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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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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Basic Protocol 1: Prepare Cell Culture Medium Using Aseptic Technique

  • 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

  • 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

  • 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

  • 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

  • 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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  •   FigureFigure 4.3.1 Various forms of contamination artistically rendered on photographs of sub‐confluent cultured adherent cells at ∼400x magnification (40x objective x 10x ocular). (A) An uncontaminated culture. (B) Adherent cells contaminated with rod bacteria. (C) Cells contaminated with cocci bacteria. (D) Cells contaminated with filamentous fungus. (E) Cells contaminated with yeast. (F) Cells contaminated with another cell line or with mycoplasma may look identical to an uncontaminated culture.
  •   FigureFigure 4.3.2 Equipment used in mammalian cell culture. (A) A laminar flow hood, also known as a biosafety cabinet, is used as a clean work space when performing cell culture procedures. The cabinet has a downward airflow that reduces the chance of contamination by airborne particles or aerosols. (B) A water‐jacketed CO2 incubator is used to maintain a temperature of 37°C and physiological pH in cell culture vessels. (C) Liquid nitrogen freezer tanks are used for long‐term storage of frozen cells. (D) An inverted phase microscope with large working distance is used for counting cells and for examining cell density and morphology within culture vessels. (E) A hemacytometer is a specialized glass slide used for counting cells within a precise volume of liquid. (F) An isopropanol freezing chamber is used to slow the cooling rate of cells in cryovials in preparation for long‐term storage in liquid nitrogen freezer tanks.
  •   FigureFigure 4.3.3 Phenol red in basal medium indicates pH. (A) At high pH, basic conditions, phenol red becomes more purple‐red in color indicating the medium is basic. This may occur if there is a problem with the CO2 level in the incubator. (B) At optimal physiological pH, phenol red in a freshly fed culture is a warm red color. (C) At low pH, acidic conditions, phenol red becomes orange and then yellow in color. Medium of this color usually indicates build up of acidic waste products, possibly from overgrowth of the cultured cells or from growth of contaminating organisms.
  •   FigureFigure 4.3.4 Vulnerable areas must be protected from contamination when practicing aseptic technique. (A) The zone above any open vessel is an area of vulnerability and must be kept free from non‐sterile items. Do not pass a hand, arm, or pipettor or any other non‐sterile object above an open vessel. (B) The rim of any cap or lid and the rim and threads on the neck of any vessel are potential avenues for contamination to enter the vessel. Avoid touching these areas. (C) Pipets must remain sterile at their tips and along the shaft. Do not allow a pipet tip or shaft to touch any non‐sterile object.
  •   FigureFigure 4.3.5 A hemacytometer is filled by capillary action. (A) The specialized hemacytometer coverslip is placed over the central counting area. (B) A counting chamber is filled by capillary action by dispensing liquid into the grove and allowing it to be drawn up between the coverslip and the hemacytometer counting surface. (C) The chamber should be filled such that the liquid just reaches the edges of the counting platform. Both chambers must be filled with liquid for proper volume measurement.
  •   FigureFigure 4.3.6 Etched lines mark 1‐mm square grids upon the surface of the hemacytometer counting chamber. (A) Markings indicating the counting region are etched on the counting platform at the tip of the grooved loading region. (B) At low magnification, nine 1‐mm square grids can be viewed, a central finely marked 1‐mm square and eight surrounding 1‐mm squares. (C) At 100× magnification (10× objective × 10× ocular = 100× total magnification) a single 1‐mm square grid fills the field of view.
  •   FigureFigure 4.3.7 Counting pattern for cells within a 1‐mm square grid of a hemacytometer. (A) Count cells within a central 1‐mm square grid. Include cells that touch border lines to the top and left, exclude cells that touch border lines on the bottom and right. In this example, 30 black‐colored cells would be counted. Violet cells would be excluded because they touch the bottom and right border lines. Green cells would be excluded because they are located outside the 1‐mm square grid. (B) To count all the cells once and avoid counting any cell two times, follow a snaking pattern within the 1‐mm square grid. For each smaller internal square, count border cells that fall on the line to the top and left and avoid cells that fall on the border line to the bottom and right.
  •   FigureFigure 4.3.8 Sparse and confluent adherent cell cultures viewed at 100× total magnification (10× objective × 10× ocular). (A) When initially plated or during the early stages of log phase growth, a culture of adherent cells will have many open spaces between cells on the culture vessel surface. (B) When available surface area becomes filled with cells, the culture is confluent. Confluent cultures will cease log phase growth and cells will begin to die.
  •   FigureFigure 4.3.9 Cell number at seeding and confluence for various sizes of culture vessel (HeLa cells). Information on seeding density of HeLa cells adapted from Invitrogen. Density for seeding and for confluence varies greatly with cell type, see Table . Useful numbers for cell culture can be found at:
  •   FigureFigure 4.3.10 Surface area and typical volume of medium used for various size of culture vessel. Areas are approximate; exact surface area varies with culture dish manufacturer.

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
  Description of biosafety levels in Biosafety in Microbiological and Biomedical Laboratories.
  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.
  Question/Answer customer support page for ATCC with convenient search mode.
  Online calculator to determine speed versus relative centrifugal force (RCF) based on rotor radius.
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