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Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Fraunhofer Institute of Interfacial Engineering and Biotechnology (IGB) Stuttgart, Germany

University of Stuttgart, Germany

At present, the production of tissue engineered cartilage requires the concurrent production of two identical transplants. One transplant is used for destructive quality control and the second one is implanted into the patient. A non-invasive characterization of such tissue engineering samples would be a promising tool to achieve a production process of just one transplant that is both implanted and tested. Raman spectroscopy is a method that satisfies this requirement by analyzing cells without lysis, fixation or the use of any chemicals. This pure optical technique is based on inelastic scattering of laser photons by molecular vibrations of biopolymers. Characteristic peaks in Raman spectra of cells could be assigned to typical biochemical molecules present in biological samples. For the analysis of chondrocytes present in cartilage transplants, the determination of the cell vitality as well as the discrimination of another cell type have been studied by Raman spectroscopy. Another bottleneck in such biological processes under GMP conditions is sterility control, as most of the commonly used methods require long cultivation times. Raman spectroscopy provides a good alternative to conventional methods in terms of time saving. In this study, the potential of Raman spectroscopy as a quality and sterility control tool for tissue engineering applications was studied by analyzing and comparing the spectra of cell and bacteria cultures.

Raman spectroscopy as a tool for quality and sterility analysis for tissue engineering applications like cartilage transplants.

Noninvasive monitoring of tissue-engineered (TE) constructs during their in vitro maturation or postimplantation in vivo is highly relevant for graft evaluation. However, traditional methods for studying cell and matrix components in engineered tissues such as histology, immunohistochemistry, or biochemistry require invasive tissue processing, resulting in the need to sacrifice of TE constructs. Raman spectroscopy offers the unique possibility to analyze living cells label-free in situ and in vivo solely based on their phenotype-specific biochemical fingerprint. In this study, we aimed to determine the applicability of Raman spectroscopy for the noninvasive identification and spectral separation of primary human skin fibroblasts, keratinocytes, and melanocytes, as well as immortalized keratinocytes (HaCaT cells). Multivariate analysis of cell-type-specific Raman spectra enabled the discrimination between living primary and immortalized keratinocytes. We further noninvasively distinguished between fibroblasts, keratinocytes, and melanocytes. Our findings are especially relevant for the engineering of in vitro skin models and for the production of artificial skin, where both the biopsy and the transplant consist of several cell types. To realize a reproducible quality of TE skin, the determination of the purity of the cell populations as well as the detection of potential molecular changes are important. We conclude therefore that Raman spectroscopy is a suitable tool for the noninvasive in situ quality control of cells used in skin tissue engineering applications.

Raman spectroscopy: a noninvasive analysis tool for the discrimination of human skin cells.

The non-destructive and label-free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non-contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400-1800 cm(-1)), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof-of-principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically-degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non-destructive and non-contact tool to probe ECM state in situ.

Article	Year
Raman spectroscopy as a tool for quality and sterility analysis for tissue engineering applications like cartilage transplants. The International journal of artificial organs\| PubMed ID: 20458692	2010
Raman spectroscopy: a noninvasive analysis tool for the discrimination of human skin cells. Tissue engineering. Part C, Methods\| PubMed ID: 21774693	2011
Raman spectroscopy for the non-contact and non-destructive monitoring of collagen damage within tissues. Journal of biophotonics\| PubMed ID: 21954177	2012

Marieke Pudlas

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