Despite increased global incidence of kidney disease, their mechanisms are yet not fully understood. Among the different segments of the nephron, the functional unit of kidneys, the proximal tubule (PT) is most susceptible for toxicant-induced injury because of its role in xenobiotic secretion and reabsorption. Moreover, genetic defects in transporters may lead to metabolic complications and tubulopathies. To gain insight in these diseases, it is of paramount importance to develop representative biomimetic in vitro models. The often-applied 2D cell models are based on the use of PT epithelial cells (PTECs) cultures. 3D (bio)printing offers new modeling alternatives to incorporate cell-extracellular matrix (ECM) interactions as it has been repeatedly shown that both ECM curvature and composition are fundamental for the adequate behavior of PTECs. Here, we applied co-axial printing to create a convoluted channel within a gelatin-based microfiber to model the convoluted structure of the PT and address the ECM-cells interaction in a diseased model. For that, we included a cystinosin-deficient (CTNS-/-) cell line to model cystinosis, a currently uncurable kidney tubulopathy.
A 3D printing system consisting of syringe pumps, heaters, coaxial needles, and a silicon holder was designed. A gelatin/alginate-based ink was formulated to allow printability while maintaining structural properties. Fine-tuning of the composition, printing temperature and feeding rate allowed an optimal biomaterial ink viscosity. Calcium chloride and microbial transglutaminase were used to stabilize the biomaterial ink. To study the stability of the hydrogel, a degradation assay was conducted. Healthy conditionally immortalized PTECs (ciPTEC), and CTNS-/- cells were seeded to mimic two genotypes of PT. Immunofluorescent stainings for cytoskeleton organization (F-actin), polarization markers (a-tubulin, Na+K+-ATPase), ECM-production (collagen IV), and barrier-formation (inulin-FITC leakage) were performed to evaluate the performance of the engineered PT.
The printed microfibers exhibited prolonged structural stability (42 days) and cytocompatibility in culture. Healthy and cystinotic cells showed homogenous cytoskeleton organization upon 14 days of culture in the helical microfibers, as indicated by staining for filamentous actin, measuring barrier-formation and assessing polarization with the apical marker a-tubulin and the basolateral marker Na+K+-ATPase. Cell viability was slightly impaired in cystinotic cells upon prolonged culturing for 14 days. Finally, CTNS-/- cells showed reduced apical transport activity by two efflux pumps, viz. breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4).
Our novel printing device showed potential to mimic a 3D environment compatible with healthy PT and tubulopathy modeling. By further improving this setup, new insights in kidney disease development and progression can be gained. This eventually aids in new treatment options.