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Derivation of snake venom gland organoids for in vitro venom production


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All previously unpublished data is included in the figures.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.


  1. 1.

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  Article  Google Scholar 

  2. 2.

    Kretzschmar, K. & Clevers, H. Organoids: modeling development and the stem cell niche in a dish. Dev. Cell 38, 590–600 (2016).

    CAS  Article  Google Scholar 

  3. 3.

    Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Gutierrez, J. M. et al. Snakebite envenoming. Nat. Rev. Dis. Primer 3, 17063 (2017).

    Article  Google Scholar 

  5. 5.

    Sells, P. G., Hommel, M. & Theakston, R. D. G. Venom production in snake venom gland cells cultured in vitro. Toxicon 27, 1245–1249 (1989).

    CAS  Article  Google Scholar 

  6. 6.

    Carneiro, S. M. et al. Venom production in long-term primary culture of secretory cells of the Bothrops jararaca venom gland. Toxicon 47, 87–94 (2006).

    CAS  Article  Google Scholar 

  7. 7.

    Yamanouye, N. et al. Long-term primary culture of secretory cells of Bothrops jararaca venom gland for venom production in vitro. Nat. Protoc. 47, 87–94 (2007).

    Google Scholar 

  8. 8.

    Post, Y. et al. Snake venom gland organoids. Cell 180, 233–247.e21 (2020).

    CAS  Article  Google Scholar 

  9. 9.

    Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

    CAS  Article  Google Scholar 

  10. 10.

    Boj, S. F. et al. Organoid models of human and mouse ductal pancreatic cancer. Cell 160, 324–338 (2015).

    CAS  Article  Google Scholar 

  11. 11.

    Schutgens, F. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat. Biotechnol. 37, 303–313 (2019).

    CAS  Article  Google Scholar 

  12. 12.

    Nicolas, J. et al. Detection of marine neurotoxins in food safety testing using a multielectrode array. Mol. Nutr. Food Res. 58, 2369–2378 (2014).

    CAS  Article  Google Scholar 

  13. 13.

    Whiteley, G. et al. Defining the pathogenic threat of envenoming by South African shield-nosed and coral snakes (genus Aspidelaps), and revealing the likely efficacy of available antivenom. J. Proteomics 198, 186–198 (2019).

    CAS  Article  Google Scholar 

  14. 14.

    Slagboom, J. et al. High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches. PLoS Negl. Trop. Dis. 14, e0007802 (2020).

    Article  Google Scholar 

  15. 15.

    Muraro, M. J. et al. A single-cell transcriptome atlas of the human pancreas. Cell Syst 3, 385–394.e3 (2016).

    CAS  Article  Google Scholar 

  16. 16.

    Baran-Gale, J., Chandra, T. & Kirschner, K. Experimental design for single-cell RNA sequencing. Brief. Funct. Genomics 17, 233–239 (2018).

    CAS  Article  Google Scholar 

  17. 17.

    Dekkers, J. F. et al. High-resolution 3D imaging of fixed and cleared organoids. Nat. Protoc. 14, 1756–1771 (2019).

    CAS  Article  Google Scholar 

  18. 18.

    Drost, J., Artegiani, B. & Clevers, H. The generation of organoids for studying WNT signaling. Methods Mol. Biol. 1481, 141–159 (2016).

    CAS  Article  Google Scholar 

  19. 19.

    Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474–1485 (2015).

    CAS  Article  Google Scholar 

  20. 20.

    Lu, Y. et al. Avian-induced pluripotent stem cells derived using human reprogramming factors. Stem Cells Dev. 21, 394–403 (2012).

    CAS  Article  Google Scholar 

  21. 21.

    Peng, L. et al. Generation of stable induced pluripotent stem-like cells from adult zebra fish fibroblasts. Int. J. Biol. Sci. 15, 2340–2349 (2019).

    CAS  Article  Google Scholar 

  22. 22.

    Pierzchalska, M., Panek, M., Czyrnek, M. & Grabacka, M. The three-dimensional culture of epithelial organoids derived from embryonic chicken intestine. Methods Mol. Biol. 1576, 135–144 (2019).

    CAS  Article  Google Scholar 

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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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Corresponding author

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

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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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Post, Y. et al. Cell 180, 233–247.e21 (2020):

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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).

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