地穴
类有机物
绿色荧光蛋白
生物
基质凝胶
肠上皮
基因
细胞培养
分子生物学
细胞生物学
转染
上皮
生物化学
遗传学
内分泌学
作者
Jialiang Sun,Zhiyang Zeng,Liang Li,Dali Li,Xueli Zhang
出处
期刊:Chinese journal of experimental surgery
日期:2018-06-08
卷期号:35 (6): 1066-1069
标识
DOI:10.3760/cma.j.issn.1001-9030.2018.06.021
摘要
Objective
To establish human intestinal organoid culture in vitro and effective gene delivery system mediating by lentivirus into organoid, it will be essential to develop colorectal cancer research and gene therapy.
Methods
(1) The biological specimen of ileum or colon by surgical resection is taken 3 to 5 cm and separated mucous membrane from muscular layer. It is digested and separated by ethylene diamine tetraacetic acid Chelation Buffer for collecting intestinal crypts. The intestinal crypts is resuspended with in Matrigel. The sample is seeded on pre-warmed96-well plate, after solidification, which is added with culture medium including growth factors. The organoid status can be observed in everyday, the medium be exchanged in each 2 or 3 days, and the organoid structure be passaged in each 6 or 7 days. (2) Packaging high-titer lentivirus carrying with reporter gene, green fluorescent protein (GFP), infect into intestinal organoid, and the expression quantity of fluorescent is observed in organoid.
Results
We could culture mini-gut organoid with budding spherical structures using sporadic intestinal crypts ex vivo, and the livability of both groups of crypt/organoid are greater than 70%. Intestinal organoid can express a protein marker about absorptive cell which is located in normal intestinal epithelium. Reporter gene mediating by lentivirus vector can be stably expressed in organoids.
Conclusion
This new intestinal organoid culture model not only is used to research gene functions in maintaining intestinal homeostasis.
Key words:
Intestinal organoid; Crypt base columnar cells; Lentivirus; Gene-editing; Gene delivery system
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