Department of Mechanical & Aerospace Engineering, The University of Alabama in Huntsville, Huntsville, AL, USA.
Department of Mechanical Engineering, Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2024;2764:205-224. doi: 10.1007/978-1-0716-3674-9_14.
Organoids are 3D cultures of self-organized adult or pluripotent stem cells with an epithelial membrane enclosing a defined fluid-filled lumen. These organoids have been demonstrated with a wide range of organotypic tissue types, but the enclosed nature of the structure restricts access to the lumen and apical surface of the cell membrane. To increase the potential applications of organoids, new technologies are required to provide access to the lumen of the organoid and apical surface of the epithelial cell membrane to enable new biomedical studies. This chapter details a method to access the lumen and apical surface of an organoid utilizing a double-barrel pulled glass capillary and pressure-based pump. The organoid perfusion system uses a three-axis micromanipulator to position the double-barrel capillary to pierce the organoid with the tip of the capillary. Each barrel of the double-barrel capillary is controlled independently with the pressure-based pump to allow injection and removal of material into and from the lumen. Additionally, the organoid is immobilized with a custom-designed PDMS organoid holder. The design of the components for the organoid perfusion system and details on their use are presented here and can be utilized as the basis to enable a wide range of organoid studies including but not limited to modifying luminal contents and apical cell membrane interactions during organoid cultures, recapitulation of physiological flow within the normally static organoid lumen, and effects of mechanical strain on organoid cell development.
类器官是由自我组织的成体或多能干细胞组成的 3D 培养物，具有包裹在定义的充满液体的腔室周围的上皮膜。这些类器官已经在广泛的器官样组织类型中得到了证明，但结构的封闭性质限制了对腔室和细胞膜顶表面的访问。为了增加类器官的潜在应用，需要新技术来提供对类器官腔室和上皮细胞膜顶表面的访问，以实现新的生物医学研究。本章详细介绍了一种利用双筒拉制玻璃毛细管和基于压力的泵来访问类器官腔室和顶膜表面的方法。类器官灌注系统使用三轴微操纵器将双筒毛细管定位到用毛细管尖端刺穿类器官。双筒毛细管的每个筒都由基于压力的泵独立控制，以允许将材料注入和从腔室中取出。此外，类器官用定制的 PDMS 类器官支架固定。本文介绍了类器官灌注系统组件的设计及其使用细节，可作为基础，以实现广泛的类器官研究，包括但不限于在类器官培养过程中改变腔内容物和顶膜细胞相互作用、在正常静态类器官腔室中再现生理流动以及机械应变对类器官细胞发育的影响。
