Biomaterials for MEMS

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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