Air–liquid and liquid–liquid interfaces influence the formation of the urothelial permeability barrier in vitro

Abstract

Optimizing culture conditions is known to be crucial for the differentiation of urothelial cell cultures and the formation of the permeability barrier. However, so far, no data exist to confirm if air-liquid (AL) and liquid-liquid (LL) interfaces are physiologically relevant during urothelial differentiation and barrier formation. To reveal the influence of interfaces on the proliferation, differentiation, and barrier formation of the urothelial cells (UCs) in vitro, we cultured UCs under four different conditions, i.e., at the AL or LL interfaces with physiological calcium concentration and without serum or without physiological calcium concentration and with serum. For each of the four models, the urothelial integrity was tested by measuring the transepithelial resistance (TER), and the differentiation stage was examined by immunolabeling of differentiation-related markers and ultrastructural analysis. We found that the UCs at a LL interface, regardless of the presence or absence of calcium or serum, form the urothelium with more cell layers and achieve a higher TER than UCs at an AL interface. However, UCs grown at an AL interface with physiological concentration of calcium in medium form only one- to two-layered urothelium of UCs, which are larger and express more differentiation-related proteins uroplakins than UCs in other models. These results demonstrate that the interface itself can play a major, although so-far neglected, role in urothelial physiology, particularly in the formation of the urothelial permeability barrier in vitro and the regulatory mechanisms related with urothelial differentiation. In the study, the culturing of UCs in three successive steps is proposed.

Co‐culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research

“…In the present study the adequate and successful isolation of the porcine urothelial cells was performed as described previously (Kreft et al, , ; Tratnjek et al, ; Visnjar et al, ; Visnjar and Kreft, , ). Then we adjusted an easy to repeat protocol for fibroblast and smooth muscle cell culture isolation of the same urinary bladder and achieved isolation of the various cell types, with no cross‐contamination.…”

“…The immunofluorescence reaction was performed as described previously (Kreft et al, 2005b). Each antibody used in this study has been previously extensively tested in our lab by performing immunofluorescence as well as immunohistochemistry by biotinylated secondary antibodies on cell cultures and on cryo‐ and paraffin‐sections of pig, mouse, rat, and human urinary bladder urothelium tissue sections (unpublished research and Kreft et al, , , , , 2010; Visnjar et al, ; Visnjar and Kreft, , ; Zupancic et al, , , ; Zupancic and Romih, ). The panel of antibodies we used was: CK 7 (mouse monoclonal antibody, diluted 1:20, Dako, Glostrup, Denmark), vimentin (rabbit polyclonal antibody, diluted 1:20, Dako), desmin (rabbit polyclonal antibody, diluted 1:40, Sigma), collagen I (mouse monoclonal antibody, diluted 1:200, Sigma), occludin (rabbit polyclonal antibody, diluted 1:20, Zymed Laboratories, San Francisco, CA, USA), E‐cadherin (mouse monoclonal antibody, diluted 1:20, Transduction Laboratories, Lexington, KY, USA), uroplakins (rabbit polyclonal antibody, diluted 1:10.000, a kind gift from Prof. dr. T.T.…”

“…The second one is DMEM and Ham's F12 supplemented with 5%, 10%, or 20% fetal bovine serum (FBS) (Fujiyama et al, ; Ehmann and Terris, ; Fossum et al, ; Larsson et al, ). The third one is UroM medium, which is composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A‐DMEM) and MCDB 153 medium supplemented with 2.5% FBS or with physiological calcium concentration of 2.5 mM (Kreft et al, , ; Visnjar et al, ; Visnjar and Kreft, , ). Therefore, the comparison of the results and faster development of urothelial cultures suitable for tissue engineering are hindered.…”

Properties of the Urothelium that Establish the Blood–Urine Barrier and Their Implications for Drug Delivery

“…Interdigitation is found at the superficial UC borders, and this membrane zipper might contribute to the barrier function. The membrane overlap does not include TJs and is thought to fasten two cells better together by providing more apical membrane surface contact, additionally increasing the electrical resistance of the junction and possibly stabilizing the TJs (Kreplak et al 2007;Višnjar et al 2012;Višnjar and Kreft 2013). TER is a measure of ion permeability (Lewis 2000), and the main route of passive ion permeation does not traverse the cells, but the cell junctions.…”

Urothelial cell expansion and differentiation are improved by exposure to hypoxia

“…Mesenchymal cells or fibroblasts used in this technique are allowed to secrete and assemble their own extracellular matrix to form a stroma that can be manipulated. Urothelial cells (UCs) seeded on top of the stroma differentiate at an air–liquid interface (Visnjar and Kreft, ) into a pseudostratified or stratified epithelium depending on the nature of the reconstructed tissue. This technique, well adapted to skin (Michel et al , ) or cornea (Carrier et al , ) reconstruction where the epithelium needs to be in contact with oxygen, is less physiological for bladder tissue regeneration where the urothelium needs to be in contact with urine.…”