(006) ADVANCING PD.SPHERE: ENHANCED PROTOCOLS FOR A MORPHOLOGICALLY AND PATHOLOGICALLY ACCURATE 3D CELLULAR MODEL IN PEYRONIE’S DISEASE RESEARCH

Abstract Introduction Peyronie's disease (PD) affects about 10% of men and is often studied using traditional monolayer fibroblast monolayer cultures and limited animal models, which do not adequately replicate the complex cellular interactions of PD. We have developed an advanced three-dimensional (3D) cellular model named PD.SPHERE using a novel 'micro-gravity' cell culture technique from human PD tissues. Significant advancements in the culturing protocol have improved the morphology and physiopathology of these models, providing an essential platform for in vitro treatment screening. Objective The aim is to refine the culturing protocol for generating PD.SPHEREs that more accurately mimics the morphological and physiological features of PD plaques and to assess the efficacy of these refined models as tools for treatment screening. Methods Fibroblasts from human PD tissues were isolated and cultured using the 'micro-gravity' technique with modifications. The PD fibroblast cells were cultured and propagated in a non-adhesive 96-well plate, with and without 10 ng/ml transforming growth factor-beta (TGF-β) supplementation, under micro-gravity conditions. The plates were then placed on an orbital shaker inside the incubator and incubated at 37°C with 5% CO2 and 95% relative humidity. We confirmed the appropriate morphology and cellular compositions, including α-smooth muscle actin (α-SMA) and cellular collagen, using microscopic assays. The refined 3D model was subsequently used to evaluate the effects of collagenase from Clostridium histolyticum (CCH) and actinidin, a collagenase-like enzyme derived from kiwi fruits. Results Our findings from the refined 3D model revealed significant changes in cellular morphology. Specifically, PD.SPHEREs developed from cells that were not initially supplemented with TGF-β, but were later cultured in micro-gravity with TGF-β supplementation, and PD.SPHEREs from cells both propagated and cultured with TGF-β were approximately 2.3 and 3.5 times larger, respectively, than those PD.SPHEREs propagated and cultured entirely without TGF-β (P < 0.0001). The collagen content was significantly highest (P < 0.01) in PD.SPHEREs that were both propagated and cultured with TGF-β, compared to all other groups. The refined model was susceptible to both CCH and actinidin. The size and collagen content of the refined PD.SPHEREs significantly decreased (P < 0.05) following treatment with CCH and actinidin. Conclusions The advancements made in the culturing protocol of PD.SPHEREs represent a significant improvement in the modeling of PD for in vitro studies. Our results demonstrate the critical role of TGF-β in the growth and morphology of PD.SPHEREs. The increase in sized collagen content of the spheroids when cultured in the presence of TGF-β suggests that this factor plays a substantial role in cellular proliferation and cellular matrix accumulation, which are key aspects of PD pathogenesis. Furthermore, the increased collagen content in PD.SPHEREs cultured with TGF-β aligns with the known pathophysiology of PD, where collagen accumulation contributes to plaque formation and disease progression. This finding underscores the utility of our model in replicating key pathological features of PD and supports the potential of PD.SPHERE as an effective tool for screening and evaluating the efficacy of treatments aimed at modulating these pathological processes. Disclosure No.