Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens
Defined serum-free culture of human infant small intestinal organoids with predetermined doses of Wnt3a and R-spondin1 from surgical specimens
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DOI：
10.1007/s00383-021-04957-4
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发表时间：
2021-07-03
影响因子：
1.8
通讯作者：
Nakamura, Tetsuya
中科院分区：
文献类型：
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作者：
Matsumoto, Yuka;Koga, Hiroyuki;Nakamura, Tetsuya
目的改进类器官技术对研究肠道生理和疾病具有重要意义。我们的目的是优化使用手术标本中预定剂量的Wnt3a和Rspo1进行人类婴儿小肠（SI）类器官培养的无血清条件。我们进一步评估了肠道标本是否可以在使用前储存作为类器官的来源。方法在无血清培养基中检测不同剂量的Wnt3a和Rspo1，建立手术切除的SI细胞多次传代生长为类器官的条件。用定量聚合酶链反应检测干细胞和分化细胞标记基因的表达。我们还研究了在切除后不同时间保存在4℃的退化肠中的细胞的类器官形成效率。结果我们确定了具有多分化潜能的婴儿SI类器官持续生长所需的Wnt3a和Rspo1剂量。我们发现，尽管干细胞的损失依赖于时间，但储存长达2天的组织保留了能够产生可扩增类器官的细胞。结论在一定条件下，SI细胞可以发育为类器官。这可以为肠道成熟及其与儿科疾病的相关性研究提供一种可重复和可定制的手术标本方法。
Purpose Refinement of organoid technology is important for studying physiology and disease of the intestine. We aimed to optimize defined serum-free conditions for human infant small intestinal (SI) organoid culture with predetermined doses of Wnt3a and Rspo1 from surgical specimens. We further assessed whether intestinal specimens could be stored before use as a source of organoids. Methods Different doses of Wnt3a and Rspo1 in a serum-free medium were tested to establish a condition in which surgically resected SI cells grew as organoids over multiple passages. The expression of marker genes for stem and differentiated cells was assessed by quantitative polymerase chain reaction. We also investigated the organoid-forming efficiency of cells in degenerating intestines stored at 4 degrees C for various intervals post-resection. Results We determined the doses of Wnt3a and Rspo1 required for the continuous growth of infant SI organoids with multi-differentiation potential. We revealed that, despite the time-dependent loss of stem cells, tissues stored for up to 2 days preserved cells capable of generating amplifiable organoids. Conclusion SI cells can be grown as organoids under defined conditions. This could provide a reproducible and customizable method of using surgical specimens for the study of intestinal maturation and their relevance to pediatric diseases.