The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, Darlinghurst, NSW, Australia.
Faculty of Medicine, St Vincent's Clinical School, UNSW Sydney, Kensington, NSW, Australia.
Methods Mol Biol. 2021;2294:27-42. doi: 10.1007/978-1-0716-1350-4_3.
Three-dimensional models of spheroid formation have been routinely used in the cancer field to test the colony forming capacity of malignant cells in an in vitro setting. Use of such a model provides a robust surrogate for in vivo testing, enabling large-scale interrogation into the effect of certain treatment conditions. This adapted protocol describes a high throughput and readily accessible composite alginate hydrogel system for spheroid formation, within a biomechanically tunable three-dimensional environment. This model therefore allows users to examine the effect of certain treatment conditions while cells are embedded within a hydrogel of defined stiffness. This is particularly important in the context of cancer where cells experience a wide range of mechanical properties within their microenvironment, driven by widespread changes in the extracellular matrix composition and architecture.This protocol describes a high-throughput method which results in homogeneous interpenetrating polymer networks of collagen and alginate. We show that this network readily supports single-cell spheroid formation in numerous malignant cell lines (breast cancer, lung cancer, and melanoma) and that these can be robustly analyzed for colony formation measures such as spheroid size, spheroid number, and overall cell viability; therefore, allowing users to undertake high-throughput, in vitro screening against a controlled biomechanical background.
三维球体形成模型已被广泛应用于癌症领域，用于测试体外环境中恶性细胞的集落形成能力。使用这种模型提供了一种强大的替代体内测试的方法，能够大规模地研究特定治疗条件的影响。本改编方案描述了一种高通量且易于获得的复合藻酸盐水凝胶系统，用于在可生物力学调节的三维环境中形成球体。因此，该模型允许用户在细胞嵌入具有特定硬度的水凝胶时检查某些治疗条件的影响。在癌症的背景下，这一点尤为重要，因为细胞在其微环境中经历广泛的细胞外基质组成和结构变化，从而导致其经历广泛的力学性质变化。本方案描述了一种高通量方法，该方法导致胶原和藻酸盐的互穿聚合物网络均匀化。我们表明，该网络可以轻松支持多种恶性细胞系（乳腺癌、肺癌和黑色素瘤）中的单细胞球体形成，并且可以对球体大小、球体数量和总体细胞活力等集落形成措施进行稳健分析；因此，允许用户在受控的生物力学背景下进行高通量的体外筛选。
