Characterization and Separation of Suspension Cells by Isoacoustic Focusing

Enabling techniques for separating target cells, such as subgroups of white blood cells and cancer
cells from blood and other body fluids, are necessary for lab work facilitation, inline integration with
high-end bioanalytical instrumentation, and for point-of-care or home testing. The work of this thesis
addresses this need by further studying an equilibrium-based ultrasonic wave-based technology called
isoacoustic focusing to characterize and gain access to cells. In this size-insensitive technique, cells are
suspended in a microchannel filled with an inhomogeneous medium where the interaction of diffusion,
gravity, and acoustic radiation shapes a smooth gradient profile for the acoustic impedance of the
media orthogonal to the flow. While flowing, the acoustic radiation force pushes cells towards their
isoacoustic point where the acoustic contrast and radiation force become zero and cells’ acoustically
induced sideways displacement ceases. The first two included papers in this thesis uncover in detail the
acoustophoretic motion of cells suspended in homogeneous and inhomogeneous media in a stop-flow
condition. Cell three-dimensional trajectories were measured by a defocused-image tracking approach,
and the technique’s applicability for tracking cells was assessed by determining the associated error
when measuring the out-of-image-plane component of the tracks. In a homogeneous medium, for cells
with near-zero acoustic contrast, strong effects of buoyancy and acoustic streaming were observed,
and small distributions of cell properties within a population resulted in large differences in the
cell motion patterns. The second article shows how cells migrate towards their iso-acoustic point in
acoustic impedance gradient media while acoustic streaming is substantially suppressed. This enabled
the readout of the effective acoustic impedance of neutrophils and K562 cancer cells. A numerical
model was introduced to estimate the acoustic energy density in the acoustic impedance gradient
media by tracking particles of known properties. To examine cell separation based on the knowledge
acquired in the two first studies, the third paper presents the use of gradient acoustic focusing and
dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole
blood in a label-free and flow-through format. By modifying the medium and thus tuning the contrast
factor of the cells, PBMCs were enriched relative to RBCs by a factor of 3600 to 11000 and with a
separation efficiency of 85%. Such a level of RBC depletion is high compared to most other microfluidic
methods and similar to density centrifugation. In the fourth Paper, we show that cell compressibility
can be determined at the isoacoustic point by an independent measurement of the density of each cell.
Cells were pre-sorted off-chip in linear, continuous, and reproducible density gradients, and fractions
with known densities were fed into an isoacoustic focusing device. The relation between density and
compressibility for two cell types was investigated, and it was found that for increasing density of
K562 cells, the compressibility decreases. For neutrophils, the compressibility was measured, and a
slight change was observed with increasing density.