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<p>Organoids are three-dimensional tissue cultures intended to replicate in vivo organs such that their function can be analyzed for applications in drug discovery, diagnostics, and research. This requires the ability to assess organoid health, development, and function on the cellular and molecular level, possibly frequently and over long periods of time. Here, we report an assay for monitoring organoid development and health by tracking the molecular cargo of small extracellular vesicles (sEV) with an integrated nanopore sensor chip. Specifically, we implement amplification-free and label-free quantification of the organoid stress marker ENO1 produced by cerebral organoid tissue. We demonstrate that mRNA levels measured non-invasively in sEVs are representative of the amounts measured by PCR measurements of the tissue cells. We also quantify the ENO1 RNA load in sEVs over the course of 15 weeks and show that ENO1 expression levels are correlated with other physiological parameters such as organoid glucose consumption. These results illustrate the capability of single molecule nanopore sensors for providing simple, continuous, quantitative assessment of organoids’ phenotypes on the molecular level. This approach can be expanded to other molecular biomarkers such as protein transcripts, multiplexed analysis, and fully integrated in-line analysis in an automated tissue culture platform.</p>

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Single molecule nanopore counting assay targeting small extracellular vesicle cargo for non-invasive monitoring of cerebral organoid development and health

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年12月12日

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  • S. M. Saiduzzaman,
  • Ruiting Xu,
  • Mohammad Julker Neyen Sampad,
  • Ryan N. Hoffman,
  • Spencer T. Seiler,
  • Quinton Brail,
  • Viktor Yurevych,
  • Zachary J. Walker,
  • Tanner N. Wells,
  • Ephraim M. Ong,
  • Thomas D. Yuzvinsky,
  • Aaron R. Hawkins,
  • Sofie R. Salama,
  • Mircea Teodorescu,
  • David Haussler,
  • Holger Schmidt

摘要

Organoids are three-dimensional tissue cultures intended to replicate in vivo organs such that their function can be analyzed for applications in drug discovery, diagnostics, and research. This requires the ability to assess organoid health, development, and function on the cellular and molecular level, possibly frequently and over long periods of time. Here, we report an assay for monitoring organoid development and health by tracking the molecular cargo of small extracellular vesicles (sEV) with an integrated nanopore sensor chip. Specifically, we implement amplification-free and label-free quantification of the organoid stress marker ENO1 produced by cerebral organoid tissue. We demonstrate that mRNA levels measured non-invasively in sEVs are representative of the amounts measured by PCR measurements of the tissue cells. We also quantify the ENO1 RNA load in sEVs over the course of 15 weeks and show that ENO1 expression levels are correlated with other physiological parameters such as organoid glucose consumption. These results illustrate the capability of single molecule nanopore sensors for providing simple, continuous, quantitative assessment of organoids’ phenotypes on the molecular level. This approach can be expanded to other molecular biomarkers such as protein transcripts, multiplexed analysis, and fully integrated in-line analysis in an automated tissue culture platform.