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Techniques for Mammalian Cell Tissue Culture

Mary C. Phelan1

1Molecular Pathology Laboratory Network, Maryville, Tennessee

Publication Name: 
Current Protocols in Cytometry
Unit Number: 
Appendix 3B
DOI: 
10.1002/0471142956.cya03bs36
Online Posting Date: 
May, 2006
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Abstract

This unit opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport.

Keywords: mammalian cells; cell culture; trypsinizing; passaging; hemacytometer

     
 
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Table of Contents

  • Unit Introduction
  • Sterile Technique
  • Culture Medium Preparation
  • Basic Protocol: Trypsinizing and Subculturing Cells from a Monolayer
  • Alternate Protocol 1: Passaging Cells in Suspension Culture
  • Support Protocol 1: Freezing Human Cells Grown in Monolayer Cultures
  • Alternate Protocol 2: Freezing Cells Grown in Suspension Culture
  • Support Protocol 2: Thawing and Recovering Human Cells
  • Support Protocol 3: Determining Cell Number and Viability with a Hemacytometer and Trypan Blue Staining
  • Support Protocol 4: Preparing Cells for Transport
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Trypsinizing and Subculturing Cells from a Monolayer

 Materials
  • Primary cultures of cells
  • HBSS (appendix 2A) without Ca2+ and Mg2+, 37°C
  • 0.25% (w/v) trypsin/0.2% EDTA solution (see recipe), 37°C
  • Complete medium with serum: e.g., DMEM supplemented with 10% to 15% (v/v) fetal bovine serum (complete DMEM-10; see recipe), 37°C
  • Sterile Pasteur pipets
  • 37°C warming tray or incubator
  • Tissue culture plasticware or glassware including pipets and 25-cm2 flasks or 60-mm petri plates, sterile

NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.

Support Protocol 1: Freezing Human Cells Grown in Monolayer Cultures

 Materials
  • Log-phase monolayer culture of cells in petri plate
  • Complete medium
  • Freezing medium: complete medium (e.g., DMEM or RPMI; see recipes) supplemented with 10% to 20% (v/v) FBS and 5% to 10% (v/v) DMSO, 4°C
  • Benchtop clinical centrifuge with 45° fixed-angle or swinging-bucket rotor

NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO2 to maintain pH 7.4.

Support Protocol 2: Thawing and Recovering Human Cells

 Materials
  • Cryopreserved cells stored in liquid nitrogen freezer
  • 70% (v/v) ethanol
  • Complete medium (e.g., DMEM or RPMI; see recipes) containing 10% to 20% FBS (see recipe), 37°C

Support Protocol 3: Determining Cell Number and Viability with a Hemacytometer and Trypan Blue Staining

 Materials
  • 70% (v/v) ethanol
  • Cell suspension
  • 0.4% (w/v) trypan blue or 0.4% (w/v) nigrosin, prepared in HBSS (appendix 2A)
  • Hemacytometer with coverslip (Improved Neubauer, Baxter Scientific)
  • Hand-held counter
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure A.3B.1
    Hemacytometer slide (Improved Neubauer) and coverslip. Coverslip is applied to slide and cell suspension is added to counting chamber using a Pasteur pipet. Each counting chamber has a 3 × 3–mm grid (enlarged). The four corner squares (1, 2, 4, and 5) and the central square (3) are counted on each side of the hemacytometer (numbers added).

    View Image

Literature Cited

Literature Cited
    Barch, M.J., Lawce, H.J., and Arsham, M.S. 1991. Peripheral Blood Culture. In The ACT Cytogenetic Laboratory Manual, 2nd ed. (M.J. Barch, ed.) pp. 17-30. Raven Press, New York.
    Freshney, R.I. 1996. Culture of Animal Cells. A Manual of Basic Techniques, 3rd ed. Wiley-Liss, New York.
    Hyclone Labs. 1996. Heat inactivation: Are you wasting your time Art to Science 15:1-5.
    Knutsen, T. 1991. The ACT Cytogenetic Laboratory Manual, 2nd ed. (M.J. Barch, ed.) pp. 563-587. Raven Press, New York.
    Lee, E.C. 1991. Cytogenetic Analysis of Continuous Cell Lines. In The ACT Cytogenetic Laboratory Manual, 2nd ed. (M.J. Barch, ed.) pp. 107-148. Raven Press, New York.
    Mortensen, R.E., Chesnut, J.D., Hoeffler, J.P., and Kingston, R.E. 1997. Selection of transfected mammalian cells. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 9.5.1-9.5.19. John Wiley & Sons, Hoboken, N.J.
    Newman, C. 2003. Serum-free cell culture—the ethical, scientific and economic choice. The Biomedical Scientist, p. 941-942.
    Priest, J.H. 1997. General cell culture principles and fibroblast culture. In The AGT Cytogenetics Laboratory Manual, 3rd ed. (M.J. Barch, T. Knutsen, and J.L. Spurbeck, eds.). Lippincott-Raven, Philadelphia.
    Rooney, D.E. and Czepulskowski, B.H. (eds). 2001. Human Cytogenetics: Constitutional Analysis: A Practical Approach, 3rd ed. Oxford University Press, New York.
    Triglia, R.P. and Linscott, W.D. 1980. Titers of nine complement components, conglutinin and C3b inactivator in adult and fetal bovine sera. Mol. Immunol. 17:741-748.
    Verma, R.S. and Babu, A. 1995. Human Chromosomes: Principles and Techniques, 2nd ed. McGraw-Hill, New York.
 Key Reference
    Lee, E.C. 1991. See above.

Contains pertinent information on cell culture requirements including medium preparation and sterility. Also discusses trypsinization, freezing and thawing, and cell counting.

 Internet Resources
    http://www.unc.edu/depts/tcf/info.html

This site contains troubleshooting information for treating tissue culture contamination, including discussion of contamination risks and recommendations.

    http://www.cdc.gov/od/ohs.pddfiles/BCS-3.pdf

Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety Cabinets, 2nd ed. Sept 2001. U.S. Dept of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention and National Institutes of Health, U.S. Government Printing Office, Washington.

    http://www.yale.edu/oehs/PDG_files/cad_07_01.pdf

Clean Air Device (Primary Containment Device) Program Guide, July 2001, Yale University, Office of Environmental Health and Safety, 135 College Street, New Haven, Conn.

    http://www.ehs.cornell.edu/bio/cabinets.htm
    http://www.yale.edu/oehs/cad2_0.htm

This site contains a comprehensive discussion of the use of biological safety cabinets including operational procedures and the advantages and disadvantages of ultraviolet lights.

     
 
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