Determination of trace elements in extracellular vesicles secreted by an in vitro model of retinal pigment epithelium cells by elemental mass spectrometry Jaime Martínez-García 1 , Ana Álvarez-Barrios 1 , Beatriz Fernández 1 , Héctor González-Iglesias 2 , Rosario Pereiro 1 1 University of Oviedo, Spain, 2 Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas (IPLA-CSIC) Spain. Age-Related Macular Degeneration (AMD) is one of the leading causes of irreversible blindness, affecting almost 200 million people worldwide [1]. The mechanisms involved in AMD onset are not fully understood, but previous studies have revealed that progressive degeneration of the retinal pigment epithelial (RPE) cells can play an important role in the development as well as further treatment of AMD. RPE cells secrete extracellular vesicles (EVs), whose cell-to-cell signaling function may be crucial during early stages of AMD. Recent studies have revealed that EVs secreted by RPE cells in AMD in vitro models are enriched in certain proteins and contribute to protein aggregates formation, one of the main causes of RPE degeneration [2]. Therefore, the study of the possible implication of EVs in metallic dyshomeostasis and RPE cell degeneration during AMD is of great research interest. The main aim of this work is to develop appropriate protocols for the purification of EVs from cell culture media, the characterisation of the purified EVs and the determination of Fe, Cu and Zn content in the EVs by elemental mass spectrometry. The samples of interest were fractions of cell culture media extracted from in vitro models of immortalized RPE cells. Two purification strategies were evaluated for EVs isolation: differential centrifugation and precipitation with a commercial kit. The characterization of EVs was done at three different stages: characterizing the size population of purified samples by dynamic light scattering, studying the size and morphology of EVs by transmission electron microscopy (different sample preparation protocols were evaluated), and identifying specific EVs markers by Western Blot. Furthermore, the total protein content (BCA protein assay) was always determined for normalisation purposes. After the isolation of EVs, Fe, Cu and Zn determination was performed by inductively coupled plasma mass spectrometry (ICP-MS) using three different sample introduction systems. The low sample volume available for ICP-MS analysis together with the low elemental concentration expected for the metals of interest hinder the use of traditional sample introduction systems. Thus, the use of micronebulizers and systems including total consumption spray chambers were evaluated in order to obtain the best analytical performances in terms of limits of detections and linear dynamic range. References 1. B. M. Williams, P. I. Burgess, Y. Zheng, “Drusen and Macular Degeneration”, Computational Retinal Image Analysis (2019) pp. 245-272. 2. M. Flores-Bellver, J. Mighty, S. Aparicio-Domingo et al ., “Extracellular vesicles released by human retinal pigment epithelium mediate increased polarized secretion of drusen proteins in response to AMD stressors”. J. Extracel. Vesicles (2021) e12165.
V15
© The Author(s), 2022
Made with FlippingBook Learn more on our blog