Lutter Anne-Helen, Scholka Jenny, Richter Heiko, Anderer Ursula
Department of Cell Biology and Tissue Engineering, Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany.
Klinikum Niederlausitz GmbH, Department Trauma Surgery and Orthopedics, Senftenberg, Germany.
Clin Hemorheol Microcirc. 2017;67(3-4):327-342. doi: 10.3233/CH-179213.
Tetrazolium-based assays are optimized to assess proliferation/toxicity of monolayer or suspension cells in microtiter plates. With regard to tissue engineering and regenerative medicine the need for in vivo like 3D microtissues has an increasing relevance. Applying tetrazolium-based assays to 3D culture systems is technically more challenging. The composed microenvironment may influence the assay standards, e.g. equal distribution of tetrazolium.
Evaluation of membrane-impermeable tetrazolium salt-based assays with regard to spheroid culture (3D) of human chondrocytes.
Chondrocytes were isolated from human articular cartilage. XTT, WST-1, and WST-8 were applied to monolayer cells (2D, varying cell numbers) and spheroids (3D, different sizes) in 96well plates. Formazan formation was measured spectrophotometrically after different incubation periods. Evaluation was done using phase contrast microsopy (toxicity), analyzing the correlation of cell number and absorbance signals (Gompertz function), and document signal over background ratio.
In monolayer culture the assays showed a correlation between seeded cell numbers and absorption data. Spheroid sizes are directly related to the starting cell number. A correlation between size and absorbance was only detectable starting from 10,000 cells/aggregate. Phase contrast microscopy of monolayer cells revealed strong toxicity effects of the WST-1 (4 h) and XTT (8 h) assay and no signs of toxicity using WST-8.
The WST-8 assay is non-toxic and revealed the highest sensitivity in comparison to the XTT or WST-1 assay. There is evidence, that only cells of the outer rim of spheroids are able to convert membrane-impermeable tetrazolium salts to formazans.
基于四氮唑的检测方法经过优化，用于评估微孔板中单层或悬浮细胞的增殖/毒性。对于组织工程和再生医学而言，构建类似体内的三维微组织的需求日益增加。将基于四氮唑的检测方法应用于三维培养系统在技术上更具挑战性。复杂的微环境可能会影响检测标准，例如四氮唑的均匀分布。
评估基于膜不透性四氮唑盐的检测方法用于人软骨细胞球体培养（三维）的效果。
从人关节软骨中分离软骨细胞。将XTT、WST-1和WST-8应用于96孔板中的单层细胞（二维，不同细胞数量）和球体（三维，不同大小）。在不同孵育时间后，通过分光光度法测量甲臜形成。使用相差显微镜进行评估（毒性），分析细胞数量与吸光度信号的相关性（Gompertz函数），并记录信号与背景比值。
在单层培养中，检测方法显示接种细胞数量与吸收数据之间存在相关性。球体大小与起始细胞数量直接相关。仅从10,000个细胞/聚集体开始，才能检测到大小与吸光度之间的相关性。单层细胞的相差显微镜检查显示WST-1（4小时）和XTT（8小时）检测方法具有强烈的毒性作用，而使用WST-8未观察到毒性迹象。
与XTT或WST-1检测方法相比，WST-8检测方法无毒且灵敏度最高。有证据表明，只有球体边缘的细胞能够将膜不透性四氮唑盐转化为甲臜。
