Presentation Title

Serum Exosome-Depletion Protocols Alter Cell Growth and Viability of Primary Astrocyte Cultures

Faculty Mentor

Massimo S. Fiandaca, MD/MBA, Howard J. Federoff, MD/PhD

Start Date

18-11-2017 9:59 AM

End Date

18-11-2017 11:00 AM

Location

BSC-Ursa Minor 69

Session

Poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Exosomes are extracellular lipid-bilayer nanovesicles of endosomal origin that range in diameter from 50-120 nm, and provide unique intercellular molecular messages via their inherent lipids, proteins, and nucleic acids. Culture media supplemented with serum provides growth factors, promotes cell adhesion, and protects primary brain cells from stress in vitro. Such media contains a substantial concentration of extracellular microvesicles (EVs), including exosomes, and thereby, potentially confounds experimental results where EVs are quantified and analyzed. Two methodologies have been developed to solve this problem: 1) An 18-hour ultracentrifugation (UC) of serum at 100,000 x g; and, 2) Exo-FBS™ (System Biosciences, Inc), a manufactured EV-depleted serum that has undergone polymer precipitation and a low speed (1,500 x g) centrifugation; with both methods capable of pelletizing and removing serum-derived EVs. This project proposed analyzing whether the EV-depletion method used (UC vs. Exo-FBS™) directly impacted primary astrocyte culture growth and viability compared to using replete serum media. Using comparative in vitro photo-microscopy, time-dependent cell counts, and cell-viability assays, statistically significant differences were confirmed between the three culture conditions (p<0.001), with the Exo-FBS™ depletion providing the greatest-fold reduction in serum-EVs, yet providing the worst cell growth and viability. Results from this and previously published reports suggest that serum EVs and other serum factors precipitated and removed may be responsible for altered cell growth and viability in culture. Future studies will be devised to unravel the differential nutritional differences provided by these EV-depletion approaches, while advancing our analyses of primary brain cell exosomes.

Keywords: Exosomes; Extracellular Vesicles; fetal bovine serum; in vitro; cell-culture

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Nov 18th, 9:59 AM Nov 18th, 11:00 AM

Serum Exosome-Depletion Protocols Alter Cell Growth and Viability of Primary Astrocyte Cultures

BSC-Ursa Minor 69

Exosomes are extracellular lipid-bilayer nanovesicles of endosomal origin that range in diameter from 50-120 nm, and provide unique intercellular molecular messages via their inherent lipids, proteins, and nucleic acids. Culture media supplemented with serum provides growth factors, promotes cell adhesion, and protects primary brain cells from stress in vitro. Such media contains a substantial concentration of extracellular microvesicles (EVs), including exosomes, and thereby, potentially confounds experimental results where EVs are quantified and analyzed. Two methodologies have been developed to solve this problem: 1) An 18-hour ultracentrifugation (UC) of serum at 100,000 x g; and, 2) Exo-FBS™ (System Biosciences, Inc), a manufactured EV-depleted serum that has undergone polymer precipitation and a low speed (1,500 x g) centrifugation; with both methods capable of pelletizing and removing serum-derived EVs. This project proposed analyzing whether the EV-depletion method used (UC vs. Exo-FBS™) directly impacted primary astrocyte culture growth and viability compared to using replete serum media. Using comparative in vitro photo-microscopy, time-dependent cell counts, and cell-viability assays, statistically significant differences were confirmed between the three culture conditions (p<0.001), with the Exo-FBS™ depletion providing the greatest-fold reduction in serum-EVs, yet providing the worst cell growth and viability. Results from this and previously published reports suggest that serum EVs and other serum factors precipitated and removed may be responsible for altered cell growth and viability in culture. Future studies will be devised to unravel the differential nutritional differences provided by these EV-depletion approaches, while advancing our analyses of primary brain cell exosomes.

Keywords: Exosomes; Extracellular Vesicles; fetal bovine serum; in vitro; cell-culture