Wei Zhiyun, Kale Shubham, El Fatimy Rachid, Rabinovsky Rosalia, Krichevsky Anna M
Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States.
Front Neurosci. 2019 Apr 18;13:361. doi: 10.3389/fnins.2019.00361. eCollection 2019.
Intercellular communication within complex biological and pathological systems via extracellular vesicles (EVs) and secreted factors is a highly attractive area of research. However, cell models enabling investigation of such communication are limited. Commonly utilized is the supplementation of hyper-concentrated EVs or other extracellular factors to the recipient cell cultures. This approach requires purification of the secreted complexes and is confounded by the contamination of media components. Two-chamber co-cultures of donor and recipient cells separated by a pore membrane may represent a more physiological and better-controlled system for the investigation of intercellular communication. Yet, distinct culture conditions for different neural cell types often make them incompatible for co-culturing. Here we optimized short-term co-cultures of patient-derived low-passage glioma-initiating stem cells with normal cells of the brain microenvironment, such as primary neurons, astrocytes, microglia, and brain endothelial cells. We demonstrate the culture compatibility of these cell types and internalization of glioma-derived extracellular RNA by the normal recipient cells. The presented protocols are valuable for the investigation of intercellular communication between glioma brain tumor and cells of its microenvironment, including but not limited to the EVs-mediated communication.
Cell-to-cell communication is essential in normal physiology and implicated in disease; however, experimental systems for its modeling are limited. Particularly, the investigation of communication between brain tumors and normal cells of the brain microenvironment has been challenged by the lack of adequate culture models. Here we developed co-cultures of glioma stem cells with various types of normal brain cells, including primary neurons, astrocytes, microglia, and brain endothelial cells, and demonstrated their utility for the study of intercellular communication. Detection of proposed markers in the recipient cells confirmed RNA transfer in these co-cultures.
通过细胞外囊泡（EVs）和分泌因子在复杂生物和病理系统内进行的细胞间通讯是一个极具吸引力的研究领域。然而，能够研究这种通讯的细胞模型有限。常用的方法是向受体细胞培养物中补充高浓度的细胞外囊泡或其他细胞外因子。这种方法需要纯化分泌的复合物，并且会受到培养基成分污染的干扰。通过孔膜分隔供体细胞和受体细胞的双室共培养可能代表了一种更符合生理且控制更好的细胞间通讯研究系统。然而，不同神经细胞类型的独特培养条件常常使它们不适合共培养。在这里，我们优化了患者来源的低传代胶质瘤起始干细胞与脑微环境正常细胞（如原代神经元、星形胶质细胞、小胶质细胞和脑内皮细胞）的短期共培养。我们证明了这些细胞类型的培养兼容性以及正常受体细胞对胶质瘤衍生细胞外RNA的内化。所提供的方案对于研究胶质瘤脑肿瘤与其微环境细胞之间的细胞间通讯非常有价值，包括但不限于细胞外囊泡介导的通讯。
细胞间通讯在正常生理学中至关重要且与疾病有关；然而，用于模拟它的实验系统有限。特别是，脑肿瘤与脑微环境正常细胞之间通讯的研究一直受到缺乏合适培养模型的挑战。在这里，我们开发了胶质瘤干细胞与各种类型正常脑细胞（包括原代神经元、星形胶质细胞、小胶质细胞和脑内皮细胞）的共培养，并证明了它们在细胞间通讯研究中的实用性。在受体细胞中检测到所提出的标志物证实了这些共培养中的RNA转移。
