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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.

View Article: PubMed Central - PubMed

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

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α-SMA expression in initial and 4-week-old cultures of HPTCs. (A) The expression levels of α-SMA (relative to GAPDH, average ± S.D.) were determined by qRT-PCR in initial cultures of HPTCs. These initial cultures contained cells freshly seeded from the vial obtained from the vendor, and the cells had not been passaged before analysis. The analysis was performed as soon as an epithelial sheet had been formed. For comparison, similar qRT-PCR analyses were also performed with confluent monolayer cultures of HEK293 and HeLa cells, and the results are shown. (B) α-SMA expression (relative to GAPDH, average ± S.D.) was determined by qRT-PCR in initial cultures of HPTCs (day 0) and 28 days later in cultures that were seeded in parallel. The cultures were not passaged during this time period, but the medium was regularly exchanged. (C) The image shows an initial culture of HPTCs (day 0) after co-immunostaining (ZO-1: green, α-SMA: red, DAPI: blue). α-SMA was not detectable. (D) The same co-immunostaining procedure was performed after 28 days with cultures seeded in parallel. Many α-SMA-expressing cells are present.
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fig09: α-SMA expression in initial and 4-week-old cultures of HPTCs. (A) The expression levels of α-SMA (relative to GAPDH, average ± S.D.) were determined by qRT-PCR in initial cultures of HPTCs. These initial cultures contained cells freshly seeded from the vial obtained from the vendor, and the cells had not been passaged before analysis. The analysis was performed as soon as an epithelial sheet had been formed. For comparison, similar qRT-PCR analyses were also performed with confluent monolayer cultures of HEK293 and HeLa cells, and the results are shown. (B) α-SMA expression (relative to GAPDH, average ± S.D.) was determined by qRT-PCR in initial cultures of HPTCs (day 0) and 28 days later in cultures that were seeded in parallel. The cultures were not passaged during this time period, but the medium was regularly exchanged. (C) The image shows an initial culture of HPTCs (day 0) after co-immunostaining (ZO-1: green, α-SMA: red, DAPI: blue). α-SMA was not detectable. (D) The same co-immunostaining procedure was performed after 28 days with cultures seeded in parallel. Many α-SMA-expressing cells are present.

Mentions: Besides substrate architecture, interactions between epithelial cells and myofibroblasts appeared to be important for tubule formation. Unresolved questions included where the myofibroblasts were derived from, whether their presence was due to contaminations of the epithelial cells and what role they played in tubule formation. Figure 9C shows that α-SMA-expressing cells were not detectable by immunostaining in the initial cultures of HPTCs. The expression levels of α-SMA, as determined by qRT-PCR, were very low in such initial cultures, and were not higher than the expression levels in HeLa cells or human embryonic kidney (HEK) 293 cells (Fig. 9A). HeLa cells are negative for α-SMA [36], and HeLa as well HEK293 cells are well-established epithelial cell lines free of contaminations with other cell types.


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)

α-SMA expression in initial and 4-week-old cultures of HPTCs. (A) The expression levels of α-SMA (relative to GAPDH, average ± S.D.) were determined by qRT-PCR in initial cultures of HPTCs. These initial cultures contained cells freshly seeded from the vial obtained from the vendor, and the cells had not been passaged before analysis. The analysis was performed as soon as an epithelial sheet had been formed. For comparison, similar qRT-PCR analyses were also performed with confluent monolayer cultures of HEK293 and HeLa cells, and the results are shown. (B) α-SMA expression (relative to GAPDH, average ± S.D.) was determined by qRT-PCR in initial cultures of HPTCs (day 0) and 28 days later in cultures that were seeded in parallel. The cultures were not passaged during this time period, but the medium was regularly exchanged. (C) The image shows an initial culture of HPTCs (day 0) after co-immunostaining (ZO-1: green, α-SMA: red, DAPI: blue). α-SMA was not detectable. (D) The same co-immunostaining procedure was performed after 28 days with cultures seeded in parallel. Many α-SMA-expressing cells are present.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4373329&req=5

fig09: α-SMA expression in initial and 4-week-old cultures of HPTCs. (A) The expression levels of α-SMA (relative to GAPDH, average ± S.D.) were determined by qRT-PCR in initial cultures of HPTCs. These initial cultures contained cells freshly seeded from the vial obtained from the vendor, and the cells had not been passaged before analysis. The analysis was performed as soon as an epithelial sheet had been formed. For comparison, similar qRT-PCR analyses were also performed with confluent monolayer cultures of HEK293 and HeLa cells, and the results are shown. (B) α-SMA expression (relative to GAPDH, average ± S.D.) was determined by qRT-PCR in initial cultures of HPTCs (day 0) and 28 days later in cultures that were seeded in parallel. The cultures were not passaged during this time period, but the medium was regularly exchanged. (C) The image shows an initial culture of HPTCs (day 0) after co-immunostaining (ZO-1: green, α-SMA: red, DAPI: blue). α-SMA was not detectable. (D) The same co-immunostaining procedure was performed after 28 days with cultures seeded in parallel. Many α-SMA-expressing cells are present.
Mentions: Besides substrate architecture, interactions between epithelial cells and myofibroblasts appeared to be important for tubule formation. Unresolved questions included where the myofibroblasts were derived from, whether their presence was due to contaminations of the epithelial cells and what role they played in tubule formation. Figure 9C shows that α-SMA-expressing cells were not detectable by immunostaining in the initial cultures of HPTCs. The expression levels of α-SMA, as determined by qRT-PCR, were very low in such initial cultures, and were not higher than the expression levels in HeLa cells or human embryonic kidney (HEK) 293 cells (Fig. 9A). HeLa cells are negative for α-SMA [36], and HeLa as well HEK293 cells are well-established epithelial cell lines free of contaminations with other cell types.

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