Development of an Open Microfluidic Platform for Oocyte One-Stop Vitrification with Cryotop Method

Abstract

Oocyte vitrification technology is widely used for assisted reproduction and fertility preservation, which requires precise washing sequences and timings of cryoprotectant agents (CPAs) treatment to relieve the osmotic shock to cells. The gold standard Cryotop method is extensively used in oocyte vitrification and is currently the most commonly used method in reproductive centers. However, the Cryotop method requires precise and complex manual manipulation by an embryologist, whose proficiency directly determines the effect of vitrification. Therefore, in this study, an automatic microfluidic system consisting of a novel open microfluidic chip and a set of automatic devices was established as a standardized operating protocol to facilitate the conventional manual Cryotop method and minimize the osmotic shock applied to the oocyte. The proposed open microfluidic system could smoothly change the CPA concentration around the oocyte during vitrification pretreatment, and transferred the treated oocyte to the Cryotop with a tiny droplet. The system better conformed to the operating habits of embryologists, whereas the integration of commercialized Cryotop facilitates the subsequent freezing and thawing processes. With standardized operating procedures, our system provides consistent treatment effects for each operation, leading to comparable survival rate, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) level of oocytes to the manual Cryotop operations. The vitrification platform is the first reported microfluidic system integrating the function of cells transfer from the processing chip, which avoids the risk of cell loss or damage in a manual operation and ensures the sufficient cooling rate during liquid nitrogen (LN2) freezing. Our study demonstrates significant potential of the automatic microfluidic approach to serve as a facile and universal solution for the vitrification of various precious cells.

Keywords: cell manipulation; open microfluidic chip; vitrification.

Figure 1

Schematic showing manual and microfluidic vitrification approaches. Vitrification involves multiple steps of cell pick-and-place before freezing in liquid nitrogen.

Figure 2

The structure diagram of the vitrification system and working principle of system. (a) The vitrification system composed of the open microfluidic chip and the companion system (syringe pumps, operation platform, transfer system and the imaging system). The operator manual placed the oocyte into the chip, completed the vitrification pretreatment and transferred with the assistance of the companion system. The real-time internal conditions of the chip on the whole operation process were presented to the operator by visual feedback. (b) Above all, Cryotop and the open microfluidic chip containing the oocyte were fixed on the operation platform. The syringes used for loading (ES, VS and air) and unloading were, respectively, connected to the chambers on both sides of the chip and PDMS sealing ring with soft silicone tubes. (c) Schematic diagram of the transfer process. After vitrification pretreatment, the operator pressed the transfer handle, and the PDMS sealing ring was pressed on the upper surface of the open microfluidic chip to form a hermetic space. The oocyte was transferred to Cryotop by external pressure.

Figure 3

The dimension detail, structure diagram and physical diagram of the open microfluidic chip, composed of capillary gap, oocyte chamber, solution exchange chamber and capillary valve. Scale bars are 2 mm.

Figure 4

The operation steps of vitrification on the open microfluidic chip: preparation, loading and unloading ES/VS and transfer.

Figure 5

Different situations of liquid flow in capillary valve. (a) The internal structure diagram of the capillary valve; the inner radius (r1: 100 $\mathsf{\mu }$m, r2: 150 $\mathsf{\mu }$m) had two sudden expansions and the channel expansion angle $\beta$ is 90${}^{\circ }$. (b) The liquid reached the capillary valve, without external pressure, and the meniscus stopped at the edge of the microchannel outlet. (c) The external pressure gradually increased, and the convexity of the meniscus and contact angle became larger. (d) When the external pressure exceeded the critical burst pressure, the capillary valve was opened and droplet was transferred to Cryotop.

Figure 6

Theoretical and experimental VS concentration around the oocyte with respect to time. (a) Simulated time-dependent sucrose concentration in the oocyte chamber under diffusion condition (initial concentration upper: 0.4 mol/L; bottom: 0 mol/L). (b) Gray: the numerically predicted sucrose concentration in the oocyte chamber over time; Red: the experimentally measured intensity of the fluorescent tracer in the oocyte chamber.

Figure 7

Numerical simulation of critical burst pressure of capillary valve. (a) The capillary valve modeling. (b) Deformation of the lower meniscus under different external pressures (liquid: pink; air: gray).

Figure 8

The microparticle was tracked by the imaging system over the entire process of system vitrification. (a) The operator placed the microparticle in the chip. (b–g) The microparticle completed vitrification pretreatment and was transferred to Cryotop. (h) The droplet on Cryotop contained microparticle. Scale bars in (a,f,g,h) are 400 $\mathsf{\mu }$m.

Figure 9

Evaluation of oocytes thawed from microfluidics vitrification via comparing with traditional manual vitrification method. (a,c) GV oocytes and (b,d) MII oocytes (with polar body extruded) were thawed from manual vitrification and microfluidics vitrification. Scale bar is 100 $\mathsf{\mu }$m.

Figure 10

JC-1 staining and ROS level measurement of the oocytes. (a) The MMP level of thawed MII oocytes was measured by JC-1 staining. (b) The red/green fluorescence ratio of oocytes was quantified between manual and microfluidic groups. (c) The ROS level of thawed GV oocytes between manual and microfluidic group. (d) The ROS levels were measured and quantified in two groups. The students’ t-test was used to compare the difference in two groups. n.s. means no significant difference between two compared groups. Scale bar is 50 $\mathsf{\mu }$m.