MICROFLUIDIC DEVICE FOR CAPTURING CIRCULATING TUMOR
CELLS
-SEPARATION BY CELL SIZE AND RIGIDITYTomoki Konishi, Hiromasa Okano, Takahiro Suzuki, Shinya Ariyasu, Toshihiro Suzuki,
Ryo Abe, Shin Aoki, and Masanori Hayase
Tokyo University of Science, JAPAN
ABSTRACT
Effect of fluid velocity on cell size sorting by a deterministic lateral displacement (DLD) device was
studied. Circulating tumor cells (CTCs) tends to be larger compared to benign blood cells, and DLD
sorting devices have been studied for CTC separation. However, high separation efficiency is not
expected by only size sorting. We focused on cell deformability, and effect of fluid velocity on
separation behavior in a simple DLD device was observed. It was found that benign lymphocytes tends to
behave like smaller cells at high velocity, while no obvious difference along fluid velocity was observed
with cultured SP2/0 tumor cells.
KEYWORDS: Circulating tumor cells, Deterministic lateral displacement, Microfluidic device
INTRODUCTION
Circulating tumor cells (CTCs) are tumor cells leaking into blood vessels from a tumor. Some CTCs
may proliferate in the distant site to establish metastases. Detection and separation of CTCs from blood is
required to understand metastases, and many studies using microfluidic devices have been published
recently. Generally, spiked cultured tumor cells were used for verifying separation performance. At the
last μTAS 2013, we reported that CTCs could be enriched from tumor-bearing mouse blood by a
deterministic lateral displacement (DLD) [1] size sorting device as shown in figure 1. But the separation
efficiency was poor, besides we noticed that large benign blood cells increased probably due to cancer
and that made the CTC separation by size sorting more difficult. It was also assumed that CTCs are
typically harder than leukocytes. In this study, we discussed to use the rigid feature of CTCs for better
separation efficiency.
DEVICE AND THEORY
The DLD devices used in this study were shown in figure 1. Specimen and buffer solution are
introduced from the inlets by syringe pumps respectively. Particles are sorted in the DLD micropost
structure, and particles larger than critical diameter flow along shifted post array and are segregated into
large section, while smaller particles go into small section.
In the DLD theory, critical separation diameter is estimated assuming a rigid sphere. But shear stress
is applied to cells by non-uniform fluid velocity or contact friction on the micropost, and cells may
become slender than stationary condition and behave as smaller diameter particles as shown in figure 2.
The deformation depends on cell rigidness, and shear stress depends on the fluid velocity. Therefore, it is
assumed that large but soft benign cells tend to be segregated into small section in large fluid velocity,
and separation efficiency of CTCs, which is assumed to be large and rigid, might be improved. In order
to examine this strategy, various fluid velocities were applied and sorting behavior by the DLD device
was observed.
Figure 1. Schematic of the DLD device. Micropost structure for DLD was formed on a Si substrate.
18th International Conference on Miniaturized
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EXPERIMENTAL
Three specimens, polystyrene beads, lymphocytes and cultured SP2/0 tumor cells, were prepared. The
polystyrene beads had a diameter of 5.9µm, and were assumed to be less deformable sphere. In order to
avoid beads aggregation, beads were dispersed into a sodium dodecyl sulfate (SDS) solution and the SDS
solution was also used as a buffer solution for the DLD device. Lymphocytes are types of white blood
cell and were tested as benign blood cells. They were collected from lymph nodes of healthy mice. As
tumor cells, SP2/0 cells ( mouse myeloma cell ) were used. The cells were dispersed in the phosphate
buffered saline (PBS) with 10 mM ethylenediaminetetraacetic acid (EDTA) to avoid cell adhesion.
Critical separation diameters of the DLD device were modulated in order for each specimen to be
segregated into both large and small sections. Four following flow conditions, (slow, normal, fast1, fast2),
were tested. In each conditions, average flow velocities at post gaps were 35, 67, 208 and 416 mm/s,
respectively. Sorting behavior was observed with a high-speed camera. Separation ratio was determined
by counting particles in recorded movies as shown in figure 3. Deformation of particle was evaluated
with the images as shown in figure 4. Although the images were not clear because of quick particle
movement and microscope’s short depth of field, the images were analyzed by the ImageJ and particle
profiles were estimated with identical criteria. Then, particle diameters were determined by "r edge - rpost".
RESULTS AND DISCUSSION
Variation of specimens separation ratio along flow velocity is shown in figure 5. In the case of polystyrene beads, separation ratios were constant at 50%. Particle diameters obtained by the image analysis
were also constant at 6µm, which indicates that the beads did not deform. It seemed that beads sorting
was not affected by the flow velocity. On the other hand, in the case of lymphocytes, the percentage of
cells segregated into the small section were increased from 60% to 88% as the flow velocity increases.
Cell diameters estimated from the images were 5.8, 5.2, 3.3 and 3.4µm at velocity conditions, slow, normal, fast1 and fast2, respectively. As we expected, it seemed that lymphocytes were flexible and behaved
as smaller particles at high flow velocity by their deformation. In the case of SP2/0 tumor cells, though
Figure 2: Schematic of deformation of flexible cells. Flexible cells such as blood cells can deform near
microposts as the fluid velocity increases.
Figure 3: A microscopic image of outlet of the device. Separation ratio was determined by counting particles in recorded movies.
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Figure 4: A microscopic image of a cell on the
micropost. Profile of the cell was extracted by image analysis and particle diameter was determined by “redge - rpost”.
(a) Polystyrene beads
(b) Lymphocytes
(c) SP2/0 tumor cells
Figure 5: Separation ratio (left figures) and diameter (right figures) of the particles at each velocity. Flow
condition was determined by taking mean velocity in post gaps. Slow, normal, fast1 and fast2 are 35, 67, 208
and 416 mm/s, respectively.
the separation ratio varied between 75% from 66.4%, there are no clear tendency along flow velocity.
Cell diameters estimated from the images were also almost constant around 10.5µm. Tumor cells are
considered to be relatively rigid, and it is reasonable for tumor cells to behave similar way to polystyrene
beads. These results support our strategy, in which separation efficiency of large and rigid CTCs is raised
by segregating large but soft benign cells into small section.
CONCLUSION
CTCs separation efficiency by cell size sorting is limited by the existence large benign blood cells. In
this study, we discussed to improve the separation efficiency by utilizing cell rigidness. In the DLD devices, shear force depending on fluid velocity is applied to dispersed particles, and it was assumed that
soft cells become slender and behave as a smaller particle. It was demonstrated that benign lymphocytes
tends to behave like smaller diameter particle at higher fluid velocity, while SP2/0 tumor cells did not
show velocity dependency. It is said that CTCs tend to be rigid, and higher separation efficiency of CTCs
is expected by optimizing fluid velocity in the DLD devices.
REFFERENCES
[1] L, R, Huang, E. C. Cox, R. H. Austin, J. C. Sturm, “Continuous Particle Separation Through Deterministic Lateral Displacement,” Science, 304, 987-990, 2004.
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