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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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Flow cytometric analysis of peritubular endothelial cells isolated by MicroBeads. (A) Flow cytometry analysis of CD146-PE positive endothelial cells before (left panel), after MicroBeads isolation (middle panel), and after MicroBeads isolation with Fc-blocking and trypsin digestion (right panel). Shaded areas are respective isotype controls. (B) Flow cytometry analysis of blood cell (CD45) contamination when CD146-PE positive endothelial cells were isolated with MicroBeads (1:20) with (right panel) or without kidney perfusion (left panel). Shaded areas are respective isotype controls. (C) After perfusion, CD146-PE positive cells before (top left panel) and after MicroBeads isolation at different MicroBeads concentration: 1:5 (top middle panel), 1:20 (top right panel), 1:30 (bottom left panel) and 1:50 (bottom right panel). Shaded areas are respective isotype controls. (G) Statistical analysis of each individual experiment (N = 3). After modification of kidney perfusion and optimization of MicroBeads concentration, the purity of CD146 positive cells increased significantly from 7.5 ± 0.3 (before separation) to as high as 83.3 ± 1.7. Data are expressed as mean ± SEM with N = 3 for each experimental group. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig1: Flow cytometric analysis of peritubular endothelial cells isolated by MicroBeads. (A) Flow cytometry analysis of CD146-PE positive endothelial cells before (left panel), after MicroBeads isolation (middle panel), and after MicroBeads isolation with Fc-blocking and trypsin digestion (right panel). Shaded areas are respective isotype controls. (B) Flow cytometry analysis of blood cell (CD45) contamination when CD146-PE positive endothelial cells were isolated with MicroBeads (1:20) with (right panel) or without kidney perfusion (left panel). Shaded areas are respective isotype controls. (C) After perfusion, CD146-PE positive cells before (top left panel) and after MicroBeads isolation at different MicroBeads concentration: 1:5 (top middle panel), 1:20 (top right panel), 1:30 (bottom left panel) and 1:50 (bottom right panel). Shaded areas are respective isotype controls. (G) Statistical analysis of each individual experiment (N = 3). After modification of kidney perfusion and optimization of MicroBeads concentration, the purity of CD146 positive cells increased significantly from 7.5 ± 0.3 (before separation) to as high as 83.3 ± 1.7. Data are expressed as mean ± SEM with N = 3 for each experimental group. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: Following isolation using anti-CD146 MicroBeads, the peritubular endothelial cells were assessed by FACS analysis. The FACS results show that before magnetic separation, the percentage of CD146-positive cells was around 8% (Figure 1A, left panel). The percentage of CD146-positive cells was around 38% after magnetic separation (Figure 1A, middle panel). To improve purity, we used Fc-blocking and typsinisation. Fc-blocking was used in our isolation of peritubular endothelial cells with anti-CD146 (rat anti-mouse) MicroBeads as described [12] to avoid non-specific binding. To obtain single-cell suspensions from MicroBeads isolation, the tubule fraction underwent digestion with optimized concentrations of trypsin to avoid co-sorting of non-endothelial cells by sorting of endothelial-non-endothelial cell aggregates. The FACS results showed that Fc-blocking and trypsin digestion increased the percentage of CD146 positive endothelial cells from 39% (Figure 1A, middle panel) to 45% (Figure 1A, right panel).Figure 1


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)

Flow cytometric analysis of peritubular endothelial cells isolated by MicroBeads. (A) Flow cytometry analysis of CD146-PE positive endothelial cells before (left panel), after MicroBeads isolation (middle panel), and after MicroBeads isolation with Fc-blocking and trypsin digestion (right panel). Shaded areas are respective isotype controls. (B) Flow cytometry analysis of blood cell (CD45) contamination when CD146-PE positive endothelial cells were isolated with MicroBeads (1:20) with (right panel) or without kidney perfusion (left panel). Shaded areas are respective isotype controls. (C) After perfusion, CD146-PE positive cells before (top left panel) and after MicroBeads isolation at different MicroBeads concentration: 1:5 (top middle panel), 1:20 (top right panel), 1:30 (bottom left panel) and 1:50 (bottom right panel). Shaded areas are respective isotype controls. (G) Statistical analysis of each individual experiment (N = 3). After modification of kidney perfusion and optimization of MicroBeads concentration, the purity of CD146 positive cells increased significantly from 7.5 ± 0.3 (before separation) to as high as 83.3 ± 1.7. Data are expressed as mean ± SEM with N = 3 for each experimental group. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Flow cytometric analysis of peritubular endothelial cells isolated by MicroBeads. (A) Flow cytometry analysis of CD146-PE positive endothelial cells before (left panel), after MicroBeads isolation (middle panel), and after MicroBeads isolation with Fc-blocking and trypsin digestion (right panel). Shaded areas are respective isotype controls. (B) Flow cytometry analysis of blood cell (CD45) contamination when CD146-PE positive endothelial cells were isolated with MicroBeads (1:20) with (right panel) or without kidney perfusion (left panel). Shaded areas are respective isotype controls. (C) After perfusion, CD146-PE positive cells before (top left panel) and after MicroBeads isolation at different MicroBeads concentration: 1:5 (top middle panel), 1:20 (top right panel), 1:30 (bottom left panel) and 1:50 (bottom right panel). Shaded areas are respective isotype controls. (G) Statistical analysis of each individual experiment (N = 3). After modification of kidney perfusion and optimization of MicroBeads concentration, the purity of CD146 positive cells increased significantly from 7.5 ± 0.3 (before separation) to as high as 83.3 ± 1.7. Data are expressed as mean ± SEM with N = 3 for each experimental group. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: Following isolation using anti-CD146 MicroBeads, the peritubular endothelial cells were assessed by FACS analysis. The FACS results show that before magnetic separation, the percentage of CD146-positive cells was around 8% (Figure 1A, left panel). The percentage of CD146-positive cells was around 38% after magnetic separation (Figure 1A, middle panel). To improve purity, we used Fc-blocking and typsinisation. Fc-blocking was used in our isolation of peritubular endothelial cells with anti-CD146 (rat anti-mouse) MicroBeads as described [12] to avoid non-specific binding. To obtain single-cell suspensions from MicroBeads isolation, the tubule fraction underwent digestion with optimized concentrations of trypsin to avoid co-sorting of non-endothelial cells by sorting of endothelial-non-endothelial cell aggregates. The FACS results showed that Fc-blocking and trypsin digestion increased the percentage of CD146 positive endothelial cells from 39% (Figure 1A, middle panel) to 45% (Figure 1A, right panel).Figure 1

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