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2009, Number 4

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Rev Inst Nal Enf Resp Mex 2009; 22 (4)

Purification, culture and characterization of human fibrocytes from peripheral blood

García-de Alba C, Becerril K, Cruz N

Language: Spanish
References: 17
Page: 288-294
PDF size: 123.52 Kb.


ABSTRACT

Background: Fibrocytes constitute a population of progenitor cells for fibroblasts and myofibroblasts first described in 1994. They co-express markers of mesenchymal cells (collagen I), monocytes (CD14), pan-leukocyte antigen (CD45) and hematopoietic stem cells (CD34). The presence of fibrocytes has been documented in diseases such as asthma and idiopathic pulmonary fibrosis. Until now, the role of fibrocytes in the molecular mechanisms of pulmonary fibrosis is largely unknown, and thus their in vivo and in vitro study is of great importance. Our objective was to establish a technique and optimal culture conditions to obtain highly enriched fibrocytes cultures.
Material and methods: Fibrocytes were obtained from leukocyte concentrates by two different methods, firstly by separating them by their adherent properties, and secondly by CD14+ cells enrichment. Characterization was done by flow cytometry, collagen synthesis assay, collagen quantification in culture supernatant, and immunocytochemistry with an antibody against collagen I. A normal lung fibroblasts cell line was used as positive controls.
Results: Flow cytometry showed that fibrocytes cultures obtained from CD14+ cells had less percentage of contaminant cells (B and T lymphocytes) than cultures obtained by adherence. In addition, a higher percentage of collagen synthesis and more collagen concentration in media cultures were observed in these cultures (CD14+). Finally, immunocytochemistry images showed more expression of collagen I in cultures of fibrocytes differentiated from CD14+ cells.
Conclusions: Our results show that the best technique to obtain highly enriched fibrocytes cultures is through separation and culture of CD14+ cells.


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