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Derivation of snake venom gland organoids for in vitro venom production

Abstract

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually ‘milking’ snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.

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Fig. 1: Overview of working with snake venom gland organoids.
Fig. 2: Establishment of snake venom gland organoids from primary tissue.
Fig. 3: Setting up experiments with snake venom gland organoids.
Fig. 4: Analytical applications of snake venom gland organoids.

Data availability

All previously unpublished data is included in the figures. Raw image files are available from the corresponding author upon request.

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Acknowledgements

We thank A. de Graaff and the Hubrecht Imaging Centre (HIC) for microscopy assistance; B. Ponsioen for providing a lentiviral H2B-RFP construct; and local snake breeders as well as W. Getreuer and SERPO for donating venom gland material.

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Correspondence to Hans Clevers.

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Competing interests

H.C. is inventor on multiple patents held by the Dutch Royal Netherlands Academy of Arts and Sciences that cover organoid technology: PCT/NL2008/050543, WO2009/022907; PCT/NL2010/000017, WO2010/090513; PCT/IB2011/002167, WO2012/014076; PCT/IB2012/052950, WO2012/168930;PCT/EP2015/060815, WO2015/173425; PCT/EP2015/077990, WO2016/083613; PCT/EP2015/077988, WO2016/083612; PCT/EP2017/054797,WO2017/149025; PCT/EP2017/065101, WO2017/220586; PCT/EP2018/086716; and GB1819224.5. H.C.’s full disclosure is given at https://www.uu.nl/staff/JCClevers/.

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Peer review information Nature Protocols thanks C. Caldeira, J. Calvete and C. M. Trim for their contribution to the peer review of this work.

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Key reference using this protocol

Post, Y. et al. Cell 180, 233–247.e21 (2020): https://doi.org/10.1016/j.cell.2019.11.038

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Puschhof, J., Post, Y., Beumer, J. et al. Derivation of snake venom gland organoids for in vitro venom production. Nat Protoc 16, 1494–1510 (2021). https://doi.org/10.1038/s41596-020-00463-4

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