Development of sorting, aligning, and orienting motile sperm using microfluidic device operated by hydrostatic pressure
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- Seo, D., Agca, Y., Feng, Z.C. et al. Microfluid Nanofluid (2007) 3: 561. doi:10.1007/s10404-006-0142-3
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In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are the most commonly used assisted reproductive technologies to overcome male infertility problems. One of the obstacles of IVF and ICSI procedures is separating motile sperm from non-motile sperm to select the most competent sperm population from any given sperm sample. In addition, orientation and separation of the head from the tail is another obstacle for ICSI. Using the self-movement of sperm against flow direction, motile and non-motile sperm can be separated with an inexpensive polymeric microfluidic system. In this paper, we describe the development of a microfluidic system obtained through low-cost fabrication processes. We report experimental results of sperm sorting using hydrostatic pressure of three different species: bull, mouse, and human. The movement of cells in these channels was observed under a microscope and recorded with a digital camera. It is shown that the hydrostatic pressure and self-movement of motile sperm can be used to solve separating, aligning and orienting sperm in the microchannel.
KeywordsMicrofluidicsMotile-sperm sortingIntracytoplasmic sperm injection
The microfabrication technologies of the integrated circuit and semiconductor industry that started in the 1960s have made it possible to integrate complex electronic and mechanical functions, providing us with even smaller and less expensive sensors and devices. With the development of micromachining technologies for fluidic systems in recent years (DeLos Santos 1999; Nadim 1999; Duffy et al. 1998; McDonald et al. 2000; Collins 2003), microfluidics have been widely applied to biomedical fields because very small amounts of fluid transport is useful for DNA analysis systems (Burns et al. 1998), chemical analysis systems (Harrison et al. 1993; Manz et al. 1994; Becker and Manz, 1997; Buettgenbach and Robohm 1998; Darlin et al. 2002; Sudarsan et al. 2005), assisted reproductive technologies (Beebe et al. 2002; Suh et al. 2006), and drug delivery systems (Chan et al. 1999). The microfluidic pumps and valves in particular have witnessed extensive research activities (Xia et al. 2006; Khoo and Liu 2000). Furthermore, research efforts are also focused on integrating many functions on one chip, the so-called labs-on-chip, to achieve two main goals: (1) to simplify the working mechanism and (2) to reduce labor and cost.
One of the biological applications of microfluidic devices is cell sorting. Bull, mouse, and human spermatozoa are widely used in biomedical research, agriculture application, and human reproductive medicine related procedures. Separating motile sperm from non-motile sperm is critical for successful intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). Cell sorting microfluidic devices have been previously reported (Krüger et al. 2002; Huh et al. 2002). Sperm sorting systems using microfluidic devices have also been recently reported (Schuster et al. 2003; Horsman et al. 2005). Among the sperm sorting microfluidic systems, Cho et al. (2003) introduced a motile human sperm sorting device. This system uses the self-movement of motile sperm to escape from the initial inlet streamline, which is generated by passive pumping systems using hydrostatic pressure, and then collects motile sperm in one of two outlet reservoirs. Even though their system works well, there are a few limitations to its usage. The pressure has to be stable during sorting and flow velocity has to be large enough to prevent motile sperm swimming against flow direction. In addition, they reported sorting results for human sperm only.
In view of the limitations of the sorting devices reported in the literature, we developed a novel sperm sorting device. Additionally, controlling the orientation of the sperm cell while being sorted is important to integrate the sperm dissection, which is another important step during ICSI. This device is based on the self-movement of the sperm in a flow. The sperm self-movement achieves sorting and orientation control at the same time. Equally significant, the fabrication procedure is based on the “soft-lithography” method proposed by Duffy et al. (1998) using an elastometric material such as poly-dimenthylsioxane (PDMS). We carried out the procedures without expensive facilities such as clean room (Seo 2002; Collins 2003). A HCl bonding method also helps to simplify the filling of the fluidic channels.
In this paper, we report the development of a novel microfluidic device. This microfluidic device design has been developed with the long-term goals of : (a) being able to detect sperm DNA with sufficient resolution to enable sorting of X and Y chromosome-bearing sperm, and (b) being able to add a “cutting laser” component to separate sperm heads and tails for ICSI procedures. Based on these goals, the a multi purpose device may be designed to allow high resolution sorting system at the expense of the throughout. We provide the mechanical principles governing the design of the device. Sperm motion in the device is observed and recorded using an inverted microscope (ECLIPSE TE200, Nikon) and a digital camera (COOLPIX 5000, Nikon). We also report the experimental results on the variability among different species’s sperm: bull, mouse, and human.
2 Motile sperm sorting microfluidic system
3 Analysis and fabrication of the channel
3.1 Analysis of the fluid flow in the microchannel
3.2 Channel design
The design of the MSMS is obtained based on the following two considerations: (1) to allow easy control of flow direction and velocity using hydrostatic pressure, and (2) to allow enough space for the sperm to swim against the flow direction without “clogging” the channel. Geometric parameters such as height, width, and length all affect the flow velocity and direction in channels. Among these parameters, the channel widths (2an) and lengths (ln) are defined by the patterns on the optical mask. The channel height is defined by the thickness of the SU-8 coating on the wafer which is used as the mold for the PDMS channel. Large channel width is desired to maximize the throughput of the sorting device. However, since the microchannels are made of polymeric material, the channels could close-up under external pressure if they are too wide.
3.3 Microchannel fabrication procedures
The complete process for fabricating the microchannel consists of five steps: (1) coating, (2) exposing, (3) etching, (4) molding, and (5) bonding. These five steps are detailed in recent reports 3–6 and our fabrication processes are similar except the bonding. We created the channels inexpensively without using a cleanroom facility. The following instruments and supplies were used to complete fabrication. A spin-coater (Model P6700, A Specialty Coating System. INC) was used to coat the silicon wafer with a photoresist (SU-8). After spin coating, an UV exposure unit (KVB – 30.KINSTEN, 55 mW/cm2) was used to expose the coated wafer. A heater with temperature control (Model PC-220, Corning) was used to pre-bake and post-bake. We used a 6-inch diameter silicon wafer, which has a polished side and an oxide side as the substrate of the mold. The coated wafer is developed lithographically to form the mold. The mask for lithography was designed on AUTOCAD 2002. This design was then printed on a transparency by a commercial printing shop (University of Illinois at Urbana-Champaign, Urbana, IL, USA).
Without using a cleanroom facility, we have taken special steps in order to create defect-free molds. It is better to use the polished side of the wafer to easily bond PDMS (SYLGARD® 184 Silcone Elastomer Base, DOW CORNING) and glass plate. Furthermore, to avoid removing SU-8 mold on the wafer during etching process, the exposed SU-8 coated wafer is etched by applying SU-8 developer drops on the surface of the coated wafer instead of immersing it. Applying drops can also reduce the amount of SU-8 developer used in the fabrication procedure.
4 Flow visualization in the channel
The MSMS was designed to maintain the desired flow direction for at least 1 h. However, in our most recent experiments, we collected sorting data after 20 min of inserting sample.
To study the variability among different species, we applied the MSMS to the sorting of bull, mouse, and human sperm. Each experiment was performed using sperm from only one species. In spite of respective differences in their own characteristics, the experimental results show that they all have similar characteristics in terms of aligning their heads opposite to the flow direction as well as swimming against the flow (Fig. 12).
Sorting rate of bull sperm using the MSMS
Approx. inlet sperm no.
Approx. inlet motile rate
Approx. outlet motile rate
Average sorting rate (ea/min)
2.68 × 105
4.20 × 105
4.90 × 105
6 Conclusions and discussions
A motile sperm sorting microfluidic system was developed using an inexpensive fabrication method. Bull, mouse, and human motile sperm can be sorted by controlling hydrostatic pressure in the microchannel. Non-motile sperm and debris can be separated and collected. Since the sorting takes place by sperm swimming against the flow in channel B, a single channel may appear to be sufficient. However, our multi-channel design allows a much lager pressure difference which may be easier to use in the clinical laboratory. For a single channel with a size similar to channel B, the pressure difference at the two ends would have to be controlled within less than 0.1 mm to avoid generating flows in the channel that would overwhelm the cell swimming ability. Either reducing the channel cross section or lengthening the channel will diminish the sorting efficiency of the channel.
Even though the current design has a relatively low throughput, it provides proof of concept that this approach works well. In addition, the current system has the advantage of orienting and aligning the sperm. The modification of the MSMS design to increase the throughput is straightforward by adding multiple channels. Moreover, the system can be applied to other integrated microfluidic devices, for example, a micro Coulter-counter or other cell sensing devices.
This work was funded by a grant from the NIH (RR1482) to JKC.