An agarose fluidic chip for high-throughputorganoid imaging.

in Lab on a chip by Sarah De Beuckeleer, Andres Vanhooydonck, Johanna Van Den Daele, Tim Van De Looverbosch, Bob Asselbergh, Hera Kim, Coen Campsteijn, Peter Ponsaerts, Regan Watts, Winnok H De Vos

TLDR

  • The study presents a novel pipeline for high-throughput organoid imaging using a transparent agarose fluidic chip, which can achieve theoretically unlimited throughput and high spatial resolution.
  • The system provides a cost-effective, accessible, and efficient solution for imaging organoids, enabling comprehensive spatial mapping of live and dead cells and revealing significant differences in DNA damage response.

Abstract

Modern cell and developmental biology increasingly relies on 3D cell culture systems such as organoids. However, routine interrogation with microscopy is often hindered by tedious, non-standardized sample mounting, limiting throughput. To address these bottlenecks, we have developed a pipeline for imaging intact organoids in flow, utilizing a transparent agarose fluidic chip that enables efficient and consistent recordings with theoretically unlimited throughput. The chip, cast from a custom-designed 3D-printed mold, is coupled to a mechanically controlled syringe pump for fast and precise sample positioning. We benchmarked this setup on a commercial digitally scanned light sheet microscope with cleared glioblastoma spheroids. Spheroids of varying sizes were positioned in the field of view with micrometer-level stability, achieving a throughput of 40 one-minute recordings per hour. We further showed that sample positioning could be automated through online feedback microscopy. The optical quality of the agarose chip outperformed FEP tubing, glass channels and PDMS casts for the clearing agents used, as demonstrated by image contrast profiles of spheroids stained with a fluorescent nuclear counterstain and further emphasized by the resolution of fine microglial ramifications within cerebral organoids. The retention of image quality throughout 500 μm-sized spheroids enabled comprehensive spatial mapping of live and dead cells based on their nuclear morphology. Finally, imaging a batch ofknockoutwildtype astrocytoma spheroids revealed significant differences in their DNA damage response, underscoring the system's sensitivity and throughput. Overall, the fluidic chip design provides a cost-effective, accessible, and efficient solution for high-throughput organoid imaging.

Overview

  • This study presents a novel pipeline for imaging intact organoids in flow, enabling efficient and consistent recordings with theoretically unlimited throughput.
  • The pipeline utilizes a transparent agarose fluidic chip cast from a custom-designed 3D-printed mold, which is coupled to a mechanically controlled syringe pump for fast and precise sample positioning.
  • The study demonstrates the feasibility of this setup on a commercial digitally scanned light sheet microscope, benchmarking it on cleared glioblastoma spheroids and benchmarking its throughput at 40 one-minute recordings per hour.

Comparative Analysis & Findings

  • The study compared the optical quality of the agarose chip with FEP tubing, glass channels, and PDMS casts and found that the agarose chip outperformed the others for the clearing agents used, as demonstrated by image contrast profiles.
  • The study demonstrated the ability to achieve micrometer-level stability in sample positioning, allowing for comprehensive spatial mapping of live and dead cells based on their nuclear morphology.
  • Imaging a batch of knockout wild-type astrocytoma spheroids revealed significant differences in their DNA damage response, underscoring the system's sensitivity and throughput.

Implications and Future Directions

  • The fluidic chip design provides a cost-effective, accessible, and efficient solution for high-throughput organoid imaging, which is crucial for various applications in modern cell and developmental biology.
  • Future studies could explore the use of this system for imaging complex systems, such as whole organs or tissues, and for investigating various disease models or developmental processes.
  • Automating sample positioning through online feedback microscopy could further enhance the throughput and efficiency of the system, making it even more suitable for high-throughput applications.
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