Li Dandan, Zheng Chunfu
Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, China.
Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, Canada.
Methods Mol Biol. 2025;2940:173-185. doi: 10.1007/978-1-0716-4615-1_16.
Infectious diseases, including recent outbreaks of H7N9 influenza, Ebola, Zika, and SARS-CoV-2, remain significant global health threats. While traditional two-dimensional (2D) cell cultures have long been the cornerstone of virology research, their inability to replicate complex in vivo microenvironments, such as cell-cell interactions, apical-basal polarity, and extracellular signaling gradients, limits their utility for studying viral pathogenesis and drug responses. Three-dimensional (3D) culture systems overcome these limitations by providing physiologically relevant platforms that better mimic native tissue environments. Among the commonly used methods, the hanging-drop method enables spheroid formation by suspending cell droplets, allowing natural cell aggregation at the liquid-air interface. The pHEMA method creates a nonadhesive surface through a poly-2-hydroxyethyl methacrylate coating, ensuring uniform spheroid sizes and minimal cell-matrix interactions. The Matrigel embedding method embeds cells in a growth factor-reduced extracellular matrix, supporting cell-matrix interactions, tissue morphogenesis, and differentiation. Finally, the inlet-well-based hanging-drop method employs a specialized inlet-well system, combining hanging-drop formation with controlled transfer to receiving wells while maintaining humidity and minimizing evaporation. These versatile methods facilitate studies on viral replication, infectivity, and antiviral screening, offering reproducible, high-fidelity models for understanding host-virus interactions and advancing therapeutic development.
包括近期爆发的H7N9流感、埃博拉、寨卡和严重急性呼吸综合征冠状病毒2（SARS-CoV-2）在内的传染病，仍然是全球重大的健康威胁。虽然传统的二维（2D）细胞培养长期以来一直是病毒学研究的基石，但它们无法复制复杂的体内微环境，如细胞间相互作用、顶-基极性和细胞外信号梯度，这限制了它们在研究病毒发病机制和药物反应方面的效用。三维（3D）培养系统通过提供能更好模拟天然组织环境的生理相关平台克服了这些限制。在常用方法中，悬滴法通过悬浮细胞液滴使球体形成，让细胞在液-气界面自然聚集。聚甲基丙烯酸-2-羟乙酯（pHEMA）法通过聚甲基丙烯酸-2-羟乙酯涂层创建一个非黏附表面，确保球体大小均匀且细胞-基质相互作用最小。基质胶包埋法将细胞包埋在生长因子减少的细胞外基质中，支持细胞-基质相互作用、组织形态发生和分化。最后，基于进样孔的悬滴法采用专门的进样孔系统，将悬滴形成与可控转移到接收孔相结合，同时保持湿度并使蒸发最小化。这些通用方法促进了对病毒复制、感染性和抗病毒筛选的研究，为理解宿主-病毒相互作用和推进治疗开发提供了可重复、高保真的模型。
