Label-free and real-time assessment of 660 nm red light photobiomodulation induced molecular alterations in human adipose derived mesenchymal stem cells using micro Raman spectroscopy

Therapeutic applications involving mesenchymal stem cells (MSCs) encounter challenges of attaining therapeutically potent and scaled up number during in-vitro batch culture. Recently, photobiomodulation (PBM) has emerged as a non-pharmacological method for enhancing MSC number, potency, and secretome production. However, the absence of a versatile, non-invasive technique to accurately identify PBM-induced biochemical alterations hinders the clinical translation of the approach. Raman spectroscopy (RS) can be a potential solution to this challenge. In this study, we demonstrate the utility of micro-RS to determine red light (∼660 nm) induced molecular alterations in human adipose tissue derived MSCs (hADMSCs) exposed to three different red light (∼660 nm) fluence; ∼3, 6 and 9 J/cm². While the immediate changes in response to ∼660 nm exposure are subtle, at 6 and 24 h, there is increase in peak intensity of reducedCytochromes c, c1 and b, phenylalanine,CN stretching, CC lipids, OPO stretchingin cells. Maximum increase in intensity of these peaks was observed at ∼6 J/cm². Raman peak at 1585 cm^-1, assigned to stretching vibration (CαCm) asymmetry of reduced Cyt c and sensitive to cellular redox status, shows notable change. Further, the intensity ratio of 1585 cm^-1 and 1452 cm^-1, a suggestive Raman biomarker for cell proliferation, is increased in cells exposed to ∼3 & ∼6 J/cm² followed by a decrease in cells exposed to ∼9 J/cm². Furthermore, both micro-RS intensity ratio (1585 cm^-1/1452 cm^-1) and MTT data on cell viability are in qualitative agreement with each other and show biphasic response to ∼660 nm exposure. While these results suggest the utility of micro-RS for label free assessment of PBM induced changes in hADMSCs, detailed studies on other cell types are necessary to validate the utility of micro-RS in this field.