Skip to main content
SpringerLink
Log in

In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers

  • Protocol
  • First Online:
Book cover Cryptosporidium

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2052))

Abstract

Cryptosporidium parvum has a complex life cycle consisting of asexual and sexual phases that culminate in oocyst formation in vivo. The most widely used cell culture platforms to study C. parvum only support a few days of growth and do not allow the parasite to proceed past the sexual stages to complete oocyst formation. Additionally, these cell culture platforms are mostly adenocarcinoma cell lines, which do not adequately model the parasite’s natural environment in the small intestine. We describe here a method to create primary intestinal epithelial cell monolayers that support long-term C. parvum growth. Monolayers were derived from mouse intestinal stem cells grown as spheroids and plated onto transwells, allowing for separate apical and basolateral compartments. In the apical chamber, the cell growth medium was removed to create an “air–liquid interface” that enhanced host cell differentiation and supported long-term C. parvum growth. The use of primary intestinal cells to grow C. parvum in vitro will be a valuable tool for studying host–parasite interactions using a convenient in vitro model that more closely resembles the natural niche in the intestine.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Arrowood MJ (2002) In vitro cultivation of Cryptosporidium species. Clin Microbiol Rev 15(3):390–400

    Article  PubMed  PubMed Central  Google Scholar 

  2. Flanigan TP, Aji T, Marshall R, Soave R, Aikawa M, Kaetzel C (1991) Asexual development of Cryptosporidium parvum within a differentiated human enterocyte cell line. Infect Immun 49(1):234–239

    Article  Google Scholar 

  3. Gut J, Petersen C, Nelson R, Leech J (1991) Cryptosporidium parvum: in vitro cultivation in Madin-Darby canine kidney cells. J Protozool 38(6):72S–73S

    CAS  PubMed  Google Scholar 

  4. Sow SO, Muhsen K, Nasrin D, Blackwelder WC, Wu Y, Farag TH, Panchalingam S, Sur D, Zaidi AK, Faruque AS, Saha D, Adegbola RA, Alonso PL, Breiman RF, Bassat Q, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ahmed S, Qureshi S, Quadri F, Hossain A, Das SK, Antonio M, Hossain MJ, Mandomando I, Nhampossa T, Acácio S, Omore R, Oundo JO, Ochieng JB, Mintz ED, O’Reilly CE, Berkeley LY, Livio S, Tennant SM, Sommerfelt H, Nataro JP, Ziv-Baran T, Robins-Browne RM, Mishcherkin V, Zhang J, Liu J, Houpt ER, Kotloff KL, Levine MM (2016) The Burden of Cryptosporidium diarrheal disease among children < 24 months of age in moderate/high mortality regions of Sub-Saharan Africa and South Asia, utilizing data from the global enteric multicenter study (GEMS). PLoS Negl Trop Dis 10(5):e0004729. https://doi.org/10.1371/journal.pntd.0004729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chalmers RM, Smith R, Elwin K, Clifton-Hadley FA, Giles M (2011) Epidemiology of anthroponotic and zoonotic human cryptosporidiosis in England and Wales, 2004–2006. Epidemiol Infect 139(5):700–712. https://doi.org/10.1017/S0950268810001688

    Article  CAS  PubMed  Google Scholar 

  6. Cacciò SM, Chalmers RM (2016) Human cryptosporidiosis in Europe. Clin Microbiol Infect 22(6):471–480. https://doi.org/10.1016/j.cmi.2016.04.021

    Article  PubMed  Google Scholar 

  7. 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

    Article  CAS  PubMed  Google Scholar 

  8. 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–e00716

    Article  PubMed  PubMed Central  Google Scholar 

  9. Miyoshi H, Ajima R, Luo C, Yamaguchi TP, Stappenbeck TS (2012) Wnt5a potentiates TGF-beta signaling to promote colonic crypt regeneration after tissue injury. Science 338(6103):108–113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Miyoshi H, Stappenbeck TS (2013) In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc 8(12):2471–2482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Moon C, VanDussen KL, Miyoshi H, Stappenbeck TS (2014) Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis. Mucosal Immunol 7(7):818–828

    Article  CAS  PubMed  Google Scholar 

  12. VanDussen KL, Marinshaw JM, Shaikh N, Miyoshi H, Moon CS, Tarr PI, Ciorba MA, Stappenbeck TS (2014) Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays. Gut 64(6):911–920. https://doi.org/10.1136/gutjnl-2013-306651

    Article  CAS  PubMed  Google Scholar 

  13. Wilke G, Ravindran S, Funkhouser-Jones L, Barks J, Wang Q, VanDussen KL, Stappenbeck TS, Kuhlenschmidt TB, Kuhlenschmidt MS, Sibley LD (2018) Monoclonal antibodies to intracellular stages of Cryptosporidium parvum define life cycle progression in vitro. mSphere 3(3):e00124–e00118. https://doi.org/10.1128/mSphere.00124-18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wang X, Yamamoto Y, Wilson LH, Zhang T, Howitt BE, Farrow MA, Kern F, Ning G, Hong Y, Khor CC, Chevalier B, Bertrand D, Wu L, Nagarajan N, Sylvester FA, Hyams JS, Devers T, Bronson R, Lacy DB, Ho KY, Crum CP, McKeon F, Xian W (2015) Cloning and variation of ground state intestinal stem cells. Nature 522(7555):173–178. https://doi.org/10.1038/nature14484

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Whitcutt MJ, Adler KB, Wu R (1988) A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. Vitro Cell Dev Biol 24(5):420–428

    Article  CAS  Google Scholar 

  17. Pezzulo AA, Starner TD, Scheetz TE, Traver GL, Tilley AE, Harvey BG, Crystal RG, McCray PB, Zabner J (2011) The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol 300(1):L25–L31. https://doi.org/10.1152/ajplung.00256.2010

    Article  CAS  PubMed  Google Scholar 

  18. O’Boyle N, Sutherland E, Berry CC, Davies RL (2017) Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface. PLoS One 12(7):e0181583. https://doi.org/10.1371/journal.pone.0181583

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nossol C, Diesing AK, Walk N, Faber-Zuschratter H, Hartig R, Post A, Kluess J, Rothkötter HJ, Kahlert S (2011) Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochem Cell Biol 136(1):103–115. https://doi.org/10.1007/s00418-011-0826-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Klasvogt S, Zuschratter W, Schmidt A, Kröber A, Vorwerk S, Wolter R, Isermann B, Wimmers K, Rothkötter HJ, Nossol C (2017) Air-liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2. Cell Death Discov Feb 27(3):17001. https://doi.org/10.1038/cddiscovery.2017

    Article  Google Scholar 

  21. Georgia Wilke, Lisa J. Funkhouser-Jones, Yi Wang, Soumya Ravindran, Qiuling Wang, Wandy L. Beatty, Megan T. Baldridge, Kelli L. VanDussen, Bang Shen, Mark S. Kuhlenschmidt, Theresa B. Kuhlenschmidt, William H. Witola, Thaddeus S. Stappenbeck, L. David Sibley, (2019) A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability. Cell Host & Microbe

    Google Scholar 

Download references

Acknowledgments

Supported by grants from the Bill and Melinda Gates Foundation (OPP1098828, OPP1139330). G. Wilke was partially supported by an Institutional Training grant to Washington University (AI007172). We are grateful to Mark Kuhlenschmidt, Theresa Kuhlenschmidt, Lisa Funkhouser-Jones, and Kelli vanDussen for their helpful comments and assistance with the initial phase of this project.

Author information

Authors and Affiliations

  1. Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA

    Georgia Wilke, Soumya Ravindran & L. David Sibley

  2. Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA

    Yi Wang & Thaddeus Stappenbeck

  3. Department of Pathobiology, University of Illinois College of Veterinary Medicine, Urbana, IL, USA

    William H. Witola

Authors
  1. Georgia Wilke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Soumya Ravindran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thaddeus Stappenbeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. William H. Witola
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. L. David Sibley
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L. David Sibley .

Editor information

Editors and Affiliations

  1. Atlanta Veterans Affairs Medical Center, Decatur, GA, USA

    Jan R. Mead

  2. Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

    Michael J. Arrowood

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wilke, G., Wang, Y., Ravindran, S., Stappenbeck, T., Witola, W.H., Sibley, L.D. (2020). In Vitro Culture of Cryptosporidium parvum Using Stem Cell-Derived Intestinal Epithelial Monolayers. 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_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9748-0_20

  • 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

Publish with us

Policies and ethics

Search

Navigation