Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7–21 d; generation of genetically manipulated clonal organoid cultures takes 14–21 d; and organoid expansion for xenotransplantation takes >4 weeks.

类器官技术彻底改变了针对个体的人体器官发育、疾病和治疗反应的研究。尽管有详细的协议可用于从各种器官生成和长期传播人体器官，但乳房组织缺乏此类方法。在这里，我们为来自正常人类乳腺组织或乳腺癌 (BC) 组织的类器官的长期培养提供了一个优化的、高度通用的方案，以及一组 45 个生物样本库样本的培养条件，包括涵盖所有主要的 BC 类器官疾病亚型（三阴性、雌激素受体阳性/孕激素受体阳性和人表皮生长受体 2 阳性）。此外，我们还提供通过 Lipofectamine 2000 进行基因操作的方法，电穿孔或慢病毒以及随后的类器官选择和克隆培养。最后，我们介绍了一种优化的小鼠原位类器官移植方法，其中包括无需手术即可注射类器官和雌激素颗粒。从组织碎片中提取类器官直到第一次分裂需要 7-21 天；基因操作的克隆类器官培养物的产生需要 14-21 天；异种移植的类器官扩增需要> 4周。