(260) Organoid Slicing for Long-Term Culture and High-Throughput Analysis 

Marina V. Pryzhkova*^1,2, Nicholas A. Chartrain*^3,4, Juliana I. Candelaria ^1,2, Kristin H. Gilchrist ^3,4, Philip W. Jordan ^1,2,5,6

*These authors contributed equally

Abstract Text:

Human organoid models have become an important tool in developmental studies, disease modeling, toxicology studies and the identification of effective treatment strategies in personalized medicine. However, maintaining organoids in long-term cultures presents challenges, such as hypoxia and nutrient deprivation due to an increase in organoid size. Cutting organoids into smaller pieces has emerged as a strategy to improve viability and functionality, but existing methods often suffer from low throughput and can compromise culture conditions. In this work, we developed a novel, efficient method for organoid slicing to improve long-term culture conditions, as well as designed a strategy for creating densely packed organoid cryosections optimized for high-throughput screening.

We utilized 3D-printing technology to create four prototypes of organoid slicing jigs, including blade guides, and tested them using human pluripotent stem cell-derived organoids. The best-performing slicing jig was continuously used for up to 5 months of culture. Organoids were maintained in mini-spin bioreactors and were cut every 3 weeks starting on day 35 of culture. Organoid health and growth were evaluated by increase in size and proliferative marker expression. Additionally, we utilized 3D-printed rigid molds to create GelMA or Geltrex-embedded organoid arrays and soft silicone molds for OCT-embedded organoid arrays.

The 3D-printed slicing jigs facilitated rapid and consistent organoid sectioning under sterile conditions. Organoid cutting improved nutrient diffusion, accelerated cell proliferation and organoid growth during long-term culture. The developed approach enables scalability and improves the uniformity of organoids regarding their size and shape. The use of molds for making densely packed organoid blocks and preparation of cryosections with evenly distributed organoid arrays facilitates consistent sample preparation and makes them suitable for high-throughput spatial transcriptomics studies. Our work aids current organoid research and extends the limits of organoid culture and analysis.