104 M. C. W. Chen and K. C. Cheung
initial cell viability after device construction, the cell viability dropped after 3 days
except in the vicinity of the microfluidic channels through which culture medium
was supplied.
Paguirigan and Beebe have used a similar molding process to create gelatin-
based microfluidic devices seeded with cells.
69
5.4.3 Droplet Encapsulation
Cells can be encapsulated on-chip within gel spheres with diameters ranging from
tens to hundreds of microns. Generation of monodisperse droplets in microfluidic
systems has already been demonstrated, for applications including micromixing,
70
reverse transcriptase PCR, molecular synthesis, and cell encapsulation.
71
Droplets can be generated using two immiscible flows, in which the hydrogel
is the dispersed aqueous phase and a solvent is the continuous phase. In the flow-
focusing design, droplets are generated through shear at the interface between
two parallel streams, where the flows pass through a constriction. The continuous
phase places viscous stress on the immiscible dispersed phase, which is balanced
by the interfacial surface tension. Viscous shear stress tends to extend the interface,
while the competing surface tension effect tends to reduce the interfacial area. In
this situation, droplets are created above a critical stress. The Capillary number
Ca = μv/γ is a dimensionless parameter. It is the ratio of viscous forces to surface
tension, where μ is the viscosity of the oil phase, v is the velocity of the droplet, and
γ is the interfacial tension between the two phases. Droplet size is a function of the
fluid viscosities, surface tension, microfluidic channel geometry, and flowrates.
72,73
Droplet size decreases with increasing flow rates, and increases with channel
diameter.
Recent work has demonstrated monodisperse bead and single-cell loading
within aqueous drops using the flow-focusing geometry.
75
A high-aspect-ratio
channel was used to achieve single-particle loading within aqueous drops due to
the hydrodynamic interactions. A high density suspension of particles, in which
the particle diameter is significant compared to the channel constriction diameter,
will become self-organized into one of two configurations when traveling rapidly
through this channel:
(a) particles will be located along one side of the channel, or
(b) particles will alternate from one side of the channel to the other. The flowrates
can be adjusted so that droplets form with the same frequency that particles
arrive at the flow constriction.
The droplet volume ranged from 14–21 pL (droplet diameter from 30–34 μm),
which encapsulated HL60 cells and 9.9 μm-diameter beads, respectively.
The flow-focusing method has been used to generate liquid NiPAAm droplets
at room temperature, which then gelled further downstream in a heated region
of the channel.
76,77
Embedded thin-film electrodes were used: as current was
applied through the heater on the bottom of the channel, the local temperature
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Hydrogel-Based Microfluidic Cell Culture 105
Figure 5.13. Droplet formation using the flow focusing design. Droplet size decreases with
increasing oil (continuous phase) flow rate relative to water (dispersed phase) flow rate.
74
in the channel increased above the gel formation temperature to 36
C. The gelled
droplets were then collected in a simple grid to form a packed bed.
Although the majority of droplet-encapsulated cells have used macroscale
extrusion, air-jet or electrostatic methods to generate droplets which gel subse-
quently in a large bath, microfluidic hydrogel droplets have also been made.
Alginate gel droplets have been generated using the flow-focusing method.
78,79
Alginate microspheres with encapsulated cells have also been generated using
microfluidic devices. Choi et al. used alginate and CaCl
2
flows in hexadecane, and
they reported blocking problems depending on the aqueous phase flowrate, due
to gelation at the interface between alginate and calcium flows.
80
Hexadecane is
highly immiscible with water and has low solubility (9.0 × 10
8
g/100 g water
at 25
C) in the aqueous phase, allowing high cell viability in beads formed in
hexadecane. However, for specific flowrates they did report successful on-chip
crosslinking and encapsulation of yeast into alginate beads. Tan and Takeuchi
used insoluble CaCO
3
particles interspersed in aqueous alginate solution, and
droplets were created in corn oil which contained acetic acid.
81
Workman et al.
have also used a flow-focusing geometry in which one phase contains sodium
alginate mixed with CaCO
3
and cells and another phase contains acetic acid in oil.
These two phases are separated by flows of plain oil, so that during laminar flow
the H
+
does not diffuse into the alginate stream. After droplet formation, the H
+
then diffuses inside the alginate droplet to release Ca
2+
, thus permitting gelation
of the droplet. This method has been used to encapsulate the PC12, HEK293, and
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