title: Potential and limitations of microscopy and Raman spectroscopy for live-cell analysis of 3D cell cultures.
abstract: 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.
authors: University of Natural Resources and Life Sciences, Department of Biotechnology, Vienna, Austria.
authors: Verena
authors: V
authors: Charwat
authors: CellTool GmbH, Bernried, Germany.
authors: Karin
authors: K
authors: Schütze
authors: Ludwig-Boltzmann-Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria.
authors: Wolfgang
authors: W
authors: Holnthoner
authors: Leibniz University of Hannover, Institute for Technical Chemistry, Hannover, Germany.
authors: Antonina
authors: A
authors: Lavrentieva
authors: CellTool GmbH, Bernried, Germany.
authors: Rainer
authors: R
authors: Gangnus
authors: Ludwig-Boltzmann-Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria.
authors: Pablo
authors: P
authors: Hofbauer
authors: Cellendes GmbH, Reutlingen, Germany.
authors: Claudia
authors: C
authors: Hoffmann
authors: Cellendes GmbH, Reutlingen, Germany.
authors: Brigitte
authors: B
authors: Angres
authors: University of Natural Resources and Life Sciences, Department of Biotechnology, Vienna, Austria. Electronic address: Cornelia.Kasper@boku.ac.at.
authors: Cornelia
authors: C
authors: Kasper
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002477:Cells / Q000166:cytology*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods*; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D013059:Spectrum Analysis, Raman / Q000295:instrumentation / Q000379:methods
keywords: 3D cell culture; 3D imaging; Image analysis; Live-cell analysis; Microscopy; Non-invasive cell analysis; Raman micro-spectroscopy