Raman and Infrared Spectroscopy Distinguishing Replicative Senescent from Proliferating Primary Human Fibroblast Cells by Detecting Spectral Differences Mainly Due to Biomolecular Alterations

Eberhardt, Katharina; Beleites, Claudia; Marthandan, Shiva; Matthäus, Christian; Diekmann, Stephan; Popp, Jürgen

doi:10.1021/acs.analchem.6b04264

Cited by 41 publications

(51 citation statements)

References 61 publications

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“…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.…”

Section: Resultssupporting

confidence: 87%

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

et al. 2020

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Skin aging is driven by intrinsic and extrinsic factors impacting on skin functionality with progressive age. One factor of this multifaceted process is cellular senescence, as it has recently been identified to contribute to a declining tissue functionality in old age. In the skin, senescent cells have been found to markedly accumulate with age, and thus might impact directly on skin characteristics. Especially the switch from young, extracellular matrix-building fibroblasts to a senescence-associated secretory phenotype (SASP) could alter the microenvironment in the skin drastically and therefore promote skin aging. In order to study the influence of senescence in human skin, 3D organotypic cultures are a well-suited model system. However, only few "aged" skinequivalent (SE) models are available, requiring complex and long-term experimental setups. Here, we adapted a previously published full-thickness SE model by seeding increasing ratios of stress-induced premature senescent versus normal fibroblasts into the collagen matrix, terming these SE "senoskin". Immunohistochemistry stainings revealed a shift in the balance between proliferation (Ki67) and differentiation (Keratin 10 and Filaggrin) of keratinocytes within our senoskin equivalents, as well as partial impairment of skin barrier function and changed surface properties. Monitoring of cytokine levels of known SASP factors confirmedly showed an upregulation in 2D cultures of senescent cells and at the time of seeding into the skin equivalent. Surprisingly, we find a blunted response of cytokines in the senoskin equivalent over time during 3D differentiation.

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Section: Resultssupporting

confidence: 87%

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

et al. 2020

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“…Because catalytic histochemical techniques were originally developed to produce quantitative data on enzyme activity in tissues (significantly predating the routine use of immunocytochemistry), the senescence-associated β galactosidase assay was readily applicable to fresh tissue sections. Interestingly, alternative techniques based on other aspects of the classic senescent fibroblast phenotype such as the auto-fluorescence associated with the accumulation of lipofuscin are being developed 12 , 13 and clearly hold considerable promise provided that users do not expect too much (‘too much’ in this instance is shorthand for “a single, perfect, rapid, easy-to-use, definitive biomarker for senescent cells that works in each and every cell type from all species under every conceivable set of conditions”).…”

Section: How Does Cell Senescence Work?mentioning

confidence: 99%

Senescence in the aging process

et al. 2017

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The accumulation of ‘senescent’ cells has long been proposed to act as an ageing mechanism. These cells display a radically altered transcriptome and degenerative phenotype compared with their growing counterparts. Tremendous progress has been made in recent years both in understanding the molecular mechanisms controlling entry into the senescent state and in the direct demonstration that senescent cells act as causal agents of mammalian ageing. The challenges now are to gain a better understanding of how the senescent cell phenotype varies between different individuals and tissues, discover how senescence predisposes to organismal frailty, and develop mechanisms by which the deleterious effects of senescent cells can be ameliorated.

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“…Applying certain solution algorithms for the above Fredholm type-I equations the G(t) can be retrieved for arbitrarily shaped pulses [8]. We can also seek solution for G(t) function for our case of i) Gaussian pulses and ii) when molecular collisions dominate the dephasing process.…”

Section: Fig 2(a) Shows Cars Signal Versus Delay Time When Raman Actmentioning

confidence: 99%

“…Spontaneous Raman version has been applied with greater focus towards detection of spectral features within cells and tissue [7]. However, the reported results have been limited to obtaining characteristic multi-line spectra and detecting relative changes in the intensities and spectral shifts with a goal to correlate those with biomolecular alterations occurring on sub-cellular level [8]. The true spectroscopic strength, that would ultimately include resolution of molecular vibration damping rates Γ (or linewidths, Δν=1/Γ) and line shapes, has not been enabled and demonstrated.…”

mentioning

confidence: 99%

Tracing molecular dephasing in biological tissue

Mokim

Carruba

Ganikhanov

2017

Applied Physics Letters

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Abstract.We demonstrate the quantitative spectroscopic characterization and imaging of biological tissue using coherent time-domain microscopy with femtosecond resolution. We identify tissue constituents and perform dephasing time (T 2 ) measurements of characteristic Raman active vibrations. This was shown in subcutaneous mouse fat embedded within collagen rich areas of the dermis and the muscle connective tissue. The demonstrated equivalent spectral resolution (<0.3 cm -1 ) is an order of magnitude better compared to commonly used frequency-domain methods for characterization of biological media. This provides with the important dimension and parameter in biological media characterization and can become an effective tool in detecting minute changes in bio-molecular composition and environment that is critical for molecular level diagnosis. 2Fundamental nonlinear optical phenomena have been shown to be useful in applications related to noninvasive characterization of biological media [1][2][3][4]. Raman scattering based techniques, both spontaneous and coherent versions, are of particular interest since their spectroscopic power can deliver molecular sensitive information that can become a key in early diagnosis of diseases. Absolute majority of the relevant applications of the techniques is, for natural reasons, in frequency domain. The coherent Raman microscopy studies were primarily applied to highlight tissue and cells constituent by producing high-contrast images at targeted Raman active vibration [5,6]. Spontaneous Raman version has been applied with greater focus towards detection of spectral features within cells and tissue [7]. However, the reported results have been limited to obtaining characteristic multi-line spectra and detecting relative changes in the intensities and spectral shifts with a goal to correlate those with biomolecular alterations occurring on sub-cellular level [8]. The true spectroscopic strength, that would ultimately include resolution of molecular vibration damping rates Γ (or linewidths, Δν=1/Γ) and line shapes, has not been enabled and demonstrated. It is worth noting that the damping rate is directly affected by inter-and intra-molecular interactions. Therefore, ability to measure Raman line shapes with a precision is absolutely crucial from that point of view. Depending on the immediate molecular environment (e.g. density, viscosity) and composition of tissue and cells, linewidths for the investigated Raman active modes can vary broadly. For instance, solute-solvent interactions in aqueous solutions can result in 0.3-7.5 cm -1 linewidth range that depend on the molar ratio [9].Both spontaneous Raman and frequency-domain have the best possible resolution of ~ 3-7 cm -1 .The limits are imposed by the detection sensitivity in spontaneous Raman spectroscopy. Laser pulse bandwidths (~3-10 cm -1 ) employed in coherent Raman microscopy does not provide a better resolution either. In addition, for the latter case the need to adjust laser wavelength in 3 point-by-point spectral measu...

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Raman and Infrared Spectroscopy Distinguishing Replicative Senescent from Proliferating Primary Human Fibroblast Cells by Detecting Spectral Differences Mainly Due to Biomolecular Alterations

Cited by 41 publications

References 61 publications

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

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

Senescence in the aging process

Tracing molecular dephasing in biological tissue

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