Human peritoneal mesothelial cells isolated from spent dialysate fluid maintain contaminating macrophages via production of macrophage colony stimulating factor

Authors

  • MELINDA M TEE,

    1. Department of Nephrology, and
    2. Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia
    Search for more papers by this author
  • GREGORY H TESCH,

    Corresponding author
    1. Department of Nephrology, and
    2. Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia
      Dr Greg Tesch, Department of Nephrology, Monash Medical Centre, 246 Clayton Road, Clayton, Vic. 3168, Australia. Email: greg.tesch@med.monash.edu.au
    Search for more papers by this author
  • DAVID J NIKOLIC-PATERSON,

    1. Department of Nephrology, and
    2. Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia
    Search for more papers by this author
  • FIONA G BROWN

    1. Department of Nephrology, and
    Search for more papers by this author

Dr Greg Tesch, Department of Nephrology, Monash Medical Centre, 246 Clayton Road, Clayton, Vic. 3168, Australia. Email: greg.tesch@med.monash.edu.au

SUMMARY:

Background:  Human peritoneal mesothelial cells (HPMC) are useful for the analysis of peritoneal reactions to various insults and to peritoneal dialysate. HPMC can be readily obtained from spent dialysis fluid, but leucocyte contamination is a major problem when using these cells for in vitro experiments. Therefore, we examined the persistence of leucocyte contamination in HPMC cultures obtained from spent dialysate.

Methods:  Cells were obtained from spent patient dialysate bags by centrifugation and analysed for specific cell phenotypes by flow cytometry at the initial collection and during sequential passages in cell culture. Cell proliferation was assessed by either bromodeoxyuridine incorporation or a dehydrogenase assay. Cytokine secretion was analysed by enzyme-linked immunosorbent assay.

Results:  Spent dialysate bags contained two major cell populations: CD45+ leucocytes and cytokeratin-8/18+ cells. Initially, most collected cells were CD45+, but their numbers decreased rapidly during the first week of culture. However, a persistent contamination of CD45+ leucocytes, approximately 20% of cells, was evident during the next three passages. This persistent CD45+ contamination was identified as CD68+ macrophages and contained bromodeoxyuridine + proliferating cells. These macrophages could be removed by fluorescence-activated cell sorting using anti-CD45 antibody, resulting in highly purified HPMC which expressed cytokeratin-8/18 and calretinin. Supernatant obtained from these purified HPMC contained macrophage colony stimulating factor and induced proliferation of bone marrow-derived macrophages.

Conclusion:  Spent dialysate contains macrophages which persist in culture and are associated with HPMC secretion of macrophage colony stimulating factor and macrophage proliferation. Therefore, contaminating macrophages should be specifically removed from HPMC preparations before performing in vitro studies.

Ancillary