Charwat Verena, Schütze Karin, Holnthoner Wolfgang, Lavrentieva Antonina, Gangnus Rainer, Hofbauer Pablo, Hoffmann Claudia, Angres Brigitte, Kasper Cornelia
University of Natural Resources and Life Sciences, Department of Biotechnology, Vienna, Austria.
CellTool GmbH, Bernried, Germany.
J Biotechnol. 2015 Jul 10;205:70-81. doi: 10.1016/j.jbiotec.2015.02.007. Epub 2015 Feb 14.
Today highly complex 3D cell culture formats that closely mimic the in vivo situation are increasingly available. Despite their wide use, the development of analytical methods and tools that can work within the depth of 3D-tissue constructs lags behind. In order to get the most information from a 3D cell sample, adequate and reliable assays are required. However, the majority of tools and methods used today have been originally designed for 2D cell cultures and translation to a 3D environment is in general not trivial. Ideally, an analytical method should be non-invasive and allow for repeated observation of living cells in order to detect dynamic changes in individual cells within the 3D cell culture. Although well-established laser confocal microscopy can be used for these purposes, this technique has serious limitations including penetration depth and availability. Focusing on two relevant analytical methods for live-cell monitoring, we discuss the current challenges of analyzing living 3D samples: microscopy, which is the most widely used technology to observe and examine cell cultures, has been successfully adapted for 3D samples by recording of so-called "z-stacks". However the required equipment is generally very expensive and therefore access is often limited. Consequently alternative and less advanced approaches are often applied that cannot capture the full structural complexity of a 3D sample. Similarly, image analysis tools for quantification of microscopic images range from highly specialized and costly to simplified and inexpensive. Depending on the actual sample composition and scientific question the best approach needs to be assessed individually. Another more recently introduced technology for non-invasive cell analysis is Raman micro-spectroscopy. It enables label-free identification of cellular metabolic changes with high sensitivity and has already been successful applied to 2D and 3D cell cultures. However, its future significance for cell analysis will strongly depend on the availability of application oriented and user-friendly systems including specific tools for easy analysis and interpretation of spectral data focusing on biological relevant information.
如今，越来越多高度复杂的、能紧密模拟体内情况的3D细胞培养形式可供使用。尽管它们被广泛应用，但能在3D组织构建体深度范围内发挥作用的分析方法和工具的开发却滞后了。为了从3D细胞样本中获取最多信息，需要适当且可靠的检测方法。然而，当今使用的大多数工具和方法最初是为2D细胞培养设计的，将其转换到3D环境通常并非易事。理想情况下，一种分析方法应该是非侵入性的，并允许对活细胞进行重复观察，以便检测3D细胞培养中单个细胞的动态变化。尽管成熟的激光共聚焦显微镜可用于这些目的，但该技术存在严重局限性，包括穿透深度和可用性。聚焦于两种用于活细胞监测的相关分析方法，我们讨论分析活的3D样本当前面临的挑战：显微镜是观察和检测细胞培养最广泛使用的技术，通过记录所谓的“z轴堆叠”已成功应用于3D样本。然而，所需设备通常非常昂贵，因此使用机会往往有限。因此，人们常常采用替代的、不太先进的方法，这些方法无法捕捉3D样本的全部结构复杂性。同样，用于定量显微图像的图像分析工具从高度专业化且昂贵到简化且廉价不等。需要根据实际样本组成和科学问题单独评估最佳方法。另一种最近引入的非侵入性细胞分析技术是拉曼显微光谱。它能够以高灵敏度对细胞代谢变化进行无标记识别，并且已经成功应用于2D和3D细胞培养。然而，其在细胞分析方面的未来意义将在很大程度上取决于面向应用且用户友好的系统的可用性，包括用于轻松分析和解释聚焦于生物学相关信息的光谱数据的特定工具。
