Belfast, Northern Ireland--Most techniques currently available for the characterization of stem cells are invasive and make the cells unusable. But a Nottingham University (Nottingham, England) team led by Ioan Notingher has developed a non-invasive Raman microspectroscopy (RMS) technique using Andor technology that phenotypically identifies live cardiomyocyte cells within highly heterogeneous cell populations with greater than 96% sensitivity and specificity.
Compared to existing techniques, Raman spectroscopy has several unique advantages for characterizing heterogeneous cell populations that makes it ideal for stem cell therapy. Conventional cell biology assays such as Western Blotting are invasive, require a large number of cells, and present averaged results representing entire cell populations. Fluorescence and magnetic cell sorting approaches rely on lineage-specific surface markers expressed on the cell membrane. However, many cell types, including cardiomyocytes, do not express these markers on the surface and are rendered unusable in a clinical environment by fixation and permeabilization. And, although transgenic strategies may be used to express surface markers, the complex genetic modifications also mean that the resulting cells are not suitable for clinical applications.
To discriminate between different types of stem cells, the Nottingham team used an Andor iDus 401A-BRDD cooled, deep-depletion, back-illuminated CCD camera attached to a purpose-built Raman microspectrometer to record spectra from individual cells derived from micrometric regions of human embryonic stem cells (hESC). By comparing with matching immunofluorescence images from the same cells, they showed that the Raman spectra correspond to the spatial distribution of biomolecules such as nucleic acids, proteins, lipids and carbohydrates, and that this can be used to discriminate between different cell types.
"We needed the shortest possible acquisition times for the Raman spectra and the Andor iDus 401A camera is ideal for this application, allowing measurements of Raman spectra from selected positions in the cells in only 0.5 seconds," says Notingher. "Also, the detectors are optimised for the spectral regions in which we work, 800-900 nm, which is vital for avoiding photodamage to the cells. Since RMS has only a minimal background signal from water, it allows repeated observations of viable cells maintained under physiological conditions. Together with Professor Denning, also of the University of Nottingham and a leader in regenerative medicine, we have validated the potential of RMS for allowing the non-invasive phenotypic identification of hESC progeny. With further development, such label-free optical techniques may enable the separation of high-purity cell populations with mature phenotypes and provide repeated measurements to monitor time-dependent molecular changes in live hESCs during differentiation in vitro."
SOURCE: Andor; www.andor.com