CUR - The Council on Undergraduate Research

Human Embryonic Kidney (HEK) Cell Exosome Isolation and Characterization

Brianna Cowan and Dr. Heather Coan, Department of Biology, Western Carolina University, 1 University Way, Cullowhee, NC, 28723

Exosomes are small extracellular vesicles that transport proteins and genetic information to and from neighboring cells allowing for intracellular communication. They appear to impact cell behavior in various manners, which raises the question of whether exosomes can be used in therapeutic settings. However, little is known about how the environmental conditions of the cells that produce exosomes might alter the content of secreted exosomes, thereby altering the function of the cells that receive the vesicles. The purpose of this research is multifaceted: 1) determine optimal conditions of isolating exosomes from HEK cells, 2) determine if exosomes isolated from two different environments (unstressed versus oxidatively stressed) alter the way the receiving cells grow in stressed and unstressed conditions, and 3) determine how to standardize dosing of exosomes to receiving cells. To this end, exosomes are isolated using a Thermo Fisher Exosome Isolation Kit. Cells are stressed via hydrogen peroxide dosing, and growth is assessed by a WST-1 cell proliferation assay. Additional characterization of exosomes will be performed by transfecting with plasmids encoding fluorescent exosome proteins (CD9, CD81, and CD63) to determine the concentration of isolated exosomes via fluorescence spectrophotometry. Preliminary results suggest that we can isolate exosomes and that they do appear to have an impact on receiving cell growth. Future experiments will further investigate these questions and determine whether we can utilize transfection systems to tag exosomes for better characterization and tracking.

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