细胞培养长期以来对于人类发育和疾病的临床前建模至关重要。然而，传统的二维 (2D) 细胞培养无法忠实地模拟体内发现的复杂性，并且在 2D 模型中显示出有希望的结果的新候选药物通常无法转化为临床。最近，三维（3D）细胞培养模型因其与体内生物学更大的生理相关性而受到欢迎。特别是，3D 球体模型因其能够模拟实体瘤而得到广泛应用，无论是结构还是从细胞外增殖层到内静止层分布的营养物的梯度。与体内肿瘤类似，在 3D 球体模型中生长的细胞系往往比 2D 培养的细胞系对抗肿瘤药物治疗具有更强的抵抗力，尽管赋予这种抵抗力的不同信号通路和基因靶标尚未得到充分探索。 RNA 干扰 (RNAi) 是阐明基因功能和在 2D 模型中发现新的可药物靶标的有效工具；然而，迄今为止，只有少数研究成功地在复杂的 3D 模型中进行 RNAi。在这里，我们在存在或不存在细胞外基质的情况下，在三种球体培养模型中使用“免转染”Dharmacon Accell siRNA 证明了有效的 RNAi 介导的敲低。这种方法有可能扩大到复杂的阵列筛选实验，这可能有助于识别比二维实验中确定的具有更大临床相关性的新型药物靶标。 © 2024 Dharmacon, Inc. 当前协议由 Wiley periodicals LLC 出版。基本方案 1：在无基质 ULA 板中生成 3D 球体 替代方案 1：生成基质胶基质嵌入的 3D 球体 替代方案 2：生成 GrowDex 水凝胶嵌入的 3D 球体 基本方案 2：递送 siRNA 并收集基质 -免费 3D 球体 替代方案 3：递送 siRNA 并收集基质嵌入的球体 基本方案 3：从球体中提取 RNA 和蛋白质，用于表征基因敲低。© 2024 Dharmacon, Inc. 当前方案由 Wiley periodicals LLC 出版。

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown.© 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC.