FTIR and Raman spectroscopy in characterization of bioreactor cultured prostate cancer extracellular vesicles

Background: Extracellular vesicles (EVs) are nano-sized cell membrane derived structures that function as intercellular messengers and hold potential for applications in diagnostics and drug delivery. There is a need to establish efficient methods of production and quality control for EV preparations. The International Society for Extracellular Vesicles has suggested the protein/lipid ratio of the vesicle components as a measure of purity for EV samples. Spectroscopic methods such as FTIR and Raman are promising options for characterization of these EV biomolecular properties. Hypoxia is a common feature of solid tumors such as prostate cancer and increases the secretion of EVs from cells. Aims: The aims for this study were to 1) establish a bioreactor cell culture protocol for 22Rv1 and RWPE-1 cell lines, 2) characterize the EVs obtained from these cell lines using Raman and FTIR spectroscopy and subsequently obtain the spectroscopic protein/lipid (P/L) ratio, and 3) to analyze the spectroscopic P/L ratio as a measure of EV composition and purity as a function of time in cell culture 4) to investigate EV secretion from these cell lines under hypoxic conditions. Methods: Prostate cancer (22Rv1 and PC3) and normal prostate cell lines (RWPE-1 and PNT2) were cultured in CELLine bioreactor conditions, and EVs were extracted from the cell culture media once per week. The collected culture media was subjected to a series of (ultra)centrifugations and size-exclusion chromatography to purify the vesicles. The purified vesicles were characterized by means of Western blotting, nanoparticle tracking analysis and Raman and ATR-FTIR spectroscopy, and the spectral protein/lipid ratios were analyzed. Culturing of 22Rv1 and RWPE-1 cell lines in hypoxic conditions was also attempted. Results: The obtained results suggest that this protocol was effective for producing purified EV preparations. However, notable variation in EV sample protein/lipid ratios between weekly samples from newly established bioreactor cultures was detected. These results highlight both the utility of spectroscopic methods in distinguishing the biomolecular compositions of different EV populations, and the volatility of EV preparations to changes in the cell culture, therefore indicating a need to carefully optimize stable EV culture conditions