Figure 1.
Manual and digital microfluidic vitrification workflow.
Schematic showing differences between manual vitrification approach, which requires manual pipetting between cryoprotectant mediums, and the digital microfluidic (DMF) approach, which moves the embryo between mediums on chip. The chip automates the high skill portion of the procedure providing labor cost savings and opportunities for parallel processing.
Figure 2.
Key device design elements and fabrication layers.
(A) Device structure and fabrication. Electrodes (1 mm×1 mm) were separated by a 20 µm gap. (B) Regions for vitrification medium dispensing and for embryo loading/extraction. The top ITO slide is placed on the device in a manner that exposes portions of the top electrodes in the dispensing reservoir and exposes portions of the leg of the T-shaped electrodes, for medium and embryo loading, respectively. (C) Embryo is input and extracted by actuating electrodes at edge of top glass slide.
Figure 3.
Droplet mixing protocol and resulting concentration profile.
(A) 1: Embryo (red circle) contained in culture medium (CM) droplet. 2: Embryo droplet mixed with VS droplet. 3: Droplet split into two droplets (left contains embryo). 4: Droplet containing embryo is kept and other droplet is sent to waste. Process is repeated to increase VS concentration. (B) Mixing profile showing the generation of ES medium and VS medium. Droplet volumes were calculated by imaging droplet boundaries and modeling as cylinders. Concentrations were then calculated using these volumes before and after each mixing step.
Figure 4.
Healthy and unhealthy embryo morphology before and after freezing, and after 24 hours culturing.
(A) Healthy and (B) unhealthy examples showing morphology changes before and after freezing. Survival rate was determined on the basis of embryo morphology after thawing vitrified embryos. Cell leakage, abnormal shapes, and membrane damage, as commonly used in vitrification studies, were counted as failure cases. Development rate was quantified by the embryo stage reached after culturing. For instance, the 8-cell stage embryo shown in (A) after vitrification, thawing, and culturing successfully developed to the blastocyst stage.
Figure 5.
Embryo cell volume monitoring.
Mouse embryo cell volume change measured on chip. (A) Vitrified using a two-step human embryo vitrification protocol (Irvine in Figure 6 with the addition of sucrose in the ES stage). (B) Vitrified using a one-step mouse embryo vitrification protocol (CMMR in Figure 6). Snapshots from recorded videos, at instances when the droplet was not moving, were used to measure volume. Volumes were calculated by modeling cells as spheres and were normalized to initial volume (this could result in errors as cells may collapse or flatten instead of shrinking symmetrically [32]. The initial volume dip in the human protocol matches the volume dip over the mouse protocol. For this experiment of volume measurement, 2-cell embryos were used to simplify image analysis. Error bars in (B) are relatively large because for this vitrification protocol, droplets are required to move quickly, which did not leave sufficient time to switch to higher microscope magnification for imaging on our digital microfluidic platform.
Table 1.
Summary of vitrification results.
Figure 6.
Comparisons of mouse and human vitrification protocols [28], [29], [42]–[47].
For those protocols that specify a timing range, the average value is used.
Figure 7.
Vitrification protocol implementation on digital microfluidic chip.
Implementation of common vitrification protocols on a digital microfluidic chip with a single dispensing reservoir. Timings and concentrations are shown in (A), and the generalized mixing curve is shown in (B).