Many
medical researchers believe that stem cell therapy will revolutionise the
treatment of human disease and may provide treatments for many currently
incurable diseases. However, one problem still to be overcome is controlling
the excessive proliferation of cells with unwanted phenotypes after
transplantation to prevent tissue overgrowth and tumour formation.
Unfortunately, most techniques currently available for the characterisation of
cells are invasive and make the cells unusable. Now, a Nottingham University
team led by Dr Ioan Notingher has developed a non-invasive Raman
microspectroscopy (RMS) technique that phenotypically identifies live
cardiomyocyte cells within highly heterogeneous cell populations with greater
than 96% sensitivity and specificity.
The 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 Dr Ioan 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."
According to Antoine Varagnat, Product Specialist at Andor, "the iDus has been
the detector of choice in the research community for many years and Dr
Notingher's breakthrough work is a fantastic testimony to the performance of
this platform. With its unique -100°C thermo-electric cooling platform and
highest sensitivity in the near-infrared (NIR), as well as an extremely compact
design for ease of integration to complex experiments, the iDus is just the
right camera for NIR micro-Raman applications.".
Compared to existing techniques, Raman microspectroscopy has several unique
advantages for characterizing heterogeneous cell populations that makes it
ideal for stem cell therapy. Conventional cell biology assays, such as PCR and
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.
Andor's modular Spectroscopy solutions encompass a wide range of high
performance CCD, ICCD and EMCCD detectors, as well as a comprehensive range of
Research-grade spectrograph platforms and versatile interfacing to microscopes.
To learn more about the iDus camera series and their use in spectroscopy,
please visit the spectroscopy section of the
Andor website (
http://www.andor.com/spectroscopy_solutions/).