Part C | 20.2
514 Part C Marine Genomics
croflow cytometer, with its present dimensions, was not
able to measure particles with sizes < 2 m.
In addition to the distinction between the different
cells in the same solution, microfluidic technology can
be used to separate cells with different characteristics
to study their functions. Sasaki et al. [20.47] developed
a microfluidic device to separate bioluminescent bacteria in function of their motility. The device is made of
PDMS and composed of two inlets connected to two
separated channels that merged in one main channel.
The end of the channels, which is 35 mm long, was
split into two channels connected to two outlets. This
configuration of the microchannel allows the formation
in the main channel of two parallel flows, each coming
from a different inlet, without mixing the two liquids.
Sasaki et al. used this property to separate luminescent
Photobacterium kishitanii bacteria according to their
motility [20.47]. The cell suspension was introduced
in one inlet and filtered liquid broth in the other. In
the main channel, motile bacteria swim and crossed the
flow to reach the liquid broth while nonmotile cells remain in the cell suspension flow. Suspensions collected
from both outlets were analyzed. The luminescent intensity of the separated samples was measured using
a luminometer. The luminescent intensity per cell was
calculated. The result was that the value from the mobile (swimmers) bacteria was larger than the nonmobile
cells (nonswimmers).
So far, very few systems have been used to study
marine cells. However, other microfluidic reactors have
been developed to analyze the cell proteins from multiple or single cells [20.37, 38, 48–51], which could be
used in marine biotechnology.
Tissues and Whole Organisms
Fish embryogenesis-based biotechnologies have various promising applications in the field of marine
biotechnology, from commercially valuable fish production to threatened marine species protection [20.52].
Conventionally, handling of embryos, treatment, and
analysis are performed manually, in 96–384 microtiter plates, [20.53–56] and combined with laborious imaging methodology [20.57]. Under such conditions, analysis and imaging of fish embryos and
juveniles in a high-throughput and high-content manner remains challenging [20.53, 55, 57, 58]. These limitations can, however, be overcome with miniaturized LOC technologies that can provide an automated platform for the manipulation and immobilization of micron-sized organisms [20.53, 59–62] and
for high-throughput screening [20.63]. Recently, several microfluidic-based methodologies have been developed that involve droplet-based microfluidic systems [20.43, 64] or continuous-flow systems [20.44,
65–68]. Droplet-based microfluidic systems were the
first technology to be used for controlled fish embryogenesis. In 2006, Funfak et al. studied the behavior of
multicellular systems, in particular embryonic development in a microfluidic system [20.43]. The system,
which is schematically represented in Fig. 20.2d, is
composed of a Teflon microtube in which fish eggs in
aqueous solution are introduced into the tube, perfused
with immiscible perfluoromethyldecalin (PP9), resulting in droplets containing one single embryo each. The
development processes of fish embryos were observed
over a time period of 80 h. Finally, after 5 days, the fish
larvae were collected from the droplets and transferred
into breeding reservoirs for further experiments. More
recently, Akagi et al. introduced a miniaturized embryo
array that automatically traps, immobilizes, and perfuses fish embryos [20.44]. The device, which is shown
in Fig. 20.2d, is composed of a serpentine channel that
contains embryo traps (where embryos are trapped and
immobilized), suction channels (to attract embryos in
the embryo trap), and hydrodynamic deflectors (to enhance embryo trapping). Embryos were loaded in the
chip one by one every 5 s. After entering the serpentine channel, embryos rolled on the bottom surface of
the main channels under the influence of drag force.
Embryos approaching an empty trap were affected by
the flow passing through the suction channel and directed toward the trap, which was designed to assure
single-embryo occupancy, and unobstructed passage of
other embryos in the main channel. The following embryo rolled freely in the serpentine channel toward the
next available trap. The process was repeated until all
traps were occupied. After loading, the chip was perfused for long-term culture (up to 72 h), and normal
and uniform development of all embryos was observed.
Although most microfluidic technology techniques for
fish embryological studies were performed on Danio rerio (a freshwater fish), one can easily transpose those
techniques to marine fish embryogenesis studies and
engineering.
20.2.2 Microfluidic Systems
for Cell Behavior Studies
In the last decade, microfluidic systems have been
widely used for the study of cell behavior, mainly
with respect to how cells sense mechanical, electrical, and chemical signals, and for the measurement
514 Part C Marine Genomics
croflow cytometer, with its present dimensions, was not
able to measure particles with sizes < 2 m.
In addition to the distinction between the different
cells in the same solution, microfluidic technology can
be used to separate cells with different characteristics
to study their functions. Sasaki et al. [20.47] developed
a microfluidic device to separate bioluminescent bacteria in function of their motility. The device is made of
PDMS and composed of two inlets connected to two
separated channels that merged in one main channel.
The end of the channels, which is 35 mm long, was
split into two channels connected to two outlets. This
configuration of the microchannel allows the formation
in the main channel of two parallel flows, each coming
from a different inlet, without mixing the two liquids.
Sasaki et al. used this property to separate luminescent
Photobacterium kishitanii bacteria according to their
motility [20.47]. The cell suspension was introduced
in one inlet and filtered liquid broth in the other. In
the main channel, motile bacteria swim and crossed the
flow to reach the liquid broth while nonmotile cells remain in the cell suspension flow. Suspensions collected
from both outlets were analyzed. The luminescent intensity of the separated samples was measured using
a luminometer. The luminescent intensity per cell was
calculated. The result was that the value from the mobile (swimmers) bacteria was larger than the nonmobile
cells (nonswimmers).
So far, very few systems have been used to study
marine cells. However, other microfluidic reactors have
been developed to analyze the cell proteins from multiple or single cells [20.37, 38, 48–51], which could be
used in marine biotechnology.
Tissues and Whole Organisms
Fish embryogenesis-based biotechnologies have various promising applications in the field of marine
biotechnology, from commercially valuable fish production to threatened marine species protection [20.52].
Conventionally, handling of embryos, treatment, and
analysis are performed manually, in 96–384 microtiter plates, [20.53–56] and combined with laborious imaging methodology [20.57]. Under such conditions, analysis and imaging of fish embryos and
juveniles in a high-throughput and high-content manner remains challenging [20.53, 55, 57, 58]. These limitations can, however, be overcome with miniaturized LOC technologies that can provide an automated platform for the manipulation and immobilization of micron-sized organisms [20.53, 59–62] and
for high-throughput screening [20.63]. Recently, several microfluidic-based methodologies have been developed that involve droplet-based microfluidic systems [20.43, 64] or continuous-flow systems [20.44,
65–68]. Droplet-based microfluidic systems were the
first technology to be used for controlled fish embryogenesis. In 2006, Funfak et al. studied the behavior of
multicellular systems, in particular embryonic development in a microfluidic system [20.43]. The system,
which is schematically represented in Fig. 20.2d, is
composed of a Teflon microtube in which fish eggs in
aqueous solution are introduced into the tube, perfused
with immiscible perfluoromethyldecalin (PP9), resulting in droplets containing one single embryo each. The
development processes of fish embryos were observed
over a time period of 80 h. Finally, after 5 days, the fish
larvae were collected from the droplets and transferred
into breeding reservoirs for further experiments. More
recently, Akagi et al. introduced a miniaturized embryo
array that automatically traps, immobilizes, and perfuses fish embryos [20.44]. The device, which is shown
in Fig. 20.2d, is composed of a serpentine channel that
contains embryo traps (where embryos are trapped and
immobilized), suction channels (to attract embryos in
the embryo trap), and hydrodynamic deflectors (to enhance embryo trapping). Embryos were loaded in the
chip one by one every 5 s. After entering the serpentine channel, embryos rolled on the bottom surface of
the main channels under the influence of drag force.
Embryos approaching an empty trap were affected by
the flow passing through the suction channel and directed toward the trap, which was designed to assure
single-embryo occupancy, and unobstructed passage of
other embryos in the main channel. The following embryo rolled freely in the serpentine channel toward the
next available trap. The process was repeated until all
traps were occupied. After loading, the chip was perfused for long-term culture (up to 72 h), and normal
and uniform development of all embryos was observed.
Although most microfluidic technology techniques for
fish embryological studies were performed on Danio rerio (a freshwater fish), one can easily transpose those
techniques to marine fish embryogenesis studies and
engineering.
20.2.2 Microfluidic Systems
for Cell Behavior Studies
In the last decade, microfluidic systems have been
widely used for the study of cell behavior, mainly
with respect to how cells sense mechanical, electrical, and chemical signals, and for the measurement