Printed Edition of the Special Issue Published in Micromachines 0LFUR1DQRÀ XLGLF'HYLFHVIRU 6LQJOH&HOO$QDO\VLV Edited by Fan-Gang Tseng and Tuhin Subhra Santra www.mdpi.com/journal/micromachines Fan-Gang Tseng and Tuhin Subhra Santra (Eds.) Micro/Nanofluidic Devices for Single Cell Analysis This book is a reprint of the special issue that appeared in the online open access journal Micromachines (ISSN 2072-666X) in 2013 (available at: http://www.mdpi.com/journal/micromachines/special_issues/single_cells_analysis). Guest Editors Fan-Gang Tseng and Tuhin Subhra Santra Department of Engineering and System Science National Tsing Hua University #101, Sec. 2, Kuang-Fu Road 30013 Hsinchu Taiwan Editorial Office MDPI AG Klybeckstrasse 64 Basel, Switzerland Publisher Shu-Kun Lin Editorial Manager Alistair Freeland 1. Edition 2015 MDPI • Basel • Beijing • Wuhan ISBN 978-3-03842-090-3 (PDF) ISBN 978-3-03842-146-7 (Hbk) © 2015 by the authors; licensee MDPI, Basel, Switzerland. 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A microfluidic system was specially designed for enucleation, and the microrobot actively controls the local flow-speed distribution in the microfluidic chip. The microrobot can adjust fluid resistances in a channel and can open or close the channel to control the flow distribution. Analytical modeling was conducted to control the fluid speed distribution using the microrobot, and the model was experimentally validated. The novelties of the developed microfluidic system are as follows: (1) the cutting speed improved significantly owing to the local fluid flow control; (2) the cutting volume of the oocyte can be adjusted so that the oocyte undergoes less damage; and (3) the nucleus can be removed properly using the combination of a microrobot and hydrodynamic forces. Using this device, we achieved a minimally invasive enucleation process. The average enucleation time was 2.5 s and the average removal volume ratio was 20%. The proposed new system has the advantages of better operation speed, greater cutting precision, and potential for repeatable enucleation. Reprinted from Micromachines. Cite as: Feng, L.; Hagiwara, M.; Ichikawa, A.; Arai, F. Micro/Nanofluidic Devices for Single Cell Analysis. Micromachines 2013 , 4 , 272-285. 1. Introduction “Dolly” is famous for being the first mammal to be successfully cloned from an adult cell [1]. Despite low success rates, several mammalian species have been successfully cloned since then [2–4]. Embryo manipulation is a potential technique for the genetic improvement of domestic animals and the preservation of genes of rare animals. Oocyte enucleation is a primary technique for the cloning process. The cloning of Dolly the sheep had a low success rate; 277 eggs were used to create 29 embryos, of which only three resulted in lambs, and eventually only one lived. Peura et al. conducted a viability test of the oocyte volume during nuclear transfer and determined that the nucleocytoplasmic ratio is an important parameter for embryo development [5]. Therefore, the low success rate of cloning techniques is a bottleneck for developing this field. Conventional techniques for the enucleation process mainly include the following: manual manipulator operation, microfluidic cutting methods in a microchip, and chemical treatment methods [6–8]. However, these methods tend to have a long operation time, low success rate, contamination, and low repeatability. Additionally, such types of complicated cell manipulation processes can only be undertaken by skilled people. During the chemical treatment processes, a person unaware of toxicities may poison the cells. Recently, researchers invented many techniques that do not require manual operation for the treatment of cells by fabricating a microrobot on a microchip. Magnetically actuated microrobots appear to be the most promising because of this method is minimally invasive to a cell, features a 6 noncontact drive, and has a low production cost [9–13]. Nelson et al. controlled the untethered microrobots using electromagnetic fields [14]. Sitti et al. designed a biologically inspired miniature robot [15]. These methods reduced the technical skills of operation and increased the throughput and repeatability. These robots can be operated precisely, but they do not have enough power to separate an oocyte. Previously, we have developed magnetically driven microtools (MMTs) in order to apply the microrobot to a wide range of cell manipulations. A permanent magnet possesses a magnetic field that drives an MMT 10–100 times more forcefully than an electromagnetic coil of the same size, effortlessly causing the output of mN-order forces. In order to reduce significantly the effective friction of the MMT, we arranged permanent magnets parallel to the driving plane [16] and piezoelectric ceramics were employed on the drive plane to induce ultrasonic vibrations so that the effective friction reduced significantly [17]. As a result, we achieved ȝ m-order positioning accuracy while maintaining a mN output force. Enucleation by a dual arm MMT was conducted previously [18]; however, it was difficult to remove the nucleus because the oocyte is a viscoelastic material. The cutting process is a complicated model because the oocyte is soft and sticky. Once the tip of the MMT blade touched the surface of the oocyte, a resistance force generated by the oocyte decreased the position accuracy. In addition, achieving the enucleation process was difficult because the oocyte flow was not well-controlled. The contribution of this paper is the development of an enucleation system using cooperation of an MMT with fluid control. Our new enucleation system contains three remarkable improvements: the oocyte enucleation process can be conducted one by one; the removal ratio in the volume is controllable, minimizing damage to the oocyte; and the nucleus can be removed with a hydrodynamic force controlled by the MMT. The methods of how to achieve these merits will be introduced sequentially in this manuscript. Figure 1a shows the concept of the enucleation chip that we used to conduct oocyte enucleation experiments. Two large chambers with a height of 300 ȝ m and a diameter of 5 mm were designed. The MMT with a height of 200 ȝ m was placed in one chamber with its blade inserted in the microchannel branch. To conduct the enucleation process, the other inlet of the Y-shaped microchannel was used to inject continually the oocytes. On the other side of the inlet is a shallow withdrawal microchannel with a height of 50 ȝ m that was used to confine the oocyte to a position to cut accurately the oocyte by a given volume. The first stage involves oocytes in a cell culture medium being injected from the inlet of the microchip. By connecting a digital pump to the outlet, the medium containing the oocytes from the inlet flowed through the microchamber towards the outlet. The tip of the MMT cutting blade was placed at the interface of the chamber and the withdrawal microchannel. The oocyte was delivered by the flow to the withdrawal microchannel, as shown in Figure 1b. Then, the tip of the MMT controls the oocyte orientation so that the nucleus comes to suction port. The delivered oocyte is obstructed at the interface because of the height limitation (50 ȝ m) of the microchannel, as shown in Figure 1f. Then, the hydraulic pressure deformed the oocyte, allowing the lower part of the oocyte to be suctioned into the withdrawal microchannel, as shown in Figure 1c. After the nucleus was in the withdrawal microchannel, the tip of the MMT was actuated to the left in order to close 7 the interface, as shown in Figure 1d. Next, under the protection of the MMT, the initial portion of the oocyte was reserved and only the separated nucleus was flushed away with the flow (Figure 1e). After the nucleus separated from the oocyte, the remaining part was also sucked out and collected from the outlet. Figure 1f shows the cross-sectional view of the microchannel; there is a height difference between the main chamber and the withdrawal microchannel. The oocyte can be stopped at the intersectional junction where the oocyte enucleation was conducted because of the height difference. Figure 1. ( a ) Overview of the enucleation microchip. ( b – e ) Concept of the oocyte enucleation process by the use of magnetically driven microtools (MMTs) in a micro À uidic chip. Blue arrows show the flow direction. ( f ) Height differences in the microchannel design. The white arrow shows the movement of the MMT.