High-Throughput Three-Dimensional Hydrogel Cell Encapsulation Assay for Measuring Matrix Metalloproteinase Activity
High-Throughput Three-Dimensional Hydrogel Cell Encapsulation Assay for Measuring Matrix Metalloproteinase Activity
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DOI：
10.1089/adt.2018.877
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发表时间：
2019-04-01
影响因子：
1.8
通讯作者：
Leight, Jennifer L.
中科院分区：
文献类型：
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作者：
Fakhouri, Abdulaziz S.;Weist, Jessica L.;Leight, Jennifer L.
三维（3D）细胞培养系统比传统的二维细胞培养方法更接近地模拟体内细胞微环境，使其成为药物筛选测定中有价值的工具。然而，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测定仅限于测量细胞活力。然而，许多其他细胞功能有助于疾病，并且是重要的药理学靶点。因此，需要新的技术，使HT测量更广泛的细胞功能，用于药物筛选。在这里，我们采用了一种水凝胶系统，使细胞能够在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，该系统的特点是在HT筛选方法中的实用性。MMPs是组织稳态的关键调节剂，并且在许多疾病中上调，例如关节炎和癌症。所开发的测定法对于酶测定法和细胞测定法分别实现了高于0.9和0.5的Z '-因子值，板内变异系数（%CV）分别低于10%和12%，并且信号测量不受二甲亚砜（治疗化合物的常用溶剂）的影响。人MMP-1、MMP-2和MMP-9导致信号强度显著增加。几种细胞类型的包封产生了高于背景噪声且在测定的线性范围内的稳健信号。已知改变MMP活性的多种药物以一定浓度范围与纤维肉瘤细胞系一起使用，以证明HT应用的测定的可行性。该检测结合了HT格式的3D细胞包封和MMP活性检测，这使得它适合于药物筛选和开发应用。
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.