Raman and infrared spectroscopy differentiate senescent from proliferating cells in a human dermal fibroblast 3D skin model

Abstract

Senescent cells contribute to tissue aging and dysfunction. Therefore, detecting senescent cells in skin is of interest for skin tumor diagnostics and therapy. Here, we studied the transition into senescence of human dermal fibroblasts (HDFs) in a three-dimensional (3D) human fibroblast-derived matrix (FDM). Senescent and proliferating cells were imaged by Raman spectroscopy (RS) and Fourier transform infrared (FTIR) spectroscopy. The obtained averaged spectra were analyzed using PLS-LDA. For these 3D cultured cells, RS and FTIR could clearly distinguish senescent from proliferating cells. For both techniques, we detected senescence-associated alterations in almost all cellular macromolecules. Furthermore, we identified different biochemical properties of 3D compared to two-dimensional (2D) cultured cells, indicating that cells in their natural, skin-like 3D environment act differently than in (2D) cell cultivations in vitro. Compared to 2D cultured cells, cells grown in 3D models displayed a sharper contrast between the proliferating and senescent state, also affecting the abundance of biomolecules including nucleic acids. The training accuracies of both vibrational spectroscopic techniques were >96%, demonstrating the suitability of these label-free measurements for detecting these cellular states in 3D skin models.

Organotypic human skin culture models constructed with senescent fibroblasts show hallmarks of skin aging

“…3b, c), among others, amide II (1480-1580 cm −1 ) and amide III (1220-1300 cm −1 ) bands corresponding to proteins, lipids, and nucleic acids, as well as peaks between 600 and 900 cm −1 referring to nucleic acids, are responsible for the differences in the analyzed spectra. These findings are consistent with differences in Raman fingerprints at the cellular level between proliferating and senescent fibroblasts 15,16 . As additional control, we analyzed the supernatants of skin equivalents with low young fibroblast cell numbers, again to exclude that lower fibroblast density might confound the results.…”

The Dual Role of Cellular Senescence in Developing Tumors and Their Response to Cancer Therapy

“…Indeed, first proof of principle for Raman- and near-infrared spectroscopy, followed by multivariate statistics has been achieved as it was able to distinguish different cell types and cellular states in a non-invasive manner. First results on different human fibroblast strains, which were cultivated in 2D and 3D and subjected to serial passaging to induce replicative senescence, are very promising and allowed classification of cells at high confidence ( 26 , 27 ). However, it needs to be determined if these methods are also applicable to other cell types, as well as to other inducers of cellular senescence.…”

Identification of senescent cells in multipotent mesenchymal stromal cell cultures: Current methods and future directions

“…Raman spectroscopy has likewise been investigated for its ability in distinguishing between young and senescent cells. This was demonstrated in breast cancer cells [121], human umbilical cord MSCs [122] and in human dermal fibroblasts [123,124]. In Raman spectroscopy, the Raman peaks provide biochemical and compositional information of the probed cells.…”

“…In particular, changes in Raman peaks are typically too subtle for easy identification. In that regard, of note is the incorporation of machine learning methods in the more recent publications [123,124] that largely facilitate the data analysis process.…”

“…Label-free Breast cancer cells [121] Human umbilical cord MSCs [122] Human dermal fibroblasts [123,124] Transduction by a retroviral vector containing the gene of human telomerase (hTERT) [148] Increases MSC lifespan via telomere length preservation…”