Generation, Isolation, and Maintenance of Human Mast Cells and Mast Cell Lines Derived from Peripheral Blood or Cord Blood

Madeleine Rådinger1, Bettina M. Jensen2, Hye Sun Kuehn1, Arnold Kirshenbaum2, Alasdair M. Gilfillan1

1 Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 2 Allergy Clinic, Copenhagen University Hospital, Copenhagen, Denmark
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.37
DOI:  10.1002/0471142735.im0737s90
Online Posting Date:  August, 2010
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Abstract

Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identify potential ways to prevent them, have primarily been conducted in rodent mast cells. However, to understand how these responses pertain to human disease, and to investigate and develop novel therapies for the treatment of human mast cell–driven disease, human mast cell models may have greater relevance. Recently, a number of systems have been developed to allow investigators to readily obtain sufficient quantities of human mast cells to conduct these studies. These mast cells release the appropriate suite of inflammatory mediators in response to known mast cell activators including antigen. These systems have also been employed to examine the signaling events regulating these responses. Proof of principle studies has also demonstrated utility of these systems for the identification of potential inhibitors of mast cell activation and growth. In this unit, techniques for the development and culture of human mast cells from their progenitors and the culture of human mast cell lines are described. The relative merits and drawbacks of each model are also described. Curr. Protoc. Immunol. 90:7.37.1-7.37.12. © 2010 by John Wiley & Sons, Inc.

Keywords: FcRI; KIT; mast cells; human mast cells; CD34+ peripheral blood progenitor cells; HMC1 cells; LAD2 cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth and Culture of Primary Human Mast Cells from Peripheral Blood-Derived CD34+ Cells
  • Support Protocol 1: Surface Staining of FcRI and Kit
  • Support Protocol 2: Toluidine Blue Staining
  • Basic Protocol 2: Generation and Culture of Cord Blood–Derived Human Mast Cells
  • Basic Protocol 3: Culturing of LAD2 Cells
  • Basic Protocol 4: Culturing of the HMC1.1 and HMC1.2 Cell Lines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Growth and Culture of Primary Human Mast Cells from Peripheral Blood-Derived CD34+ Cells

 Materials
  • Frozen aliquot of cells (1 × 107 CD34+ cells) in cryotubes obtained previously from cord blood or CD34+ progenitors obtained from peripheral blood
  • Human mast cell culture medium (see recipe)
  • 37°C water bath
  • 15- and 50-ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • Tabletop centrifuge
  • 175-cm2 culture flasks (e.g., Nunc)
  • 37°C, 5% CO2 humidified incubator 1 ml

Support Protocol 1: Surface Staining of FcRI and Kit

 Materials
  • Cells (see Basic Protocol 1)
  • Phosphate-buffered saline (PBS; appendix 2A) containing 0.1% BSA (4°C)
  • Anti-FcRI-APC (eBioscience)
  • Mouse IgG-APC (eBioscience)
  • Anti-CD117-PE (BD Pharmingen)
  • Mouse IgG1-PE (BD Pharmingen)
  • Centrifuge
  • 5-ml polystyrene round-bottom tubes
  • Flow cytometer

Support Protocol 2: Toluidine Blue Staining

 Materials
  • Cells (see Basic Protocol 1)
  • Phosphate-buffered saline (PBS; appendix 2A) containing 0.1% BSA (4°C)
  • Mota's fixative (see recipe)
  • Toluidine blue solution (see recipe)
  • Permount
  • Centrifuge
  • Cytospin slides
  • Cytocentrifuge (cytospin/Shandon)
  • Coverslips

Basic Protocol 2: Generation and Culture of Cord Blood–Derived Human Mast Cells

 Materials
  • 100 ml heparinized cord blood obtained within 30 min of newborn delivery
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Ficoll-Paque (GE Healthcare)
  • CD133 MicroBead kit (Miltenyi Biotech) containing:
    • FcR blocking reagent
    • CD133 MicroBeads
  • MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA), 4°C
  • 0.5% trypan blue stain in saline
  • Human cord blood mast cell culture medium (see recipe)
  • Fetal bovine serum
  • Tabletop centrifuge
  • 15- and 50-ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • LS+ separation column (Miltenyi Biotech)
  • MACS separator for LS columns
  • 175-cm2 culture flasks (e.g., Nunc)

Basic Protocol 3: Culturing of LAD2 Cells

 Materials
  • Frozen aliquot of cells in pZerve
  • 0.4% trypan blue solution
  • LAD2 cell culture medium (see recipe)
  • pZerve cryopreservative supplemented with 200 ng/ml SCF
  • Liquid nitrogen
  • Hemacytometer
  • 15-ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • Sterile 6-well plates
  • Platform shaker
  • 37°C incubator
  • Tabletop centrifuge with plate holder
  • 75-cm2 culture flasks
  • Cryotubes
  • Nalgene cryo container

Basic Protocol 4: Culturing of the HMC1.1 and HMC1.2 Cell Lines

 Materials
  • Frozen aliquot of cells
  • HMC1.1 and 1.2 cell culture medium (see recipe)
  • 15-ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • Tabletop centrifuge
  • 75-cm2 culture flasks
  • 37°C, 5% CO2 humidified incubator
  • Hemacytometer
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Figures

Videos

Literature Cited

Literature Cited
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    Butterfield, J.H., Weiler, D., Dewald, G., and Gleich, G.J. 1988. Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk. Res. 12:345-355.
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    Holm, M., Andersen, H.B., Hetland, T.E., Dahl, C., Hoffmann, H.J., Junker, S., and Schiotz, P.O. 2008. Seven week culture of functional human mast cells from buffy coat preparations. J. Immunol. Methods 336:213-221.
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    Kuehn, H.S., Radinger, M., and Gilfillan, A.M. 2010. Measuring mast cell mediator release. Curr. Protoc. Immunol. In press.
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    Qiao, H., Andrade, M.V., Lisboa, F.A., Morgan, K., and Beaven, M.A. 2006. FcR1 and toll-like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells. Blood 107:610-618.
    Rivera, J. and Gilfillan, A.M. 2006. Molecular regulation of mast cell activation. J. Allergy Clin. Immunol. 117:1214-1225.
    Ryzhov, S., Goldstein, A.E., Biaggioni, I., and Feoktistov, I. 2006. Cross-talk between G(s)- and G(q)-coupled pathways in regulation of interleukin-4 by A(2B) adenosine receptors in human mast cells. Mol. Pharmacol. 70:727-735.
    Sundstrom, M., Vliagoftis, H., Karlberg, P., Butterfield, J.H., Nilsson, K., Metcalfe, D.D., and Nilsson, G. 2003. Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-KIT proto-oncogene. Immunology 108:89-97.
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