In the first attempt, we used a hollow fiber tube (HFT) 16 and an OptiCellTM device. Mouse 2-cell embryos were inserted into the HFT, which was then inserted into a pipette tip attached to a 1-ml syringe and then immersed in LN2 (Fig. 1A–D). The frozen embryos in the HFT were inserted into the OptiCellTM immediately after thawing (Fig. 1E, F) and then washed and cultured through exchange with the rewarming solution and culture medium respectively. Those solutions were delivered using a 10-ml syringe (Fig. 1G) and then placed in an incubator (37°C, 5% CO2) for 4 days. The HFT in the OptiCellTM achieved 88% survival of thawed embryos.
Blastocytes eveloped from 81% of the 2-cell embryos after 4 days in culture, which closer to non-frozen control embryos (92%) (Table 1). Generally, thawing frozen embryos is strictly time-limited and requires intensive practice. However, this method could be performed without any practice, therefore it will be use in animal research facilities and infertility clinics. However, if the HFT was located on the inside edge of the OptiCellTM, the embryos were difficult observe. Although hard shaking moved the HFT from edge to the center of the OptiCellTM, this was difficult. Furthermore, the culture area of the OptiCellTM (100 cm2) was too large, making it difficult to find the HFT inside the OptiCellTM using a microscope. The astronauts performing this experiment on the ISS must be able to easily to find the HFT inside the device, which requires limiting the culture area.
We next used a Mini-plate developed by the Japan Aerospace Exploration Agency (JAXA) instead of the OptiCellTM. The structure of the Mini-plate is similar to that of the OptiCellTM, but much smaller (Fig. 1H, I). When fresh embryos in the HFT were inserted into the Mini-plate as a control and cultured for 4 days in the CO2 incubator, 98% of the embryos developed into blastocysts, demonstrating that the Mini-plate was not cytotoxic (Table 1). However, the HFT more often resided on the edge of this device and was difficult to move to the center, even after vigorous shaking. Furthermore, the port of Mini-plate is too tight, requiring the use of a metal needle instead of pipette tip to insert the HTF (Fig. 1J). However, when the HTF was frozen inside the metal needle, the HFT cracked after thawing, and all embryos were lost during washing. To avoid cracking the HFT in the needle, the HFT was inserted into the Mini-plate before immersion in the vitrification solution. However, we were unable to perform vitrification within the 2-min limit, and none of the thawed embryos survived.
Cell Experimental Unit (CEU)
We next used a Cell Experimental Unit (CEU) developed by JAXA in place of the Mini-plate (Fig. 1K). The CEU can be assembled so that the HFT harboring embryos can be fixed to its center. When non-frozen embryos in the HFT were cultured in the CEU, 95% developed into blastocysts after 4 days of culture in the CO2 incubator (Table 1), indicating that the CEU was not toxic to the embryos. However, because of the complex structure of the CEU (Fig. 1L), the HFT containing frozen embryos melted before CEU assembly was completed, even on dry ice. We attempted to place the HFT containing embryos into the vitrification solution onto the plate of CEU before freezing and then assembly, after which the CEU was immersed in LN2. Unfortunately, we were unable to complete the process within the time limit, despite practice. We therefore abandoned the use of the CEU.
New Cell Experimental Unit (NCEU)
Next, we designed a new device based on the CEU, which we called the NCEU (Fig. 1M). The NCEU, which tolerates freezing in LN2, is equipped with a silicone flipper that can pinch the HFT onto the center of plate (Fig. 1N). The culture area of the NCEU was easily covered by a gas-permeable membrane, which was attached using a nontoxic adhesive. When the HFT with fresh embryos was pinched onto the center of the NCEU and the medium was changed three times (Fig. 1O), the HFT could not be flushed out, and 100% of the embryos developed to blastocysts (Fig. 1P, Table 1) after 4 days of culture in the CO2 incubator. When OptiCellTM method was used, sometimes HFT was located on the inside edge and became difficult to observe embryos. However, this method allows the HFT to be fixed in the center and at the bottom of plate, making it easy to observe all embryos. Thus, we concluded that the NCEU was possibly the best device for thawing, washing, and observing embryos and that the procedures could be successfully performed without practice.
Restriction of freezer on ISS
Even if the frozen embryos are transported by rocket to the ISS, frozen embryos must be stored on the ISS for about a month before start experiment because the astronauts are too busy immediately after the rocket's arrival. During the development of these devices, we were informed that liquid nitrogen was not available on the ISS. When using general vitrification method, the -95°C freezer at ISS cannot preserve frozen embryos, even for short periods of time. Therefore, we decided to use the high osmolarity vitrification (HOV) method, which allows storage of the embryos at −80°C for >1 month 17. However, because the pore size of the membrane of the HFT was very small (5–15 nm), the vitrification solution (EFS42.5) used in the HOV method is highly viscous and cannot be exchanged with the thawing solution (0.75 M sucrose-PB1) through the HFT membrane within time limit. Therefore, we decided to start developing a new method that does not use HFT.
Frozebag® using a mesh bag
To achieve exchange of the viscous solution without flushing out the embryos, we used a mesh sheet (Fig. 2A), which was converted into a bag using a heat sealer (hereafter “mesh bag”) (Fig. 2B). A Frozebag® (Fig. 2C) served as the embryo culture device. First, to exam toxicity, we directly inserted fresh embryos into the Frozebag®, filled it with culture (CZB) medium, and the heat-sealed it. However, none of the embryos developed to the blastocysts, likely because of the bag's impermeability to gas (Table 2). Therefore, we developed a new embryo culture system, in which CZB medium was equilibrated with 5% CO2 in a CO2 incubator before use (hereinafter referred to as eCZB medium) (Figure 2D) 18. Next, fresh embryos were directly placed in the Frozebag®, or inserted into the mesh bag on the scoop (Fig. 2E), placed in the Frozebag®, and then cultured for 4 days in eCZB medium (Fig. 2F). Under these conditions, 86-94% of embryos developed into blastocysts (Table 2), which demonstrated that the Frozebag®, the mesh bag, and the scoop were not cytotoxic.
Next, the mesh bag on the scoop was filled with EFS42.5, and 2-cell embryos were inserted into the mesh bag, frozen in LN2, and the scoop then was placed in the Frozebag®, heat-sealed, and stored at −80°C for a few days. When the Frozebag® containing frozen embryos was thawed, washed two times via a port using syringes (Fig. 2G), and then cultured for 4 days, 50% of the embryos were recovered from the mesh bag, and none formed blastocysts. Furthermore, no embryo reached the blastocyst stage even when embryos were immediately harvested from the Frozebag® after thawing and cultured on a Petri dish for 4 days (37°C, 5% CO2) (Table 2). This outcome may be explained by the possibility that the Frozebag® method failed to allow exchange of the vitrification solution with the dilution solution within the restricted time limit. Furthermore, the Frozebag® contained a 10-fold greater volume of EFS42.5 solution compared with the HOV method.
Frozebag® using a mesh cap
To avoid introducing a large volume of EFS42.5 solution into the Frozebag, we attempted to freeze the embryos directly on the inner surface of the Frozebag® in the presence of a small amount of EFS42.5 solution. To prevent flushing out the embryos during solution exchange, the mesh sheet was converted to a cap shape (hereafter “mesh cap”) and attached to the inner port of the Frozebag® (Fig. 3A). When fresh embryos were inserted into the Frozebag®, the medium was exchanged twice. After 4 days of culture, 15 of 60 embryos were collected, among which 80% developed into blastocysts (Table 3). When embryos were frozen on the surface of the Frozebag®, thawed, washed, and cultured for 4 days, 23% were recovered and none survived (Table 3).
Frozebag® equipped with a cryotube and mesh wall
We assumed that the HOV method could be performed in a cryotube 17. The material and thickness of the Frozebag® are completely different from those of the cryotube, and the difference in thermal conductivity during freezing may have damaged the embryos. Therefore, we used cryotubes to freeze the embryos and placed them in a Frozebag®, which was stored at −80°C for several days. The embryo recovery rate using a mesh cap from the Frozebag® ranged from 23% to 25%. We after testing several other methods, we decided to use a heat sealer to attach a mesh sheet to the middle of the Frozebag® to serve as a barrier (hereafter “mesh wall”) to prevent flushing out the embryos (Fig. 3B). When fresh embryos were inserted into cryotubes, which were placed in this modified Frozebag® with mesh wall and cultured for 4 days, 80% of the embryos were recovered. Blastocytes were produced by 91% of these embryos (Table 3).
Next, embryos were frozen in cryotubes, placed in a Frozebag®, and stored at −80°C for several days. When the Frozebag® was thawed, washed by exchanging the solutions, and cultured for 4 days in eCZB medium, we obtained two blastocysts (2%). Although the morphological quality of those blastocysts was quite poor (Fig. 3C), this was the first successful attempt to culture frozen embryos to produce blastocytes without directly manipulating the embryos.
The low yield of blastocysts may be explained by insufficient removal of the EFS42.5 solution from the Frozebag®, even after multiple exchanges of the solution. Next, we opened the Frozebag® immediately after thawing, collected the embryos, and cultured them on a Petri dish in the CO2 incubator. This procedure yielded 48 of 50 embryos, among which 16 (55%) developed into blastocysts (Table 3). Hereafter, we called this modified Frozebag® as an Embryo Thawing and Culturing unit (ETC).
ETC using V-tube
To improve the efficiency of exchanging the EFS42.5 solution with eCZB medium, the size of the cryotubes was reduced to the extent possible (Fig. 3D). We called the smallest cryotube a “V-tube.” We simultaneously attempted to increase the number of medium exchanges to completely remove residual vitrification/dilution solution from ETC (Fig. 3E). Embryos were frozen using the V-tube, placed in the ETC, stored at −80°C for a few days, thawed with an additional medium exchange 1-h later, and cultured for 4 days. We recovered 61 of 98 embryos (62%), and 30 (59%) developed into good-quality blastocysts (Fig. 3F, Table 3). We succeeded in obtaining good-quality blastocysts, as described in the next paragraph, when one or two additional medium exchanges were performed 2-h later or 30 min and 2-h later, although the rate of survived embryo decreased to15%–20%.
To evaluate the quality of the embryos obtained using this system, morulae/blastocysts cultured for 3 days in ETC were transferred to mice. These embryos yielded 21 (31%) offspring (Fig. 3G, Table 4), which is same rate achieved when embryos cultured in vitro were transferred in my laboratory 19. This result shows that the ETC generated normal morulae/blastocysts.
Preservation period of embryos at −80°C and reproducibility of this method
When the µG experiment is conducted on the ISS, storage in the −95°C freezer may exceed 1 month. Therefore, we determined our ability to preserve frozen embryos at −80°C in the ETC. When the ETC was stored for 1 month at −80°C, 17 of 52 (32.7%) embryos developed into blastocysts. The blastocyst generation rate decreased with longer storage, although some embryos developed into blastocysts after storage for 3 months (Table 5). Furthermore, we determined whether inexperienced people were able to obtain blastocysts using the ETC. For this purpose we used 2 ETCs with 180 vitrified embryos. Two people thawed and cultured the embryos. Although the rate of blastocyst generation was not high, both person obtained good blastocysts (Table 5).