Department of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Madhya Pradesh, India.
Department of Chemistry, Indian Institute of Technology Indore, Madhya Pradesh, India.
Curr Protoc. 2021 Jul;1(7):e199. doi: 10.1002/cpz1.199.
In recent years, 3D culture of tumor spheroids has managed to revolutionize cancer research and drug discovery. 2D monolayer cells grown in cell culture flasks undergo radical changes in cell behavior, structure, and function owing to varying environmental cues and are unable to provide predictive data for preclinical evaluation. 3D tumor spheroids can better recapitulate tumor architecture, cell-cell and cell-matrix connectivity, and the tissue complexity of tumors grown in animal models. However, many of the existing techniques to culture 3D spheroids are time-consuming and ineffective and produce irregular-shaped spheroids that cannot be easily incorporated in biological assays. The set of protocols described herein makes use of a commercial hair brush as a template to create concave micro-well impressions in agarose. This technique is easy, inexpensive, and adaptable and also has the ability to produce uniform, homogenous cancer spheroids, with large diameter (∼1000 μm) and thickness (∼250 μm), within 24 to 48 hr after cell seeding. The 3D spheroids produced using the agarose micro-well platform function as an excellent 3D in vitro model for understanding the extent of penetration, uptake, and distribution of targeted cargos such as a diagnostic or therapeutic agents for identification and treatment of cancer. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Fabrication of agarose micro-well scaffold for growing tumor spheroids using a commercial hair brush Basic Protocol 2: Formation of homogenous tumor spheroids in agarose micro-well platform Basic Protocol 3: Assessing viability of 3D tumor spheroids grown in agarose micro-wells using confocal microscopy Basic Protocol 4: Analyzing uptake and penetration of targeted fluorescent bioconjugate in 3D tumor spheroids using two-photon imaging.
近年来，肿瘤球体的 3D 培养成功地推动了癌症研究和药物发现的发展。在细胞培养瓶中生长的 2D 单层细胞由于环境线索的变化，其细胞行为、结构和功能发生了根本性变化，无法为临床前评估提供预测数据。3D 肿瘤球体能够更好地重现肿瘤结构、细胞-细胞和细胞-基质连接以及动物模型中生长的肿瘤的组织复杂性。然而，许多现有的 3D 球体培养技术既耗时又低效，并且产生的球体形状不规则，难以纳入生物测定。本文所述的一系列方案利用商用发刷在琼脂糖中创建凹微井印痕。这种技术简单、廉价且适应性强，还能够在 24 至 48 小时内产生均匀、同质的癌症球体，其直径（约 1000μm）和厚度（约 250μm）较大。使用琼脂糖微井平台产生的 3D 球体是了解靶向载药（如诊断或治疗剂）渗透、摄取和分布程度的优秀体外 3D 模型，可用于癌症的诊断和治疗。© 2021 威立出版社。基础方案 1：使用商用发刷制造用于生长肿瘤球体的琼脂糖微井支架基础方案 2：在琼脂糖微井平台上形成均匀的肿瘤球体基础方案 3：使用共聚焦显微镜评估在琼脂糖微井中生长的 3D 肿瘤球体的活力基础方案 4：使用双光子成像分析靶向荧光生物缀合物在 3D 肿瘤球体中的摄取和穿透。
