Abstract
Scalable fabrication concepts of 3D kidney tissue models are required to enable their application in pharmaceutical high-throughput screenings. Yet the reconstruction of complex tissue structures remains technologically challenging. We present a novel concept reducing the fabrication demands, by using controlled cellular self-assembly to achieve higher tissue complexities from significantly simplified construct designs. We used drop-on-demand bioprinting to fabricate locally confined patterns of renal epithelial cells embedded in a hydrogel matrix. These patterns provide defined local cell densities (cell count variance <11%) with high viability (92 ± 2%). Based on these patterns, controlled self-assembly leads to the formation of renal spheroids and nephron-like tubules with a predefined size and spatial localization. With this, we fabricated scalable arrays of hollow epithelial spheroids. The spheroid sizes correlated with the initial cell count per unit and could be stepwise adjusted, ranging from Ø = 84, 104, 120–131 µm in diameter (size variance <9%). Furthermore, we fabricated scalable line-shaped patterns, which self-assembled to hollow cellular tubules (Ø = 105 ± 22 µm). These showed a continuous lumen with prescribed orientation, lined by an epithelial monolayer with tight junctions. Additionally, upregulated expression of kidney-specific functional genes compared to 2D cell monolayers indicated increased tissue functionality, as revealed by mRNA sequencing. Furthermore, our concept enabled the fabrication of hybrid tubules, which consisted of arranged subsections of different cell types, combining murine and human epithelial cells. Finally, we integrated the self-assembled fabrication into a microfluidic chip and achieved fluidic access to the lumen at the terminal sites of the tubules. With this, we realized flow conditions with a wall shear stress of 0.05 ± 0.02 dyne cm−2 driven by hydrostatic pressure for scalable dynamic culture towards a nephron-on-chip model.
Highlights
Novel 3D cell culture models with enhanced physiological properties are desirable to increase predictability of organ responses in vitro compared to 2D cell monolayers or animal models [1, 2]
To establish a process that provides reproducible, spatial, and temporal control over epithelial cell selfassembly of three-dimensional (3D) spheroids and nephron-like tubule structures, we investigated a compatible set of a scaffold material for an artificial extracellular matrix materials (ECMs), a technology for bioprinting that enables the precise and automated deposition of cells, and a suitable cell containing bioink for printing
Epithelial cells showed changing morphologies compared to Day 0, with self-assembly of spheroid-like structures in Matrigel, Collagen and Fibrin with all tested concentrations
Summary
Novel 3D cell culture models with enhanced physiological properties are desirable to increase predictability of organ responses in vitro compared to 2D cell monolayers or animal models [1, 2]. Key structural objectives for the reconstruction of an artificial nephron tubule are (a) a hollow lumen in an extracellular matrix material, lined by (b) a closed renal epithelial cell layer, and (c) optional culture with luminal flow, to fully simulate physiological conditions. This is achieved by integration into microfluidic devices, as nephron-on-chip models [4,5,6]. For successful in vitro application, the models must be (d) scalable, reproducible, and parallelizable, with appropriate technological effort to fabricate large numbers of replicates
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