Multifocal Raman Spectrophotometer for Examining Drug-Induced and Chemical-Induced Cellular Changes in 3D Cell Spheroids

Cell spheroids offer alternative in vitro cell models to monolayer cultured cells because they express complexities similar to those of in vivo tissues, such as cellular responses to drugs and chemicals. Raman spectroscopy emerged as a powerful analytical tool for detecting chemical changes in living cells because it nondestructively provides vibrational information regarding a target. Although multiple iterations are required in drug screening to determine drugs to treat cell spheroids and assess the inter-spheroid heterogeneity, current Raman applications used in spheroids analysis allow the observation of only a few spheroids owing to the low throughput of Raman spectroscopy. In this study, we developed a multifocal Raman spectrophotometer that enables simultaneous analysis of multiple spheroids in separate wells of a regular 96-well plate. By utilizing 96 focal spots excitation and parallel signal collection, our system can improve the throughput by approximately 2 orders of magnitude compared to a conventional single-focus Raman microscope. The Raman spectra of HeLa cell spheroids treated with anticancer drugs and HepG2 cell spheroids treated with free fatty acids were measured simultaneously, and concentration-dependent cellular responses were observed in both studies. Using the multifocal Raman spectrophotometer, we rapidly observed chemical changes in spheroids, and thus, this system can facilitate the application of Raman spectroscopy in analyzing the cellular responses of spheroids

Label-Free Monitoring of Drug-Induced Cytotoxicity and Its Molecular Fingerprint by Live-Cell Raman and Autofluorescence Imaging

Simultaneous observation of drug distribution at the effector site and subsequent cell response are essential in the drug development process. However, few studies have visualized the drug itself and biomolecular interactions in living cells. Here, we used label-free Raman microscopy to investigate drug-induced cytotoxicity and visualize drug uptake and subcellular localization by its specific molecular fingerprint. A redox-sensitive Raman microscope detected the decrease of reduced cytochrome c (cyt c) after Actinomycin D (ActD) treatment in a time-dependent and dose-dependent format. Immunofluorescence staining of cyt c suggested that the release of cyt c was not the major cause. Combining Raman microscopy with conventional biological methods, we reported that the oxidization of cyt c is an early cytotoxicity marker prior to the release of cyt c. Moreover, as the spectral properties of ActD are sensitive to the surrounding environment, subcellular localization of ActD was visualized sensitively by the weak autofluorescence, and the intercalation of ActD into DNA was detected by shifted Raman peaks, allowing for parallel observation of drug uptake and the mechanism of action. In this research, we achieved simultaneous observation of cytotoxicity and cellular drug uptake by Raman microscopy, which could facilitate a precise understanding of pharmacological effects and predict potential drug toxicity in the future