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Automatic Single-Cell Transfer Module
PDMS chip via a Teflon tube (outer diameter 0.5 mm, inner diameter 0.3
mm). The main route starting from the dock side port (outlet port) to the
glass microtube was on the same line. Therefore, flow speed and flow direction in the microchannel, as well as the speed on the tip of the glass microtube, were handled by this pump. During the experiments, we realized that
small-diameter tubes decrease the possibility of creating cell stacks on the
intersection point of the inlet port and the Teflon tube. Hence, the glass microtube and the cell delivery channel were connected using another Teflon tube
(outer diameter 0.3 mm, inner diameter 0.2 mm). We used a glass microtube
(0.30 mm outer diameter, 0.18 mm inner diameter) as a suction mouth for
the oocyte cells. A glass microtube can easily vacuum single cells without
inflicting any damage. In addition, it has a noncomplex fabrication procedure that makes it easy to apply for common procedures. For the fibroblast,
the glass microtube was heated to decrease its diameter (inner diameter ~50
μm). The tip of the glass microtube was processed by a microforge (MF-830,
Narishige Inc., Tokyo, Japan) and polishing machine (EG-44, Narishige Inc.).
7.2.4 Manufacture of the Microfluidic Chip
The microfluidic chip employed in this research has been designed to perform two main functions: aligning aspirated cells in the docking area and
transferring them to the next module. We designed two different types of
microfluidic chips that have two- and three-layer structures. A two-layer
microfluidic chip has two molds, the main mold carrying the fluidic channel and the valves. The molds of the second fluidic chip are the main mold,
valves (second mold), and air chamber (third mold), which were patterned
on a silicon wafer with different heights using SU8-based photolithography,
which has been described elsewhere (Zhang, Tan, and Gong, 2001). In the
main mold, channels for delivering cells were 200 μm wide and 150 μm deep,
dimensions sufficient to contain an oocyte. The main mold has a “Y”-shaped
character and is completed with a snake-like dock, as shown in Figure 7.3.
After preparation of the mold, the PDMS device and the valves were fabricated using a classic multilayer soft lithography technique. Firstly, a thin layer
of PDMS (thickness: ~300 μm, the first layer) was spin-coated on the main mold
and then cured for less than 20 minutes at 100°C in an oven. The same method
was repeated for the second layer, which also contained the valve. Incorrect
placement of the second layer (thickness: ~250 μm), containing a thin membrane, may obstruct flow permanently. The first and second layers were carefully aligned together. The valve layer was completed by adding a PDMS slab
containing an air chamber (depth: 500 μm). Then, the combined PDMS layers
were treated by air plasma using an expanded plasma cleaner for 1 minute
together with a glass slide rinsed in ethanol. The glass slide was placed on the
prosthesis and pressure was applied until they were firmly bound to each other.
Finally, the microfluidic chip was sealed with PDMS and cured a second time.
Automatic Single-Cell Transfer Module
PDMS chip via a Teflon tube (outer diameter 0.5 mm, inner diameter 0.3
mm). The main route starting from the dock side port (outlet port) to the
glass microtube was on the same line. Therefore, flow speed and flow direction in the microchannel, as well as the speed on the tip of the glass microtube, were handled by this pump. During the experiments, we realized that
small-diameter tubes decrease the possibility of creating cell stacks on the
intersection point of the inlet port and the Teflon tube. Hence, the glass microtube and the cell delivery channel were connected using another Teflon tube
(outer diameter 0.3 mm, inner diameter 0.2 mm). We used a glass microtube
(0.30 mm outer diameter, 0.18 mm inner diameter) as a suction mouth for
the oocyte cells. A glass microtube can easily vacuum single cells without
inflicting any damage. In addition, it has a noncomplex fabrication procedure that makes it easy to apply for common procedures. For the fibroblast,
the glass microtube was heated to decrease its diameter (inner diameter ~50
μm). The tip of the glass microtube was processed by a microforge (MF-830,
Narishige Inc., Tokyo, Japan) and polishing machine (EG-44, Narishige Inc.).
7.2.4 Manufacture of the Microfluidic Chip
The microfluidic chip employed in this research has been designed to perform two main functions: aligning aspirated cells in the docking area and
transferring them to the next module. We designed two different types of
microfluidic chips that have two- and three-layer structures. A two-layer
microfluidic chip has two molds, the main mold carrying the fluidic channel and the valves. The molds of the second fluidic chip are the main mold,
valves (second mold), and air chamber (third mold), which were patterned
on a silicon wafer with different heights using SU8-based photolithography,
which has been described elsewhere (Zhang, Tan, and Gong, 2001). In the
main mold, channels for delivering cells were 200 μm wide and 150 μm deep,
dimensions sufficient to contain an oocyte. The main mold has a “Y”-shaped
character and is completed with a snake-like dock, as shown in Figure 7.3.
After preparation of the mold, the PDMS device and the valves were fabricated using a classic multilayer soft lithography technique. Firstly, a thin layer
of PDMS (thickness: ~300 μm, the first layer) was spin-coated on the main mold
and then cured for less than 20 minutes at 100°C in an oven. The same method
was repeated for the second layer, which also contained the valve. Incorrect
placement of the second layer (thickness: ~250 μm), containing a thin membrane, may obstruct flow permanently. The first and second layers were carefully aligned together. The valve layer was completed by adding a PDMS slab
containing an air chamber (depth: 500 μm). Then, the combined PDMS layers
were treated by air plasma using an expanded plasma cleaner for 1 minute
together with a glass slide rinsed in ethanol. The glass slide was placed on the
prosthesis and pressure was applied until they were firmly bound to each other.
Finally, the microfluidic chip was sealed with PDMS and cured a second time.