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

Abstract

New strategies for culturing and co-culturing of the main types of urinary bladder cells are essential for successful establishment of biomimetic in vitro models, which could be applied for research into, and management of, diverse urological disorders. Porcine normal urothelial cells are available in nearly unlimited amounts and have many properties equivalent to human urothelial cells. In the present study, we established normal differentiated porcine urothelial cells in co-cultures with porcine urinary bladder normal fibroblasts and/or smooth muscle cells. The optimal culture medium for establishment of differentiated urothelial cells, demonstrated by positive immunofluorescence of uroplakins, cytokeratins (CK 7, CK 20), zonula occludens 1 (ZO-1), claudin 4, claudin 8, and E-cadherin, was the medium composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A-DMEM) and MCDB 153 medium with physiological calcium concentration of 2.5 mM and without fetal bovine serum, named UroM (+Ca - S). This medium was also proven to be suitable for culturing of bladder fibroblasts and smooth muscle cells and co-culturing of urothelial cells with these mesenchymal cells. Urothelial cell differentiation was optimal in UroM (+Ca - S) medium in all co-culture conditions and when compared to all conditioned-media combinations. To summarize, these strategies for culturing and co-culturing of urinary bladder urothelial cells with mesenchymal cells could be used as new in vitro models for future basic and applicable research of the urinary bladder and thus potentially also for translational tissue engineering studies.

Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells

“…The experiments were approved by the Veterinary Administration of the Slovenian Ministry of Agriculture and Forestry in compliance with the Animal Health Protection Act and the Instructions for Granting Permits for Animal Experimentation for Scientific Purposes. Normal porcine urothelial cells from VII to XII passages were cultured in UroM medium, which consisted of equal parts of MCDB153 medium and Advanced-Dulbecco’s modified essential medium (Thermo Fisher Scientific, Invitrogen, Austria), and was supplemented with 0.1 mM phosphoethanolamine 15 μg/ml adenine 0.5 μg/ml hydrocortisone 5 μg/ml insulin), 4 mM glutamax (Thermo Fisher Scientific), 0.9 mM calcium, 2.5% FBS (Thermo Fisher Scientific), 100 μg/ml streptomycin, and 100 U/ml penicillin 33 . T24 cell line of cancer cells originated from human invasive urothelial neoplasm (ATTC, Manassas, VA, USA) was cultured in Advanced-Dulbecco’s modified essential medium and medium F12 (1:1), 5% FBS, 100 μg/ml streptomycin, and 100 U/ml penicillin.…”

Human Amniotic Membrane Enriched with Urinary Bladder Fibroblasts Promote the Re-Epithelization of Urothelial Injury

“…The provided culture conditions were optimal for the growth of the NPU cells. In the UroM medium, the BFs remains viable; however, their proliferation rate significantly decreases 24 . In our situation, this was appreciated, since it prevented BFs to expand excessively.…”

Innervation: the missing link for biofabricated tissues and organs

“…3m). Engineering a functional bladder would require culture of urothelial cells along with the bladder smooth muscle cells, which can be harvested from different layers of bladder tissue from rodent, porcine, or human sources [190][191][192][193][194] . Moreover, both urothelial cells 195,196 and bladder smooth muscle cells 197,198 can be induced from human PSCs.…”

“…Moreover, both urothelial cells 195,196 and bladder smooth muscle cells 197,198 can be induced from human PSCs. An underlying layer of bladder smooth muscle cells would facilitate growth and maturation of urothelial cells 190,191 . This can be achieved by initial culture of bladder smooth muscle cells on a scaffold followed by plating of urothelial cells to mimic the native architecture of the bladder wall.…”