Media for Culture of Mammalian Cells

J. Denry Sato1, Mikio Kan2

1 Adirondack Biomedical Research Institute, Lake Placid, New York, 2 Texas A&M University, Houston, Texas
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 1.2
DOI:  10.1002/0471143030.cb0102s00
Online Posting Date:  May, 2001
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Abstract

When mammalian cells are cultured in vitro, the investigator is attempting to reproduce the physiological environment in order to maintain and analyze normal functions and responses. The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This unit provides guidelines for design of serum‐containing and serum‐free media, selective andspecialty media, and media for growth under special conditions such as soft‐agar growth.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Serum‐Containing Media
  • Basic Protocol 2: Preparing Media for Reduced‐Serum or Serum‐Free Growth
  • Basic Protocol 3: Preparation of Selective Media: HAT Medium
  • Alternate Protocol 1: Preparation of Selective Media: Cholesterol‐Free, Serum‐Free Medium
  • Basic Protocol 4: Growth of Transformed Cells in Soft Agar
  • Support Protocol 1: pH Control in Media
  • Support Protocol 2: Use of Antibiotics in Media
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Serum‐Containing Media

  Materials
  • Basal nutrient medium, such as DMEM, Ham's F‐12, or RPMI 1640 ( appendix 2B; see for discussion of medium selection)
  • HEPES (e.g., Research Organics)
  • Sodium bicarbonate (e.g., J.T. Baker)
  • Glutamine and pyruvic acid (e.g., Sigma)
  • Penicillin G and streptomycin sulfate (e.g., Sigma)
  • 5 N NaOH (e.g., J.T. Baker)
  • Serum (e.g., Hyclone, Sigma, UBI; see for discussion of fetal bovine serum)
  • Filter sterilization units (e.g., Nalge Nunc, Corning, Gelman)
  • 0.2‐µm‐pore‐size filters (e.g., Nalge Nunc, Corning, Gelman)
  • Additional reagents and equipment for culture of mammalian cells (unit 1.1)

Basic Protocol 2: Preparing Media for Reduced‐Serum or Serum‐Free Growth

  Materials
  • Basal nutrient medium, such as DMEM, Ham's F‐12, or RPMI 1640 ( appendix 2B; see for discussion of medium selection)
  • Nutrients: inorganic salts, amino acids, and vitamins (e.g., Sigma)
  • Trace elements (e.g., Sigma)
  • Supplements: growth factors and hormones (e.g., Sigma, UBI, Becton Dickinson Labware) and other assorted medium components (Table 1.2.2)
    Table 1.2.2   Materials   Components of Reduced‐Serum and Serum‐Free Medium b   Components of Reduced‐Serum and Serum‐Free Medium

    Component Final concentration Stock concentration Suggested supplier(s)
    Undefined supplements
    Sera c 5% to 20% (v/v) 100% (v/v) Hyclone, Sigma, or UBI
    Pituitary extract 5 to 100 µg/ml 1 to 2 mg/ml UBI, Clonetics
    Conditioned medium 5% to 50% (v/v) 100% (v/v) Not commercially available d
    Energy sources
    Glucose 1 to 4.5 g/liter None Sigma
    Glutamine 1 to 2 mM None Sigma
    Attachment factors
    Collagen type I 10 to 50 µg/ml 3 to 4 mg/ml UBI, Sigma
    Fibronectin 1 to 10 µg/ml 0.5 to 1 mg/ml Sigma
    Vitronectin 1 to 10 µg/ml 0.5 to 1 mg/ml Sigma
    Hormone
    Insulin 1 to 10 µg/ml 1 mg/ml Sigma, UBI
    Carrier proteins
    Transferrin 5 to 30 µg/ml 1 mg/ml Sigma, UBI
    BSA, fatty acid–free 0.5 to 4 mg/ml 50 mg/ml Bayer
    Trace element
    Selenium, sodium salt 1 to 20 nM 2 µM Sigma
    Lipids and lipid precursors
    Ethanolamine 1 to 20 µM 2 mM Sigma
    Fraction V BSA 0.05 to 5 mg/ml 50 mg/ml Sigma
    Unsaturated fatty acids 1 to 10 µg/ml 20 to 50 mg/ml Sigma
    Sterols 1 to 20 µg/ml 2 to 4 mg/ml Steraloids, Sigma
    Low‐density lipoprotein 1 to 20 µg/ml 1 to 2 mg/ml Chemicon International

     bNonsterile stock solutions should be sterilized by filtration. Add glucose and glutamine as dry powder (or frozen aliquots that have been thawed) to reconstituted powdered medium.
     cSee for discussion of FBS.
     dConditioned medium must be made in the investigator's laboratory, and the choice of cells used depends on the investigator's purpose.
  • Additional reagents and equipment for culture of mammalian cells (unit 1.1)

Basic Protocol 3: Preparation of Selective Media: HAT Medium

  Materials
  • Spleen cell × myeloma fusion products (10:1)
  • RD medium (Life Technologies) with 10% FBS (Hyclone; see recipe)
  • 4 × 10−5 M aminopterin (A solution; 100× stock in 0.1 N NaOH)
  • 1 × 10−5 M hypoxanthine/1.6 × 10−3 M thymidine in water (HT solution; 100× stock)
  • HAT medium: RD/10% FBS/1× A solution/1× HT solution
  • 96‐well tissue culture plates
  • Additional reagents and equipment for culture of mammalian cells (unit 1.1)

Alternate Protocol 1: Preparation of Selective Media: Cholesterol‐Free, Serum‐Free Medium

  Materials
  • Spleen cell × NS‐1 myeloma fusion products (10:1)
  • RD medium with 5F supplement (see reciperecipes)
  • 100× BSA–oleic acid conjugate solution: fatty acid−free BSA (e.g., Bayer; 50 mg/ml) conjugated with oleic acid (e.g., Sigma; 500 µg/ml) in PBS
  • 96‐well tissue culture plates
  • Additional reagents and equipment for culture of mammalian cells (unit 1.1)

Basic Protocol 4: Growth of Transformed Cells in Soft Agar

  Materials
  • 2% (w/v) agar (e.g., Difco; see recipe)
  • Basal nutrient medium, such as DMEM, Ham's F‐12, or RPMI 1640 ( appendix 2B; see for discussion of medium selection), with 24.6% and 20% FBS
  • Single‐cell suspension
  • 12‐well culture plates (e.g., Corning Costar)
  • 15‐ml polycarbonate conical centrifuge tubes (e.g., Sarstadt), sterile

Support Protocol 1: pH Control in Media

  Materials
  • Powdered medium without NaHCO 3 or HEPES
  • HEPES (e.g., Research Organics)
  • NaHCO 3 (e.g., J.T. Baker)

Support Protocol 2: Use of Antibiotics in Media

  Materials
  • Antibiotic
  • Sterile solvent
  • 0.2‐µm‐pore‐size sterilizing filter
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Figures

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Literature Cited

Literature Cited
   Barnes, D.W. 1987. Serum‐free animal cell culture. BioTechniques 5:534‐541.
   Barnes, D.W. and Sato, G.H. 1980. Serum‐free cell culture: A unifying approach. Cell 22:649‐655.
   Barnes, D.W., Sirbasku, D.A., and Sato, G.H. (eds.) 1984. Cell Culture Methods for Cell Biology, Vols. 1‐4. Alan R. Liss, New York.
   Baserga, R. (ed.) 1989. Cell Growth and Division: A Practical Approach. Oxford University Press, Oxford.
   Bettger, W.J. and McKeehan, W.L. 1986. Mechanisms of cellular nutrition. Physiol. Rev. 66:1‐35.
   Bottenstein, J., Hayashi, I., Hutchings, S., Masui, H., Mather, J., McClure, D.B., Ohasa, S., Rizzino, A., Sato, G., Serrero, G., Wolfe, R., and Wu, R. 1979. The growth of cells in serum‐free hormone‐supplemented media. Methods Enzymol. 58:94‐109.
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   Ham, R.G. 1984. Formulation of basal nutrient media. In Cell Culture Methods for Cell Biology, Vol. 1 (D.W. Barnes, D.A. Sirbasku, and G.H. Sato, eds.) pp. 3‐21. Alan R. Liss, New York.
   Ham, R.G. and McKeehan, W.L. 1979. Media and growth requirements. Methods Enzymol. 58:44‐93.
   Horibata, K. and Harris, A.W. 1970. Mouse myeloma and lymphomas in culture. Exp. Cell Res. 60:61‐77.
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   Kawamoto, T., Sato, J.D., McClure, D.B., and Sato, G.H. 1983. Development of a serum‐free medium of growth of NS‐1 mouse mycloma cells and its explication to the isolation of NS‐1 hybridomes. Anal. Biochem. 130:445‐453.
   Littlefield, J.W. 1964. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145:709‐710.
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   Myoken, Y., Okamoto, T., Osaki, T., Yabumoto, M., Sato, G.H., Takada, K., and Sato, J.D. 1989. An alternative method for the isolation of NS‐1 hybridomas using cholesterol auxotrophy of NS‐1 mouse myeloma cells. In Vitro Cell Dev. Biol. 25:477‐480.
   Perlman, D. 1979. Use of antibiotics in cell culture media. Methods Enzymol. 58:110‐116.
   Sanford, K.K., Earle, W., and Likely, G.D. 1948. The growth in vitro of single isolated tissue cells. J. Natl. Cancer Inst. 9:229‐246.
   Sato, J.D., Kawamoto, T., McClure, D.B., and Sato, G.H. 1984. Cholesterol requirement of NS‐1 mouse myeloma cells for growth in serum‐free medium. Mol. Biol. Med. 2:121‐134.
   Sato, J.D., Kawamoto, T., and Okamoto, T. 1987. Cholesterol requirement of P3‐X63‐Ag8 and X63‐Ag8.653 myeloma cells for growth in vitro. J. Exp. Med. 165:1761‐1766.
   Sato, J.D., Cao, H.‐T., Kayada, Y., Cabot, M.C., Sato, G.H., Okamoto, T., and Welsh, C.J. 1988. Effects of proximate cholesterol precursors and steriod hormones on mouse myeloma growth in serum‐free medium. In Vitro Cell Dev. Biol. 24:1223‐1228.
   Sato, J.D., Hayashi, I., Hayashi, J., Hoshi, H., Kawamoto, T., McKeehan, W.L., Matsuda, R., Matsuzaki, K., Mills, K.H.G., Okamoto, T., Serrero, G., Sussman, D.J., and Kan, M. 1994. Specific cell types and their requirements. In Basic Cell Culture: A Practical Approach (J.M. Davis, ed.) pp. 181‐222. Oxford University Press, Oxford.
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