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bioRxiv · 10.64898/2026.01.15.699659

Integration of early-stage cryopreservation and cell cycle modulation into a flexible kidney organoid differentiation system

Yan, X.·Wang, J.·Xu, M.·Hu, C.·Chen, S.·Zhao, Y.·Rong, R.·Zhu, T.·Zhang, W.
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Abstract

Kidney organoids derived from human pluripotent stem cells (hPSCs) represent a promising platform for modeling nephrogenesis and renal diseases. However, conventional differentiation protocols are continuous and time-sensitive, limiting their scalability and reproducibility. Here, we developed a method to pause and resume organoid formation through cryopreservation at an early differentiation stage using a chemically defined formulation that maintains high post-thaw viability and differentiation potential. We further found that synchronizing hPSCs in the G1 phase with PD-0332991 enhanced post-thaw organoid formation and transcriptional fidelity, while G2 phase enrichment with Ro-3306 promoted the development of SLC12A3-positive distal convoluted tubules. The post-thaw organoids exhibited well-organized nephron architecture and function comparable to uninterrupted cultured controls. This platform proved effective for modeling BK polyomavirus (BKV) infection, drug-induced nephrotoxicity, and renal fibrosis. Together, our cryopreservation and cell cycle synchronization strategy provides a flexible, practical framework to advance organoid-based research and translation.

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Yan, X., Wang, J., Xu, M., Hu, C., Chen, S., Zhao, Y., Rong, R., Zhu, T., Zhang, W.. 2026-01-16. Integration of early-stage cryopreservation and cell cycle modulation into a flexible kidney organoid differentiation system. https://doi.org/10.64898/2026.01.15.699659

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