Title : Non-destructive quality control of 2D and 3D cell cultures using Raman-Trapping-Microscopy (RTM)
Abstract:
Advanced cell culture technologies and especially three-dimensional (3D) cell cultivation combined with modern
biotechnological methods are increasingly applied to regenerate damaged and diseased tissue, such as skin, cartilage or bones. In order to monitor and follow 3D cell cultures, analytical technologies are required that are fast, reliable and sensitive. They should especially be label-free and non-destructive. Most of the microscopic methods are limited with regard to thickness and opaqueness of cell materials. Raman trapping microscopy is a unique method to follow cell development and tissue growth but to also warrant quality of the explants.
The principle of spontaneous Raman spectroscopy will shortly be described. Raman spectroscopy records scattered photons caused by collision of photons incident derived from a near infrared laser with target molecules, which ultimately results in an energy transfer. This so-called “inelastic” scattering is a very rare event, and only one of ten million impinging photons will result in a Raman scattered photon. The benefit and great potential of integrated trapping features for measurement cells directly within liquid cell cultures will be demonstrated. We also will explain the rich chemical information and uniqueness that Raman spectra provide from individual cells and tissue.
RTM analysis helps to find best culturing conditions of stem cells, finds differences between pure and cocultured fibroblasts within collagen matrix or discriminates activated from non-activated gingival fibroblasts within opaque mucoderm matrix.
A skin graft matrix constructed on a 3D hydrogen scaffold was used as a model of 3D-cell culture. In a first step, the purity of patient derived, keratinocytes and fibroblasts expanded in selection media was measured using single cell Raman trapping spectroscopy. Secondly, Raman spectra of keratinocytes and fibroblasts within separate layers of the graft were taken, monitoring composition, functionality, and quality of the cells. Cluster analysis of Raman data reveals cross-contamination between the layers of the graft.
Furthermore, RTM can follow cell development within beating cardiomyocytes and provides direct information about penetration of drugs into spheroids. Spheroids have emerged recently as an attractive 3D cell-model representing tissue biology, complexity, and architecture of 3D invitro systems.
These results indicate that Raman trapping microscopy is able to detect the biochemical profile of cells even in the
depth of tissues and microspheres, providing the opportunity to trace and evaluate changes of cells in response to
environmental impact or to compound treatment.
