

     
 
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Table of Contents

  • 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
  • Support Protocol 2: Trypsinization of Vaccinia Virus Stocks
  • Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts
  • Basic Protocol 5: Preparation of MVA Stock
  • Support Protocol 3: Titration of MVA Stocks by Immunostaining
  • Basic Protocol 6: Purification of Vaccinia Virus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Culture of Monolayer Cells

  Materials
  • Frozen vial of cells (Table 16.16.2): BS‐C‐1 (ATCC #CCL26), HuTK 143B (ATCC #CRL8303), or BHK‐21 (ATCC #CCL10)
  • 70% ethanol
  • Start‐up medium (Table 16.16.2): complete MEM‐16, complete DMEM‐16, or complete MEM‐16/BrdU (see reciperecipes), 37°C
  • Maintenance medium (Table 16.16.2): complete MEM‐8, complete DMEM‐8, or complete MEM‐8/BrdU (see reciperecipes), 37°C
  • PBS ( appendix 22), optional
  • Trypsin/EDTA: 0.25% (w/v) trypsin/0.02% (w/v) EDTA, 37°C
  • 37°C water bath
  • 50‐ml centrifuge tubes
  • Benchtop centrifuge and Sorvall H 6000A rotor or equivalent
  • 75‐ and 162‐cm2 tissue culture flasks
  • 37°C, 5% CO 2 humidified incubator
  • Aspirator

Basic Protocol 2: Culture of Cells in Suspension

  Materials
  • Frozen vial of HeLa S3 cells (ATCC #CCL2.2)
  • 70% ethanol
  • Complete MEM‐8 (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
  • 37°C water bath
  • 50‐ml centrifuge tubes
  • 75‐cm2 tissue culture flask
  • Centrifuge and Sorvall H‐6000A rotor (or equivalent)
  • 37°C, 5% CO 2 humidified incubator
  • Aspirator
  • 100‐ or 200‐ml vented spinner bottles and caps with filters (Bellco)
  • 37°C incubator without CO 2
  • Additional reagents and equipment for counting cells with a cell counter or hemacytometer ( appendix 3F; Phelan, )

Basic Protocol 3: Preparation of a Vaccinia Virus Stock

  Materials
  • HeLa S3 cells from suspension culture (see protocol 2)
  • Complete MEM‐8 and ‐2.5 (see recipe), 37°C
  • Vaccinia virus (ATCC #VR1354 or equivalent)
  • Dry ice/ethanol
  • Refrigerated centrifuge and Sorvall H‐6000A rotor (or equivalent)
  • 162‐cm2 tissue culture flask
  • 37°C, 5% CO 2 humidified incubator
  • Cup sonicator (e.g., Ultrasonic Processor VCX‐750 from Sonics and Materials)
  • 50‐ml screw‐cap plastic tubes, sterile
  • 37°C water bath
  • Additional reagents and equipment for counting cells with a cell counter or hemacytometer ( appendix 3F; Phelan, )

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

  Materials
  • Confluent monolayer of BS‐C‐1 cells (see protocol 1)
  • Complete MEM‐8 and ‐2.5 media
  • Virus stock (see protocol 3)
  • Complete MEM‐2.5 containing 0.5% methylcellulose
  • 0.1% (w/v) crystal violet (Sigma) in 20% ethanol (store indefinitely at room temperature)
  • 6‐well, 35‐mm2 tissue culture dishes
  • 37°C, 5% CO 2 humidified incubator
  • Additional reagents and equipment for counting cells with a cell counter or hemacytometer ( appendix 3F; Phelan, )

Support Protocol 2: Trypsinization of Vaccinia Virus Stocks

  Materials
  • Virus stock (see protocol 3)
  • 0.25 mg/ml trypsin (2× crystallized and salt‐free; Worthington; filter sterilize and store at –20°C)
  • Vortexer
  • 37°C water bath

Basic Protocol 4: Preparation of Chicken Embryo Fibroblasts

  Materials
  • Ten 10‐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
  • FBS
  • Complete MEM‐8 (Table 16.16.2; see recipe), 37°C
  • Sterile dissecting scissors and forceps
  • 100‐cm2 sterile petri dishes
  • 6‐ml syringes
  • Sterile conical trypsinizing flask with spout available from Thomas Scientific and magnetic stir bar
  • 37° and 31°C, 5% CO 2 humidified incubators
  • Sterile 500‐ml beaker with two layers of gauze taped over top
  • Refrigerated centrifuge with Sorvall H‐6000A rotor and 250‐ml centrifuge bottles (or equivalent)
  • 50‐ml centrifuge tubes
  • 162‐cm2 tissue culture flasks

Basic Protocol 5: Preparation of MVA Stock

  Materials
  • 162‐cm2 tissue culture flasks of nearly confluent CEF (see protocol 6) or BHK‐21 cells (see protocol 1)
  • Complete MEM‐8 and ‐2.5 (see recipe), 37°C
  • Modified vaccinia virus Ankara (MVA; ATCC #VR‐1508)
  • 162‐cm2 tissue culture flasks
  • 37°C, 5% CO 2 humidified incubator
  • Sonicator
  • Cell scraper, optional
  • Refrigerated centrifuge with Sorvall H‐6000A rotor and sterile 250‐ml centrifuge bottles (or equivalent)
  • 37°C water bath
  • Additional reagents and equipment for trypsinizing cells (see protocol 1)

Support Protocol 3: Titration of MVA Stocks by Immunostaining

  Materials
  • CEF (see protocol 6) or BHK‐21 cells (see protocol 1) in 162‐cm2 tissue culture flasks
  • Complete MEM‐8 and ‐2.5 (see recipe), 37°C
  • MVA stock (see protocol 7)
  • 1:1 (v/v) acetone/methanol
  • PBS ( appendix 22), with and without 3% FBS
  • Rabbit anti‐vaccinia antibody (e.g., Pierce via ThermoScientific or see Linscott, )
  • Horseradish peroxidase–conjugated whole anti‐rabbit Ig antibody (HRP‐anti‐rabbit; Pierce via ThermoScientific)
  • Dianisidine, or premade peroxidase substrate kit (Vector Laboratories)
  • PBS/H 2O 2 (add 10 μl of 30% H 2O 2 to 10 ml PBS immediately before use)
  • 6‐well, 35‐mm2 tissue culture dishes
  • 37°C, 5% CO 2 humidified incubator
  • Sonicator
  • 37°C water bath
  • Microcentrifuge
  • Microscope
  • Additional reagents and equipment for trypsinizing cells (see protocol 1)
CAUTION: Dianisidine is carcinogenic and the powder should be manipulated with gloves in a fume hood. The 30% H 2O 2 is caustic to skin and should be handled with gloves.

Basic Protocol 6: Purification of Vaccinia Virus

  Materials
  • Vaccinia virus stock (see protocol 3)
  • HeLa S3 cells growing in suspension culture (see protocol 2)
  • Complete spinner medium‐5 (see recipe)
  • 10 mM and 1 mM Tris·Cl, pH 9.0 ( appendix 22)
  • 36% (w/v) sucrose solution in 10 mM Tris·Cl, pH 9.0
  • 40%, 36%, 32%, 28%, and 24% (w/v) sucrose solutions in 1 mM Tris·Cl, pH 9.0
  • Cup sonicator (e.g., Ultrasonic Processor VCX‐750 from Sonics and Materials)
  • 250‐ml conical centrifuge tubes
  • Sorvall centrifuge with H‐6000A rotor (or equivalent)
  • 2‐liter vented spinner flasks (microcarrier type; Bellco)
  • 15‐ or 40‐ml Dounce homogenizer, glass with tight pestle
  • Light microscope
  • Refrigerated ultracentrifuge with Beckman SW 27 or SW 28 rotor (or equivalent) and sterile 38.5‐ml ultracentrifuge tubes
  • Gradient Master (BioComp Instruments), optional
  • Sterile pipets
  • Sterile 15‐ml tubes
  • Spectrophotometer
  • ViroCyt virus counter, optional
  • Additional reagents and equipment for tissue culture and counting cells ( appendix 3F; Phelan, ) and titering virus (see protocol 4)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly. Incubations should be performed in a 37°C, 5% CO 2 humidified incubator unless otherwise specified.
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Literature Cited

Literature Cited
  Antoine, G., Scheiflinger, F., Dorner, F., and Falkner, F.G. 1998. The complete genomic sequence of the modified vaccinia Ankara strain: Comparison with other orthopoxviruses. Virology 244:365‐396. Corrigendum: Virology 350:501‐502. doi: 10.1006/viro.1998.9123.
  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. doi: 10.1006/viro.1997.8845.
  Elroy‐Stein, O. and Moss, B. 2001. Gene expression using the vaccinia virus/T7 RNA polymerase hybrid system. Curr. Protoc. Mol. Biol. 43:16.19.1‐16.19.11. doi: 10.1002/0471142727.mb1619s43.
  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. doi: 10.1007/BF01641272.
  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. doi: 10.1099/0022‐1317‐72‐5‐1031.
  Moss, B. 2013. Poxviridae. In Fields Virology, Sixth ed., Vol. 2 (D.M. Knipe and P.M. Howley, eds.) pp. 2129‐2159. Wolters Kluwer/Lippincott Williams &Wilkins, Philadelphia.
  Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18. doi: 10.1002/0471142727.mba03fs74.
  Sutter, G. and Moss, B. 1992. Nonreplicating vaccinia virus efficiently expresses recombinant genes. Proc. Natl. Acad. Sci. U.S.A. 89:10847‐10851. doi: 10.1073/pnas.89.22.10847.
   Wyatt, L.S., Earl, P.L., and Moss, B. 2015. Generation of recombinant vaccinia viruses. Curr. Protoc. Micribiol. 117:16.17.1‐16.17.18. doi: 10.1002/cpmb.32.
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