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Generation of easily accessible human kidney tubules on two-dimensional surfaces in vitro.

Zhang H, Lau SF, Heng BF, Teo PY, Alahakoon PK, Ni M, Tasnim F, Ying JY, Zink D - J. Cell. Mol. Med. (2010)

Bottom Line: However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture.Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology.The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption.

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Affiliation: Institute of Bioengineering and Nanotechnology, The Nanos, Singapore, Singapore.

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The process of tubule formation on 2D surfaces. (A)–(D) and (H) show images obtained by epifluorescence microscopy after immunodetection of zonula occludens-1 (ZO-1) (green) and α-SMA (red). Nuclei were counterstained with 4′,6′-diamidino-2′-phenylindole (DAPI) (blue). The other panels show images obtained by (E, F) differential interference contrast microscopy and (G) bright-field microscopy. In all cases, the HPTCs were cultured on the bottom of the wells of 24-well plates. (A) First, a well-differentiated epithelial monolayer is formed. (B) Subsequently, myofibroblast aggregates that are strongly positive for α-SMA appear. (C, E) The monolayer then retracts on the one side of the myofibroblast aggregates, leaving a surface devoid of cells (left half in C). (D, F) The monolayer subsequently retracts on the other side of the myofibroblast aggregates. This leads to the formation of cell stripes, which include myofibroblast aggregates (note: myofibroblast aggregates are labelled with arrowheads in E, F and G). (G, H) Finally, large renal tubules are formed on the 2D surface. Several images were stitched together in order to cover the entire tubule shown in (G). Scale bars: (A) 100 μm, (B–F, H) 200 μm, and (G) 1 mm.
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fig01: The process of tubule formation on 2D surfaces. (A)–(D) and (H) show images obtained by epifluorescence microscopy after immunodetection of zonula occludens-1 (ZO-1) (green) and α-SMA (red). Nuclei were counterstained with 4′,6′-diamidino-2′-phenylindole (DAPI) (blue). The other panels show images obtained by (E, F) differential interference contrast microscopy and (G) bright-field microscopy. In all cases, the HPTCs were cultured on the bottom of the wells of 24-well plates. (A) First, a well-differentiated epithelial monolayer is formed. (B) Subsequently, myofibroblast aggregates that are strongly positive for α-SMA appear. (C, E) The monolayer then retracts on the one side of the myofibroblast aggregates, leaving a surface devoid of cells (left half in C). (D, F) The monolayer subsequently retracts on the other side of the myofibroblast aggregates. This leads to the formation of cell stripes, which include myofibroblast aggregates (note: myofibroblast aggregates are labelled with arrowheads in E, F and G). (G, H) Finally, large renal tubules are formed on the 2D surface. Several images were stitched together in order to cover the entire tubule shown in (G). Scale bars: (A) 100 μm, (B–F, H) 200 μm, and (G) 1 mm.

Mentions: In accordance with our previous results, we observed initial formation of a flat and well-differentiated epithelial monolayer when HPTCs were cultured in multiwell plates (Fig. 1A). Subsequently, increasing amounts of α-SMA-expressing myofibroblasts appeared. These myofibroblasts formed large aggregates (Fig. 1B). In the surroundings of such aggregates, the epithelium became reorganized. Firstly, highly coordinated and simultaneously directed movements of large numbers of cells led to retraction of the monolayer on one side of myofibroblast aggregates, leaving behind the largely empty surface of the well (Fig. 1C, E). Subsequently, the monolayer retracted on the other side of the myofibroblast aggregates (Fig. 1D, F). These highly coordinated cell movements led to the formation of stripes of cells, with a length of up to several millimetres or even centimetres (Fig. 1F). The stripes included the myofibroblast aggregates.


Generation of easily accessible human kidney tubules on two-dimensional surfaces in vitro.

Zhang H, Lau SF, Heng BF, Teo PY, Alahakoon PK, Ni M, Tasnim F, Ying JY, Zink D - J. Cell. Mol. Med. (2010)

The process of tubule formation on 2D surfaces. (A)–(D) and (H) show images obtained by epifluorescence microscopy after immunodetection of zonula occludens-1 (ZO-1) (green) and α-SMA (red). Nuclei were counterstained with 4′,6′-diamidino-2′-phenylindole (DAPI) (blue). The other panels show images obtained by (E, F) differential interference contrast microscopy and (G) bright-field microscopy. In all cases, the HPTCs were cultured on the bottom of the wells of 24-well plates. (A) First, a well-differentiated epithelial monolayer is formed. (B) Subsequently, myofibroblast aggregates that are strongly positive for α-SMA appear. (C, E) The monolayer then retracts on the one side of the myofibroblast aggregates, leaving a surface devoid of cells (left half in C). (D, F) The monolayer subsequently retracts on the other side of the myofibroblast aggregates. This leads to the formation of cell stripes, which include myofibroblast aggregates (note: myofibroblast aggregates are labelled with arrowheads in E, F and G). (G, H) Finally, large renal tubules are formed on the 2D surface. Several images were stitched together in order to cover the entire tubule shown in (G). Scale bars: (A) 100 μm, (B–F, H) 200 μm, and (G) 1 mm.
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fig01: The process of tubule formation on 2D surfaces. (A)–(D) and (H) show images obtained by epifluorescence microscopy after immunodetection of zonula occludens-1 (ZO-1) (green) and α-SMA (red). Nuclei were counterstained with 4′,6′-diamidino-2′-phenylindole (DAPI) (blue). The other panels show images obtained by (E, F) differential interference contrast microscopy and (G) bright-field microscopy. In all cases, the HPTCs were cultured on the bottom of the wells of 24-well plates. (A) First, a well-differentiated epithelial monolayer is formed. (B) Subsequently, myofibroblast aggregates that are strongly positive for α-SMA appear. (C, E) The monolayer then retracts on the one side of the myofibroblast aggregates, leaving a surface devoid of cells (left half in C). (D, F) The monolayer subsequently retracts on the other side of the myofibroblast aggregates. This leads to the formation of cell stripes, which include myofibroblast aggregates (note: myofibroblast aggregates are labelled with arrowheads in E, F and G). (G, H) Finally, large renal tubules are formed on the 2D surface. Several images were stitched together in order to cover the entire tubule shown in (G). Scale bars: (A) 100 μm, (B–F, H) 200 μm, and (G) 1 mm.
Mentions: In accordance with our previous results, we observed initial formation of a flat and well-differentiated epithelial monolayer when HPTCs were cultured in multiwell plates (Fig. 1A). Subsequently, increasing amounts of α-SMA-expressing myofibroblasts appeared. These myofibroblasts formed large aggregates (Fig. 1B). In the surroundings of such aggregates, the epithelium became reorganized. Firstly, highly coordinated and simultaneously directed movements of large numbers of cells led to retraction of the monolayer on one side of myofibroblast aggregates, leaving behind the largely empty surface of the well (Fig. 1C, E). Subsequently, the monolayer retracted on the other side of the myofibroblast aggregates (Fig. 1D, F). These highly coordinated cell movements led to the formation of stripes of cells, with a length of up to several millimetres or even centimetres (Fig. 1F). The stripes included the myofibroblast aggregates.

Bottom Line: However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture.Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology.The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioengineering and Nanotechnology, The Nanos, Singapore, Singapore.

Show MeSH
Related in: MedlinePlus