Microfluidics-Enabled Robotic System for Embryo Vitrification With Real-Time Observation: Design, Method, and Evaluation

Embryo vitrification is an essential technique for in vitro fertilization (IVF), which avoids the formation of ice crystals during cryopreservation by dehydrating cells in cryoprotectant agents (CPAs) and ultrarapidly freezing them in liquid nitrogen ( $\rm LN_{2}$ ). In manual vitrification, the embryo will be repeatedly aspirated and released in different CPAs and finally transferred onto the vitrification straw under the microscope, which is time-consuming and requires intensive training of skilled operators. Robotic cell manipulation has been considered the solution to enhance the processing efficiency while reducing the failure rate for the manual vitrification protocols; however, the time-critical treatment with CPAs and transfer of the embryo to the straw still remain challenging in ongoing studies. In this article, we report a microfluidics-enabled robotic system for the emerging automatic embryo vitrification. Based on the strategy of microfluidic manipulation in our early works, an open microfluidic chip was designed to confine the embryo within a specific region while manipulating the surrounding flow field for solution exchange through the capillary effect. The embryo chamber was zoned into safe and danger zones based on the numerically simulated local velocity toward the capillary gap, which represents the risk of embryo loss and damage. A vision-based switch controller was adopted to manipulate the embryo trajectory during the solution exchange process, which selected the optimized flow rate according to the zoning of real-time embryo. The challenge of embryo transfer was addressed by a two-stage capillary valve, which switches open and allows the embryo to pass through under a certain pressure. The vitrification experiments with porcine embryos validate the reliable positioning of the embryo in each step, the satisfactory success rate of the robotic system, and the high quality of the robotic-vitrified embryos after thawing. Integrated with new manipulation strategies, our intelligent mechatronics system provides a promising solution for IVF featuring an embryologist-centered configuration and standardized robotic operation.