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This study shows for the first time the ability to rewarm cryopreserved zebrafish embryos that grow into adult fish capable of breeding normally. The protocol employs a single injection of cryoprotective agents (CPAs) and gold nanorods (GNRs) into the yolk and immersion in a precooling bath to dehydrate the perivitelline space. Then embryos are encapsulated within CPA and GNR droplets, plunged into liquid nitrogen, cryogenically stabilized, and rewarmed by a laser pulse. Postlaser nanowarming, embryos (n = 282) exhibit intact structure by 1 h (40%), continued development after 3 h (22%), movement after 24 h (11%), hatching after 48 h (9%), and swimming after Day 5 (3%). Finally, from fish that survives till Day 5, two larvae are grown to adulthood and spawned, yielding survival comparable to an unfrozen control. Future efforts will focus on improving the survival to adulthood and developing methods to cryopreserve large numbers of embryos for research, aquaculture, and biodiversity preservation.
Bibliographical noteFunding Information:
This work was funded by NIH SBIR Phase I (1R41OD024430‐01) and Phase II (9R44MH122118‐02) grants. The authors would like to thank Mr. Marc Tye and Dr. Mark Masino of University of Minnesota Zebrafish Core for their support for the zebrafish studies. The authors would also like to thank Dr. Robert Evans III at the University of Minnesota for helping image zebrafish embryos in vitrified or crystalized state.
- gold nanoparticles
- photothermal heating
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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