病毒学研究
在病毒学研究
中使用活细胞成像
病毒学是研究病毒和病毒性疾病的生物学。作为专性细胞内寄生虫，病毒劫持宿主细胞的分子机制并利用它产生新的病毒颗粒。
尽管活细胞成像由于其体积小 (20 – 300 nm) 和光学显微镜本身施加的限制而不能用于病毒的直接可视化，但这种技术极大地促进了对复杂病毒-宿主相互作用的理解。活细胞显微镜可用于可视化和量化由病毒感染引起的形态和功能变化，称为病毒细胞病变效应 (CPE)。一些病毒只会引起细微的细胞变化，而其他病毒会裂解细胞并破坏宿主细胞单层。受感染细胞的连续成像可以在体外检测到这些病毒诱导的细胞变化的全部范围，从而可以研究病毒感染的进展以及筛选新的抗病毒药物和疫苗。
BSL-3/4 实验室的活细胞成像
生物安全级别（BSL）-3和4级实验室是专为研究高致病性病毒和细菌而设计的高防护设施。其中包括 SARS-CoV-2、COVID-19 的病原体、结核分枝杆菌、埃博拉病毒和马尔堡病毒等。 BSL-3 和 4 设施在针对世界上最危险微生物的诊断和治疗开发中发挥着核心作用，但是在这些实验室中工作存在许多挑战。尽管采取了健康和安全措施，研究人员仍然面临感染危及生命的疾病的巨大风险。此外，一次性防护设备的高成本以及与执行基于 BSL-3/4 的实验相关的时间和劳动力，对研究人员施加了进一步的限制。细胞培养的远程监控减少了在 BSL-3/4 实验室中花费的时间。
它也是一种更具成本效益的替代方案，因为在远程分析细胞培养物时无需使用昂贵的保护设备。一旦感染了病毒，细胞培养物就可以放置在培养箱内，并通过 CytoSMART 活细胞成像设备进行安全分析，最大限度地减少研究人员对生物危害的暴露。可以通过 CytoSMART Cloud 立即访问图像和延时视频，提供有关细胞培养和运行实验的实时更新。
病毒性细胞病变效应 (CPE)（链接 – CytoSMART CPE 文章）通常包括贴壁感染细胞的变圆、分离和结块，导致在汇合的细胞单层中形成孔洞。 CPE 分析的传统方法（例如斑块和病灶形成分析）通常是终点病毒学分析，涉及使用明场显微镜手动检查 CPE，使其成为一种劳动密集型、耗时且依赖于用户的方法。 CytoSMART 设备（LINK – CytoSMART 产品），包括 CytoSMART Omni (LINK) 和 Lux3 FL (LINK)，可以使用明场和荧光成像实时分析 CPE。 CPE 的发生可以一次监测数天或数周，而不会错过病毒诱导的 CPE 发展的任何关键阶段。此外，图像和延时电影是自动获取和分析的。例如，可以使用基于 AI 的汇合算法（LINK – 应用程序/细胞汇合）测量宿主细胞计数的减少，从而消除结果解释中的任何主观性和偏见。
Videos
Appnotes
Cell Viability Analysis
Cell Culture Optimization
Cell Culture Monitoring - Lux2
Cell Viability Analysis
Cell viability, growth and cytotoxicity studies can be performed using metabolic activity assays. The overall metabolic activity of the cell is indicated by the enzymatic cleavage of colorimetric or fluorescent substrates.
While these assays are relatively straightforward and cheap, they are dependent on culture conditions and intrinsic metabolic activity of the cell type that is being investigated. Furthermore, depletion of the metabolic substrate can lead to a plateau in the fluorescent signal, making assay output unreliable. To overcome these limitations cell viability could be determined optically using confluency measurements.
In the study described here the performance of confluency measurements to assess cell viability were compared to a metabolic activity assay: cell titer blue. Confluency was visualized using automated bright-field microscopy and subsequently analyzed using image analysis algorithms. Images were collected inside a CO2-incubator, keeping the culture at optimal conditions. For the cell titer blue assay resazurin was added to the medium and incubated for 3 hours. The fluorescent signal was normalized to the control to obtain the relative metabolic activity as a measure of cell viability. The comparison between the methods was performed for two pancreatic cancer cell lines, PACO7 and POCA43.
Cell Culture Optimization
Mammalian cells are being used in many research fields including tissue engineering, regenerative medicine and drug discovery. To ensure manageable research, cell cultures should be robust and experimentally reproducible. Therefor, optimization of culture conditions is an essential part of lab work. Media composition, temperature, O2 levels and seeding density influence the cell growth. To monitor the cell cultures, researchers regularly observe cell growth using bright-field microscopy.
In the study performed here, an alternative method for manual inspection of cell growth has been explored. Automated live-cell imaging was used to examine a 48-well plate containing CHO-K1 cells at varying seeding densities (n=6) from inside a CO2-incubator.
Cell Culture Monitoring - Lux2
Setting up cell cultures is easy enough. However monitoring your cultures and optimization is time-consuming and cumbersome. Waiting for the ideal confluency, quickly studying effects of various media means taking your cells in and out of the incubator more often than you would like.
Visualizing cell cultures from inside an incubator using a compact microscope that facilitates live cell imaging can overcome these issues. While live cell imaging has been restricted to costly, high-end devices, the CytoSMART Lux2 offers an affordable and easy-to-use alternative for virtually any lab. The CytoSMART Lux2 can be set up in minutes, enabling untrained users to quickly perform their own time-lapse recordings.
Images and videos can be easily accessed and retrieved from the CytoSMART cloud portal. Advanced functions, such as reporting of cell confluency, cell migration analysis and the option to use automatic confluency email alerts, can be applied to inform the user when certain culture conditions are reached (for example, once the cell culture has reached the desired confluency). Hence, the CytoSMART Lux2 can be used in many different ways to facilitate cell culture work and research.
In the following appnote several examples of applications of the CytoSMART Lux2 will be shown.
- Culturing cells in hypoxic conditions
- Standardizing cell culturing conditions
- The effect of confluency on transfection efficiency