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Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.

Zhao Y, Zhao H, Zhang Y, Tsatralis T, Cao Q, Wang Y, Wang Y, Wang YM, Alexander SI, Harris DC, Zheng G - BMC Cell Biol. (2014)

Bottom Line: The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%.Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yzha7726@uni.sydney.edu.au.

ABSTRACT

Background: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.

Results: Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.

Conclusion: In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

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Assessments of purity of cultured endothelial cells. (A) Flow cytometry analysis of CD11c, Lyve-1, CD45, F4/80, FSP-1 and PDGFR-β positive cells in MicroBeads isolated peritubular endothelial cells after 2 days of culture. Shaded areas are respective isotype controls. (B) Flow cytometry analysis of the MicroBeads isolated peritubular endothelial cells by CD146, CD31 and PV-1 after 2 days of culture with statistical analysis (N = 3 independent experiments) (bottom panel).
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Fig2: Assessments of purity of cultured endothelial cells. (A) Flow cytometry analysis of CD11c, Lyve-1, CD45, F4/80, FSP-1 and PDGFR-β positive cells in MicroBeads isolated peritubular endothelial cells after 2 days of culture. Shaded areas are respective isotype controls. (B) Flow cytometry analysis of the MicroBeads isolated peritubular endothelial cells by CD146, CD31 and PV-1 after 2 days of culture with statistical analysis (N = 3 independent experiments) (bottom panel).

Mentions: After incubation with the optimal concentration of MicroBeads (1:30) and following immunomagnetic separation, the percentage of CD146-positive cells remained stable at about 80%. Magnetically isolated peritubular endothelial cells were cultured for two days and then analyzed with antibodies for different cell surface markers to determine the purity of endothelial cells and possible contamination of other cells by FACS. The peritubular endothelial cells were incubated with antibodies for lymphatic vessel endothelial cell marker Lyve-1, dendritic cell marker CD11c, hematopoietic cell marker CD45, macrophage cell marker F4/80, fibroblast cell marker FSP-1, pericyte cell marker PDFGR-β, and endothelial cell markers CD146 and CD31. To distinguish from glomerular endothelial cells, PV-1 was used as peritubular endothelial marker [13]. The FACS results showed that the respective percentage for other cell type was less than 1.83% (Figure 2A). The percentage of CD31 positive cells was 78% and that of CD146 positive cells was 92% (Figure 2B). These results could be due to CD31 being trypsin sensitive [12] while CD146 is trypsin resistant. However, the percentage of contaminating cells could potentially be higher if their surface markers were trypsin sensitive. The results after 2 days culture suggested that culturing of isolated peritubular endothelial cells further improved their purity, possibly through separation of adherent endothelial cells from other non-adherent cells. Over 2 million cells were obtained from each mouse.Figure 2


Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.

Zhao Y, Zhao H, Zhang Y, Tsatralis T, Cao Q, Wang Y, Wang Y, Wang YM, Alexander SI, Harris DC, Zheng G - BMC Cell Biol. (2014)

Assessments of purity of cultured endothelial cells. (A) Flow cytometry analysis of CD11c, Lyve-1, CD45, F4/80, FSP-1 and PDGFR-β positive cells in MicroBeads isolated peritubular endothelial cells after 2 days of culture. Shaded areas are respective isotype controls. (B) Flow cytometry analysis of the MicroBeads isolated peritubular endothelial cells by CD146, CD31 and PV-1 after 2 days of culture with statistical analysis (N = 3 independent experiments) (bottom panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260259&req=5

Fig2: Assessments of purity of cultured endothelial cells. (A) Flow cytometry analysis of CD11c, Lyve-1, CD45, F4/80, FSP-1 and PDGFR-β positive cells in MicroBeads isolated peritubular endothelial cells after 2 days of culture. Shaded areas are respective isotype controls. (B) Flow cytometry analysis of the MicroBeads isolated peritubular endothelial cells by CD146, CD31 and PV-1 after 2 days of culture with statistical analysis (N = 3 independent experiments) (bottom panel).
Mentions: After incubation with the optimal concentration of MicroBeads (1:30) and following immunomagnetic separation, the percentage of CD146-positive cells remained stable at about 80%. Magnetically isolated peritubular endothelial cells were cultured for two days and then analyzed with antibodies for different cell surface markers to determine the purity of endothelial cells and possible contamination of other cells by FACS. The peritubular endothelial cells were incubated with antibodies for lymphatic vessel endothelial cell marker Lyve-1, dendritic cell marker CD11c, hematopoietic cell marker CD45, macrophage cell marker F4/80, fibroblast cell marker FSP-1, pericyte cell marker PDFGR-β, and endothelial cell markers CD146 and CD31. To distinguish from glomerular endothelial cells, PV-1 was used as peritubular endothelial marker [13]. The FACS results showed that the respective percentage for other cell type was less than 1.83% (Figure 2A). The percentage of CD31 positive cells was 78% and that of CD146 positive cells was 92% (Figure 2B). These results could be due to CD31 being trypsin sensitive [12] while CD146 is trypsin resistant. However, the percentage of contaminating cells could potentially be higher if their surface markers were trypsin sensitive. The results after 2 days culture suggested that culturing of isolated peritubular endothelial cells further improved their purity, possibly through separation of adherent endothelial cells from other non-adherent cells. Over 2 million cells were obtained from each mouse.Figure 2

Bottom Line: The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%.Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Transplant and Renal Research, Westmead Millennium Institute, The University of Sydney, Sydney, NSW, Australia. yzha7726@uni.sydney.edu.au.

ABSTRACT

Background: Endothelial-mesenchymal transition (EndoMT) has been shown to be a major source of myofibroblasts, contributing to kidney fibrosis. However, in vitro study of endothelial cells often relies on culture of isolated primary endothelial cells due to the unavailability of endothelial cell lines. Our recent study suggested that peritubular endothelial cells could contribute to kidney fibrosis through EndoMT. Therefore, successful isolation and culture of mouse peritubular endothelial cells could provide a new platform for studying kidney fibrosis. This study describes an immunomagnetic separation method for the isolation of mouse renal peritubular endothelial cells using anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain endothelial phenotype.

Results: Flow cytometry showed that after isolation and two days of culture, about 95% of cells were positive for endothelial-specific marker CD146. The percentage of other cells, including dendritic cells (CD11c) and macrophages (F4/80), was less than 1%. Maintenance of endothelial cell phenotype required vascular endothelial growth factor (VEGF) and co-culture with mouse proximal tubular epithelial cells.

Conclusion: In this study, we established a method for the isolation of mouse renal peritubular endothelial cells by using immunomagnetic separation with anti-CD146 MicroBeads, followed by co-culture with mouse renal proximal tubular epithelial cells to maintain phenotype.

Show MeSH
Related in: MedlinePlus