Kelm Jens M, Timmins Nicholas E, Brown Catherine J, Fussenegger Martin, Nielsen Lars K
Laboratory for Biological Engineering, Department of Chemical Engineering, University of Queensland, 4072 Brisbane, Australia.
Biotechnol Bioeng. 2003 Jul 20;83(2):173-80. doi: 10.1002/bit.10655.
Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue.
多细胞肿瘤球体（MCTS）被用作正常和实体瘤组织的器官型模型。传统的生成MCTS的技术，如在非粘附表面、悬浮液或支架上生长，存在许多缺点，包括需要人工选择以获得均匀的群体以及使用非生理性基质化合物。在本研究中，我们描述了一种生成MCTS的温和方法，其中单个球体在微量滴定板悬挂的液滴中形成。该方法已成功应用于广泛的细胞系，效率接近100%（即每滴一个球体）。使用肝癌细胞系HepG2，悬滴法生成了形状圆润、大小分布狭窄的MCTS（变异系数[CV]为10%至15%，相比之下，在非粘附表面生长的变异系数为40%至60%）。对HepG2和乳腺腺癌细胞系MCF-7组成的球体进行结构分析，发现其具有高度组织化的三维组织样结构以及广泛的细胞外基质。悬滴法是生成MCTS的一种有吸引力的替代方法，因为它温和，可应用于多种细胞系，且无需筛选或人工选择就能产生大小均匀的球体。该方法可应用于生理学和代谢、肿瘤生物学、毒理学、细胞组织以及生物人工组织开发等基础研究。
