Techniques for Mammalian Cell Tissue Culture

Mary C. Phelan1

1 Molecular Pathology Laboratory Network, Maryville, Tennessee
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Appendix 3B
DOI:  10.1002/0471142727.nsa03bs38
Online Posting Date:  January, 2007
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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

  • Sterile Technique
  • Culture Medium Preparation
  • Basic Protocol 1: 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 1: Trypsinizing and Subculturing Cells from a Monolayer

  Materials
  • Primary cultures of cells
  • HBSS (See recipe) without Ca2+ and Mg2+, 37°C
  • 0.25% (w/v) trypsin/0.2% EDTA solution (see recipe), 37°C
  • Complete medium (e.g., complete DMEM; see recipe) supplemented with 10% to 15% (v/v) fetal bovine serum, 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% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

Alternate Protocol 1: Passaging Cells in Suspension Culture

  Materials
  • See protocol 1
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

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., complete DMEM or RPMI; see reciperecipes) 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% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

Alternate Protocol 2: Freezing Cells Grown in Suspension Culture

  Materials
  • Cells in suspension culture
  • Centrifuge tubes, sterile
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

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 reciperecipes) containing 10% to 20% FBS (see recipe), 37°C
CAUTION: Protective clothing, particularly insulated gloves and goggles, should be worn when removing frozen vials or ampules from the liquid nitrogen freezer. The room containing the liquid nitrogen freezer should be well‐ventilated. Care should be taken not to spill liquid nitrogen on the skin.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.

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 (See recipe)
  • Hemacytometer with coverslip (Improved Neubauer, Baxter Scientific)
  • Hand‐held counter
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Figures

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.
   Newman, C. 2003. Serum‐free cell culture—the ethical, scientific and economic choice. The Biomedical Scientist, pp. 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, 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.pdffiles/BSC‐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.cdc.gov/od/ohs/symp5/jyrtext.htm
  The 1, 2, 3's of Biosafety Levels by J.Y. Richmond. Adapted from Richmond, J.Y. and McKinney, R.W. 1993. Biosafety in Microbiological and Biomedical Laboratories, 3rd ed. U.S. Department of Health and Human Services, CDC/NIH, U.S. Government Printing Office, Washington, D.C.
   http://www.ehs.cornell.edu/bio/cabinets.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.
   http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4aa.htm
  This site contains information pertaining to design, function, and use of biological safety cabinets, including comparison of Class I, II, and III safety cabinets.
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