Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology (IGB), Stuttgart, Germany.
Tissue Eng Part C Methods. 2011 Oct;17(10):1027-40. doi: 10.1089/ten.tec.2011.0082. Epub 2011 Jul 20.
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.
在体外成熟或植入后体内对组织工程（TE）构建体进行非侵入性监测对于移植物评估非常重要。然而，用于研究工程组织中的细胞和基质成分的传统方法，如组织学、免疫组织化学或生物化学，需要进行侵入性组织处理，从而需要牺牲 TE 构建体。拉曼光谱技术提供了独特的可能性，可以仅基于细胞表型特异性生物化学指纹图谱对活细胞进行无标记的原位和体内分析。在这项研究中，我们旨在确定拉曼光谱技术在非侵入性识别和分离原代人皮肤成纤维细胞、角质形成细胞和黑素细胞以及永生化角质形成细胞（HaCaT 细胞）方面的适用性。细胞类型特异性拉曼光谱的多元分析能够区分活的原代和永生化角质形成细胞。我们进一步非侵入性地区分了成纤维细胞、角质形成细胞和黑素细胞。我们的研究结果对于体外皮肤模型的工程和人工皮肤的生产尤其重要，因为活检和移植都包含多种细胞类型。为了实现 TE 皮肤的可重复性质量，确定细胞群体的纯度以及检测潜在的分子变化非常重要。因此，我们得出结论，拉曼光谱技术是用于皮肤组织工程应用中细胞的非侵入性原位质量控制的合适工具。
