Isolation of Human Intestinal Mucosal Mononuclear Cells

Claudio Fiocchi1, Kenneth R. Youngman2

1 Case Western Reservn University School of Medicine, Cleveland, Ohio, 2 Standford University School of Medicine, Palo Alto, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.30
DOI:  10.1002/0471142735.im0730s19
Online Posting Date:  May, 2001
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The aim of this procedure is to obtain large numbers of isolated, viable, and functional mononuclear cells that are representative of the lymphoid population present in the mucosa of the human gastrointestinal tract under physiological and pathological conditions. The basic protocol is based on the use of surgically resected small and large bowel specimens, and consists of two basic stages: (1) a combination of chemical, enzymatic, and mechanical treatments to dissociate intestinal tissue and free the mononuclear cells from the surrounding interstitial framework; and (2) separation, isolation, and purification of viable mucosal lamina propria mononuclear cells from other cellular and amorphous components. The proportion of viable cells obtained can be increased by including extra separation steps using nylon wool columns or Percoll gradients, as described.

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Table of Contents

  • Support Protocol 1: Removal of Dead Cells by Nylon Wool Filtration
  • Reagents and Solutions
  • Commentary
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Basic Protocol 1:

  • Fresh specimen of large or small bowel
  • Hanks balanced salt solution (HBSS), calcium‐ and magnesium‐free, pH 7.2 ( appendix 2A)
  • recipe0.15% (w/v) DTT/HBSS (prepared fresh; see recipe)
  • recipe1 mM EDTA/HBSS (diluted from 10 mM stock; see recipe)
  • recipeEnzymatic digestive solution (see recipe)
  • recipeComplete RPMI‐10 medium, supplemented (see recipe)
  • Ficoll‐Hypaque solution, density 1.076 to 1.078 g/ml (unit 7.1)
  • recipe30% Percoll solution (see recipe)
  • Disposable 250‐ml flat‐bottom plastic containers with lids (Corning)
  • Curved fine forceps and curved iris scissors
  • 100‐ and 50‐mm plastic petri dishes
  • Smooth, rounded, 5‐cm‐long Teflon‐coated magnetic stir‐bars
  • Magnetic stir‐plates, room temperature and 37°C
  • 250‐ml polycarbonate Erlenmeyer flasks with caps (Corning)
  • 50‐ml conical centrifuge tubes
  • Polystyrene ring (5‐cm diameter, 1‐cm height)
  • Nitex cloth (100‐mm grid; Tetko), cut in 10‐cm squares
  • Surgical gloves, sterile
  • Additional reagents and equipment for cell counting ( appendix 3A), trypan blue exclusion ( appendix 3B), nylon wool filtration (see protocol 2), Ficoll‐Hypaque gradient centrifugation (unit 7.1), and Percoll gradients (unit 3.8)

Support Protocol 1: Removal of Dead Cells by Nylon Wool Filtration

  • 20‐ml nylon wool column, 3‐way stopcock, and ring stand (unit 3.2)
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Literature Cited

Literature Cited
   Bland, P.W., Richens, E.R., Britton, D.C., and Lloyd, J.V. 1979. Isolation and purification of human large bowel mucosal lymphoid cells: Effect of separation technique on functional characteristics. Gut 20:1037‐1046.
   Bull, D.M. and Bookman, M.A. 1977. Isolation and functional characterization of human intestinal mucosal lymphoid cells. J. Clin. Invest. 59:966‐974.
   Clancy, R. 1976. Isolation and kinetic characteristics of mucosal lymphocytes in Crohn's disease. Gastroenterology 70:177‐180.
   Fiocchi, C., Battisto, J.R., and Farmer, R.G. 1979. Gut mucosal lymphocytes in inflammatory bowel disease. Isolation and preliminary functional characterization. Dig. Dis. Sci 24:705‐717.
   Goodacre, R., Davidson, R., Singal, D., and Bienenstock, J. 1979. Morphological and functional characteristics of human intestinal lymphoid cells isolated by a mechanical technique. Gastroenterology 76:300‐308.
   MacDermott, R.P., Franklin, G.O., Jenkins, K.M., Kodner, I.J., Nash, G.S., and Weinrieb, I.J. 1980. Human intestinal mononuclear cells. I. Investigation of antibody‐dependent, lectin‐induced, and spontaneous cell‐mediated cytotoxic capabilities. Gastroenterology 78:47‐56.
Key References
   Bull and Bookman, 1977. See above.
  Original report concerning the enzymatic methodology for isolating human intestinal mucosal mononuclear cells.
   Fiocchi, C. 1985. Lymphoid cells of the gastrointestinal tract. Isolation procedures. Acta. Chirugica Scand. Suppl. 525:11‐23.
  Further detailed description of the methodology.
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