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bioRxiv · 10.1101/2025.07.10.664241

Formulation and Characterization of Primary Tissue-Derived Microfluidic Droplet-Engineered Organoids

Wang, W.·Cai, Y.·Dai, X.·Yang, H.·Khutsishvili, D.·Li, J.·Zhu, Y.·Wang, J.·Yan, X.·Wang, Z.·Ma, S.
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Abstract

Organoid technology offers a powerful platform for modeling human tissues, studying disease mechanisms, and developing personalized therapies. However, widespread clinical application is hindered by challenges in scalability, reproducibility, and the handling of ultra-small tissue samples typical of clinical biopsies. Here, we present a comprehensive and automated protocol for the formulation and characterization of microfluidic droplet-engineered organoids (DEOs) derived from primary tissue samples. This protocol integrates 3 key stages: (1) Extraction and purification of viable primary cells from ultra-small tissue specimens using the small-Tissue Extraction Device (sTED); (2) High-throughput fabrication of uniform cell-laden microspheres using an integrated microfluidic bioprinter (OrgFab), capable of generating over 100 organoids from just 10 L of bioink; and (3) Rapid organoid characterization using lamination-based processing for single-cell analysis while preserving spatial context. The automated workflow minimizes manual intervention, reducing variability and enhancing reproducibility, making it suitable for high-throughput applications such as drug screening and disease modeling. Our method allows for the generation of patient-derived organoids that closely mimic the native tissue microenvironment, including diverse cell types and structural features, within a significantly reduced timeframe. This 3 stage protocol enhances the use of organoids in personalized medicine by enabling the rapid assessment of drug efficacy in patient-specific models. Here, the integration of advanced techniques supports existing organoid protocols, providing a valuable resource for researchers and clinicians seeking to improve patient outcomes through more efficient and precise organoid-based applications.

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Wang, W., Cai, Y., Dai, X., Yang, H., Khutsishvili, D., Li, J., Zhu, Y., Wang, J., Yan, X., Wang, Z., Ma, S.. 2025-07-16. Formulation and Characterization of Primary Tissue-Derived Microfluidic Droplet-Engineered Organoids. https://doi.org/10.1101/2025.07.10.664241

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