Real time analysis of microvesiculation using a Quartz Crystal Microbalance

Introduction: Characterization of microvesicles (MVs) is essential for
understanding their mechanisms of action and biological importance.
Stimulated MVs (sMVs) are released through the activation of cells by
a multitude of factors. We aimed to use a quartz crystal microbalance
(QCM) or piezoelectric quartz resonator able to determine small mass
changes, to monitor MV release and to determine MV mass. Methods:
A QCM (Q-Sence E1) was used to analyse MV release from THP-1
leukaemic promonocytes. The cells in RPMI and 2 mM Ca2_ were
applied to the QCM to establish a steady baseline. The sample on the
sensor was stimulated to microvesiculate with 10% exosome- and
MV-free normal human serum. The QCM was then able to monitor
sample density and fluid rigidity. Over the same time frame, the level
of apoptosis of cells releasing MVs was assessed by staining with
annexin V and 7-aminoactinomycin D (Guava Nexin Reagent). Using
the QCM we were also able to measure MV mass directly by
measuring their ability to quench the oscillating momentum of the
QCM. Results: Using the QCM, we were able to monitor deposition of
cells on the crystal and then sMV release from cells, in the absence of
any labelling or fluorescent probe, by measuring cell mass change.
Cells (105) were deposited onto the QCM electrodes, and the
frequency decreases over the first 1000s indicating attachment. The
cells were then stimulated with 10% EV-free NHS in RPMI and Ca2_
(2 mM) or, as a control, with heat inactivated NHS. During the ensuing
6.5 min, the resonate frequency remained stable. Then, over the
following 10 min there was a 30 Hz increase indicating a loss in mass,
consistent with the high rate of sMV observed. Given the crystal
constant, C as 17.7, ^f as 19 Hz and v (the third overtone) as 3, and
with the crystal area at 0.2 cm2, using the Sauerbrey equation we
calculated the mass loss to be 23 ng which corresponded to 0.25 pg
per MV given that 0.92 _ 105 MVs were released. The 16 min period
over which MVs continue to be released as determined on the QCM
coincides with the MV increase measured by FACS and with an
increase in early apoptosis from 4% plateauing at 10%, levels of
late apoptosis remaining at 1_3%. We also looked at deposition of
sMV on the sensor. Given a Df of 27197 Hz for the deposition
of 1.3_106 sMVs, we estimate the mass of an sMV by this approach
as 0.24190.006 pg. Summary/conclusion: Using the QCM we were
able to measure a significant change in cellular mass, beginning at 6.5
min post-stimulus and peaking at 1000 s post-stimulus. The QCM also
detected a decrease in media fluidity, attributed to the process of
membrane blebbing on THP-1 and MV release. The QCM was able
to provide an accurate measurement of sMV mass (0.25 pg) by
calculating the loss in mass of the stimulated cells. By measuring the
quenching of the oscillating momentum on the QCM as sMVs are
deposited on the sensor, we were also able to calculate the mass of
an sMV as 0.24 pg.