However, more than 50% of PGCs remained discernible when suspended in CPA containing 20% EG and 1 M sucrose (Supplementary Table 1 and Supplementary Fig. Without using CPA, all PGCs were ruptured and indiscernible after freeze-thawing (Supplementary Table 1 and Supplementary Fig. Sucrose was used to reduce the risk of intracellular ice crystal formation due to dehydration 19. We examined their morphology immediately after freeze-thawing (Supplementary Table 1 and Supplementary Fig. PGCs were immersed in CPAs that all contained either ethylene glycol (EG), dimethyl sulfoxide (DMSO), or glycerol (G), along with sucrose at various concentrations. We first optimized the chemical composition of the CPA. PGCs were collected from donor embryos at the blastoderm stage (stage 5) using a thin glass needle and subsequently suspended in cryoprotectant agent (CPA) for cryopreservation in liquid nitrogen (LN 2) (see Methods). When transplanted into host animals, PGCs can differentiate into gametes that are subsequently fertilized to produce offspring 17, 18. In order to overcome this critical limitation, we sought to cryopreserve primordial germ cells (PGCs) instead of ovaries and embryos. Consequently, these methods are not practical for preserving a variety of Drosophila strains 16. However, these methods are laborious, have variable success rates, and have poor reproducibility. Hence, cryopreservation techniques are urgently needed for long-term storage of Drosophila strains to prevent accidental loss of genetic strains due to labor shortages induced by the pandemic and decrease the risk of genetic alteration.ĭrosophila strains can be preserved by freezing ovaries or embryos 13, 14, 15. Cryopreservation is widely used to preserve genetic strains of model organisms, although not for flies 6, 7, 8, 9, 10, 11, 12. However, long-term maintenance of Drosophila strains as living stocks increases the risk of unwanted second mutations, which can cause phenotypes to be altered or important strains to be lost. The number of Drosophila strains harboring mutations and transgenes continues to increase, and strains have been maintained as living cultures in both individual laboratories and Drosophila stock centers. The importance of this animal to biological research is due largely to its ease of culture and genetic manipulation 4, 5. Use of the fly has enabled key advances in our understanding of human diseases as well as a wide range of phenomena in developmental, cellular, and evolutionary biology 1, 2, 3. The fruit fly, Drosophila melanogaster, is a classical model animal for basic biology research. Model organisms that carry chromosomal aberrations, mutations, and transgenic constructs are critical for studies of gene functions in animal systems.

Germ the poducer