Mammalian Cell Tissue Culture Techniques

Katy Phelan1, Kristin M. May2

1 Florida Cancer Specialists and Research Institute, Fort Myers, Florida, 2 Genetic Diagnostic Laboratory, Children's Hospital at Erlanger, Chattanooga, Tennessee
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Appendix A.3F
DOI:  10.1002/cpmb.31
Online Posting Date:  January, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Cultured mammalian cells are used extensively in molecular biology studies. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This appendix 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. © 2017 by John Wiley & Sons, Inc.

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

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

Table of Contents

  • Introduction
  • Aseptic Technique and Biosafety Practices
  • Culture Medium Preparation
  • Continuous Cell Lines
  • 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 Cells Grown in Monolayer Cultures
  • Alternate Protocol 2: Freezing Cells Grown in Suspension Culture
  • Support Protocol 2: Thawing and Recovering 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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Establishing Primary Cultures from Tissue

  Materials
  • Fresh tissue
  • Complete RPMI (see recipe)
  • Complete RPMI with Fungizone (optional; recommended if tissue was not collected aseptically; 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 without Ca2+ and Mg2+ (e.g., Life Technologies), 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 Cells Grown in Monolayer Cultures

  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

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

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.
  Hussain, T. and Mulherkar, R. 2012. Lymphoblastoid cell lines: A continuous in vitro source of cells to study carcinogen sensitivity and DNA repair. Int. J. Mol. Cell Med. 1:75‐87.
  Masters, J.R.W. and Palsson, B. (eds.) 2002a. Human Cell Culture Vol I Cancer Cell Lines Part 1. Kluwer Academic Publishers, New York.
  Masters, J.R.W. and Palsson, B. (eds.) 2002b. Human Cell Culture Vol II Cancer Cell Lines Part 2. Kluwer Academic Publishers, New York.
  Masters, J.R.W. and Palsson, B. (eds.) 2002c. Human Cell Culture Vol III Cancer Cell Lines Part 3: Leukemia and Lymphoma. Kluwer Academic Publishers, 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. Biomed. Sci. 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.
  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. doi: 10.1016/0161‐5890(80)90144‐3.
  U.S. Department of Health and Human Services. 2009. Centers for Disease Control and Prevention. Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS Publication No. (CDC) 21‐1112. Governmental Printing Office, Washington D.C. Available at http://cdc.gov/biosafety/publications/bmbl5/BMBL.pdf.
  Uphoff, C. C. and Drexler, H. G. 2014. Detection of mycoplasma contamination in cell cultures. Curr. Protoc. Mol. Biol. 106:28.4.1‐28.4.14.
  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. doi: 10.1016/j.mcp.2011.01.002.
Key Reference
  Freshney, R.I. 2010. See above.
  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 non‐profit 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 technical 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).
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library