Bone-marrow mesenchymal stem cells (BM-MSCs) are a promising cell source for regenerative medicine.
However, it is challenging to determine whether isolated BM-MSC populations are free of fibroblasts. We
employed traditional methods and Raman spectroscopy to distinguish BM-MSCs and human dermal
fibroblasts (hDFs). Although in vitro differentiation assays revealed the multipotent character of BM-MSCs,
long culture periods are a major disadvantage. Using Raman spectroscopy, we could quickly distinguish
between BM-MSCs and hDFs. Therefore we conclude that this method is sufficient for the rapid detection of
fibroblastic contaminations in BM-MSC cultures.
In the field of cell culture and tissue engineering is an increasing need for non-invasive methods to analyze
living cells in vitro. One important application is the cell characterization in tissue engineering products.
Raman spectroscopy is a method which analyzes cells without lysis, fixation or the use of any chemicals and
do not affect cell vitality adversely if suitable laser powers and wavelength are used. This purely optical
technique is based on inelastic scattering of laser photons by molecular vibrations of biopolymers. Basically
Raman spectra of cells contain typical fingerprint regions and information about cellular properties.
Characteristic peaks in Raman spectra could be assigned to biochemical molecules like proteins, nucleic acid
or lipids. The distinction of cell types by a multivariate analysis of Raman spectra is possible due to their
biochemical differences. As this method allows a characterization of cells without any cell damage it is a
promising technology for the quality control of cells in tissue engineering or cell culture applications.
Monitoring the sterility of cell or tissue cultures is of major concern, particularly in the fields of regenerative medicine
and tissue engineering when implanting cells into the human body. Our sterility-control system is based on a Raman
micro-spectrometer and is able to perform fast sterility testing on microliters of liquid samples. In conventional sterility
control, samples are incubated for weeks to proliferate the contaminants to concentrations above the detection limit of
conventional analysis. By contrast, our system filters particles from the liquid sample. The filter chip fabricated in
microsystem technology comprises a silicon nitride membrane with millions of sub-micrometer holes to retain particles
of critical sizes and is embedded in a microfluidic cell specially suited for concomitant microscopic observation. After
filtration, identification is carried out on the single particle level: image processing detects possible contaminants and
prepares them for Raman spectroscopic analysis. A custom-built
Raman-spectrometer-attachment coupled to the
commercial microscope uses 532nm or 785nm Raman excitation and records spectra up to 3400cm-1. In the final step,
the recorded spectrum of a single particle is compared to an extensive library of GMP-relevant organisms, and
classification is carried out based on a support vector machine.
Sterility testing of cell or tissue cultures is an essential task in the fields of regenerative medicine and tissue engineering.
Especially in case of Good manufacturing practice (GMP) of cell and tissue based transplants. We present a system
based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the
solution for analysis. A Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman
spectra, and a data acquisition and analysis tool for identification of the particles. Identification of critical particles like
microorganisms via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of
those particles is then carried out with an excitation wavelength of 785 nm. However an automated image analysis of
small particles from supernatant of biopsies on a filter chip with tiny holes is a difficult task. Especially for the
discrimination of small particles like cell debris and bacteria, which have a quite similar range of size. Because of that
particles in the supernatant and microorganisms have to be discriminated by means of Raman spectroscopy. We present
here a Raman based method to discriminate between cells, microorganisms and particles in cell culture.
The fast and direct identification of possibly pathogenic microorganisms in air is gaining increasing interest due to their
threat for public health, e.g. in clinical environments or in clean rooms of food or pharmaceutical industries. We present
a new detection method allowing the direct recognition of relevant germs or bacteria via fluorescence-labeled antibodies
within less than one hour. In detail, an air-sampling unit passes particles in the relevant size range to a substrate which
contains antibodies with fluorescence labels for the detection of a specific microorganism. After the removal of the
excess antibodies the optical detection unit comprising reflected-light and epifluorescence microscopy can identify the
microorganisms by fast image processing on a single-particle level. First measurements with the system to identify
various test particles as well as interfering influences have been performed, in particular with respect to autofluorescence
of dust particles. Specific antibodies for the detection of Aspergillus fumigatus spores have been established. The
biological test system consists of protein A-coated polymer particles which are detected by a fluorescence-labeled IgG.
Furthermore the influence of interfering particles such as dust or debris is discussed.
Monitoring the sterility of cell or tissue cultures is an essential task, particularly in the fields of regenerative medicine
and tissue engineering when implanting cells into the human body. We present a system based on a commercially
available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis, a
Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition
and analysis tool for identification of the particles. In contrast to conventional sterility testing in which samples are
incubated over weeks, our system is able to analyze milliliters of supernatant or cell suspension within hours by filtering
relevant particles and placing them on a Raman-friendly substrate in the microfluidic cell. Identification of critical
particles via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those
particles is then carried out with an excitation wavelength of 785 nm. The potential of this setup is demonstrated by
results of artificial contamination of samples with a pool of bacteria, fungi, and spores: single-channel spectra of the
critical particles are automatically baseline-corrected without using background data and classified via hierarchical
cluster analysis, showing great promise for accurate and rapid detection and identification of contaminants.