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Characterization of exosomes from glioma cells under hypoxia

Tomazin, Katarina (2015) MOBN29 20141
Degree Projects in Molecular Biology
Abstract
Exosomes are cell-secreted nanosized (30-200 nm) membrane vesicles that contain cytosol proteins and nucleic acids (mRNA, miRNA and mDNA). They have been characterized to be released by most cell types and thus, represent an important part of cell-cell communication. Especially they might play an important role in cell adaptation processes to stress conditions such as hypoxia or oxidative stress. The main difficulty in studying exosomes is however their purification and characterization process which is still a major challenge in the field of exosome research. In this study, we set up the method for isolation of exosomes with differential centrifugation and two characterization techniques nanoparticle tracking analysis (NTA) and... (More)
Exosomes are cell-secreted nanosized (30-200 nm) membrane vesicles that contain cytosol proteins and nucleic acids (mRNA, miRNA and mDNA). They have been characterized to be released by most cell types and thus, represent an important part of cell-cell communication. Especially they might play an important role in cell adaptation processes to stress conditions such as hypoxia or oxidative stress. The main difficulty in studying exosomes is however their purification and characterization process which is still a major challenge in the field of exosome research. In this study, we set up the method for isolation of exosomes with differential centrifugation and two characterization techniques nanoparticle tracking analysis (NTA) and bicinchoninic acid assay (BCA). With the latter techniques we studied whether glioma cells exposed to stress conditions produce more exosomes as communication messengers compared to standard environment. The study so far did not answer this question, our data nevertheless show the initial evidence that exosomes reflect the inner metabolic status of cells. (Less)
Popular Abstract
Characterization of exosomes

All multicellular living systems rely on cellular communication in order to form functional organisms. This communication is dependent on physical contact between cell membranes, free diffusion of small cell-secreted soluble molecules and cell-secreted lipid vesicles. Among those are also exosomes which have been found to be released by most cells types. They vary in size between 30 and 200 nm and contain proteins and nucleic acids.

Because exosomes are very small in size and their density can overlap with other particles in the samples, their isolation and purification is still a major challenge in the field of exosome research. Generally they are isolated with differential centrifugation which consists... (More)
Characterization of exosomes

All multicellular living systems rely on cellular communication in order to form functional organisms. This communication is dependent on physical contact between cell membranes, free diffusion of small cell-secreted soluble molecules and cell-secreted lipid vesicles. Among those are also exosomes which have been found to be released by most cells types. They vary in size between 30 and 200 nm and contain proteins and nucleic acids.

Because exosomes are very small in size and their density can overlap with other particles in the samples, their isolation and purification is still a major challenge in the field of exosome research. Generally they are isolated with differential centrifugation which consists of a step-by-step removal of bigger contaminating particles until the final ultra-centrifugation to pellet exosomes. To characterize exosomes and check whether they are pure one has to determine the presence of exosomal markers and contaminants, the size-distribution of particles in the sample and the density gradient with sucrose. I set up the methods for exosome isolation, size distribution and protein measurements. These measurements showed that exosomes were present in the sample but were not highly purified. Further analysis with sucrose gradient centrifugation, electron microscopy and specific protein identification will be performed in the future.

Release of exosomes from glioma cells under hypoxia and oxidative stress
In this study I investigated whether glioma cells release more exosomes under stress compared to standard environment. Glioma is a type of cancer that arises from glial cells in the brain. Two glioma cell lines, U87-MG (high-grade glioma) and H4 (low-grade glioma), were exposed to mimicked hypoxia (i.e. low oxygen) and oxidative stress (i.e. high presence of reactive oxygen species).

The qualitative analysis of the exosomal proteasome (i.e. protein content) showed the initial evidence that exosomes reflect the inner metabolic status of cells. Exosomes isolated from hypoxia-treated cells were enriched in ribosomal proteins, which represent a higher metabolical activity under stress. Furthermore, the exosomes were depleted in proteins involved in the modulation of extracellular matrix (ECM) which might indicate a differential need for those proteins due to morphological changes that cells experience under stress.

Overall the study did not provide definite and clear answers to the influence of stress on exosomes release and composition and more experiments are required before drawing final conclusions.

Supervisor: Georg Kuhn
Master’s Degree Project in Neurobiology, 45 ECTS, 2015
Research group Georg Kuhn, Sahlgrenska academy, Göteborg. (Less)
Please use this url to cite or link to this publication:
author
Tomazin, Katarina
supervisor
organization
course
MOBN29 20141
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
5271385
date added to LUP
2015-04-10 09:24:36
date last changed
2015-04-10 09:24:36
@misc{5271385,
  abstract     = {Exosomes are cell-secreted nanosized (30-200 nm) membrane vesicles that contain cytosol proteins and nucleic acids (mRNA, miRNA and mDNA). They have been characterized to be released by most cell types and thus, represent an important part of cell-cell communication. Especially they might play an important role in cell adaptation processes to stress conditions such as hypoxia or oxidative stress. The main difficulty in studying exosomes is however their purification and characterization process which is still a major challenge in the field of exosome research. In this study, we set up the method for isolation of exosomes with differential centrifugation and two characterization techniques nanoparticle tracking analysis (NTA) and bicinchoninic acid assay (BCA). With the latter techniques we studied whether glioma cells exposed to stress conditions produce more exosomes as communication messengers compared to standard environment. The study so far did not answer this question, our data nevertheless show the initial evidence that exosomes reflect the inner metabolic status of cells.},
  author       = {Tomazin, Katarina},
  language     = {eng},
  note         = {Student Paper},
  title        = {Characterization of exosomes from glioma cells under hypoxia},
  year         = {2015},
}