Garcia Tânia Martins, Navis Marit, Wildenberg Manon E, van Elburg Ruurd M, Muncan Vanesa
Department of Gastroenterology and Hepatology, Tytgat Institute for Intestinal and Liver Research, Amsterdam UMC, AG&M, University of Amsterdam.
Department of Pediatrics, Amsterdam UMC, University of Amsterdam.
J Vis Exp. 2019 Nov 15(153). doi: 10.3791/60470.
At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.
在哺乳期结束时，许多哺乳动物的肠道上皮会发生重大变化，这些变化与消化固体食物的能力有关。这个过程被称为从哺乳到断奶的转变，其结果是新生儿上皮被成人上皮所取代，这与代谢和形态学调整相伴发生。这些复杂的发育变化是肠道上皮细胞内在遗传程序的结果，但在一定程度上可以受到外在因素的调节。从胎儿后期开始对小鼠原代肠道上皮细胞进行长期培养，可在体外重现从哺乳到断奶的转变。在这里，我们描述了一种最适合在体外模拟这一过程的小鼠胎儿肠道类器官培养的详细方案。我们描述了几种有用的检测方法，旨在监测与从哺乳到断奶转变相关的肠道功能随时间的变化。此外，我们还给出了一个能够在体内影响从哺乳到断奶转变的外在因素的例子，作为在体外调节从哺乳到断奶转变时间的一种表现。这种体外方法可用于研究从哺乳到断奶转变的分子机制以及这一过程的调节因子。重要的是，就研究中的动物伦理而言，用这种体外模型取代体内模型有助于改进动物实验，并可能减少用于研究肠道成熟过程的动物数量。
