Department of Biomedical Engineering, School of Medical Technology, Beijing Institute of Technology, Beijing 100081, China.
Department of Thoracic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
Anal Chem. 2024 Oct 22;96(42):17027-17036. doi: 10.1021/acs.analchem.4c05111. Epub 2024 Oct 14.
Genetically sequencing patient-derived organoids (PDOs) at the single-cell level has emerged as a promising method to infer cell-level heterogeneity of original organs and improve cancer precision medicine. Unfortunately, because of the limited starting quantity and uncontrolled establishing process of PDOs, the existing single-cell sequencing technologies, either manual-operation-based or microfluid-based, are inefficient in processing PDOs originating from clinical tissue samples. To address such issues, this study presents a microfluidic chip-based automatic system for sequencing organoids at the single-cell level, named as MASSO. By performing all required procedures, including PDO establishment/culturing/digesting and single-cell isolation/lysis/whole-genome amplification, in a single microfluidic chip, the possible loss of precious PDO is avoided, and the high quality of on-chip whole-genome amplification of a single PDO cell is ensured. By automating the entire operation process, possible human error is eliminated, and the data repeatability is improved, therefore bridging the technical gap between laboratorial proof-of-concept studies and clinical practices. After characterizing the organoid single-cell whole-genome amplification chip (named as OSA-Chip) and the MASSO, the first successful attempt, to the best of our knowledge, on whole-genome sequencing lung cancer PDO at the single-cell level was performed by MASSO. The results reveal that the MASSO is capable of not only identifying common cancer-related mutations but also discovering specific mutations that affect drug responses, therefore laying the technical foundation for efficiently understanding the cell-level heterogeneities of PDOs and corresponding original organs.
在单细胞水平上对患者来源的类器官（PDO）进行基因测序已成为推断原始器官细胞异质性和提高癌症精准医学的一种有前途的方法。不幸的是，由于 PDO 的起始数量有限且建立过程不可控，现有的单细胞测序技术（基于手动操作或微流控）在处理源自临床组织样本的 PDO 时效率低下。为了解决这些问题，本研究提出了一种基于微流控芯片的用于单细胞水平测序类器官的自动系统，称为 MASSO。通过在单个微流控芯片中执行所有必需的程序，包括 PDO 的建立/培养/消化以及单细胞分离/溶解/全基因组扩增，避免了珍贵 PDO 的可能损失，并确保了单个 PDO 细胞的高质量的芯片上全基因组扩增。通过自动化整个操作过程，消除了人为错误的可能性，提高了数据的可重复性，从而缩小了实验室概念验证研究与临床实践之间的技术差距。在对类器官单细胞全基因组扩增芯片（命名为 OSA-Chip）和 MASSO 进行表征后，我们首次成功地尝试使用 MASSO 对单细胞水平的肺癌 PDO 进行全基因组测序。结果表明，MASSO 不仅能够识别常见的癌症相关突变，还能够发现影响药物反应的特定突变，从而为有效地理解 PDO 和相应原始器官的细胞异质性奠定了技术基础。
