Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis
Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis
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DOI：
10.1177/1087057106292763
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发表时间：
2006-12-01
影响因子：
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通讯作者：
Kubbies, Manfred
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文献类型：
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作者：
Ivascu, Andrea;Kubbies, Manfred
球体广泛用于生物学，因为它们提供体外三维（3D）模型来研究肿瘤中细胞的增殖、细胞死亡、分化和代谢以及肿瘤对放疗和化疗的反应。产生球状体的方法受到尺寸异质性、长培养时间或用于更高通量方式的机械可达性的限制。作者提出了一种在单个威尔斯孔中悬浮培养产生单个球状体的快速方法。将1000至20，000个范围内的规定数量的细胞接种到标准培养基中的聚HEMA包被的96孔圆底或锥底平板的威尔斯孔中，并以1000 g离心10 min。该程序在24小时培养时间内在每个孔中产生单个球状体，其具有均匀的尺寸、形态和边缘中增殖细胞的分层以及核心区域中垂死细胞的分层。由于大量肿瘤细胞系在3D培养时仅形成松散的聚集体，因此作者还对培养基添加剂进行了筛选，以实现从聚集体到球体形态的转变。少量的基底膜提取物基质胶，加入到培养基中，离心前，最有效地诱导致密的球状体形成。在真正的悬浮培养物中，早在离心后24小时，致密的球状体形态就很明显。不同谱系的二十种肿瘤细胞系已被用于在96孔板中成功地产生具有均匀尺寸的紧凑的单个球状体，并且易于获得用于随后的功能分析。
Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round- or conical-bottom plates in standard medium and centrifuged for 10 min at 1000g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.