Abstract: PO0511
Biomimetic Platform for Quantitative Drug Screening of Podocyte Cytoskeletal Dynamics and Morphology
Session Information
- Bioengineering: Organoids and Organs-on-a-Chip
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Bhattacharya, Smiti, Columbia University, New York, New York, United States
- Haydak, Jonathan C., Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Stern, Alan, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Wiener, Robert, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Wong, Nicholas Joseph, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Defronzo, Stefanie, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Iyengar, Ravi, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Gordon, Ronald, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Costa, Kevin D., Icahn School of Medicine at Mount Sinai, New York, New York, United States
- He, John Cijiang, Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Hone, James C., Columbia University, New York, New York, United States
- Azeloglu, Evren U., Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background
Foot process effacement is driven by dysregulation of cytoskeletal dynamics. Currently, there are no drugs that primarily target the podocyte cytoskeleton due to dearth of in vitro model systems. To address this limitation, we designed a 3D drug discovery platform with a morpho-mimetic milieu that allows high-throughput quantitative measurements of drug-induced cytoskeletal changes in podocytes.
Methods
Immortalized human podocytes were differentiated on micropatterned surfaces fabricated via photolithography. High-resolution microscopy including confocal, TIRF, SEM and atomic force microscopy (AFM) were used to characterize morphometric and biophysical properties. Protein expression was quantified using automated immunofluorescence, cell proliferation via EdU, and basal motility via fluorescent live-cell imaging. High-throughput analytical capabilities of the system were tested with cytoskeletal dose response against puromycin aminonucleoside (PAN).
Results
Cells cultured in 3D micropatterns for up to 14 days show a significant reduction in morphometric variability compared to unpatterned controls. During differentiation, micropatterned podocytes achieve cell cycle arrest faster and more robustly with reduced motility. Furthermore, the increased speed and the extent of cell cycle arrest is also observed in low serum, suggesting that the effects of morpho-mimetic culture are independent from biochemical stimuli. AFM elastography shows primarily a peripheral distribution of stiff actin fibers in micropatterned podocytes, mimicking in situ conditions. A cohesive cytoskeletal dose response was observed against PAN in the micropatterned cells only.
Conclusion
3D micropatterns increase the speed and efficiency of podocyte differentiation while reducing cell-to-cell variability by up to 5-fold. Through its increased reproducibility, our automated system allows for quantitative in vitro study of podocyte morphology, cytoskeleton and biomechanics in response to drugs and pathogens with high fidelity and reproducibility.
Funding
- NIDDK Support