On-chip enucleation of an oocyte by untethered microrobots

We propose a novel on-chip enucleation of an oocyte with zona pellucida by using a combination of untethered microrobots. To achieve enucleation within the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into the microfluidic chip. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage. The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e. the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated by using grayscale lithography, which allows fabrication of three-dimensional microstructures. The microgripper has a gripping function that is independent of the driving mechanism. On-chip enucleation was demonstrated, and the enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. The results show that the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%. The technique will contribute to complex cell manipulation such as cell surgery in lab-on-a-chip devices.

donor cell. Details of the cloning process are summarized as follows. Firstly, an oocyte is aspirated and held by a glass capillary (figure 1(i)). Secondly, the oocyte is penetrated and cut by a glass needle (figure 1(ii)). Thirdly, the nucleus included in the cytoplasm is pushed out of the zona pellucida (ZP) (figure 1(iii)). A donor cell is then injected into the oocyte (figure 1(iv)). Finally, the oocyte and donor cell are fused by applying voltage. Enucleation without the use of toxic fluorescent dye increases the production rate and is a minimally invasive operation. A polar body is used as an enucleation target without using any fluorescent dye, because the polar body is most likely next to the nucleus of the oocyte, which is in maturation stage II [5]. Therefore, removal of the polar body and a small ambient area are sufficient to complete the enucleation.
The conventional cloning process is carried out using glass capillaries attached to mechanical micromanipulators as end effectors that have various functions, such as holding and cutting. In addition, a mechanical micromanipulator with robot technology is capable of precise operation, high output force and multiple degrees of freedom (multi-DOF). Consequently, the complicated cloning process is mainly conducted manually by operators. Although mechanical micromanipulators with multi-DOF can manipulate a cell by using complex procedures, the manipulation requires highly skilled operators because the manipulators must be controlled in multi-DOF. Therefore, the success rate, repeatability and productivity of manipulations tend to depend on the operator's skill, especially for complex procedures like cloning. Moreover, the manipulators are placed outside the cell culture environment, where they are exposed to air. This can lead to several problems, including contamination.
On the other hand, lab-on-a-chip devices employ a closed environment, such as microchannels and microchambers. This type of configuration prevents cell contamination and restricts the position of cells in a two-dimensional plane. Moreover, the mass production of disposable microfluidic chips lowers their cost. Therefore, we proposed on-chip cloning and reported the effectiveness of on-chip enucleation using fluid force in a previous study [6]. The enucleation was automated by utilizing real-time feedback control of the robotic pump based on image analysis [7]. This automation system achieved high throughput with high repeatability. However, the method required removal of the ZP from the oocyte [7,8]. High speedenucleation using a microrobot was also performed with a non-ZP oocyte [9]. The ZP plays vital roles during oogenesis, fertilization and preimplantation development [10]. Therefore, an enucleation method that includes the ZP is highly required for biomedical applications.
In this paper, we propose a novel on-chip enucleation of an oocyte with ZP by untethered microrobots. To achieve on-chip enucleation, we integrate the advantages of a mechanical manipulator and lab-on-a-chip devices. The untethered microrobots, which have a gripping or knife function, are packaged in a microfluidic chip and actuated by the external magnetic force generated by permanent magnets placed on the robotic stage. In section 2, the details of each untethered microrobot are described by considering the requirements for on-chip enucleation. Section 3 outlines the evaluation results of the microrobots. Moreover, on-chip enucleation of a bovine oocyte with ZP is demonstrated by the combination of the untethered microrobots in a microfluidic chip. Finally, the conclusions and our future plans are presented in section 4.

Untethered microrobots for on-chip enucleation
Enucleation, as described in section 1, is mainly divided into three processes, (a) holding, (b) penetrating and cutting an oocyte, and (c) removing the nucleus and some cellular cytoplasm. The most important requirement is the output force, because an output force on the order of several hundred micronewtons is required to deform an oocyte. There are several different types of microactuator that can be applied in the confined space of microchannels, including bubble actuators driven by thermocapillary flow [11], optical tweezers [12] and magnetic microactuators [13]. Among the many different microactuation mechanisms, magnetic actuation has certain advantages over other methods for an on-chip microactuator due to the suitable source of the force [14,15]. Thus, considerable research has been carried out on magnetic actuators for cell manipulators. Previously [8,9,13,[16][17][18], we proposed a magnetically driven microtool (MMT) for on-chip cell manipulation. Magnetic actuation enables manipulation of cells in a closed microfluidic chip by forces on the order of millinewtons. Therefore, we applied a magnetically driven method to actuate the untethered microrobots because of its noncontact actuation capability and high output force for onchip enucleation.
A conceptual view of the on-chip enucleation system is shown in figure 2. The system contains two motorized stages, a microfluidic chip and a sensing part that is based on a CCD camera attached to the optical microscope. The microfluidic chip has two untethered microrobots, a microgripper and a microknife, which are driven by the magnetic force produced by the permanent magnets placed on the motorized stages. Oocytes are transported to the manipulation point in a microfluidic chip by the flow. The oocytes are then enucleated by the two microrobots. The enucleation process is subdivided into three steps: (a) holding, (b) slitting, and (c) removing the nucleus. The required DOF of the microrobot in each step is summarized as follows.
(a) Holding: the microgripper requires at least 2-DOF motion in a plane to approach the oocyte. An additional 1-DOF actuation is required to accomplish the gripping function. (b) Slitting: the microknife requires at least 2-DOF motion to approach the nucleus of the oocyte. The slitting function is achieved with relative 1-DOF motion between the microknife and microgripper, as shown in figure 3; although the slitting function is based on the penetration and cutting motion. (c) Removing the nucleus: the microrobot should be actuated with at least 1-DOF motion to push the nucleus with cyto-plasm out of the ZP. This 1-DOF motion can be achieved using the griping motion of the microgripper.
The concept and design of each microrobot is described in the following sections.

Microknife
As mentioned in section 2.1, the microknife requires 2-DOF motion to approach the nucleus of the oocyte. Furthermore, in the case of a microrobot having a knife probe, a slitting function is possible with relative 1-DOF motion. Concept images of the cutting sequence are shown in figures 3(a)-(c). Firstly, a microrobot approaches the nucleus of the oocyte ( figure 3(a)). The microrobot subsequently penetrates the ZP by using the sharp tip ( figure 3(b)). While the microrobot is penetrating the ZP, the knife-like structure slits open the ZP (figure 3(c)). To design the microknife, a model of slitting is considered, as shown in figure 4. The most important parameter of the knife is the relationship between the angle of the knife tip ϕ and the required manipulation stroke L. Here, we considered the case that L is maximized when the cytoplasm of the oocyte is located at the edge of the ZP. Moreover, we assumed that the deformation of the oocyte can be ignored to consider the case of maximized L. When ϕ′ is small < − ( ) L D l , 2 2 the end point of the slitting is on the cutting edge of the microknife. On the other hand, when ϕ′ is large the end point is on the tip of the microknife. Therefore, the following relationship is satisfied between ϕ and L. (1) where, D is the diameter of the oocyte, d is the diameter of the cytoplasm, t is the thickness of the ZP and δ is a parameter determined by equation (2).
The relationship is calculated as shown in figure 5.

Microgripper
The microgripper is actuated by external magnetic force to achieve noncontact actuation with high output force, as shown in figure 2. Specifically, four driving points of permanent magnets are vertically arranged in the microgripper for 2-DOF actuation. These magnets are driven by the external magnetic force of magnets on the motorized stage. To achieve the additional 1-DOF gripping motion, a horizontally arranged permanent magnet is placed at the center of the microgripper. This magnet is driven by the magnet field of the electromagnetic coil, which is placed on the motorized stage. Thus, the microgripper picks up target cells one by one and pushes the cells using a gripping motion.

Design of the gripping mechanism.
The gripping mechanism is performed by the deformation of beams because it is difficult to achieve an ideal microscale hingeand-link structure. Figure 6(a) shows the deformation model of the gripping mechanism when the driving force F is applied to the horizontally arranged permanent magnet. First, to obtain the gripper's tip deformation δ X and δ Y caused by applied force F, the gripping mechanism is considered as half of the frame structure. Point A is the base of the frame connected the gripper's body, point B is the tip of the gripper and point C is driven by the magnetic force, as shown in figure 6(b). F x , and M are the virtual load along the x axis for point A, and the moment around point A, respectively.
Here, the structure of the microgripper is considered an elastic material. The elastic strain energy U is expressed by equation (1) under the assumption that the deformation due to compression and the torsion of each beam is ignored.
Where U AB and U BC are the elastic strain energy of the beams, and expressed as follows: Where r 1 , r 2 l 1 and l 2 are divided segments along the beams, and the total length of the beams, respectively. M AB and M BC are the moments around point A and C, respectively. M AB and M BC are described as follows: Here the f is the vertical component of force F/2 as shown in figure 6(b).The displacement of the gripper tip is given as equation (7) under the boundary condition expressed by equation (8). Where, θ, E and I are the angle between the beams of the tip, Young's module of the frame and the second moment of the beam, respectively.
Therefore, δ X and δ Y are obtained by equation (9).   The analytical results are shown in figure 7. In this calculation, the value of l 1 and l 2 are designed as 6.5 mm and 3.3 mm, respectively, because the light of the microscope through from the bottom of the microrobot and the electrical magnet interrupts the light. Considering that the bovine oocyte is approximately 150 µm in diameter, we designed the distance between the tip of the gripping mechanism to be 160 µm. To close the tip of the gripper perfectly, δ X must be over 80 µm. Therefore, we designed the angle to be 13 °.

Fabrication of the microrobots and the microfluidic chip
To create the microfluidic chip containing the microrobots, the fabrication process is based on a simple two-step procedure. First, the microgripper, microknife and microchannel were fabricated identically. The components of the microfluidic chip were then assembled and packaged. Figure 8 shows the fabrication processes of the microgripper, microknife and microfluidic chip. (c) Permanent magnets were assembled on the hole patterns. Vertically arranged magnets were adopted as the driving part for 2-DOF lateral motion and a horizontally arranged magnet was adopted for the gripping motion.

2.4.2.
Fabrication of the microknife. (d) OFPR was patterned using grayscale lithography after making the hole pattern in the same manner as the microgripper. Grayscale lithography is a photolithography technique for fabricating three-dimensional (3D) microstructures [19].
(e) The 3D shape of the microknife was transcribed by DRIE because of the difference in the etching rate between the Si substrate and the photoresist.     (h) Permanent magnets were assembled on the hole patterns for 2-DOF lateral motion.

Evaluation of the microgripper
The fabricated microgripper was evaluated in terms of the manipulation stroke, which is the tip displacement of the microgripper. The manipulation stroke was measured as a function of the applied voltage of the electromagnet coil (RZ-48 W, B5, Fujitsu Ltd), as shown in figure 10. The vertical axis shows the tip displacement on one side. The line shows the average value of four measurements, and the upper  and lower error bar shows the maximum and minimum values, respectively. The displacement was measured by the image taken from the CCD camera attached to the microscope. The results indicate that the gripper has a hysteresis between the gripping phase and release phase and that there is a relatively linear relationship between the tip displacement and applied voltage when the applied voltage is between -20 V and 20 V. The causes of the hysteresis are the friction between the bottom of the microgripper and the surface of the glass plate of the microfluidic chip. In addition, the maximum and minimum errors are -3.5 µm and 4.3 µm, respectively. From these results, we concluded that the manipulation stroke is 160 µm during the gripping phase, which was sufficient for on-chip enucleation of the bovine oocyte.

On-chip enucleation of the oocyte
Photographs of typical results for the on-chip enucleation are shown in figure 11. Each process was carried out under bright field observation without the use of fluorescent reagents. The target oocyte was transported to the manipulation point in a microchannel. The microgripper then grasped the oocyte. The ZP of the oocyte was slit by the microknife tip and the cytoplasm was pushed out of the ZP. The enucleated oocytes are shown in figure 12(a). The enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm the success of enucleation using this method, we investigated the viability of oocytes after enucleation. The enucleated oocytes were cultured in 10% FBS with M199 with 6DMAP for 4 h, and then transplanted into a cloned nucleus and cultured for 5 d. Presently, the production rate, i.e. the ratio between the number of oocytes that reach the blastocyst stage and the number of bovine oocytes after nucleus transfer, is 100%.

Conclusion
We proposed novel on-chip enucleation of an oocyte with ZP by a combination of untethered microrobots. Enucleation is a cloning technique that can be subdivided into three steps: (a) holding, (b) slitting, and (c) removing the nucleus. To achieve these steps in the closed space of a microfluidic chip, two microrobots, a microknife and a microgripper were integrated into a microfluidic chip by considering the required DOF of motion in each step. These microrobots were actuated by an external magnetic force produced by permanent magnets placed on the robotic stage.
The tip of the microknife was designed by considering the biological geometric feature of an oocyte, i.e., the oocyte has a polar body in maturation stage II. Moreover, the microknife was fabricated using grayscale lithography, which allows the fabrication of 3D microstructures. The microgripper was designed to have a gripping function that is independent of the driving mechanism. The gripping function is performed by the deformation of beams because it is difficult to achieve an ideal microscale hinge-and-link structure. The fabricated microgripper was evaluated and the results show that the measured standard deviation of the positioning accuracy is less than 5 µm, and that there is a relatively linear relationship between the tip displacement and applied voltage when the applied voltage is between -20 V and +20 V.
On-chip enucleation was demonstrated under bright field observation without the use of fluorescent reagents. The enucleated oocytes are spherical, indicating that the cell membrane of the oocytes remained intact. To confirm successful enucleation using this method, we investigated the viability of oocytes after enucleation. Presently, the production rate is 100%.
The proposed untethered microrobots will contribute to complex cell manipulation, such as cell surgery, in the closed space of lab-on-a-chip devices because the microrobots are disposable and permit noncontact actuation. In the future, we will develop an automated enucleation system for highthroughput cloning.