Using a Dielectrophoretic Microfluidic Biochip Enhanced Fertilization of Mouse Embryo in Vitro

Abstract

Droplet microfluidics has appealed to many interests for its capability to epitomize cells in a microscale environment and it is also a forceful technique for high-throughput single-cell epitomization. A dielectrophoretic microfluidic system imitates the oviduct of mammals with a microchannel to achieve fertilization in vitro (IVF) of an imprinting control-region (ICR) mouse. We applied a microfluidic chip and a positive dielectrophoretic (p-DEP) force to capture and to screen the sperm for the purpose of manipulating the oocyte. The p-DEP responses of the oocyte and sperm were exhibited under applied bias conditions (waveform AC 10 V_pp, 1 MHz) for trapping 1 min. The insemination concentration of sperm nearby the oocyte was increased to enhance the probability of natural fertilization through the p-DEP force trapping. A simulation tool (CFDRC-ACE+) was used to simulate and to analyze the distribution of the electric field. The DEP microfluidic devices were fabricated using poly (dimethylsiloxane) (PDMS) and ITO (indium tin oxide)-glass with electrodes. We discuss the requirement of sperm in a DEP microfluidic chip at varied concentrations to enhance the future rate of fertilization in vitro for an oligozoospermia patient. The result indicates that the rate of fertility in our device is 17.2 ± 7.5% (n = 30) at about 3000 sperms, compatible with traditional droplet-based IVF, which is 14.2 ± 7.5% (n = 28).

Keywords: dielectrophoresis; fertilization in vitro; oocyte; single-cell; sperm.

Figure 1

Fabrication of the designed DEP microfluidic biochip; (A) actual device of size 5 cm × 3 cm. Bonding of PDMS (microchannel and micro-structures) with the ITO electrode chip. (B) The concept of the experiment is that the mouse oocytes and sperm become trapped on the electrodes for about 1 min with the p-DEP. (C) Co-incubated 1 h in the micro-structures for natural insemination.

Figure 2

(A) Three pairs of parallel linear ITO-glass electrodes. (B) Actual device (5 cm × 3 cm). Each gap between the electrodes has width 100 μm; each electrode has width 150 μm.

Figure 3

(A) Rates of fertilization in vitro under KSOM-AA (50.2 ± 6.6%; n = 96), HTF (45.0 ± 4.8%; n = 94), and DEP buffer solution (45.1 ± 7.9%; n = 37). There is no significantly statistical difference among conditions (p > 0.1). (B) Blastocyst rates under KSOM-AA (37.4 ± 8.0%; n = 34), HTF (28.6 ± 12.2%; n = 26), and DEP buffer solution (30.9 ± 13.2%; n = 30), respectively; there is no significantly statistical difference (p > 0.1). (C) Tracking of embryo development of these three groups with an optical microscope at six periods (E1.5, E2.0, E2.5, E3.0, E3.5, E4.0, and E4.5 days); magnification 40×. (All sizes of embryo pictures are about 100 μm 100 μm).

Figure 4

Illustration of the experimental process in the DEP microfluidic chip. (A) The mouse oocytes and sperm were loaded into a microchannel with volume flow rate 0.5–1.0 μL·min⁻¹. (B) At the applied AC voltage 10 V_pp at 1 MHz, the oocytes and sperm were trapped with the positive dielectrophoretic force between the two electrode pads. The local sperm concentration increased to 100 × 10⁶ sperm·mL⁻¹, regardless of the original sperm concentration. The white arrow shows the direction of the flow. (C) On release of the p-DEP force after trapping for 1 min, the oocytes and sperm were trapped with the microstructure behind the microchannel for natural insemination for 1 h. (D) A second polar body and two pronuclei were observed after 4–10 h.

Figure 5

(A) Rate of fertilization in vitro of a DFP microfluidic chip compared with traditional IVF at sperm concentrations of 3000–240,000 sperm. (B) Tracking of embryo development observed with an optical microscope; magnification 40×. The blastocyst rate was 33.6 ± 15.0% (n = 26) of the DEP microfluidic IVF chip, compared with 37.4 ± 8.0% (n = 34) of the traditional droplet-based IVF.