BACKGROUND Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus. RESULTS Our chemically defined, serum-free, human pancreas organoid culture medium supports the generation and expansion of hPOs with high efficiency from both fresh and cryopreserved primary tissue. hPOs can be expanded from a single cell, enabling their genetic manipulation and generation of clonal cultures. hPOs expanded for months in vitro maintain their ductal morphology, biomarker expression and chromosomal integrity. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. Crucially, our medium also supports the establishment and expansion of hPOs in a chemically defined, modifiable and scalable, biomimetic hydrogel. CONCLUSIONS hPOs can be expanded long-term, from both fresh and cryopreserved human pancreas tissue in a chemically defined, serum-free medium with no detectable tumorigenicity. hPOs can be clonally expanded, genetically manipulated and are amenable to culture in a chemically defined hydrogel. hPOs therefore represent an abundant source of pancreas ductal cells that retain the characteristics of the tissue-of-origin, which opens up avenues for modelling diseases of the ductal epithelium and increasing understanding of human pancreas exocrine biology as well as for potentially producing insulin-secreting cells for the treatment of diabetes.

中文翻译：

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成人胰腺类器官的长期扩增、基因组稳定性和体内安全性。


背景技术最近已经描述了用于体外培养来自小鼠和人类的胰腺导管细胞的胰腺类器官系统。小鼠胰腺类器官表现出无限的扩增潜力，而之前报道的人胰腺类器官 (hPO) 培养物在化学成分确定的无血清培养基中不能长期有效地扩增。我们试图建立一个 3D 培养系统，用于长期扩增人类胰腺导管细胞作为 hPO，作为研究人类胰腺导管上皮、外分泌胰腺疾病和开发基因组稳定的糖尿病替代细胞疗法的基础。结果 我们的化学成分确定的无血清人胰腺类器官培养基支持从新鲜和冷冻保存的原代组织中高效生成和扩增 hPO。 hPO 可以从单个细胞扩增，从而能够进行基因操作和克隆培养物的产生。 hPO 在体外扩增数月后仍能保持其导管形态、生物标志物表达和染色体完整性。当移植到免疫缺陷小鼠的胰腺中时，hPO 的异种移植物可以在体内长期存活。值得注意的是，小鼠原位移植没有表现出致瘤性的迹象。至关重要的是，我们的介质还支持在化学定义的、可修改和可扩展的仿生水凝胶中建立和扩展 hPO。结论 hPO 可以在化学成分确定的无血清培养基中从新鲜和冷冻保存的人胰腺组织中长期扩增，且未检测到致瘤性。 hPO 可以进行克隆扩增、基因操作，并且适合在化学成分确定的水凝胶中培养。 因此，hPO 代表了胰腺导管细胞的丰富来源，保留了起源组织的特征，这为建立导管上皮疾病模型、增加对人类胰腺外分泌生物学的了解以及潜在地产生胰岛素分泌开辟了途径。细胞用于治疗糖尿病。