细胞外囊泡（EVs）是细胞自然分泌的纳米级颗粒，用于细胞间的通信，并利用脂质双层封装生物活性分子货物，如蛋白质和RNA。肿瘤细胞源性EVs（tdEVs）尤其是癌症研究中有很大应用前景，因为它们的内容反映了起源细胞。在大多数研究中，tdEVs是从以静止状态培养的癌细胞中获得的，因此缺乏重现体内细胞微环境的能力。最近，可渗透细胞培养系统的发展允许氧气和营养梯度模拟生理和细胞微环境。然而，由于这些系统通过在统一结构内循环培养基进行灌流，因此分别在每个时间点独立收集细胞和EVs进行分析存在局限性。在本研究中，设计了一个用于灌注和实时收集EV的模块化细胞培养系统。该系统由三个可拆卸的仓室组成，分别用于新鲜培养基、细胞培养和EV收集。新鲜培养基通过注射泵注入的水压控制流速从培养基室流到培养室。当含有EV的培养基超过室内一定体积时，它会溢出到收集室收集EVs。这些紧凑和模块化的仓室与实验室中使用的传统细胞培养模式高度互操作，因此可以进行各种基于EV的分析。

Extracellular vesicles (EVs) are nanometer-sized particles naturally secreted by cells for intercellular communication that encapsulate bioactive cargo, such as proteins and RNA, with a lipid bilayer. Tumor cell-derived EVs (tdEVs) are particularly promising biomarkers for cancer research because their contents reflect the cell of origin. In most studies, tdEVs have been obtained from cancer cells cultured under static conditions, thus lacking the ability to recapitulate the microenvironment of cells in vivo. Recent developments in perfusable cell culture systems have allowed oxygen and a nutrient gradient to mimic the physiological and cellular microenvironment. However, as these systems are perfused by circulating the culture medium within the unified structure, independently harvesting cells and EVs at each time point for analysis presents a limitation. In this study, a modularized cell culture system is designed for the perfusion and real-time collection of EVs. The system consists of three detachable chambers, one each for fresh medium, cell culture, and EV collection. The fresh medium flows from the medium chamber to the culture chamber at a flow rate controlled by the hydraulic pressure injected with a syringe pump. When the culture medium containing EVs exceeds a certain volume within the chamber, it overflows into the collection chamber to harvest EVs. The compact and modularized chambers are highly interoperable with conventional cell culture modalities used in the laboratory, thus enabling various EV-based assays.