Poster BB2 - Investigating the impact of matrix material properties on kidney development using a novel 3D hydrogel embedding explant culture system

Introduction: Complex tissue-level organization established during development is critical for adult kidney function. Despite ongoing interest in genetic and molecular factors shaping the urinary drainage network and nephrons, their interplay with tissue-level organization, geometry, and mechanics remains unclear. Understanding these factors is pivotal for generating kidney replacement tissues through developmental mimicry. Tissue-level dynamics are often modeled in air-liquid interface (ALI) culture, which flattens the explant and renders it unfit for live imaging of tissue geometry and mechanics. To bridge this gap, we recently developed a 3D culture method compatible with live imaging, which allows us to examine kidney morphogenesis dynamics in 3D for the first time. In vivo, the embryonic kidney is wrapped by a layer of renal capsule which provides physical support, mimicked in 3D culture by suspending the explant in a hydrogel droplet. Studies done with ALI culture suggest that the surrounding matrix composition influences kidney morphogenesis. However, the effect of specific material properties on kidney development is poorly understood. We hypothesize that the mechanical stress and/or adhesion conditions at the organ surface influence kidney tubule organization and nephron formation. Our approach is to modulate the matrix mechanical/adhesive properties with both extracellular matrix (ECM)-derived and synthetic hydrogels.

Materials and

Methods: Culture device fabrication: Silicon rings are created with 10% Polydimethylsiloxane (PDMS) and then plasma-bonded to the glass-bottom 12-well plate. The outer space between the PDMS and the well is filled with DI water. Kidney preparation: Kidneys are microdissected from pregnant CD1 mouse embryos and labeled with fluorophore-conjugated antibodies before culture. 3D culture assembly: Dissected kidneys are suspended in 10uL of hydrogel in the center of the silicon rings. After 30mins of gelatin at 37 °C, the inside of the ring is filled with culture medium. The whole workflow is shown in Figure 1A. ECM-derived hydrogel: Collagen I is neutralized on ice and labeled with NHS-ester with Alexa 555. Neutralized collagen and h-ESC Matrigel are then mixed at a 1:1 ratio to make composite ECM-gel. Synthetic hydrogel: Acrylated hyaluronic acid (AHA) hydrogel is crosslinked by RGD peptides and MMP-degradable peptides at 37 °C and pH 8. Stiffness measurement: Hydrogel stiffness is measured using microindentation. Immunofluorescence and tissue clearing: Samples are fixed, stained, and cleared with the Scale system as described by Prahl et al. Imaging and image analysis: Both live and fixed samples are imaged on a spinning-disk confocal. Tubule tip and collagen fiber movements are tracked using Trackmate in FIJI. Kidney outlines and structures are also annotated in FIJI. Statistics: Student’s t-test is performed on quantitative data to determine significance.

Results, Conclusions, and Discussions: We focus on quantifying the dynamics of the ureteric bud (UB) which later forms the urinary drainage network and the formation of nephrons. We find that explants in 3D culture closely resemble the UB tip:tip distance of freshly dissected embryonic day (E) 14 kidneys compared to ALI culture (Figure 1C). UB tips in 3D also dynamically rotate and reposition as they develop, unlike the persistent radial expansion in ALI culture (Figure 1D). We have previously published a model describing the coupling between tubule tip orientation and density with available organ surface area. Our experimental data suggests that in vivo-like competition among UB tips for organ surface area also exists in 3D culture. We also assess how surface confinement, matrix stiffness, and adhesive ligand density affect development in 3D culture using AHA hydrogels. Preliminary data finds that kidneys cultured in soft (~1.5 kPa) AHAs phenocopy ECM-mimetic Matrigel/collagen 1 gels, and those in stiff (6 kPa) AHA adopt irregular morphologies, highlighting the importance of mechanical constraint on overall organ morphogenesis (Figure 1E). Our ongoing work is investigating how matrix stiffness and adhesion impact UB branching, genetic mutation penetrance, and nephron:UB tip balance. This work presents a novel tool for recapitulating 3D kidney morphogenesis with live imaging and investigating the effect of genetic and mechanical perturbations on ex vivo development. Results from this study will provide the first input-output atlas between the mechanical microenvironment and kidney development and provide a new tissue-scale understanding of mechanical regulation of embryonic kidney patterning. This work will also offer opportunities for recapitulating in vivo-like development through engineering the matrix in human stem-cell-derived organoids.

Acknowledgements (Optional): We thank all members of the Hughes lab at Penn for helpful discussions and assistance. Special thanks to Louis Prahl and John Viola for their support with the animal work and imaging. Also, thanks to Karen Xu and Yuqi Zhang from the Mauck lab for their contribution to the hydrogel work. This work is supported by NIH NIGMS MIRA R35GM133380, NIH NIDDK R01DK132296, NSF CAREER award 2047271, and Penn Center for Precision Engineering for Health (CPE4H) pilot grant.