Lee Bo-Ram, Yang Hyeon, Lee Sang-In, Haq Inamul, Ock Sun-A, Wi Hayeon, Lee Hwi-Cheul, Lee Poongyeon, Yoo Jae-Gyu
Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju-gun 55365, Korea.
Department of Animal Biotechnology, Kyungpook National University, Sangju-si 37224, Korea.
Animals (Basel). 2021 Jul 16;11(7):2115. doi: 10.3390/ani11072115.
Intestinal organoids offer great promise for disease-modelling-based host-pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem cells in livestock species compared to other species. Intestinal crypts derived from intestinal organoids under a 3D culture system from the small intestine in adult bovine were successfully established and characterised for functionality testing, including the cellular potentials and genetic properties based on immunohistochemistry, immunocytochemistry, epithelial barrier permeability assay, QuantSeq 3' mRNA-Seq. data and quantitative reverse transcription-polymerase chain reaction. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts, and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium, and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen and revealed that the genetic properties of bovine intestinal organoids were highly similar to those of in vivo. Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from the small intestine and robust 3D expansion of intestinal organoids in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo systems and will be facilitated as the practical model to replace animal experiments for various purposes in the fields of animal biotechnology.
肠道类器官在基于疾病建模的宿主-病原体相互作用、家畜饲料效率测量的营养研究以及治疗目的的再生医学方面具有巨大潜力。然而，与其他物种相比，关于家畜物种成体干细胞的功能特性和三维（3D）扩增的研究非常有限。在三维培养系统下，成功地从成年牛小肠中建立了源自肠道类器官的肠隐窝，并对其进行了功能测试，包括基于免疫组织化学、免疫细胞化学、上皮屏障通透性测定、QuantSeq 3' mRNA-Seq数据和定量逆转录-聚合酶链反应的细胞潜能和遗传特性。肠道类器官经过多次传代长期培养，隐窝的模拟能力没有丧失，并且它们具有几种参与肠干细胞、肠上皮和营养吸收的特异性标志物的特异性表达。此外，它们对高达4 kDa的FITC-葡聚糖化合物具有高通透性，而40 kDa的FITC-葡聚糖未能进入类器官腔，这显示了牛肠道类器官的遗传特性与体内高度相似。总的来说，这些结果提供了一种从成年牛小肠中有效分离肠隐窝和在体外对肠道类器官进行强大三维扩增的可靠方法，并证明了体外三维类器官模拟了体内组织拓扑结构和功能。最后，肠道类器官是体内系统的潜在替代物，将作为替代动物实验的实用模型，促进动物生物技术领域各种目的的实现。
