IEEE Robotics & Automation Magazine - June 2015 - 38

achieved reliably. After removing the excess medium from
around the embryo, the straw is plunged into liquid nitrogen for
freezing. The vitrification straw is then sealed with a plastic cap
and put in a liquid nitrogen tank for preservation.
Results and Discussion
In the experiments, mouse embryos were gathered from the
Canadian Mouse Mutant Repository in the Toronto Centre
for Phenogenomics (Toronto, Ontario). Embryos were produced by superovulating a female and collected ~1.5-2.5 days
after conception, which corresponds to the embryos being in
the with KSOM medium (EMD Millipore, Billerica, United
States) in a 35-mm Petri dish and covered with mineral oil to
prevent evaporation.
The VS typically contains antifreezing agents or cryoprotectants such as DMSO, small molecular-sized glycols (e.g., ethylene glycol), or sucrose. In our experiments, the VS was made by
diluting DMSO in KSOM medium at 20% concentration. The
ES was at half the concentration of the VS (i.e., 10% DMSO). A
multiwell plate (Repro Plate, Kitazato Corporation) was loaded
with the ES and the VS for embryo washing. A standard vitrification straw (Cryotop, Kitazato Corporation) was used as the
physical carrier to freeze embryos in liquid nitrogen. All vitrification experiments followed the Kitazato protocol by washing
embryos in the ES and the VS for 12 min and 90 s, respectively.
The robotic system can be readily reprogrammed to implement
other vitrification protocols.
System Performance
The system throughput was evaluated by processing the mouse
embryos at the two-, four-, and eight-cell stages. The capability
of automated pick and place of single embryos enabled the robotic system to perform vitrification of multiple embryos in an
Micropipette

Vitrification Straw

Embryo
200 nm

Initial Location
(a)

(b)
Vitrification Straw

Micropipette

Fd
(c)

Figure 5. A processed embryo placed on the vitrification straw
tip. (a) An embryo is deposited onto the straw by dispensing
the VS medium out of the micropipette. (b) The excess medium
removed by micropipette aspiration under a threshold flow rate
to keep the embryo in place. (c) The schematic showing the
embryo dynamics during micropipette aspiration.

IEEE ROBOTICS & AUTOMATION MAGAZINE

June 2015

optimally scheduled sequence. Since embryo equilibration in
the ES costs minutes, after the first embryo was dispensed into
the ES for equilibration, the robotic system moved back to the
culture dish to pick up the next embryo and place it into the ES
of another bath. Repeating this step to process six embryos in
the ES, the system then retrieved the equilibrated embryos
from the ES and washed them in the VS one by one, again following a prescheduled sequence. As a result, the system was
able to process six embryos within 24 min. In comparison, in
manual implementation of the same vitrification protocol, it
was only possible to process two embryos in the same time period, and the operator was fully occupied in the process.
The success rate was also quantitatively evaluated. An experiment was defined to be successful when the system successfully
processed the embryo within the given period of time for each
step of a vitrification protocol. Manual vitrification experiments
were also performed by three operators. The experimental
results showed that the robotically vitrified group had a significantly higher success rate than the manual group (90% versus
83.3%, shown in Table 1). In manual vitrification, embryos
could easily escape from operators' monitoring when they
floated upward in the VS, which was the major cause of failure.
Embryo loss was effectively avoided by the system's capability of
3-D embryo tracking in RoboVitri. However, failure in RoboVitri arose when an embryo floated in the VS solution and happened to drift into blind regions of the multiwell plate. The
Repro Plate used in the experiments has inclined sidewalls that
produce a dark blind region. Multiwell plates with vertical sidewalls can help reduce blind regions and, hence, system failure.
Embryo Volume Measurement
In RoboVitri, the system is able to track embryos in 3-D space
and monitor their volume change in real time for analyzing each
individual embryo's response to the VSs. Figure 6 shows the
tracked embryo position and the volume change of three different embryos in the VS, measured by the robotic system. When
washed in the VS, the embryos first experience a dehydration
stage in which water molecules are drawn out of the cell, causing
the embryo to shrink. When the embryo reaches its minimum
volume, the toxic cryoprotectant solutes (e.g., DMSO) start to
penetrate the cell membrane. The embryo ideally should be
transferred out of the VS at its minimum volume.
The experimental results summarized in Figure 6(b) demonstrate that the embryos reach their minimum volume in
the VS at different time points. This suggests heterogeneity in
embryo dehydration timing even in the same VS. All existing
vitrification protocols stipulate a fixed washing time for all
embryos because measuring the individual embryo's volume
Table 1. Embryo vitrification experimental results.
Method Success Rate

Survival Rate

Development Rate

Control N/A

100% (15/15)

93.3% (14/15)

Manual 83.3% (15/18) 73.3% (11/15) 90.9% (10/11)
Robotic 90% (18/20)

88.9% (16/18) 93.8% (15/16)

Table of Contents for the Digital Edition of IEEE Robotics & Automation Magazine - June 2015