Wilke Georgia, Wang Yi, Ravindran Soumya, Stappenbeck Thaddeus, Witola William H, Sibley L David
Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA.
Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.
Methods Mol Biol. 2020;2052:351-372. doi: 10.1007/978-1-4939-9748-0_20.
Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite's natural environment in the small intestine. We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an "air-liquid interface" that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host-parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.
微小隐孢子虫具有复杂的生命周期，包括无性和有性阶段，最终在体内形成卵囊。目前用于研究微小隐孢子虫的最广泛使用的细胞培养平台仅支持几天的生长，并且不允许寄生虫进入有性阶段以完成卵囊形成。此外，这些细胞培养平台大多是腺癌细胞系，不能充分模拟寄生虫在小肠中的自然环境。我们在此描述一种创建支持微小隐孢子虫长期生长原发性肠上皮细胞单层的方法。单层细胞来源于作为球体生长的小鼠肠道干细胞，并接种到Transwell上，形成独立的顶端和基底外侧区室。在顶端腔室中，去除细胞生长培养基以创建“气液界面”，从而增强宿主细胞分化并支持微小隐孢子虫的长期生长。使用原发性肠细胞在体外培养微小隐孢子虫将成为一个有价值的工具，用于利用更类似于肠道自然生态位的便捷体外模型研究宿主 - 寄生虫相互作用。
