人类肠道微生物群越来越被认为是稳态和疾病的关键因素。缺乏生理相关的体外模型来研究宿主-微生物相互作用被认为是微生物群研究的实质性瓶颈。类器官代表了一个有吸引力的模型系统,因为它们来自原始组织并体现了天然肠腔的关键特性;然而,进入类器官腔进行实验扰动是具有挑战性的。这里,我们报告了高通量类器官显微注射系统的开发和验证,该系统用于向类器官腔内输送货物和高含量采样.
使用现成的和三维打印组件设计了微注射平台。微注射针针对垂直轨迹和可重复的注射量进行了修改。计算机视觉(CVis)和微制造的CellRaft阵列(细胞微系统,研究三角公园,NC)用于增加通量并实现模拟细菌群落的高含量采样。使用COMSOLMultiphysics平台进行的建模预测了低氧腔环境,该环境在功能上已通过粪便来源的微生物群落移植和非孢子性厌氧菌的单一培养得到了验证。
CVis鉴定并记录适合注射的类器官的位置。可以以大约90个类器官/小时的速度将0.2nL的可复制载荷微注射到类器官腔中。CVis分析并证实在18小时内注射的货物在约500个类器官中的保留,并且显示需要对类器官生长进行归一化以准确评估屏障功能。CVis分析了绿色荧光蛋白或Discosomasp的模拟群落的生长动力学。红色荧光蛋白表达细菌,即使在存在抗生素以控制培养基污染的情况下,它也会在类器官腔内生长。来自粪便样品的复杂微生物群落在结肠样腔中存活并生长,而复杂性没有明显变化。
对类器官的高通量显微注射代表了研究胃肠腔生理学和胃肠微生物群的下一代体外方法。
The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling.
A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe.
CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity.
High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.