Abstract
Conventional cell cultures utilizing transformed or immortalized cell lines or primary human epithelial cells have played a fundamental role in furthering our understanding of Cryptosporidium infection. However, they remain inadequate with respect to their inability to emulate in vivo conditions, support long-term growth, and complete the life cycle of the parasite. Previously, we developed a 3D silk scaffold-based model using transformed human intestinal epithelial cells (IECs). This model supported C. parvum infection for up to 2Â weeks and resulted in completion of the life cycle of the parasite. However, transformed IECs are not representative of primary human IEC.
Human intestinal enteroids (HIEs) are cultures derived from crypts that contain Lgr5+ stem cells isolated from human biopsies or surgical intestinal tissues; these established multicellular cultures can be induced to differentiate into enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and tuft cells. HIEs better represent human intestinal structure and function than immortalized IEC lines. Recently, significant progress has been made in the development of technologies to culture HIEs in vitro. When grown in a 3D matrix, HIEs provide a spatial organization resembling the native human intestinal epithelium. Additionally, they can be dissociated and grown as monolayers in tissue culture plates, permeable supports or silk scaffolds that enable mechanistic studies of pathogen infections. They can also be co-cultured with other human cells such as macrophages and myofibroblasts. The HIEs grown in these novel culture systems recapitulate the physiology, the 3D architecture, and functional diversity of native intestinal epithelium and provide a powerful and promising new tool to study Cryptosporidium–host cell interactions and screen for interventions ex vivo. In this chapter, we describe the 3D silk scaffold-based model using transformed IEC co-cultured with human intestinal myofibroblasts and 2D and 3D HIE-derived models of Cryptosporidium, also co-cultured with human intestinal myofibroblasts.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Zhou W, Chen Y, Roh T, Lin Y, Ling S, Zhao S, Lin JD, Khalil N, Cairns DM, Manousiouthakis E, Tse M, Kaplan DL (2018) Multifunctional bioreactor system for human intestine tissues. ACS Biomater Sci Eng 4(1):231–239. https://doi.org/10.1021/acsbiomaterials.7b00794
Castellanos-Gonzalez A, Cabada MM, Nichols J, Gomez G, White AC Jr (2013) Human primary intestinal epithelial cells as an improved in vitro model for Cryptosporidium parvum infection. Infect Immun 81(6):1996–2001. https://doi.org/10.1128/IAI.01131-12
Varughese EA, Bennett-Stamper CL, Wymer LJ, Yadav JS (2014) A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum. J Microbiol Meth 106:47–54. https://doi.org/10.1016/j.mimet.2014.07.017
Miller CN, Josse L, Brown I, Blakeman B, Povey J, Yiangou L, Price M, Cinatl J Jr, Xue WF, Michaelis M, Tsaousis AD (2018) A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology. Int J Parasitol 48(3–4):197–201. https://doi.org/10.1016/j.ijpara.2017.10.001
DeCicco RePass MA, Chen Y, Lin Y, Zhou W, Kaplan DL, Ward HD (2017) Novel bioengineered three-dimensional human intestinal model for long-term infection of Cryptosporidium parvum. Infect Immun 85(3):e00731-16. https://doi.org/10.1128/IAI.00731-16
Morada M, Lee S, Gunther-Cummins L, Weiss LM, Widmer G, Tzipori S, Yarlett N (2016) Continuous culture of Cryptosporidium parvum using hollow fiber technology. Int J Parasitol 46(1):21–29. https://doi.org/10.1016/j.ijpara.2015.07.006
Bhalchandra S, Cardenas D, Ward HD (2018) Recent breakthroughs and ongoing limitations in Cryptosporidium research. F1000Res 7:F1000. https://doi.org/10.12688/f1000research.15333.1
Larregieu CA, Benet LZ (2013) Drug discovery and regulatory considerations for improving in silico and in vitro predictions that use Caco-2 as a surrogate for human intestinal permeability measurements. AAPS J 15(2):483–497. https://doi.org/10.1208/s12248-013-9456-8
Sun D, Lennernas H, Welage LS, Barnett JL, Landowski CP, Foster D, Fleisher D, Lee KD, Amidon GL (2002) Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm Res 19(10):1400–1416
Koo BK, Clevers H (2014) Stem cells marked by the R-spondin receptor LGR5. Gastroenterology 147(2):289–302. https://doi.org/10.1053/j.gastro.2014.05.007
Zachos NC, Kovbasnjuk O, Foulke-Abel J, In J, Blutt SE, De Jonge HR, Estes MK, Donowitz M (2015) Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology. J Biol Chem 291(8):3759–3766. https://doi.org/10.1074/jbc.R114.635995
Yu H, Hasan NM, In JG, Estes MK, Kovbasnjuk O, Zachos NC, Donowitz M (2017) The contributions of human mini-intestines to the study of intestinal physiology and pathophysiology. Annu Rev Physiol 79:291–312. https://doi.org/10.1146/annurev-physiol-021115-105211
Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265. https://doi.org/10.1038/nature07935
Rouch JD, Scott A, Lei NY, Solorzano-Vargas RS, Wang J, Hanson EM, Kobayashi M, Lewis M, Stelzner MG, Dunn JC, Eckmann L, Martin MG (2016) Development of functional microfold (M) cells from intestinal stem cells in primary human enteroids. PLoS One 11(1):e0148216. https://doi.org/10.1371/journal.pone.0148216
Foulke-Abel J, In J, Kovbasnjuk O, Zachos NC, Ettayebi K, Blutt SE, Hyser JM, Zeng XL, Crawford SE, Broughman JR, Estes MK, Donowitz M (2014) Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract. Exp Biol Med 239(9):1124–1134. https://doi.org/10.1177/1535370214529398
Heo I, Dutta D, Schaefer DA, Iakobachvili N, Artegiani B, Sachs N, Boonekamp KE, Bowden G, Hendrickx APA, Willems RJL, Peters PJ, Riggs MW, O’Connor R, Clevers H (2018) Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nat Microbiol 3(7):814–823. https://doi.org/10.1038/s41564-018-0177-8
Ramani S, Crawford SE, Blutt SE, Estes MK (2018) Human organoid cultures: transformative new tools for human virus studies. Curr Opin Virol 29:79–86. https://doi.org/10.1016/j.coviro.2018.04.001
Mahe MM, Aihara E, Schumacher MA, Zavros Y, Montrose MH, Helmrath MA, Sato T, Shroyer NF (2013) Establishment of gastrointestinal epithelial organoids. Curr Prot Mouse Biol 3:217–240. https://doi.org/10.1002/9780470942390.mo130179
Saxena K, Blutt SE, Ettayebi K, Zeng XL, Broughman JR, Crawford SE, Karandikar UC, Sastri NP, Conner ME, Opekun AR, Graham DY, Qureshi W, Sherman V, Foulke-Abel J, In J, Kovbasnjuk O, Zachos NC, Donowitz M, Estes MK (2015) Human intestinal enteroids: a new model to study human rotavirus infection, host restriction, and pathophysiology. J Virol 90(1):43–56. https://doi.org/10.1128/JVI.01930-15
In J, Foulke-Abel J, Zachos NC, Hansen AM, Kaper JB, Bernstein HD, Halushka M, Blutt S, Estes MK, Donowitz M, Kovbasnjuk O (2016) Enterohemorrhagic reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell Mol Gastroenterol Hepatol 2(1):48–62 e43. https://doi.org/10.1016/j.jcmgh.2015.10.001
Zou WY, Blutt SE, Crawford SE, Ettayebi K, Zeng XL, Saxena K, Ramani S, Karandikar UC, Zachos NC, Estes MK (2017) Human intestinal enteroids: new models to study gastrointestinal virus infections. Meth Mol Biol. https://doi.org/10.1007/7651_2017_1
Chen Y, Zhou W, Roh T, Estes MK, Kaplan DL (2017) In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses. PLoS One 12(11):e0187880. https://doi.org/10.1371/journal.pone.0187880
Zachos NC, Kovbasnjuk O, Foulke-Abel J, In J, Blutt SE, de Jonge HR, Estes MK, Donowitz M (2016) Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology. J Biol Chem 291(8):3759–3766. https://doi.org/10.1074/jbc.R114.635995
Heijmans J, van Lidth de Jeude JF, Koo BK, Rosekrans SL, Wielenga MC, van de Wetering M, Ferrante M, Lee AS, Onderwater JJ, Paton JC, Paton AW, Mommaas AM, Kodach LL, Hardwick JC, Hommes DW, Clevers H, Muncan V, van den Brink GR (2013) ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response. Cell Rep 3(4):1128–1139. https://doi.org/10.1016/j.celrep.2013.02.031
Vinayak S, Pawlowic MC, Sateriale A, Brooks CF, Studstill CJ, Bar-Peled Y, Cipriano MJ, Striepen B (2015) Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523(7561):477–480. https://doi.org/10.1038/nature14651
Chen Y, Lin Y, Davis KM, Wang Q, Rnjak-Kovacina J, Li C, Isberg RR, Kumamoto CA, Mecsas J, Kaplan DL (2015) Robust bioengineered 3D functional human intestinal epithelium. Sci Rep 5:13708. https://doi.org/10.1038/srep13708
Le Blancq SM, Khramtsov NV, Zamani F, Upton SJ, Wu TW (1997) Ribosomal RNA gene organization in Cryptosporidium parvum. Mol Biochem Parasitol 90(2):463–478
Cabada MM, White AC Jr (2010) Treatment of cryptosporidiosis: do we know what we think we know? Curr Opin Infect Dis 23(5):494–499. https://doi.org/10.1097/QCO.0b013e32833de052
Acknowledgments
Work in the authors’ laboratories was supported by NIH U19AI131126 (to HW, Project 3; to DK, Core); NIH R21AI120932 (to HW); NIH R21AI128342 (to HW, DK); Bill and Melinda Gates Foundation OPP1164543 (to HW); NIH U19AI16497 (to ME); NIH P30DK56338 (to Hashem El-Serag).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Cardenas, D. et al. (2020). Two- and Three-Dimensional Bioengineered Human Intestinal Tissue Models for Cryptosporidium. In: Mead, J., Arrowood, M. (eds) Cryptosporidium. Methods in Molecular Biology, vol 2052. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9748-0_21
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9748-0_21
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9747-3
Online ISBN: 978-1-4939-9748-0
eBook Packages: Springer Protocols