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.

Active nutrient supply and waste product removal are key requirements for the fabrication of long-term viable and functional tissue constructs of considerable size. This work aims to contribute to the fabrication of artificial perfusable networks with a bioprinting process, based on drop-on-demand (DoD) printing of primary endothelial cell (EC) suspension bioink (25 × 10⁶ ± 3 × 10⁶ cells/ml). The process results in prescribed lumen between two hydrogel layers, allowing its integration in common layering based bioprinting processes. Low volume bioink droplets (appr. 10 nl) as building blocks were deposited between two fibrin or Collagen I layers to realize shapeable, cell-rich aggregates. Unattainable with manual positioning, DoD printing allowed precise fabrication of various designs, such as spheroidal-, line-shaped, and Y-branch cellular structures, with a mean lateral extension of 285 ± 81 μm. For basic characterization, the cell suspension building blocks were systematically compared with preformed spheroids of the same cell type, passage, and number. Post printing investigations of initially loose cell arrangements showed self-assembly and formation of central lumen with a mean cross-sectional area of Ø_lumen = 6,400 μm² at Day 3, lined by a single layer of CD31 positive ECs, as evaluated by confocal microscopy. Originating from this main lumen smaller, undirected side branches (Ø_branches = 740 μm²) were formed by sprouting cells, inducing a first step towards a simplistic hierarchically organized network. These lumen could prospectively help for tissue construct perfusion in vitro or, potentially, as niche for angiogenesis of host vascularization in implants.

Diabetic kidney disease is a major complication in diabetes mellitus, and the most common reason for end-stage renal disease. Patients suffering from diabetes mellitus encounter glomerular damage by basement membrane thickening, and develop albuminuria. Subsequently, albuminuria can deteriorate the tubular function and impair the renal outcome. The impact of diabetic stress conditions on the metabolome was investigated by untargeted gas chromatography–mass spectrometry (GC-MS) analyses. The results were validated by qPCR analyses. In total, four cell lines were tested, representing the glomerulus, proximal nephron tubule, and collecting duct. Both murine and human cell lines were used. In podocytes, proximal tubular and collecting duct cells, high glucose concentrations led to global metabolic alterations in amino acid metabolism and the polyol pathway. Albumin overload led to the further activation of the latter pathway in human proximal tubular cells. In the proximal tubular cells, aldo-keto reductase was concordantly increased by glucose, and partially increased by albumin overload. Here, the combinatorial impact of two stressful agents in diabetes on the metabolome of kidney cells was investigated, revealing effects of glucose and albumin on polyol metabolism in human proximal tubular cells. This study shows the importance of including highly concentrated albumin in in vitro studies for mimicking diabetic kidney disease.