Controlled tubulogenesis from dispersed ureteric bud-derived cells using a micropatterned gel

Abstract

Developmental engineering is a potential option for neo-organogenesis of complex organs such as the kidney. The application of this principle requires the ability to construct a tubular structure from dispersed renal progenitor cells with defined size and geometry. In this present study we report the generation of tubular structures from dispersed ureteric bud cells in vitro by using a micropatterned gel. Dispersed CMUB-1 cells, a mouse ureteric bud-derived cell line, or mIMCD cells, a mouse collecting duct-derived cell line, were suspended in collagen I and seeded into an agarose-based micropatterned gel. We found that within 24-36 h of incubation, the cells developed a tubular structure that conformed to the geometry of the micropattern of the gel. The lumen formation of the tubular structure was confirmed by immunohistochemical staining and observed by confocal microscopy. We found that higher concentrations of collagen I negatively influenced the efficiency of tubular formation. Tubule formation in CMUB-1, but not mIMCD, cells was positively influenced by the addition of aldosterone (10, 50 and 200 μg/ml), FGF (50 and 100 μg/ml) and fibronectin (10 and 50 μg/ml) to the growth medium. We further demonstrated the functionality of the generated tubes by in vitro budding, which was induced by growth factors, such as glial cell-derived neurotrophic factor (GDNF) or fibroblast growth factor 7 (FGF7), in the presence of beads soaked with the activin A inhibitor follistatin. Our current study thus demonstrates the possibility of constructing a functional tubular structure from dispersed ureteric bud cells in vitro in a controlled manner.

Rapid optical tissue clearing using poly(acrylamide‐co‐styrenesulfonate) hydrogels for three‐dimensional imaging

“…Three‐dimensional fluorescence imaging of the SS75‐cleared tumor tissue stained with DAPI was also successfully performed (Figure ). DAPI imaging in tissues is quite useful for various applications, such as analysis of apoptosis, detection of neurons in brain and retina, and imaging of tubular lumen structure in some organs . Our result suggests that DNA was not affected by the poly(AAm‐co‐SS) hydrogel.…”

“…DAPI imaging in tissues is quite useful for various applications, such as analysis of apoptosis, detection of neurons in brain and retina, and imaging of tubular lumen structure in some organs. [22][23][24][25][26][27] Our result suggests that DNA was not affected by the poly(AAm-co-SS) hydrogel. However, there is concern that proteins might be affected by the polymer, because some previous reports showed protein denaturation by poly(sodium p-styrenesulfonate).…”

OrganIn VitroCulture: What Have We Learned about Early Kidney Development?

“…However, using a micropatterned hydrogel, tubular structures have been generated from dispersed mIMCD-3 cells and from CMUB-1, a mouse ureteric bud-derived cell line. These generated tubular structures exhibited lumen formation and in vitro budding towards growth factor soaked beads [ 104 ]. On the other hand, investigated MM derived cell lines, BSN, rat inducing metanephric mesenchyme (RIMM-18) and cultured primary MM cells were not competent for signals from freshly isolated UB and tubulogenesis did not occur; these cell lines were also unable to induce UB branching [ 103 ].…”

Self-Organogenesis from 2D Micropatterns to 3D Biomimetic Biliary Trees

“…The idea is to restrict cell spreading and cell volume, mimicking to some extent physiological environments [ 17 ]. 2D and 3D micropatterning with substrates such as natural hydrogels [ 18 ], synthetic polymers [ 19 ], extracellular matrix components (ECM) [ 20 ], and growth factors [ 21 ] have been used in a wide range of geometries and designs including tube-like shapes and networks [ 18 , 20 , 22 ]. For instance, Hauser and colleagues generated functional tubes from renal progenitor cells and ureteric bud cells on agarose gel-micropatterns [ 18 ].…”