Poster M17 - A 3D Bioprinted Proximal Tubule Model for the Investigation of Hypokalemia Induced Ammoniagenesis

Introduction: Ammoniagenesis, the generation of ammonium from glutamine, is a key mechanism in the renal system for excretion of acid and conservation of sodium and potassium ions. While hypokalemia, low potassium concentration, is associated with upregulated ammoniagenesis, the underlying mechanism is currently poorly understood and challenging to study in vitro due to the limitations of current proximal convoluted tubule (PCT) models. PCT transwell models currently used to study renal physiology do not provide uniform flow direction, shear stress, or 3D microenvironment required to induce and maintain PCT maturity across the tissue. In this study, we designed an optimized geometry to normalize flow and shear stress applied by an orbital shaker based on computational fluid dynamic (CFD) simulations. Here we report how proximal tubule (PT) cell maturity is affected by static vs dynamic culture when seeded onto control (flat), radial channel, and optimized models. Specifically, we demonstrate that cellular function, approximated by albumin uptake, is regionally affected by model design and across the tissue by culture conditions.

Materials and

Methods: Flat (Fig1A), radial channel (Fig1D), and optimized models were designed using Fusion 360 and the shear stress at the surface was simulated using Ansys Fluent simulations. Microporous collagen scaffolds were fabricated using freeform reversible embedding of suspended hydrogel (FRESH) 3D bioprinting on a custom bioprinter equipped with a Replistruder 5 extruder. Printed models were placed into transwell inserts and seeded with opossum kidney (OK) or human renal proximal tubule endothelial cells (RPTEC; ATCC CRL-4031) and cultured under static conditions until a monolayer formed. After monolayer formation scaffolds were cultured under static or dynamic conditions on an orbital shaker up to 10 additional days with regular media changes. PT cell function was assessed by albumin uptake on seeded scaffolds after 5 and 10 days of dynamic culture. To ensure appropriate morphology and maturation of PT cells within our system we utilized immunofluorescence microscopy to quantify expression and PT cell morphology by probing for nuclei, phalloidin, a-tubulin, megalin, AQP1, SGLT2, and ATPA1.

Results, Conclusions, and Discussions:

Results:

Optical coherence tomography (OCT) imaging was employed to verify dimensional accuracy of printed scaffold architecture (Fig1B,E). Analysis of OCT images revealed that scaffolds were printed with accurate outer diameter ~12.4 mm (12.5 mm expected) and low surface roughness ~70um across the flat surface (Fig1C). Furthermore, radial channels were accurately fabricated in the printed scaffold (Fig1F). CFD modeling of flat 3D bioprinted and 2D transwell models the optimal shear stress was observed at 90 rpm at the edge of the transwell (0.18 – 0.41 dyne/cm2). However, at the same rpm, the shear for channeled 3D models was significantly lower (0.005 – 0.018 dyne/cm2). Equivalent shear stress was generated within the channeled model by doubling the orbital rotation rate (Fig 1G). OK cells adhered well to both printed models during static culture and had high cell viability after 4 days (Fig 1H). Rotational shear stress further promoted OK PT cell adhesion and proliferation in the circumferential channel model but caused cell detachment from the flat model. Interestingly the OK cells not only formed a monolayer along the channel surface but also displayed evidence of ECM remodeling and growth into the bulk of the 3D bioprinted collagen. After 10 days of dynamic culture OK PT cells effectively absorbed albumin after 30 minutes of incubation (Fig 1I). Scaffolds seeded with RPTEC cells behaved similarly to those seeded observed with OK PT cells. Utilizing human RPTEC cells allowed further interrogation of cellular remodeling due to the increased availability of antibodies for human gene targets compared to opossum.



Conclusion/

Discussion:

In conclusion, our 3D bioprinted PT model will allow us to study kidney specific cell types in a more physiologically accurate environment to identify the key mechanisms controlling ammoniagenesis. Our initial data suggests that collagen-I is sufficient for OK cell adhesion and proliferation; however, by changing the ECM composition, we can vary our PCT models mechanical properties, adhesion molecules, growth factors, and other elements of the ECM that are essential to further optimize the proximal tubule model.

Acknowledgements (Optional):