Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at −80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.
Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4 days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1 g control on the International Space Station and ground-1 g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.
Our previous spaceflight experiment CERISE showed that gene and protein expression levels of muscular components, cytoskeleton, and mitochondrial enzymes are altered in space flown wild-type C. elegans.To confirm and clarify whether the C. elegans muscle fibers and mitochondrial network are physically altered in response to microgravity, this Nematode Muscles project was designed with wild-type and several mutant lines with GFP expression.This investigation also studied whether microgravity could affect the insulin/ IGF-1 (Insulin-like growth factor -1) and/or TGF-β signaling by imaging DAF-16::GFP fusion protein.Wild-type and several mutants were grown in a culture bag kept under microgravity or 1G centrifuge conditions on board ISS for 4 days starting from L1 larva.All samples were fixed on board and recovered, to be analyzed on the earth.The worms did not grow well in the μG culture bag probably due to unexpected air bubbles.Therefore, DAF-16 activation observed in larval worms in μG and not in 1G may be attributed to starvation instead of μG response.In 1G samples, we could successfully find normal mitochondrial network.We also found that chemical fixation using CFA is an effective method for preservation of GFP containing C. elegans in space environment.
Abstract Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at −80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although embryonic development could not be observed in the ETC, thawed embryos formed blastocysts after 4 days of culture without direct contact. This ETC will enable untrained astronauts to thaw and culture frozen embryos on the ISS, as well as to serve as an important advance in fields such as clinical infertility and animal biotechnology.
