Moorefield Emily C, Blue R Eric, Quinney Nancy L, Gentzsch Martina, Ding Shengli
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 111 Mason Farm Road, 6340C MBRB, CB #7545, Chapel Hill, NC, 27599-7545, USA.
Marsico Lung Institute/Cystic Fibrosis Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
BMC Cell Biol. 2018 Aug 15;19(1):15. doi: 10.1186/s12860-018-0165-0.
Conditional reprogramming has enabled the development of long-lived, normal epithelial cell lines from mice and humans by in vitro culture with ROCK inhibitor on a feeder layer. We applied this technology to mouse small intestine to create 2D mouse intestinal epithelial monolayers (IEC monolayers) from genetic mouse models for functional analysis.
IEC monolayers form epithelial colonies that proliferate on a feeder cell layer and are able to maintain their genotype over long-term passage. IEC monolayers form 3D spheroids in matrigel culture and monolayers on transwell inserts making them useful for functional analyses. IEC monolayers derived from the Cystic Fibrosis (CF) mouse model CFTR ∆F508 fail to respond to CFTR activator forskolin in 3D matrigel culture as measured by spheroid swelling and transwell monolayer culture via Ussing chamber electrophysiology. Tumor IEC monolayers generated from the Apc mouse intestinal cancer model grow more quickly than wild-type (WT) IEC monolayers both on feeders and as spheroids in matrigel culture.
These results indicate that generation of IEC monolayers is a useful model system for growing large numbers of genotype-specific mouse intestinal epithelial cells that may be used in functional studies to examine molecular mechanisms of disease and to identify and assess novel therapeutic compounds.
条件重编程通过在饲养层上与ROCK抑制剂进行体外培养，使得从小鼠和人类中开发出长寿的正常上皮细胞系成为可能。我们将这项技术应用于小鼠小肠，从基因小鼠模型中创建二维小鼠肠上皮单层（IEC单层）用于功能分析。
IEC单层形成上皮集落，在饲养细胞层上增殖，并能够在长期传代过程中保持其基因型。IEC单层在基质胶培养中形成三维球体，在Transwell小室中形成单层，这使其可用于功能分析。通过球体肿胀和经Ussing小室电生理学的Transwell单层培养测量，源自囊性纤维化（CF）小鼠模型CFTR ∆F508的IEC单层在三维基质胶培养中对CFTR激活剂福斯高林无反应。由Apc小鼠肠道癌模型产生的肿瘤IEC单层在饲养层上以及在基质胶培养中作为球体生长时，比野生型（WT）IEC单层生长得更快。
这些结果表明，IEC单层的产生是一个有用的模型系统，可用于培养大量基因型特异性的小鼠肠上皮细胞，这些细胞可用于功能研究，以研究疾病的分子机制，并识别和评估新型治疗化合物。
