Affiliations: 1) University of Minnesota- Twin Cities, Department of Mechanical Engineering, Minneapolis, MN; 2) University of Minnesota-Twin Cities, ATP-Bio, Minneapolis, MN; 3) University of Minnesota-Twin Cities, University of Minnesota Genomics Center, Minneapolis, MN; 4) University of Minnesota-Twin Cities, Department of Genetics, Cell Biology, and Development, Minneapolis, MN; 5) University of Minnesota-Twin Cities, Department of Biomedical Engineering, Minneapolis, MN
Keywords: a. microscopy; l. computational algorithms
Cryopreservation of Drosophila melanogaster has been an area of interest since the 1990s when methods of embryo vitrification protocols were introduced by Stepnokus et al. (1990) and Mazur et al. (1992). However, there has still not been widespread adoption of these methods due to the difficulty of the protocols. Recent publications from Zhan et al. (2021) and Asaoka et al. (2021) have shown successful development of an alternative vitrification methods for cryopreservation of embryos and a novel cryopreservation method based on transplantation of cryopreserved primordial germ cells, respectively. We are testing an alternative method that involves the slow cooling of embryonic nuclei and regeneration of stocks by embryonic nuclear transplantation (ENT). Using confocal Raman spectroscopy is beneficial for label-free imaging of nuclei and the distribution of water and CPA. Osmolyte solutions consisting of sucrose, glycerol, and other buffering agents that aid in the isolation of Drosophilanuclei act as quality CPAs in the slow-cooling cryopreservation process. Studies at 4°C show that certain CPAs, such as glycerol, can permeate the nuclear membrane while other CPAs, like sucrose, cannot penetrate the nuclear membrane. Using a controlled rate temperature-cooling stage, we can image the nucleus at -50°C with varying cooling rates and nucleation temperatures. We are also using an RT-qPCR assay for hsp70 induction to monitor nuclei health and viability throughout the isolation, cryopreservation, and recovery process. This functional assay will aid in determining the optimal parameters of Drosophilacryopreservation with the use of a differential evolution (DE) algorithm. The DE algorithm uses experimental observations (CPA composition, cooling rates, nucleation temperatures, and post thaw viability) to modify existing population vectors and predicts solutions that may be more favorable. This DE algorithm could also be applied to other Drosophila cryopreservation methods to optimize CPA and freezing parameters. Finally, we have been attempting to create clones or chimeric embryos derived from isolated nuclei using ENT. ENT has previously been demonstrated in Drosophila and avoids issues associated with the impermeability of Drosophila embryos to cryoprotectants. We report progress in developing this novel method which has the potential to provide additional options for the practical cryopreservation of Drosophila stocks.