ABSTRACT
Cells release extracellular vesicles (EVs) that can be detected both in vivo and in cell culture medium. Among EVs, exosomes are 50-150 nm vesicles that are systematically packaged into multivesicular bodies for release into the external environment. In cancer, these intentionally packaged exosomes carry a payload of proteins such as RNAs and surface receptors that facilitate the reprogramming of proximal cells to assemble a protumor microenvironment. Exosomes have been implicated as an important intermediary extracellular communication pathway between cells, including in melanoma. Human melanoma-derived exosomes (HMEX) have been demonstrated to modulate the extracellular environment and inhibit immune cell activation. There are many methods to isolate and enrich for exosomes and the method applied can impact yield and purity of the isolates. In this chapter we describe the REIUS (rapid exosome isolation using ultrafiltration and size exclusion chromatography) method to isolate HMEX from melanoma cell cultures and then demonstrate their enrichment using molecular and microscopic approaches.
Subject(s)
Exosomes/chemistry , Melanoma/chemistry , Cell Line, Tumor , Chromatography, Gel , Humans , UltrafiltrationABSTRACT
The cellular response to mineral trioxide aggregate (MTA) is important for the repair and regeneration of periradicular tissues. The purpose of this study was to analyze the response of human alveolar bone cells to MTA. A human alveolar bone chip was obtained from an oral surgical procedure and explant cultures harvested after 3 to 4 weeks of outgrowth in alpha-minimum essential medium supplemented with fetal calf serum. Cells in early passage were seeded onto preset ProRoot (gray) MTA, tooth-colored (white) MTA, and MTA prepared with local anesthetic solution. Scanning electron microscopy showed cells were attached and spread out onto MTA within 24 hours, and proliferated to form a matrix-like layer within 7 days. Cell attachment and cell-surface interactions with the gray and white MTA, and with the MTA prepared with local anesthetic were comparably propagated for 14 days. The surgically derived human alveolar bone cells provided a clinically relevant model that demonstrated the capacity of both ProRoot and tooth-colored MTA to support cell attachment, proliferation, and matrix formation.
