Derivation of snake venom gland organoids for in vitro venom production.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
03 2021
03 2021
Historique:
received:
22
04
2020
accepted:
12
11
2020
pubmed:
29
1
2021
medline:
7
4
2021
entrez:
28
1
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33504990
doi: 10.1038/s41596-020-00463-4
pii: 10.1038/s41596-020-00463-4
doi:
Substances chimiques
Antivenins
0
Snake Venoms
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1494-1510Subventions
Organisme : Wellcome Trust
ID : 200517/Z/16/Z
Pays : United Kingdom
Références
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
doi: 10.1038/nature07935
Kretzschmar, K. & Clevers, H. Organoids: modeling development and the stem cell niche in a dish. Dev. Cell 38, 590–600 (2016).
doi: 10.1016/j.devcel.2016.08.014
Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).
doi: 10.1016/j.cell.2016.05.082
Gutierrez, J. M. et al. Snakebite envenoming. Nat. Rev. Dis. Primer 3, 17063 (2017).
doi: 10.1038/nrdp.2017.63
Sells, P. G., Hommel, M. & Theakston, R. D. G. Venom production in snake venom gland cells cultured in vitro. Toxicon 27, 1245–1249 (1989).
doi: 10.1016/0041-0101(89)90033-0
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).
doi: 10.1016/j.toxicon.2005.10.006
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).
Post, Y. et al. Snake venom gland organoids. Cell 180, 233–247.e21 (2020).
doi: 10.1016/j.cell.2019.11.038
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).
doi: 10.1053/j.gastro.2011.07.050
Boj, S. F. et al. Organoid models of human and mouse ductal pancreatic cancer. Cell 160, 324–338 (2015).
doi: 10.1016/j.cell.2014.12.021
Schutgens, F. et al. Tubuloids derived from human adult kidney and urine for personalized disease modeling. Nat. Biotechnol. 37, 303–313 (2019).
doi: 10.1038/s41587-019-0048-8
Nicolas, J. et al. Detection of marine neurotoxins in food safety testing using a multielectrode array. Mol. Nutr. Food Res. 58, 2369–2378 (2014).
doi: 10.1002/mnfr.201400479
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).
doi: 10.1016/j.jprot.2018.09.019
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).
doi: 10.1371/journal.pntd.0007802
Muraro, M. J. et al. A single-cell transcriptome atlas of the human pancreas. Cell Syst 3, 385–394.e3 (2016).
doi: 10.1016/j.cels.2016.09.002
Baran-Gale, J., Chandra, T. & Kirschner, K. Experimental design for single-cell RNA sequencing. Brief. Funct. Genomics 17, 233–239 (2018).
doi: 10.1093/bfgp/elx035
Dekkers, J. F. et al. High-resolution 3D imaging of fixed and cleared organoids. Nat. Protoc. 14, 1756–1771 (2019).
doi: 10.1038/s41596-019-0160-8
Drost, J., Artegiani, B. & Clevers, H. The generation of organoids for studying WNT signaling. Methods Mol. Biol. 1481, 141–159 (2016).
doi: 10.1007/978-1-4939-6393-5_15
Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474–1485 (2015).
doi: 10.1038/nprot.2015.088
Lu, Y. et al. Avian-induced pluripotent stem cells derived using human reprogramming factors. Stem Cells Dev. 21, 394–403 (2012).
doi: 10.1089/scd.2011.0499
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).
doi: 10.7150/ijbs.34010
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).
doi: 10.1007/7651_2016_15