National Center for Advancing Translational Sciences (NCATS), National Institutes of Health (NIH), 9800 Medical Center Drive, Rockville, MD 20850, USA.
Department of Life Sciences and Facility Management, Institute for Chemistry and Biotechnology (ICBT), Zurich University of Applied Sciences (ZHAW), 8820 Wädenswil, Switzerland.
Cells. 2022 Jul 6;11(14):2131. doi: 10.3390/cells11142131.
The neuroglial extracellular matrix (ECM) provides critical support and physiological cues for the proper growth, differentiation, and function of neuronal cells in the brain. However, in most in vitro settings that study neural physiology, cells are grown as monolayers on stiff surfaces that maximize adhesion and proliferation, and, therefore, they lack the physiological cues that ECM in native neuronal tissues provides. Macromolecular crowding (MMC) is a biophysical phenomenon based on the principle of excluded volume that can be harnessed to induce native ECM deposition by cells in culture. Here, we show that MMC using two species of Ficoll with vitamin C supplementation significantly boosts deposition of relevant brain ECM by cultured human astrocytes. Dopaminergic neurons cocultured on this astrocyte-ECM bed prepared under MMC treatment showed longer and denser neuronal extensions, a higher number of pre ad post synaptic contacts, and increased physiological activity, as evidenced by higher frequency calcium oscillation, compared to standard coculture conditions. When the pharmacological activity of various compounds was tested on MMC-treated cocultures, their responses were enhanced, and for apomorphine, a D2-receptor agonist, it was inverted in comparison to control cell culture conditions, thus emulating responses observed in in vivo settings. These results indicate that macromolecular crowding can harness the ECM-building potential of human astrocytes in vitro forming an ultra-flat 3D microenvironment that makes neural cultures more physiological and pharmacological relevant.
神经胶质细胞细胞外基质（ECM）为大脑中神经元细胞的正常生长、分化和功能提供了关键的支持和生理信号。然而，在大多数研究神经生理学的体外环境中，细胞以单层形式生长在坚硬的表面上，这种表面最大限度地促进了细胞的黏附和增殖，因此，它们缺乏天然神经元组织中 ECM 提供的生理信号。大分子拥挤（MMC）是一种基于排斥体积原理的生物物理现象，可以被利用来诱导细胞在培养中沉积天然 ECM。在这里，我们展示了使用两种 Ficoll 与维生素 C 补充剂的 MMC 可以显著促进培养的人星形胶质细胞中相关脑 ECM 的沉积。在 MMC 处理下的这种星形胶质细胞-ECM 床层上共培养的多巴胺能神经元表现出更长和更密集的神经元延伸、更多的前突触和后突触接触，以及更高的生理活性，表现为更高的钙振荡频率，与标准共培养条件相比。当在 MMC 处理的共培养物上测试各种化合物的药理活性时，它们的反应增强了，并且对于阿扑吗啡（一种 D2 受体激动剂），与对照细胞培养条件相比，其反应被反转，从而模拟了体内观察到的反应。这些结果表明，大分子拥挤可以在体外利用人星形胶质细胞的 ECM 构建潜力，形成超平的 3D 微环境，使神经培养更接近生理和药理学相关。
