7.2.1 Cell Types and Preparation

In this chapter, we primarily focus on somatic cell cloning. Experiments were performed using fibroblasts as donor cells and oocyte cells as recipient cells. Oocytes were isolated from bovine. Ova were cultured approximately 24 hours after harvesting from the ovary. In order to detach cumulus cells from the harvested ova, cells were treated with hyaluronidase (Nacalai Tesque Inc., Kyoto, Japan). The pellucid zone of the ova was removed by pronase treatment and near-circular oocyte cells were isolated The diameter of isolated oocytes was about 100-150 urn (Figure 7.2).

Fibroblasts 10-30 urn in diameter were isolated from female bovine ears (Figure 7.2). The fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) in 35-mm dishes in a 5% CO₂ incubator. When cells became confluent, the culture medium was changed to 0 5% FBS-supplemented media Fibroblasts were harvested from the dishes by treatment with trypsin-ethylenediaminetet-raacetic acid (EDTA).

7.2.2 Penicillin-Streptomycin and Bottom Surface Treatment

Fibroblasts readily adhere to the surface of plastic culture dishes We detached fibroblasts using trypsin-EDTA (Wako Pure Chemical Industry, Osaka, Japan) treatment (Tuan and Lo 2000) and then waited over 30 minutes to attempt to aspirate the fibroblasts. However, over time, detached cells readhere to the culture dish and cannot be aspirated To avoid this problem, we treated the culture dish by painting the dish with a small amount of liquid PDMS (without the use of catalyst) for use in the following experiments. This method is also known as siliconizing (Gunnar and Bähr 2008); that is, coating a surface with silicon The other method was to pour PDMS (using a catalyst) and cover the dish with a thin, solid PDMS layer In the case of nontreated containers, less than half of the fibroblasts could be aspirated. On the other hand, the cells in the PDMS-coated dish were all easily aspirated

7.2.3 Cell Suction System

In the acquisition process of a single cell, first the rotary pump (ISMATech Inc, Wertheim-Mondfeld, Germany) was connected to the dock side of the 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 micro-tube 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 um) 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 um wide and 150 um 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 um, 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 um), 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 um) 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.

7.2.5 Valve Control Principle

Within the PDMS chip, the valve region is closed by applying air pressure to the air chamber and obstructing the microchannel (Figure 7.4). The air pressure control valves for open and close actions are activated by a syringe pump (KDS270, Kd Scientific, Holliston, MA, USA). The syringe pump links two valves so that one becomes closed if the other becomes open. In addition, the flow behavior of the "Y"-shaped channel can be observed by the use of color pigments when the pump actuates. The control of the current direction in the microchannel is handled by the rotary motion of a high-precision rotary pump (ISMATech). Although the fabrication process of a three-layer microfluidic chip is more difficult than that for a two-layer chip, it has a better performance, in particular for response time. Table 7.1 shows a comparison of both valve designs

7.2.6 Vision Systems

Two camera systems were assigned to monitor two important sections of the entire mechanism. The first camera (compact optical setup with Point grey Dragonfly camera, Point Grey Research, Richmond, BC, Canada) was placed on the cell container in order to detect the position of the oocyte or donor cells in the container. The second camera was boarded on the microfluidic chip with the purpose of actuating valves and changing flow direction.

The compact vision system provides good image quality, allowing data on the oocyte cell and donor cell to be extracted from the acquired images (Uvet et al. 2008). In this second version, we changed the optical setup and designed a task specific system that has a 1. 5-mm monitoring area and light source on the same side as the complementary metal-oxide-semiconductor (CMOS) sensor. The specifications are given in Table 7.2.

The camera can be placed and aligned on a chip with the aid of xyz micro-stages. As shown in Figure 7.5, the new system is small and can be easily combined with the microfluidic chips. This makes it possible to observe the cell container and microfluidic chip simultaneously in a short distance. Otherwise, we would have to use two commercial microscopes, which requires a large workspace and the use of a long Teflon tube to connect the glass microtube to the inlet port of the PDMS chip

In this study, the modulation transfer function (MTF) was also used as a measurement parameter of optical quality (Ray 2002; Smith 2000). MTF graphs show modulation of image contrast at different spatial frequencies Diffraction limits the contrast in every optical system Diffraction limited modulation transfer functions are given in MTF graphs with dashed curves for the purpose of comparison with perfect lens case In this MTF analysis, defocusing, which causes blurry image, is set as 0 00 MTF performance was tested under a 587.6 nm wavelength. Four different MTF graphs plot the percentage of transferred contrast according to the distance from the center of the objective plane (center of the image). The MTF results at given radiuses are shown in Figure 7.6. The modulation (also known as contrast) is expressed in terms of a percentage (1.0 = 100%).

7.2.7 Cell Detection/Tracking and Control

The detection/tracking and control algorithm was mainly programmed for single-cell applications. Its execution steps can be described as follows:

• Start the first camera and detect the position of single cells in the cell container

• Align the glass microtube to the cell position and begin suction (ISMATech rotary pump)

• Toggle to the second camera and count the number of cells that pass the cross section in the PDMS chip

• If the desired number of cells passes the cross point, switch valves and let the cells flow to the next module.

• Switch valves and cameras if a second group of cells is required

A background subtraction algorithm was employed throughout the detection phase in order to eliminate redundant artifacts and to surpass optics-based aberrations The background subtraction method was essentially applied to moving regions, and the object positions were automatically found after input images were compared with a background image In this way, it is also possible to detect and track multiple objects After the edge is identified, the algorithm makes a circular approximation to the edge of the object and draws a circle around it, which is taken as the diameter of the object

It is important to note that the relative size of each pixel in a digital image is very important. To determine accurate diameter, the ratio between a single-cell image reconstructed on an image sensor and corresponding total pixel number must be calculated The pixel ratio for each cell was calibrated by Olympus Ronchi Ruling glass (Olympus, Toyko, Japan) (100 lp/mm). A donor cell of ~15 urn corresponds to about 30 pixels. In addition, we consider the boundary of any moving region as an edge of the object, because cells always come into the observation area one by one Once the background is fixed, any moving object is filtered according to its size and dimensions and labeled as a donor or oocyte Thirty pixels are considered as a minimum detection size and an object below 30 pixels is omitted. We essentially applied the same idea to the two camera systems, but cells are only floating in the container viewed by the microscopic camera. In the case of the compact vision system, however, the program was plagued by some environmental problems such as bubbles and other unwanted objects, which appear similar to the fibroblast or the oocyte.

Becaus the oocytes are approximately ten times larger than the fibroblasts, requiring a deep channel depth (150 um), the fibroblasts can easily go out of focus range and hence the image becomes blurry. Furthermore, the donor cell is very transparent under visible light. Even though its shape is nearly circular, because of the light conditions, in most cases it is complicated to extract its features from the background (Figure 7.7). After entering the microchannel, according to light dispersion over the donor cell, high-density parts become darker, whereas light areas frequently converge to the background color Though the system could often capture the edges of the donor cell through consecutive frames, in some cases the system could not locate the edges of the fibroblast.

7.3.1 Oocyte and Fibroblast Suction

After a number of treatments, explained in the previous section, oocytes and fibroblasts were dispersed in a random manner in different containers (Figure 7.8). Before dispersion, the required flow speed must be defined in order to stabilize cell detection. In the case of fibroblasts, the ideal flow speed is determined by changing the rotary pump speed as in Table 7.3.

After determining the optimum speed, we programmed an initial desired number of cells for suction The program automatically starts searching for cells in the container if there is no cell on the monitoring area (Figure 7.9a). As soon as the program detects a cell or a cell group, it takes the position of the cells, draws circles around each detected cell, and displays their positional information (see Figure 7.8). The suction mouth of the glass microtube aligns with the nearest cell on the bottom surface, which is numbered 0, and then the rotary pump starts to flow. The required absorption time from the tip of the glass microtube to node A on the microchip is shown in Figure 7.10. According to the absorption time, the pump adjusts its speed automatically by decreasing the speed to the optimum detection level (see Table 7.3).

Once the suction of cell 0 is completed, the suction mouth returns to the original starting point, takes off from the surface to a safe height, and carries on suction of the medium without cells. After 6 seconds (an experimentally calculated delay used in order to put a specific distance between each cell) the suction mouth returns to the bottom surface and moves to the next cell, numbered 1

This procedure is repeated until the last cell is vacuumed from the screen view If the program reaches the total number of desired cells, it stops searching, moves to a safe height, and continues with the suction, even if there are still some cells on the screen The aspirated cells are batched in the docking area with a specific distance. The time interval between two connected cells is given in Figure 7.9b for suction mode and supply mode. Although the approximate absorption time for one cell to reach node A was found based on the speed of the rotary pump and tube dimensions, the experimental elapsed time values were different (Figure 7.11). The delay occurred due to the drag resistance of cells as they move through the continuous fluid flow in the Teflon tube.

7.3.2 Direction Control

Once aspiration of the desired number of cells is completed, the camera system on the cell container is toggled to the compact vision system placed on the cross section of the "Y"-character microchannels (see Figure 7.11). The task of the compact system is to ensure that the collected cells are gathered at the dock and transported to the next module, by switching the valves When a new cell shows up in the upper right-hand corner, the program detects cells and starts tracking. To avoid environmental failure in the PDMS chip and to maintain efficiency, four control parameters were added to the cell detection/ tracking and control program These parameters are shown on the screen as sequential lines. The first three lines (green) confirm that a detected cell safely passes to the dock side The second line, on the upper left-hand side (blue), counts the number of cells transferred In the beginning, three green and one blue parameter toggle as false As soon as the center of the detected circular area of the cell passes a single line, it toggles to true After it passes three green lines in order, the program counts this as one cell and waits for the second cell. When the desired total number of cells is reached, cells in the dock are transported to the next module Each time a single cell passes the blue line, this is recorded for final confirmation. To start cell transfer to the next module, the system runs as follows:

• The rotary pump connected to the end of the "Y"-shaped channel stops the flow.

• The syringe pump applies pressure to the right pneumatic valve (infusion) while releasing the left valves (withdraw) In the experiments, the time elapsed during valve switching was measured at approximately 3 seconds

• The main flow direction is reversed.

• The rotary pump starts running again and transfers cells in the batch to the microchannel on the left