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Abstract: SA-PO321

Probing Mechanical Regulation of Kidney Morphogenesis Through a Novel 3D Explant Culture Technique

Session Information

Category: Development, Stem Cells, and Regenerative Medicine

  • 600 Development, Stem Cells, and Regenerative Medicine

Authors

  • Huang, Aria (Zheyuan), University of Pennsylvania School of Engineering and Applied Science, Philadelphia, Pennsylvania, United States
  • Hughes, Alex, University of Pennsylvania School of Engineering and Applied Science, Philadelphia, Pennsylvania, United States
Background

Achieving proper tissue organization during kidney development plays a vital role in its subsequent functions. For example, congenital anomalies of the kidney and urinary tract can manifest as underdeveloped or disorganized tissue compartments that cause dysfunction or disease. While underlying genetic and molecular factors have been studied, there is little understanding of their interplay with tissue-level cell composition, geometry, and mechanics that together set the final organ structure. Here we address the limited capability to observe developing kidney tissues in a 3D context and seek to uncover the physical principles that set kidney architecture during embryonic development.

Methods

We incorporated a hydrogel droplet into a silicone ring device to encapsulate mouse embryonic day 13 (E13) kidneys for subsequent 3D culture. We tested different organotypic hydrogels (Matrigel, Collagen I) as embedding materials, and characterized their mechanical properties using microindentation. E13 kidneys grown in 3D gels and at in traditional air-liquid interface cultures were imaged every hour and fixed after 4 days.

Results

Our data showed that kidney morphogenesis proceeded in ECM-derived gel droplets, not just in air-liquid interface cultures. Both methods yielded similar numbers of ureteric bud branches and nephrons, as confirmed by immunofluorescence. Notably, our live branch tracking analysis revealed dynamic growth and rearrangement of ureteric tubules in unflattened kidneys for the first time. The mechanical properties of the embedding gel also impacted overall explant morphology. Low concentration Collagen I (0.3-1mg/ml, E<2kPa) best mimicked in vivo development. Matrigel (E<1kPa) failed to maintain the 3D integrity of the explant, resulting in partially flattened tissue. High concentration Collagen I (3mg/ml, E=4.2kPa) impeded development and lead to enlarged ureters in some samples, resembling certain congenital defects.

Conclusion

We present a 3D culture technique that not only supports ex vivo morphogenesis but also enables live imaging of mouse embryonic kidneys. Our data suggest organ ex vivo development is influenced by its mechanical environment. This work aims to transform long-term explant culture techniques and clarify the role of the mechanical environment in kidney development.