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


CONCLUSIONS
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.
Tee
Melinda M
MM
Department of Nephrology, and Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia.
Tesch
Gregory H
GH
Nikolic-Paterson
David J
DJ
Brown
Fiona G
FG
D015703:Antigens, CD / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D064032:Calbindin 2; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D017077:Culture Media, Conditioned / Q000378:metabolism; D015314:Dialysis Solutions; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D053538:Keratin-18 / Q000032:analysis; D053533:Keratin-8 / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000032:analysis; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis*; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000378:metabolism*; D019899:Paracrine Communication; D010530:Peritoneal Dialysis; D010537:Peritoneum / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D010641:Phenotype; D064030:S100 Calcium Binding Protein G / Q000032:analysis; D013997:Time Factors; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D064032:Calbindin 2; D017077:Culture Media, Conditioned; D015314:Dialysis Solutions; D053538:Keratin-18; D053533:Keratin-8; D017493:Leukocyte Common Antigens; D016173:Macrophage Colony-Stimulating Factor; D064030:S100 Calcium Binding Protein G; C576764:CALB2 protein, human; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C516769:PTPRC protein, human