A microfluidic lab chip for the manipulation and co-culturing of embryos with stromal cells

Highlights

• The embryo lab chip takes advantage of passive trapping for embryo manipulation and dynamic perfusion for co-cultivation.

• The embryos were co-cultured for sake of providing the biomimetic microenvironment for in-vitro embryo development.

• The mid-blastocyst embryos out of our co-culture chip were transplanted back into the uterus of the female mouse.

• 18.5 days after transplantation, embryos were verified to be successfully implanted in the uterus of the female mouse.

Abstract

The combination of In Vitro Fertilization (IVF) and microfluidic technology might provide a solution to infertility via increasing the success rate of IVF. Our microfluidic embryo lab chip takes advantage of passive trapping for embryo manipulation and dynamic perfusion for co-cultivation. The embryos were gently captured one by one by the passive trapping system to the groove. The medium in the liquid-pushing channels pushed the captured embryos to the G-shape co-culture chambers. The embryo manipulation was designed to push the 2-cell embryo and the mature embryo forwards and backward in/out of the co-culture chamber on desire. The perfused channels provided the thrust for the embryo manipulation and the dynamic perfusion through the holes on the middle-layer porous PDMS membrane. The embryos were co-cultured with stromal cells to provide the biomimetic microenvironment for embryo growth/development. We observed at least a several-hours faster growth/development rate of embryos for on-chip culture than the traditional droplet culture method. The blastocyst development rate of our on-chip embryo co-culture group increased by 16.1% than that of the off-chip co-culture group on E3.5. The mid-blastocyst embryos in our on-chip co-culture group were transplanted back into the uterus of the female mouse for confirmation of our chip development.

Introduction

The increase of infertility patients motivates the IVF research. Increasing the ratio of blastocyst development rate has been a significant challenge. In addition to the traditional droplet culture method, many methods to stimulate embryo growth have been studied. Tilting culture medium [1], [2] and mechanical agitation [3] have been reported to help embryo growth. Some studies have pointed out that the shear stress in the fluid field caused damage to the embryo [4], [5]. Therefore, the control of flow rate is essential for embryo culture. The low substrate stiffness of the culture environment has also been studied to be suitable for preimplantation embryos [6]. On the other hand, chemicals are more effective in stimulating embryo growth. The co-cultivation of embryos with other cells has also been heatedly discussed because other cells secret hormones and growth factors [7], [8], [9], [10], [11]. Culture medium was modified for culturing in the derived embryos [12]. Based on these factors, the combination of microfluidics technology has brought a leap in the development of IVF.

The static culture platform combined with microfluidic technology is relatively easy to develop, suitable for embryo culture. Melin et al. used microfluidic chips to accurately quantify the sub-microliter volume for embryo culture [13]. Microwells were fabricated in the microfluidic chip for trapping the embryos and giving embryos in a fresh culture medium [14], [15], [16], [17], [18], [19]. The structure in the microchannel was designed to be smaller than the embryo so that the embryo could be trapped and cultivated between the microchannel [20], [21]. Reducing the volume of the culture medium and stabilizing the culture environment are indeed the static culture platform that provides potential benefits for culturing the embryos. However, the dynamic culture platform exchanges the waste generated by the embryos and the micro-stimulation required for embryo growth.

Electrowetting on a dielectric (EWOD) was demonstrated to move a single droplet for culturing an embryo in vitro. The static droplet culture method allowed embryos to be-come dynamically cultured due to the movement of the droplets [22], [23]. The fresh culture medium was diffused from the below-microchannel and passed through the holes of the porous membrane. The dynamic culture medium was provided nutrients to the embryo co-cultured with the other cells and tiny stimulation to the embryo growth [24], [25]. Open microfluidics was designed to analyze heterogeneity within a single embryo, such as protein and mRNA [26]. The embryo was pushed through the contracted microfluidic channel, and the embryo received back pressure from the channel wall. The back pressure of the microchannel wall was stimulated to develop the embryo [27], [28], [29], [30]. The embryo in the zygote stage with cumulus was removed by the suction of the microchannel [31]. In addition, the dynamic perfusion of the micro-fluidic chip could be precisely controlled, which did not damage mature embryos [32], [33], [34], [35]. IVF was conducted successfully within microfluidic channels by lower total numbers and concentrations of sperm were required [36], [37].

Most microfluidic systems were designed to focus on the post-fertilization stage, such as embryo culture. However, about 40% of the causes of infertility come from a male. In-vitro fertilization, sperm selection is also a major challenge, which was determined by biology. Traditional sperm selection methods were used, such as swimming and densitometer. Human operation and proficiency greatly affected the damage of the sample and the selection of sperm. In response to these challenges, microfluidic technology had developed related research. Researches and techniques, such as semen analysis for male infertility diagnosis [38], [39] and sperm selection by motility [40], [41] and facilitated by chemotaxis, [42] rheotaxis, [43] thermotaxis, [44] and inertial focus [45], have been developed.

In this study, we developed a microfluidic embryo lab chip, mainly containing a passive trapping system and a G-shape co-culture chamber. The embryo lab chip takes advantage of passive trapping for embryo manipulation and dynamic perfusion for co-cultivation. The 2-cell embryos were gently captured by the passive trapping system to the groove. The embryo manipulation via hydrodynamics allowed embryo co-cultivation in the biomimicking microenvironment with dynamic perfusion to provide the rolling mechanism to enhance embryo growth and development.