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The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.
Bibliographical noteFunding Information:
We thank Dr. Amanda Neisch for productive discussions surrounding the genetics and variation in cryopreservation. We thank Zonghu Han and Anthony Chen for serving as volunteers to test the robustness of the developed cryopreservation protocol. We thank Dr. Michael Etheridge for his critical reading and comments to the manuscript. Funding from the US National Institutes of Health (NIH) (1R21OD028758) is gratefully acknowledged. T.H. further acknowledges NIH GM R01GM044757 and J.B. acknowledges NSF EEC 1941543. L.Z. acknowledges the support of a Doctoral Dissertation Fellowship at the University of Minnesota.
© 2021, The Author(s).
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't
- Research Support, U.S. Gov't, Non-P.H.S.
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- 1 Active
ATP-Bio: NSF Engineering Research Center for Advanced Technologies for the Preservation of Biological Systems (ATP-Bio)
9/1/20 → 8/31/25
Project: Research project