Optimized Culture Media Extends Human Kidney Organoid Longevity to 6 Months

Background: Although kidney organoids, derived from human pluripotent stem cells (hPSC), mature in vitro in approximately 3 weeks, there is an interest in using this model to study the effects of microgravity on the kidney during long-term space flight. The most widely used kidney organoid protocols consider their structures mature between day 16 and day 28 of differentiation, generally completing experimental assays at that time or shortly thereafter. Methods: Standard kidney organoid differentiation was performed until cultures reached the renal vesicle stage. We then supplemented our standard differentiation media with organoid tubular enhancing media (OTEM) which contains components proposed to promote epithelial cell growth and limit de-differentiation. Organoid counts were obtained using full-well brightfield images and nephron segments were visualized using immunostaining. Viability was assessed with a colorimetric assay. Transcriptomic analysis was performed via single-cell RNA sequencing of kidney organoid cultures at days 25 and 180 of differentiation. Results: OTEM drastically improved the longevity of kidney organoids, whereas no kidney organoids survived past day 100 when treated with standard media. After six months of culture, structural and transcriptomic analyses showed viable podocytes, proximal tubules, and distal tubules present in OTEM treated kidney organoids. We also identified an early glomerular epithelial cell cluster that grew relative to other cell types over the course of the experiment. At day 26 of differentiation, cultures treated with OTEM had four-fold more kidney organoids than those grown in standard media. Furthermore, the OTEM-treated cultures had less stromal overgrowth and contained twice as many viable cells compared to controls. Conclusion: Our findings indicate that OTEM is improving the stability of kidney organoids and supporting the proliferation of cell types that indicate increasing maturity of the structures. This is a simple yet powerful change to our differentiation protocol that sets the stage for future studies of organoid aging on earth and in space. Funding: Other U.S. Government SupportTimecourse of OTEM-treated kidney organoids