Spheroids of Bladder Smooth Muscle Cells for Bladder Tissue Engineering

Abstract

Cell-based tissue engineering (TE) has been proposed to improve treatment outcomes in end-stage bladder disease, but TE approaches with 2D smooth muscle cell (SMC) culture have so far been unsuccessful. Here, we report the development of primary bladder-derived 3D SMC spheroids that outperform 2D SMC cultures in differentiation, maturation, and extracellular matrix (ECM) production. Bladder SMC spheroids were compared with 2D cultures using live-dead staining, qRT-PCR, immunofluorescence, and immunoblotting to investigate culture conditions, contractile phenotype, and ECM deposition. The SMC spheroids were viable for up to 14 days and differentiated rather than proliferating. Spheroids predominantly expressed the late myogenic differentiation marker MyH11, whereas 2D SMC expressed more of the general SMC differentiation marker α-SMA and less MyH11. Furthermore, the expression of bladder wall-specific ECM proteins in SMC spheroids was markedly higher. This first establishment and analysis of primary bladder SMC spheroids are particularly promising for TE because differentiated SMCs and ECM deposition are a prerequisite to building a functional bladder wall substitute. We were able to confirm that SMC spheroids are promising building blocks for studying detrusor regeneration in detail and may provide improved function and regenerative potential, contributing to taking bladder TE a significant step forward.

Improved contractile potential in detrusor microtissues from pediatric patients with end stage lower urinary tract dysfunction

“…Sphericalplate 5D ® (Kugelmeiers, Zurich, Switzerland) was used for SMC microtissue production according to established protocols ( Gerwinn et al, 2021 ). The Sphericalplate 5D ® is a 24 well plate of which 12 wells have a micro structured well bottom with 750 non-adhesive micro cavities per well, where SMCs gather to form the microtissues.…”

“…The SMCs were seeded in 2 ml of culture medium with a calculated 1500SMCs/microtissue. Following our previous experience ( Gerwinn et al, 2021 ), microtissues were harvested and used for analysis on the 2nd day of culture.…”

“…3D cell culture thus seems to be a potent tool to better understand the underlying pathology on a cellular level. We recently reported on the successful generation of 3D spheroids from rat derived detrusor SMCs ( Gerwinn et al, 2021 ).…”

Biomedical Application of Decellularized Scaffolds

“…Common reagents are acetic acid, peracetic acid, hydrochloric acid, sulfuric acid, and ammonium hydroxide. They effectively destroy cell membranes and internal organelles. − Poornejad et al studied the effects of five decellularized agents (sodium hydroxide, peracetic acid, Triton X-100, sodium lauryl sulfate, and trypsin) on kidney tissue of pigs. The results showed that sodium hydroxide was the most effective cell scavenger, and although it caused the most significant damage to glycosaminoglycan, the cells treated with it had the highest activity and proliferation capacity after recellularizating.…”

Generating human skeletal myoblast spheroids for vascular myogenic tissue engineering

“…Cardiac muscle spheroids consisting of cardiomyocytes and supporting cell types have been successfully generated [49,50]. Also, the engineering of smooth muscle spheroids has been explored for applications in bladder tissue engineering [51]. Nonetheless, the generation of skeletal muscle spheroids seems to be mainly restricted to the use of porcine primary cells and murine C2C12 cell line [52][53][54].…”