Three-dimensional (3D) cell culture systems more closely mimic the in vivo cellular microenvironment than traditional two-dimensional cell culture methods, making them a valuable tool in drug screening assays. However, 3D environments often make analysis of cellular responses more difficult, so most high-throughput (HT) 3D assays have been limited to measurements of cell viability. Yet, many other cell functions contribute to disease and are important pharmacological targets. Therefore, there is a need for new technologies that enable HT measurements of a wider range of cell functions for drug screening. Here, we have adapted a hydrogel system that enables cells to be cultured in a 3D environment and allows for the simultaneous detection of matrix metalloproteinase (MMP) and metabolic activities. This system was then characterized for utility in HT screening approaches. MMPs are critical regulators of tissue homeostasis and are upregulated in many diseases, such as arthritis and cancer. The developed assay achieved Z'-factor values above 0.9 and 0.5 for enzymatic and cellular assays, respectively, intraplate coefficients of variation (%CV) below 10% and 12%, respectively, and signal measurement was unaffected by dimethyl sulfoxide, a common solvent of therapeutic compounds. Human MMP-1, -2, and -9 resulted in a significant increase in signal intensity. Encapsulation of several cell types produced robust signals above background noise and within the linear range of the assay. Multiple drugs that are known to alter MMP activity were utilized in a range of concentrations with a fibrosarcoma cell line to demonstrate the feasibility of the assay for HT applications. This assay combines 3D cellular encapsulation and MMP activity detection in HT format, which makes it suitable for drug screening and development applications.

中文翻译：

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高通量三维水凝胶细胞封装测定法，用于测量基质金属蛋白酶活性。

三维（3D）细胞培养系统比传统的二维细胞培养方法更能模仿体内细胞微环境，使其成为药物筛选测定中的重要工具。但是，3D环境通常使细胞反应的分析更加困难，因此大多数高通量（HT）3D分析仅限于细胞活力的测量。然而，许多其他细胞功能也会导致疾病，并且是重要的药理学靶标。因此，需要能够对更广泛的细胞功能进行HT测量以进行药物筛选的新技术。在这里，我们采用了一种水凝胶系统，该系统可使细胞在3D环境中培养，并允许同时检测基质金属蛋白酶（MMP）和代谢活性。然后将该系统表征为可用于HT筛选方法。MMP是组织动态平衡的关键调节剂，在许多疾病（如关节炎和癌症）中均被上调。对于酶和细胞分析，开发的分析方法的Z'因子值分别高于0.9和0.5，板内变异系数（％CV）分别低于10％和12％，信号测量不受二甲亚砜（一种常见溶剂）的影响治疗化合物。人MMP-1，-2和-9导致信号强度显着增加。几种细胞类型的封装产生了高于背景噪声且在测定的线性范围内的稳健信号。在纤维肉瘤细胞系中以一定浓度使用多种已知可改变MMP活性的药物，以证明该方法可用于HT应用。该测定法将3D细胞封装和HT格式的MMP活性检测结合在一起，使其适用于药物筛选和开发应用。