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Abstract The development of a widely adopted cryopreservation method remains a major challenge inDrosophilaresearch. Here we report a robust and easily implemented cryopreservation protocol ofDrosophila melanogasterembryos. 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.
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Scalable cryopreservation of infectious Cryptosporidium hominis oocysts by vitrification
Cryptosporidium hominisis a serious cause of childhood diarrhea in developing countries. The development of therapeutics is impeded by major technical roadblocks including lack of cryopreservation and simple culturing methods. This impacts the availability of optimized/standardized singular sources of infectious parasite oocysts for research and human challenge studies. The humanC.hominisTU502 isolate is currently propagated in gnotobiotic piglets in only one laboratory, which limits access to oocysts. Streamlined cryopreservation could enable creation of a biobank to serve as an oocyst source for research and distribution to other investigators requiringC.hominis. Here, we report cryopreservation ofC.hominisTU502 oocysts by vitrification using specially designed specimen containers scaled to 100 μL volume. Thawed oocysts exhibit ~70% viability with robust excystation and 100% infection rate in gnotobiotic piglets. The availability of optimized/standardized sources of oocysts may streamline drug and vaccine evaluation by enabling wider access to biological specimens.
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- Award ID(s):
- 1941543
- PAR ID:
- 10471506
- Editor(s):
- Striepen, Boris
- Publisher / Repository:
- NSF
- Date Published:
- Journal Name:
- PLOS Pathogens
- Volume:
- 19
- Issue:
- 6
- ISSN:
- 1553-7374
- Page Range / eLocation ID:
- e1011425
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.ppat.1011425
