Exosomes therefore, it is very important to characterise

Exosomes are nanoscale,
extracellular vesicles secreted by most of the cells 1 and differ in
their size, shape and molecular composition from other vesicles released from
the cell 2. Exosomes have a
round to cup shaped morphology with a lipid bilayer membrane and released into
the extracellular spaces and the size of exosomes varies from 30nm to 150nm 3,4. Besides their
nano size morphology other defining  characteristics of exosomes include density
gradient of 1.13-1.21g/ml 5 and protein
markers such as the tetraspanins (CD9, CD63, CD81), Alix, Hsp70, Tsg101 6. It has been
established that cells can release various types of extracellular vesicles such
as exosomes, ectosomes, micro-vesicles and recently identified large oncosome 7 therefore, it is very
important to characterise exosomes prior to any exosomal investigation 8. There are
several methods available to identify and characterise exosomes including
electron microscopy (EM), atomic force microscopy (AFM), nano particle tracking
analysis (NTA), Dynamic light scattering (DLS), western blot and flow cytometry
9. In the past few
years, there have been huge interest on exosomes due to their use as a
biomarker tool for cancer research.10. Several studies
have suggested the existence of exosomes with distinct composition and
biological information in the same population, which have different effects on
recipient cells 2,11. In recent years,
proteins of tumour derived exosomes have been investigated by mass spectrometry
based proteomics to search for potential biomarkers 12. For example, in
a study on melanoma patients, the expression of CD63+, a tetraspanin
super family member shown significant increase compared to healthy control 13. In another
study, one more member of the tetraspanin protein family CD81 was found
elevated in their expression in chronic hepatitis C patients 14. In any proteomic
study, protein concentration of the sample may be crucial especially if the
experiment is based on gel electrophoresis and the amount of exosomal protein
is correlated with the exosome number 15 but knowing the
total amount of proteins is sufficient for most of the times, but it has been
reported that some applications require the exact number of exosome in the
solution such as the use of exosome as a vehicle for drug delivery as it is
essential to standardise the number and size of exosomes for accurate doses of
drug delivery. Besides being a new tool for biomarker research, exosomes have
also been exploited as a delivery vehicle for therapeutic agents, due to their
stability, ability to cross blood brain barrier (BBB) and availability in most
body fluids. The number of exosomes is important as it helps to identify the
relationships with the parent cell of the exosome, state of the cells and to
understand the exosome signals 16. For example,
increased number of exosome and their miRNA cargo was observed in an alcoholic
hepatitis when tested on a mice model fed with alcohol 17.

Cells release
exosomes in the biological fluids during their growth phase to perform the
biological function such as transferring genetic materials and cell to cell
communication, immune response, removing unnecessary materials out of the cell 18. These exosomes
are taken up by other cells from the microenvironment 19. The time
exosomes stays in the biological fluid varies between cell types, for example, the
half-life of the exosomes from B16 melanoma cells is 30 minutes and this was
observed when exosomes were labelled with fluorescent dye to check their
stability 20. Whereas, exosomes
from human platelet concentrate showed a half-life of 5.5 hours 21. Within these
short period of times, exosome caries various proteins and RNAs depending on
the cell of origin 22.

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The aim of this
study was to isolate and characterise exosomes from three different types                                                of
cancer cells lines, the lung cancer cell line H358, leukemic cell line THP1 and
breast cancer cell line MCF7, quantify the number of exosomes and exosomal
protein pattern during their cellular growth.

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