Next-generation breast organoids capture human organogenesis with high-resolution live imaging

Organoids have emerged as a powerful tool for modeling tissue growth and diseases. In this study, we introduce a groundbreaking organotypic culture technique that replicates the morphology, scale, and heterogeneity of human breast tissue, and includes a mesenchymal-like stromal component. A standout feature of this approach is the use of long-term live imaging at high temporal resolution to directly observe stem cell dynamics during organogenesis, from single cells to mature organ tissue. The system is adaptable for high throughput applications and allows for genetic manipulation of the cells. Real-time imaging of ex-vivo tissue formation reveals a non-canonical process of ductal-lobular morphogenesis and branching, and de-novo generation of a supportive stroma. Incorporating patient-derived single cells from multiple donors offers an enhanced representation of the spectrum of individual responses and the impacts of distinct exposures. While developed for breast tissue, the principles of this technology can serve as a model for the development of similar systems in other tissues, where organoids do not merely reproduce the tissue, but where their regeneration can also be observed and studied. In addition, this model provides a quantitative experimental system to study mechanisms of embryogenesis, development, and tissue organization where biomechanics plays an important role. Overall design: Primary human breast epithelial single cells were grown in a next generation ECM hydrogel for 19 days and analyzed using scRNA-seq

类器官（Organoids）已成为模拟组织生长与疾病进程的强有力研究工具。本研究报道了一项突破性的器官型培养技术，该技术可复现人类乳腺组织的形态特征、组织规模与异质性，并包含间充质样基质组分。该技术的核心特色在于采用高时间分辨率的长期活细胞成像手段，可直接观测器官发生过程中从单细胞到成熟器官组织的干细胞动态变化。本培养体系适配高通量应用场景，且支持对细胞进行遗传操作。对离体组织形成过程的实时成像揭示了一条非经典的导管-小叶形态发生与分支通路，并实现了支持性基质的从头生成。纳入多供体的患者来源单细胞，可更全面地反映个体应答谱及不同暴露因素的生物学影响。尽管本技术最初针对乳腺组织开发，但其技术原理可作为其他组织构建同类系统的参考范式——此类类器官不仅能够复现目标组织的特征，还可对其再生过程进行观测与研究。此外，该模型提供了一套可量化的实验体系，用于研究胚胎发生、组织发育与组织结构构建中发挥关键作用的生物力学机制。整体实验设计：将原代人乳腺上皮单细胞接种于新一代细胞外基质（Extracellular Matrix, ECM）水凝胶中培养19天，随后通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）完成分析。