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Sriphutkiat Y, et al

where p 0 is an acoustic pressure,  is an acoustic contrast the width of 3 mm, and the height of 100 μm. The PDMS
factor given by cavity was degassed in a vacuum chamber (3608-1CE,
ThermoFisher Scientific, Waltham, MA, USA) at 60 °C

(4) for 4 h. Then the PDMS cavity was bonded directly on

LiNbO 3 by oxygen plasma (Harrick Plasma, Ithaca, NY,
The transverse motion of cells across the channel width USA) treatment and then rest at 60 °C in the vacuum
under the action of the acoustic radiation force is governed chamber for 10 min.
by Newton’s second law. As the cells are much smaller
than the dimension of the microchannel, their longitudinal 2.3 Experimental Setup
motion is assumed to follow the fluid streamlines. Particle The PDMS cavity was punched with two holes for inlet
motion was simulated by solving the ordinary differential and outlet. Prior to loading cells, the PDMS cavity was
equation (ODE) above using the fourth order Runge-Kutta filled with 2% bovine serum albumin (BSA, Thermo
method in Matlab (MathWorks, Natick, MA, USA). Material Fisher Scientific) for 15 min to coat the cavity surface in
properties used in the simulation are listed in Table 1, and order to reduce the cell adhesion. Many pressure nodes in
the schematic diagram is same as that in our previous the shape of grid with the size of half wavelength, which
[42]
study . is determined by the excitation frequency of SAW and
Table 1. Material properties used in simulation at the speed of sound propagating in the LiNbO 3 wafer, are
temperature of 27 °C generated inside the PDMS cavity after SAW excitation.
density, ρ w 997 kg/m 3 The cells suspension was filled into a 3 mL syringe that was
speed of sound, c w 1497 m/s
Water driven by a syringe pump (NE-1000, New era pump systems,
viscosity, μ w 0.890 mPa.s Farmingdale, NY, USA) to the PDMS cavity through the
−1
compressibility, κ w 448 TPa inlet. The accumulation of cells and formation of cell
3
density, ρ p 1075 kg/m spheroids in the cavity was observed under an optical
Biological cells speed of sound, c p 1600 m/s microscope (CKX-41, Olympus, Tokyo, Japan) at 40×
−1
compressibility, κ p 428 TPa magnification and captured by a digital camera (QIC-F-
Poly-dimethylsiloxane density,ρ PDMS 920 kg/m 3 CLR-12-C, QImaging, Surrey, BC, Canada), and the size
(PDMS, 10:1) speed of sound, c PDMS 1076.5 m/s of formed cell spheroid was quantitatively determined
3
Lithium niobate density,ρ LNB 4650 kg/m using digital image software (ImageJ, National Institute
(LiNbO 3) speed of sound, c LNB 3997 m/s of Health, Bethesda, MD, USA). A sinusoidal signal of
at the frequency of 10.4 or 23.8 MHz was generated
2.2 Device Fabrication (AFG3000, Tektronix, Beaverton, OR, USA), amplified

Two pairs of identical interdigital transducers (IDTs) aligned (25A250A, Amplifier Research, Souderton, PA, USA)
perpendicular to each other were fabricated by positive and supplied to these two pairs of IDTs at an output
photoresist lift-off process. The process started with power of 0.7 Watt for the acoustic excitation. During the
hexamethyldisilazane (HMDS) treatment followed by excitation of SSAW for about 30 min, the device (PDMS
coating the photoresist (AZ9260, MicroChemicals GmbH, cavity on the LiNbO 3 wafer) was placed on a lab-made
Germany) in the thickness of about 5-µm on the surface cooling plate to reduce the generated excessive heat. The
of the LiNbO 3 wafer in the thickness of 500-μm (Y-128 cooling plate consists of a Peltier plate (thermoelectric
propagating, University Wafer, Boston, MA, USA). The cooler in the size of 4×4 cm, Robot R Us, Singapore), heat
LiNbO 3 wafer was cured with UV to weaken the photoresist sink, and 5V DC brushless fan (Robot R Us). After the
which was further developed with AZ-developer (400K, formation, the cell spheroids were transferred out from the
MicroChemicals GmbH, Germany). After that, the wafer PDMS cavity by pumping 1×phosphate-buffered saline
was sputtered with a layer of 20 nm-Cr and 400 nm-Au, (PBS) solution through the inlet at a flow rate of 2 μL/min.
and the photoresist was removed by acetone (Aik Moh, Then the collected cell spheroids from the outlet were
Singapore). There are 20 strips in the width of 150 μm in observed under the same optical microscope.
each IDT with an aperture size of 2 cm. 2.4 Cell Preparation
The poly-dimethylsiloxane (PDMS) microfluidic cavity
was fabricated using the soft-lithography and mould-replica HepG2 cells, immortalized human liver carcinoma cell line
®
techniques. PDMS (Sylgard 184, Dow Corning, Midland, (HB-8065™, ATCC , Manassas, VA, USA), were cultured
MI, USA) was fixed with elastomer base in a ratio of in HyClone TM Dulbecco’s modified eagle’s medium
10:1 and then poured on the mould in the length of 3 mm, (DMEM, GE Healthcare Life Sciences, HyClone Laboratories,

International Journal of Bioprinting (2018)–Volume 4, Issue 1 3