80 EmbryoCHIP: a new microfluidic device for in vitro pre-implantation embryo development

Conventional in vitro embryo production (IVEP) system, while successful in mimicking the biochemical oviducal environment, lacks the physiological mechanical stimulation occurring during embryo transport. In contrast, dynamic microfluidic devices replicate the natural oviducal environment by enabling continuous media flow, which delivers a consistent supply of vital factors to the embryos, optimizing conditions for embryo viability. Nevertheless, available microfluidic devices predominantly focus on generating flow forces or small-volume static cultures, neglecting to mimic the movement experienced by embryos. Recognising this limitation, we designed the EmbryoCHIP, a microfluidic device that mimics the intricate movements that take place in the oviduct. To that end, a tubular-tortuous structure was designed composed of two sections representing the ampulla (1.2 × 1.4 × 150 mm) and isthmus (1.2 × 1.0 × 520 mm) segments. The EmbryoCHIP was fabricated using stereolithography to print a three-dimensional, two-piece mould, which was cast with polydimethylsiloxane and sealed in a two-stage procedure. For IVEP, cumulus–oocyte complexes were obtained from bovine ovaries and, after standard in vitro maturation and IVF, the resulting presumptive zygotes (PZ) were randomly divided into three groups: control (four-well plate, CT), static EmbryoCHIP (staEC), and perfusion EmbryoCHIP (perEC, 15 µL/h). On Day 4 of the culture, the cleavage rate was assessed and embryo quality was evaluated through apoptosis (Caspase 3) and oxidative stress (CellROX) assays in embryos (≥8-cell). Experiments were conducted in three replicates (a total of 60 PZ/group). Images were analysed using ImageJ (version 1.53t) and data analysis was conducted using a generalized linear mixed model, and a Tukey post hoc test in R. To assure no high shear stress (SS) was induced in the embryos, which could induce apoptosis, a Comsol Multiphysics simulation (version 6.1) was performed. As result, the maximum SS was 4.8e-7 dyne/cm² (at 15 µL/h). Differently from what was expected, the PZ did not move through the device at 15 µL/h, being found at the beginning of the tubular segment on Day 4. No difference in cleavage rate was observed between CT, staEC, and perEC groups, 81 ± 8%, 81 ± 5%, and 67 ± 21%, respectively (P > 0.05). Nevertheless, lower oxidative stress levels were identified in embryos from staEC (22.7 ± 15.3 arbitrary units [AU]) than the CT (44.9 ± 11.3 AU, P = 0.008) and similar to perEC (31.9 ± 16.2 AU, P > 0.05), while higher apoptosis rate was identified in perEC (8.3 ± 11.8%) than the CT (1.9 ± 1.6%, P = 0.04) and similar to staEC (1.4 ± 2.5%, P > 0.05). The future perspective is to evaluate different flow rates or coating the device surface to support embryo movement. Therefore, by integrating these essential components, the EmbryoCHIP has the potential to enhance the quality and yield of IVEP, bridging the gap between the artificial and natural reproductive processes. This groundbreaking approach might allow embryos to experience mechanical cues and to go beyond mere fluidic flow by incorporating mechanical stimulation.

The research was supported by FAPESP (19/25982–7, 20/02500–4, 22/12169–9).