A Marker-free Detection Method For Replicative Senescence Of Mesenchymal Stem Cells

Mesenchymal stem cells?MSCs?have highly proliferative,self-renewal and multi-differentiation potential,and thus become a research hotspot worldwide and have been widely used in the field of biomedicine.Generally,cell subculture in vitro is essential to provide a sufficient number of MSCs for a successful clinical application.However,this process is inescapably accompanied by replicative aging,which has a detrimental effect on the cell-based therapy.Therefore,it is important to determine the aging state of mesenchymal stem cells accurately.Traditional methods are mostly biochemical methods.Most of these methods are invasive and destructive,so they are not conducive to the application and research of mesenchymal stem cells.Based on the fact that optical technology has the advantages of label-free and non-invasive,we detected the mesenchymal stem cells with different degrees of senescence using fluorescence spectroscopy and Raman spectroscopy respectively.The purpose of our work is to explore the characteristic optical parameters that can characterize cellular senescence.This work is the basis to provide a label-free method for cellular senescence assessment.It is also beneficial to the further study of the senescence mechanism of mesenchymal stem cells.Firstly,Fluorescence spectroscopy was used to detecte tryptophan?Trp?,NADH?NADH?and flavin adenine dinucleotide?FAD?,which are the endogenous fluorescent substance in the cells.Their corresponding excitation wavelength are 280nm,340nm and 450nm,respectively.By analyzing the changes of the fluorescence intensities,it was found that the fluorescence intensities of Trp,NADH and FAD increased with the aging of cells,and showed significant differences between the young?P5?and aging?P20?cells.However,this method could not show a high sensitivity and specificity between the cells having slight changes in terms of aging,and thus not suitable for use as a spectral indicator for the characterization of mesenchymal stem cell senescence.As an alternative,Raman microspectroscopy was used to monitor the aging of mesenchymal stem cells.The results showed that no single Raman peak can achieve a high enough sensitivity for the cell aging assessment.However,the intensity ratio of Raman peaks at 1157cm^-1 and 1174cm'1 exhibited a monotonic decline with the cell aging.More importantly,the ratio I1157/I1174 showed statistical differences between the cells having slight changes in terms of aging.The results indicated that Raman spectroscopy can act as a label-free and non-invasive method for the cell aging assessment of mesenchymal stem cells,and the ratio I1157/I1174 can be used as an important characteristic spectral marker.Based on the Raman spectral results,a Raman data automatic analysis software was designed,which can achieve the functions of automatic smooth,reduce noise,remove the baseline and peak finding.This software provided a convenient,fast and accurate analysis tool for Raman data processing.