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A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation

Nature Protocols volume 12, pages 1387–1399 (2017)Cite this article

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Abstract

The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for microorganism invasion and mediate food tolerance by transcellular transport of intestinal microbiota and antigens. Their ability to transcytose intact particles can be used to develop oral drug delivery and oral immunization strategies. This protocol describes a reproducible and versatile human M-cell-like in vitro model. This model can be exploited to evaluate M-cell transport of microparticles and nanoparticles for protein, drug or vaccine delivery and to study bacterial adherence and translocation across M cells. The inverted in vitro M-cell model consists of three main steps. First, Caco-2 cells are seeded at the apical side of the inserts. Second, the inserts are inverted and B lymphocytes are seeded at the basolateral side of the inserts. Third, the conversion to M cells is assessed. Although various M-cell culture systems exist, this model provides several advantages over the rest: (i) it is based on coculture with well-established differentiated human cell lines; (ii) it is reproducible under the conditions described herein; (iii) it can be easily mastered; and (iv) it does not require the isolation of primary cells or the use of animals. The protocol requires skills in cell culture and microscopy analysis. The model is obtained after 3 weeks, and transport experiments across the differentiated model can be carried out over periods of up to 10 h.

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Figure 1
Figure 2: Images of monocultures and cocultures obtained by TEM and SEM microscopy.
Figure 3: Quantification and visualization of transported nanoparticles.
Figure 4: Adherence to and translocation of different bacterial species across M-like-cell cocultures.

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Acknowledgements

A.B. is a postdoctoral researcher from the Belgian Fonds National de la Recherche Scientifique (FRS–FNRS), Belgium. A.d.R. is a research associate of the FRS–FNRS, Belgium. We thank T. Ahmad for assistance with interpreting the microbiology studies at University College Dublin. The research leading to some of these results received funding from the European Union Seventh Framework Programme (FP7) from 2007 through 2013 under grant agreement no. 281035.

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Authors and Affiliations

  1. Department of Advanced Drug Delivery and Biomaterials,

    Ana Beloqui, Véronique Préat & Anne des Rieux

  2. Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium

    Ana Beloqui, Véronique Préat & Anne des Rieux

  3. Veterinary Biosciences Section, School of Veterinary Medicine and Conway Institute, University College Dublin, Dublin, Ireland

    David J Brayden

  4. Department of Pharmacy, Uppsala University, Uppsala, Sweden

    Per Artursson

  5. Science for Life Laboratory, Uppsala University, Uppsala, Sweden

    Per Artursson

  6. Institute of the Condensed Matter and Nanosciences,

    Anne des Rieux

  7. Université catholique de Louvain, Louvain-la-Neuve, Belgium

    Anne des Rieux

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  1. Ana Beloqui
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  2. David J Brayden
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  3. Per Artursson
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  4. Véronique Préat
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Contributions

A.B. and A.d.R. wrote the draft protocol, excluding the discussion of data for the transport of bacteria. D.J.B. wrote the discussions of experimental data related to the transport of bacteria and viruses across M cells, including Box 4, and edited the English language. All authors contributed to the composition, correction and editing of the protocol and have jointly supervised the work.

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Correspondence to Anne des Rieux.

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Beloqui, A., Brayden, D., Artursson, P. et al. A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation. Nat Protoc 12, 1387–1399 (2017). https://doi.org/10.1038/nprot.2017.041

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