Cell-based assays are crucial in disease modeling and drug discovery. However, traditional 2D cultures often fail to capture the complex interactions between cell types and the extracellular matrix (ECM) that are characteristic of native tissues. This limitation is especially significant in the context of the glomerulus, the kidney's filtration unit, where injury to any component can lead to impaired filtration and chronic kidney disease. Existing 2D models are simplistic, while 3D organoid approaches lack scalability and compatibility with industrial drug screening methods. The high-throughput nature of early drug discovery relies heavily on 2D models, despite their limitations, leading to a compounding of false positives and inefficiency in the development pipeline. Advanced 3D models are often reserved for later stages, but early integration of more representative models is essential for optimizing the process. 3D models can be broadly categorized into scaffold-based and non-scaffold-based approaches. Non-scaffold approaches, based on self-aggregation in specialized environments, offer advantages in miniaturization and high-throughput screening, but most existing examples focus on tumor cells or stem cells. Models derived from mature cells, offering a direct path towards differentiated physiological tissue structures, are currently lacking. The glomerulus is a complex structure involving glomerular endothelial cells (GEnCs), podocytes, and a specialized basement membrane. Conditionally immortalized human podocyte and GEnC cell lines are standard in vitro tools, although they are typically used in 2D monocultures, which can lead to phenotypic drift. Kidney organoids derived from induced pluripotent stem cells hold promise but are hampered by lengthy protocols and high experimental variability. Therefore, a need exists for robust, consistent in vitro models of glomerular disease that enable testing of specific injuries and their downstream consequences, particularly in immune-mediated nephrotic syndrome (NS) where circulating factors damage podocytes. Currently, animal models and human biopsies provide the most comprehensive data, but their low throughput makes them unsuitable for early drug development stages. This necessitates the development of more representative in vitro models to aid in the screening of thousands of compounds during preclinical testing. This research presents GlomSpheres, a scalable spheroid model designed to address these limitations, focusing on 3D ECM dysregulation, podocyte loss, and high-throughput compound screening.

The literature highlights the shortcomings of current 2D and 3D models of the glomerulus. 2D models oversimplify the complex interactions within the glomerular filtration barrier, failing to replicate the intricate interplay between different cell types and the ECM. 3D organoids, while more representative, face limitations in scalability and compatibility with established drug screening methodologies. The use of conditionally immortalized human podocytes and GEnCs is a step forward in representing glomerular structure and function, but their use in 2D monocultures has led to a degree of phenotypic drift and limited the capacity to investigate the complexities of glomerular disease and fibrosis in vitro. Studies on ECM interactions, particularly in tumor cells, have demonstrated the importance of a 3D microenvironment in cell behaviour and response to therapeutic agents. Existing 3D models, while promising, often lack the high-throughput potential required for efficient drug discovery, especially during the early stages of the process. This review underscores the urgent need for a superior 3D model that can recapitulate the complexities of glomerular structure and disease processes while maintaining the scalability necessary for effective drug screening.

GlomSpheres were created using a magnetic spheroid formation technique. 5000 GEnCs formed a core, which was then surrounded by 5000 podocytes. Over 10 days, the podocytes migrated to completely encapsulate the GEnC core. Initial experiments using a homogenous cell mixture demonstrated self-organization into a core-peripheral structure within 72 hours. The layered approach was adopted to improve reproducibility. Immunofluorescent staining of paraffin-embedded sections revealed a basement membrane-like layer of collagen IV at the podocyte-GEnC interface. Mature isoforms of collagen IV (a3) and laminin (a5) were upregulated in GlomSpheres compared to monocultures, indicating an ECM switch akin to glomerular development. Scanning and transmission electron microscopy (SEM and TEM) confirmed podocyte foot process interdigitation and the formation of a vessel-like network. Western blotting and immunofluorescence confirmed the upregulation of podocyte (podocin, nephrin, podocalyxin) and GEnC (PECAM-1) markers in GlomSpheres relative to 2D cultures. To induce disease phenotypes, GlomSpheres were incubated with TGF-β1 (a profibrotic agent) and Adriamycin (a nephrotoxic agent). This treatment caused podocyte loss, which was attenuated by Nintedanib (an antifibrotic agent). Incubation with patient plasma from nephrotic syndrome (NS) patients during relapse versus remission demonstrated podocyte loss and ECM dysregulation during relapse, again attenuated by Nintedanib. Finally, a high-throughput screen using an InCell analyzer was performed using a podocin-mutant podocyte cell line to test the efficacy of various compounds in rescuing podocin function. This involved quantifying podocyte attachment as a surrogate for podocin function, comparing wild-type and mutant cells with and without treatment. A standard 2D adhesion assay was also performed for comparison. Immunofluorescence staining utilized various antibodies including Collagen IV pan, Col IVa1 and a3, Laminin a5, fibronectin, podocin, nephrin, PECAM-1, and podocalyxin. Western blotting was conducted for podocalyxin, PECAM-1, Collagen IVa1, and Collagen IVa3. qPCR analysis was performed for nephrin and podocin mRNA levels. Statistical analysis used one-way ANOVA and Tukey's multiple comparisons.

GlomSpheres, a 3D co-culture model, successfully recapitulated key features of the glomerulus, including the deposition of an organized basement membrane composed of mature isoforms of collagen IV (a3) and laminin (a5). This was accompanied by a significant upregulation of key podocyte markers (podocin, nephrin, and podocalyxin) and GEnC markers (PECAM-1) compared to 2D monocultures. SEM and TEM imaging confirmed the presence of podocyte foot processes and endothelial vessel-like structures. The model accurately reflected disease pathogenesis, exhibiting podocyte loss and ECM dysregulation upon exposure to profibrotic agents (TGF-β1 and Adriamycin) in a manner attenuated by Nintedanib. Experiments using patient plasma confirmed the model's ability to capture the effects of circulating factors in nephrotic syndrome, showing podocyte loss and ECM dysregulation in relapse conditions compared to remission. Nintedanib attenuated the podocyte loss but not ECM dysregulation. Finally, a high-throughput screen using an InCell analyzer and podocin-mutant cells demonstrated the model's potential for drug screening. This showed varying degrees of rescue of podocyte attachment, highlighting the efficacy of specific compounds, and revealed a greater dynamic range compared to a standard 2D adhesion assay.

GlomSpheres represent a significant advancement in in vitro glomerular modeling. The upregulation of mature ECM proteins and the formation of foot processes and vessel-like structures indicate a higher degree of physiological relevance compared to previous 2D models. The model effectively captured two key aspects of glomerulosclerosis: podocyte loss and ECM dysregulation. The ability to modulate these phenotypes and observe the effects of antifibrotic agents demonstrates its utility in drug discovery. The use of patient plasma added further clinical relevance, highlighting the model's ability to respond to a complex disease milieu. The high-throughput screening approach showcased the model's scalability and potential for rapid assessment of therapeutic compounds. The improved dynamic range of the 3D assay, compared to the traditional 2D adhesion assay, suggests a greater sensitivity in detecting therapeutic effects.

GlomSpheres provide a robust, scalable, and reproducible 3D in vitro model of the kidney glomerulus. This model exhibits a more complete phenotypic representation of the glomerulus, including the generation of a mature ECM and the ability to accurately model podocyte loss and ECM dysregulation. The successful use of patient plasma and a high-throughput drug screen further strengthens its potential for drug development. Future research should focus on incorporating other glomerular cell types (e.g., mesangial cells) and potentially incorporating flow to enhance the model's complexity and functionality.

The current GlomSphere model does not include glomerular mesangial cells, which could influence the signaling environment and ECM composition. It does not directly assess glomerular function, such as filtration efficiency. Although some primitive vascularization is observed, the absence of flow may limit the extent to which capillary structure and function are replicated. The use of conditionally immortalized cell lines, while convenient, may introduce some limitations in reflecting the full spectrum of in vivo cellular heterogeneity.