Mammalian Cell Culture

Lisa Sandell1, Daisuke Sakai2

1 Stowers Institute for Medical Research, Kansas City, Missouri, 2 Nara Institute of Science and Technology, Nara, Japan
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 4.3
DOI:  10.1002/9780470089941.et0403s5
Online Posting Date:  July, 2011
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Abstract

Mammalian cell culture is the process of growing animal cells in vitro in a flask or dish. This unit describes the methods, equipment, supplies, and reagents used in a cell culture laboratory. Because preventing contamination and maintaining purity of cell cultures is arguably the greatest challenge in animal cell culture, the principles of aseptic technique in mammalian cell culture are presented in detail. Cell culture media and additives are discussed. A method of counting cells and assessing their viability with a hemacytometer is illustrated. The steps needed to maintain an adherent cell line by feeding and passing cells from one culture vessel to another are presented and variations for passing suspension‐grown cells are also outlined. Long‐term cryogenic storage of cells in liquid nitrogen is discussed and a protocol for freezing cells is provided, as are details for thawing and recovering viable cells from frozen stocks. Curr. Protoc. Essential Lab. Tech. 5:4.3.1‐4.3.32. © 2011 by John Wiley & Sons, Inc.

Keywords: cell culture; aseptic technique; culture medium; passage; adherent cells; cryopreservation; liquid nitrogen freezer; hemacytometer

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Prepare Cell Culture Medium Using Aseptic Technique
  • Basic Protocol 2: Counting Cell Number and Assessing Cell Viability
  • Basic Protocol 3: Passage of Adherent Cells
  • Alternate Protocol 1: Passage of Cells Grown in Suspension
  • Basic Protocol 4: Freezing Cells
  • Basic Protocol 5: Thawing and Recovering Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Prepare Cell Culture Medium Using Aseptic Technique

  Materials
  • 70% ethanol spray bottle
  • DMEM basal medium
  • Calf serum
  • 100× penicillin/streptomycin
  • Glutamine (stable dipeptide form)
  • Biosafety cabinet (laminar flow hood)
  • 5‐ and 25‐ml sterile, individually wrapped, plugged pipets
  • 100‐ml sterile bottles
  • Pipettor
  • 500‐ml sterile filter unit with 0.2‐µm filter
  • Vacuum source

Basic Protocol 2: Counting Cell Number and Assessing Cell Viability

  Materials
  • 70% ethanol
  • Cells suspended in medium
  • 0.4% (w/v) trypan blue in PBS
  • Hemacytometer with coverslip (improved Neubauer)
  • Micropipettor and tips
  • Inverted microscope with 10× objective
  • Counter
  • Kimwipes

Basic Protocol 3: Passage of Adherent Cells

  Materials
  • Complete culture medium containing serum and antibiotics
  • Wash solution: a balanced salt solution without serum, calcium, or magnesium (e.g., PBS, HBSS, or basal medium past expiration)
  • 70% ethanol
  • 0.25% trypsin/1 mM EDTA (frozen in 5‐ or 10‐ml aliquots stored up to 2 years at −20°C)
  • Cells growing in 37°C incubator
  • 37°C water bath
  • Unplugged pipets (plastic or glass Pasteur pipets)
  • Vacuum waste flask and vacuum source
  • 5‐, 10‐, and 25‐ml plugged plastic pipets (disposable, individually wrapped)
  • 15‐ml tubes
  • Centrifuge
  • Hemacytometer
  • New, sterile culture flasks or dishes

Alternate Protocol 1: Passage of Cells Grown in Suspension

  Materials
  • Freezing medium (see recipe)
  • Complete medium (see recipe)
  • DMSO
  • Healthy culture of cells in log phase
  • Cryotubes
  • Isopropanol freezing chamber or Styrofoam box
  • −80°C freezer
  • Liquid nitrogen freezer
CAUTION: Handle DMSO with caution as it can penetrate gloves and skin.

Basic Protocol 4: Freezing Cells

  Materials
  • Fresh complete medium (see recipe), pre‐warmed to 37°C
  • Frozen cells
  • 70% ethanol
  • 37°C water bath
  • Container of dry ice for transport of cells, optional
  • Sterile culture dish or flask
  • Cell culture incubator
  • Inverted microscope
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Figures

Literature Cited

   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   U.S. Department of Health and Human Services, 2009. Biosafety in Microbiological and Biomedical Laboratories, 5th ed. ( L.C. Chosewood and D.E. Wilson, eds.) Centers for Disease Control and Prevention. National Institutes of Health. U.S. Government Printing Office, Washington, D.C.
Key Reference
   Freshney, R.I. 2005. Culture of Animal Cells: A Manual of Basic Technique, 5th ed. Wiley‐Liss, New York.
  An extensive compendium of information covering all aspects of animal cell culture.
Internet Resources
   http://www.cdc.gov/biosafety/publications/bmbl5/BMBL5_sect_IV.pdf
  Description of biosafety levels in Biosafety in Microbiological and Biomedical Laboratories.
  http://www.atcc.org/
  Homepage for the biological repository known as ATCC. Many cell lines are available for purchase. Information such as the animal source, culture conditions, and growth kinetics are available for many cell lines.
  http://atcc.custhelp.com/app/home
  Question/Answer customer support page for ATCC with convenient search mode.
   http://www.sciencegateway.org/tools/rotor.htm
  Online calculator to determine speed versus relative centrifugal force (RCF) based on rotor radius.
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