Basic Techniques in Mammalian Cell Tissue Culture

Katy Phelan1, Kristin M. May2

1 Florida Cancer Specialists, Fort Myers, Florida, 2 Children's Hospital at Erlanger, Chattanooga, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Appendix 3B
DOI:  10.1002/cptx.13
Online Posting Date:  November, 2016
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Abstract

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc.

Keywords: mammalian cells; tissue culture; aseptic technique; medium; passaging cells; freezing cells

     
 
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Table of Contents

  • Introduction
  • Aseptic Technique and Biosafety Practices
  • Culture Medium Preparation
  • Basic Protocol 1: Establishing Primary Cultures from Tissue
  • Basic Protocol 2: 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
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Establishing Primary Cultures from Tissue

  Materials
  • Fresh tissue
  • Transport RPMI with Fungizone, serum free (see recipe; optional)
  • Transport RPMI, serum free (see recipe)
  • Complete α‐MEM medium (see recipe)
  • Collagenase Type I, lyophilized, from Clostridium histolyticum, activity greater than 125 U/mg (Life Technologies/Gibco or other source)
  • Sterile petri dishes: 60 × 15 mm or 100 × 15 mm
  • Sterile forceps
  • Sterile plastic serological pipets (1, 5, and 10 ml)
  • Disposable safety scalpels
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 10‐ml sterile syringe with 21‐G needle (Luer‐lok tip and blunt‐fill needle)
  • Nalgene syringe filter (0.2 μm)
  • Clinical centrifuge
  • 25‐cm2 (T‐25) culture flasks
  • Inverted microscope
  • NOTE: This is an aseptic procedure. All precautions must be taken to ensure the protection of the technologist and to maintain sterility of the specimen and reagents. Protective clothing must be worn at all times when handling specimens.

Basic Protocol 2: Trypsinizing and Subculturing Cells from a Monolayer

  Materials
  • Primary cultures of cells ( protocol 1)
  • 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% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Alternate Protocol 1: Passaging Cells in Suspension Culture

  Materials
  • Log‐phase monolayer culture of cells in petri plate
  • Complete medium
  • Freezing medium: complete medium (e.g., 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.

Support Protocol 1: Freezing Human Cells Grown in Monolayer Cultures

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

Alternate Protocol 2: Freezing Cells Grown in Suspension Culture

  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
  • NOTE: A disposable plastic hemacytometer, the INCYTO C‐Chip, has exactly the same grid pattern as the Improved Neubauer. It is a single‐use device available through several distributors.
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Figures

Literature Cited

Literature Cited
  Burgener, J. 2006. Position paper on the use of ultraviolet lights in biological safety cabinets. Appl. Biosaf. 11:228‐230.
  Coté, R. J. 1998. Aseptic technique for cell culture. Curr. Protoc. Cell Biol. 00:1.3.1‐1.3.10. doi: 10.1002/0471143030.cb0103s00.
  Coté, R.J. 1999a. Sterilization and filtration. Curr. Protoc. Cell Biol. 1:1.4.1‐1.4.21. doi: 10.1002/0471143030.cb0104s01.
  Coté, R. 1999b. Assessing and controlling microbial contamination in cell cultures. Curr. Protoc. Cell Biol. 1:1.5.1‐1.5.18. doi: 10.1002/0471143030.cb0105s01.
  Freshney, R.I. 2010. Culture of Animal Cells. A Manual of Basic Technique and Specialized Applications, 6th ed. Wiley‐Blackwell, New York.
  Meechan, P.J. and Wilson, C. 2006. Use of ultraviolet lights in biological safety cabinets: A contrarian view. Appl. Biosaf. 11:222‐227.
  Newman, C. 2003. Serum‐free cell culture—the ethical, scientific and economic choice. The Biomedical Scientist Sept: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.). pp. 173‐197. Lippincott‐Raven, Philadelphia.
  Rooney, D.E. (ed). 2001. Human Cytogenetics: Constitutional Analysis: A Practical Approach, 3rd ed. Oxford University Press, New York.
  Sato, J. D. and Kan, M. 1998. Media for culture of mammalian cells. Curr. Protoc. Cell Biol. 00:1.2.1‐1.2.15. doi: 10.1002/0471143030.cb0102s00.
  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.
  Uphoff, C.C. and Drexler, H.G. 2014. Eradication of mycoplasma contaminations from cell cultures. Curr. Protoc. Mol. Biol. 106:28.5:28.5.1‐28.5.12. doi: 10.1002/0471142727.mb2805s106.
  U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 21‐1112. Washington DC, U.S. Governmental Printing Office:2009. Available at http://cdc.gov/biosafety/publications/bmbl5/BMBL.pdf.
  Volokhov, D.V., Graham, L.J., Brorson, K.A., and Chizhikov,V.E. 2011. Mycoplasma testing of cell substrates and biologics: Review of alternative non‐microbiological techniques. Mol. Cell. Probes 25:69‐77.
Key Reference
  Freshney, R.I. 2010. Culture of Animal Cells. A Manual of Basic Technique and Special Applications, 6th ed. Wiley‐Blackwell, New York
  Contains pertinent information on cell culture requirements and techniques for many tissue types.
Internet Resources
  http://www.atcc.org
  The Web site of the American Type Culture Collection, a nonprofit biological resource center, has an excellent document library for various cell culture topics.
  http://www.coriell.org
  The Web site for the Coriell Institute for Medical Research
  http://www.lifetechnologies.com/us/en/home/technical‐resources/technical‐reference‐library.html
  This Web site has documents about general cell culture, protocols, and troubleshooting.
  http://www.lifescience.roche.com
  The document resources section of the Roche Life Science site contains a technique guide for culture of animal cells.
  http://www.phe‐culturecollections.org.uk
  The Web site for Public Health England includes four separate collections of cell lines and microbial strains. A free handbook on cell culture is available in the section for the European Collection of Cell Cultures (ECACC).
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