GlomSpheres as a 3D co-culture spheroid model of the kidney glomerulus for rapid drug-screening

Abstract

The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. Current two and three dimensional (2D and 3D) models that attempt to recreate structure and interplay between glomerular cells are imperfect. Most 2D models are simplistic and unrepresentative, and 3D organoid approaches are currently difficult to reproduce at scale and do not fit well with current industrial drug-screening approaches. Here we report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Co-cultured using a magnetic spheroid formation approach, conditionally immortalised (CI) human podocytes and glomerular endothelial cells (GEnCs) deposited mature, organized isoforms of collagen IV and Laminin. We demonstrate a dramatic upregulation of key podocyte (podocin, nephrin and podocalyxin) and GEnC (pecam-1) markers. Electron microscopy revealed podocyte foot process interdigitation and endothelial vessel formation. Incubation with pro-fibrotic agents (TGF-β1, Adriamycin) induced extracellular matrix (ECM) dysregulation and podocyte loss, which were attenuated by the anti-fibrotic agent Nintedanib. Incubation with plasma from patients with kidney disease induced acute podocyte loss and ECM dysregulation relative to patient matched remission plasma, and Nintedanib reduced podocyte loss. Finally, we developed a rapid imaging approach to demonstrate the model’s usefulness in higher throughput pharmaceutical screening. GlomSpheres therefore represent a robust, scalable, replacement for 2D in vitro glomerular disease models.

3D engineered tissue models for studying human-specific infectious viral diseases

“…In the case of a 3D in vitro model using 3D printing, it is possible to fabricate a model more scalable and consistent than the existing model. This allows for advanced screening of various drug candidates and verifying the effects of drugs [ 236 , 237 ]. Recent 3D bioprinting technology demonstrates the possibility of artificial organs with the real-scale [ 238 ] that are potentially applicable to the mechanistic studies of spatio-temporal emergence and the progression of pathogenesis as well as transplantation.…”

STORM Imaging Buffer with a Refractive Index Matched to Standard Immersion Oil

“…However, such an intentionally introduced refractive index mismatch between the aqueous specimen and the immersion oil induces spherical aberration, reducing the achievable imaging depth to a few microns above the microscope coverslip. Therefore, it is not suitable for imaging thick samples like cell spheroids, tissues, and small animals, although important for more comprehensive biological studies. , …”

STORM imaging buffer with refractive index matched to standard immersion oil

“…However, such an intentionally introduced refractive index mismatch between the aqueous specimen and the immersion oil induces spherical aberration, reducing the achievable imaging depth to a few microns above the microscope coverslip. Therefore, it is not suitable for imaging thick samples like cell spheroids, tissues, and small animals, although important for more comprehensive biological studies [8, 9].…”