Raman spectroscopy as novel marker for sensitive

DECHEMA 2015
ATMP 2015 - Issues and challenges from bench to bedside
Raman spectroscopy as novel marker for sensitive stem cell
identification
Schütze, K., CellTool GmbH, Bernried, Germany; Marino D., Tissue Biology Research
Unit, Department of Surgery, University Children`s Hospital, Zürich, Switzerland;
Meyer S., Tissue Biology Research Unit, Department of Surgery, University Children`s
Hospital, Zürich, Switzerland
Introduction
Raman spectroscopy (RS) is a highly sensitive analytical method for marker-free and noninvasive identification and characterization of cells. We demonstrate feasibility of RS in 3dimensional setups such as differentiated Human Gingival Fibroblasts cultured within
collagen matrices (mucoderm®) and autologous dermo-epidermal skin grafts derived from
human skin. We could show that RS can discriminate matrix-cultured normal versus
differentiated fibroblasts and skin-graft cultured fibroblasts, melanocytes and
keratinocytes, even in a depth of 200µm and more.
Material & Methods
Human Gingival Fibroblasts (ProVitro) were cultured for 6 weeks on a 1-2 mm thick
collagen matrix (mucoderm®) For activation cells were incubated with differentiation
medium (ProVitro) for 7 days. Afterwards, samples were fixed with 4% paraformaldehyde
for analysis with Raman spectroscopy.
The grafts were cultivated with human fibroblasts, keratinocytes and melanocytes for 10
days. Later the grafts were fixed with 4% paraformaldehyde for analysis with Raman
spectroscopy.
Raman measurements were carried out with the BioRam system (CellTool GmbH,
Bernried). At least 60 cells of each group were measured and compared. Data processing
was performed with customized BioRam-software followed by statistical Principal
Component Analysis (PCA) to find the major differences of differentiated to undifferentiated
fibroblasts.
Results
RS can clearly distinguish differentiated from undifferentiated fibroblasts and the PCA
analysis identifies Collagen Type I, proteins and lipids as major key molecules. In the skingraft setup the different cell types can also be characterized. The penetration depth in
both setups is up to 200µm, still resulting in meaningful spectra.
These two examples show Raman as label-free and non-invasive method providing highly
specific molecular information where cells remain vital and unaffected for further
downstream applications.
Discussion
RS is a photonic marker for gentle yet highly specific detection of cells in engineered
tissue. It provides information about the entire metabolome of a single cell even in a
matrix setup with a depth of 200µm that is as characteristic as a “fingerprint”. Most
importantly, RS can be used for quality assessment of cell cultures or engineered tissue
without impairing cell viability.
CellTool GmbH / www.celltool.de
November 2015