van der Hee B, Loonen L M P, Taverne N, Taverne-Thiele J J, Smidt H, Wells J M
Host-Microbe Interactomics, Animal Sciences Group, Wageningen University, De Elst 1, Wageningen, The Netherlands; Molecular Ecology, Laboratory of Microbiology, Wageningen University, Stippeneng 4, Wageningen, The Netherlands.
Host-Microbe Interactomics, Animal Sciences Group, Wageningen University, De Elst 1, Wageningen, The Netherlands.
Stem Cell Res. 2018 Apr;28:165-171. doi: 10.1016/j.scr.2018.02.013. Epub 2018 Feb 20.
An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.
干细胞衍生的肠道类器官的三维几何结构存在一个重要的实际限制，即它阻碍了对顶端上皮进行食物成分、微生物、生物活性和有毒化合物测试的便捷途径。为此，我们在此报告一种新的可靠方法，该方法使用改良的培养条件，从酶解猪类器官的单细胞悬液中生成汇合的肠道细胞单层。通过这种方法，细胞接种密度可以标准化，克服了基于类器官机械解离方法的问题。汇合的单层在三天内形成紧密连接，具有高跨上皮电阻，并且可用于长达两周的实验。通过免疫组织化学和细胞特异性转录本的RT-qPCR证明了回肠干细胞的多谱系分化，也明确证实了猪小肠中潘氏样细胞存在这一有争议的情况。这里描述的方法有助于标准化从肠道类器官形成原代上皮单层，并允许对肠道生理学进行快速而可靠的研究。
