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Generating an In Vitro Gut Model with Physiologically Relevant Biophysical Mucus Properties

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Abstract

Introduction

Gastrointestinal (GI) in vitro models have received lasting attention as an effective tool to model drug and nutrient absorption, study GI diseases, and design new drug delivery vehicles. A complete model of the GI epithelium should at a minimum include the two key functional components of the GI tract: mucus and the underlying epithelium. Mucus plays a key role in protecting and lubricating the GI tract, poses a barrier to orally administered therapies and pathogens, and serves as the microenvironment for the GI microbiome. These functions are reliant on the biophysical material properties of the mucus produced, including viscosity and pore size.

Methods

In this study, we generated in vitro models containing Caco-2 enterocyte-like cells and HT29-MTX goblet-like cells and determined the effects of coculture and mucus layer on epithelial permeability and biophysical properties of mucus using multiple particle tracking (MPT).

Results

We found that mucus height increased as the amount of HT29-MTX goblet-like cells increased. Additionally, we found that increasing the amount of HT29-MTX goblet-like cells within culture corresponded to an increase in mucus pore size and mucus microviscosity, measured using MPT. When compared to ex vivo mucus samples from mice and pigs, we found that a 90:10 ratio of Caco-2:HT29-MTX coculture displayed similar mucus pore size to porcine jejunum and that the mucus produced from 90:10 and 80:20 ratios of cells shared mechanical properties to porcine jejunum and ileum mucus.

Conclusions

GI coculture models are valuable tools in simulating the mucus barrier and can be utilized for a variety of applications including the study of GI diseases, food absorption, or therapeutic development.

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References

  1. Aksoy, N., A. Corfield, and J. Sheehan. Preliminary study pointing out a significant alteration in the biochemical composition of MUC2 in colorectal mucinous carcinoma. Clin Biochem. 33:167–173, 2000. https://doi.org/10.1016/S0009-9120(00)00058-8.

    Article  Google Scholar 

  2. Araújo, F., and B. Sarmento. Towards the characterization of an in vitro triple co-culture intestine cell model for permeability studies. Int. J. Pharm. 458(1):128–134, 2013. https://doi.org/10.1016/j.ijpharm.2013.10.003.

    Article  Google Scholar 

  3. Artursson, P., and J. Karlsson. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 175(3):880–885, 1991. https://doi.org/10.1016/0006-291x(91)91647-u.

    Article  Google Scholar 

  4. Artursson, P., K. Palm, and K. Luthman. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46(1–3):27–43, 2001. https://doi.org/10.1016/s0169-409x(00)00128-9.

    Article  Google Scholar 

  5. Atuma, C., V. Strugala, A. Allen, and L. Holm. The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am. J. Physiol. Gastroint. Liver Physiol. 280(5):G922–G929, 2001. https://doi.org/10.1152/ajpgi.2001.280.5.G922.

    Article  Google Scholar 

  6. Atuma, C., V. Strugala, A. Allen, and L. Holm. The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo. Am. J. Physiol.-Gastrointest. Liver Physiol. 280(5):922–929, 2001.

    Article  Google Scholar 

  7. Bajka, B. H., N. M. Rigby, K. L. Cross, A. Macierzanka, and A. R. Mackie. The influence of small intestinal mucus structure on particle transport ex vivo. Coll. Surf. B Biointerfaces. 135:73–80, 2015. https://doi.org/10.1016/j.colsurfb.2015.07.038.

    Article  Google Scholar 

  8. Bansil, R., and B. S. Turner. The biology of mucus: composition, synthesis and organization. Adv. Drug Deliv. Rev. 124:3–15, 2018.

    Article  Google Scholar 

  9. Beterams, A., K. De Paepe, L. Maes, et al. Versatile human in vitro triple coculture model coincubated with adhered gut microbes reproducibly mimics pro-inflammatory host-microbe interactions in the colon. FASEB J. 35(12):e21992, 2021. https://doi.org/10.1096/fj.202101135R.

    Article  Google Scholar 

  10. Briske-Anderson, M. J., J. W. Finley, and S. M. Newman. The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells. Proc. Soc. Exp. Biol. Med. 214(3):248–257, 1997. https://doi.org/10.3181/00379727-214-44093.

    Article  Google Scholar 

  11. Camilleri, M. Leaky gut: mechanisms, measurement and clinical implications in humans. Gut. 68(8):1516–1526, 2019. https://doi.org/10.1136/gutjnl-2019-318427.

    Article  Google Scholar 

  12. Chen, X. M., I. Elisia, and D. D. Kitts. Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design. J. Pharmacol. Toxicol. Methods. 61(3):334–342, 2010. https://doi.org/10.1016/j.vascn.2010.02.004.

    Article  Google Scholar 

  13. Cone, R. A. Chapter 4 - Mucus. In: Mucosal Immunology (Third Edition), edited by J. Mestecky, M. E. Lamm, J. R. McGhee, J. Bienenstock, L. Mayer, and W. Strober. Cambridge: Academic Press, 2005, pp. 49–72.

    Chapter  Google Scholar 

  14. Cone, R. A. Barrier properties of mucus. Adv. Drug Deliv. Rev. 61(2):75–85, 2009. https://doi.org/10.1016/j.addr.2008.09.008.

    Article  Google Scholar 

  15. Cornick, S., A. Tawiah, and K. Chadee. Roles and regulation of the mucus barrier in the gut. Tissue Barriers. 3(1–2):e982426–e982426, 2015. https://doi.org/10.4161/21688370.2014.982426.

    Article  Google Scholar 

  16. Crocker, J. C., and D. G. Grier. Methods of digital video microscopy for colloidal studies. J. Coll. Interface Sci. 179(1):298–310, 1996.

    Article  Google Scholar 

  17. Duncan, G. A., J. Jung, A. Joseph, et al. Microstructural alterations of sputum in cystic fibrosis lung disease. JCI Insight. 1(18):e88198–e88198, 2016. https://doi.org/10.1172/jci.insight.88198.

    Article  Google Scholar 

  18. Duncan, G. A., J. Jung, A. Joseph, et al. Microstructural alterations of sputum in cystic fibrosis lung disease. JCI Insight. 2016. https://doi.org/10.1172/jci.insight.88198.

    Article  Google Scholar 

  19. Engevik, A. C. Using microfluidics to model mucus. Cell. Mol. Gastroenterol. Hepatol. 9(3):551–552, 2020. https://doi.org/10.1016/j.jcmgh.2019.12.003.

    Article  Google Scholar 

  20. Engle, M. J., G. S. Goetz, and D. H. Alpers. Caco-2 cells express a combination of colonocyte and enterocyte phenotypes. J. Cell Physiol. 174(3):362–369, 1998. https://doi.org/10.1002/(sici)1097-4652(199803)174:3%3c362::aid-jcp10%3e3.0.co;2-b.

    Article  Google Scholar 

  21. Ensign, L. M., A. Henning, C. S. Schneider, et al. Ex vivo characterization of particle transport in mucus secretions coating freshly excised mucosal tissues. Mol. Pharm. 10(6):2176–2182, 2013. https://doi.org/10.1021/mp400087y.

    Article  Google Scholar 

  22. Ermund, A., J. K. Gustafsson, G. C. Hansson, and Å. V. Keita. Mucus properties and goblet cell quantification in mouse, rat and human ileal Peyer’s patches. PLoS ONE.8(12):e83688, 2013.

    Article  Google Scholar 

  23. Ferraretto, A., M. Bottani, P. de Luca, et al. Morphofunctional properties of a differentiated Caco2/HT-29 co-culture as an in vitro model of human intestinal epithelium. Biosci. Rep. 38(2):1497, 2018. https://doi.org/10.1042/bsr20171497.

    Article  Google Scholar 

  24. Forstner, J. Intestinal mucins in health and disease. Digestion. 17(3):234–263, 1978.

    Article  Google Scholar 

  25. Gagnon, M., A. Zihler Berner, N. Chervet, C. Chassard, and C. Lacroix. Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion. J. Microbiol. Methods. 94(3):274–279, 2013. https://doi.org/10.1016/j.mimet.2013.06.027.

    Article  Google Scholar 

  26. Georgiades, P., P. D. Pudney, D. J. Thornton, and T. A. Waigh. Particle tracking microrheology of purified gastrointestinal mucins. Biopolymers. 101(4):366–377, 2014.

    Article  Google Scholar 

  27. Gupta, V., N. Doshi, and S. Mitragotri. Permeation of insulin, calcitonin and exenatide across Caco-2 monolayers: measurement using a rapid, 3-day system. PLoS ONE. 8(2):e57136–e57136, 2013. https://doi.org/10.1371/journal.pone.0057136.

    Article  Google Scholar 

  28. Hansson, G. C. Mucins and the microbiome. Annu. Rev. Biochem. 89:769–793, 2020.

    Article  Google Scholar 

  29. Hill, D. B., P. A. Vasquez, J. Mellnik, et al. A biophysical basis for mucus solids concentration as a candidate biomarker for airways disease. PLoS ONE.9(2):e87681, 2014.

    Article  Google Scholar 

  30. Howard, R. L., M. Markovetz, Y. Wang, et al. Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus. Biophys. J. 120(23):5384–5394, 2021. https://doi.org/10.1016/j.bpj.2021.10.024.

    Article  Google Scholar 

  31. Johansson, M. E., and G. C. Hansson. Immunological aspects of intestinal mucus and mucins. Nat. Rev. Immunol. 16(10):639–649, 2016.

    Article  Google Scholar 

  32. Johansson, M. E. V., J. M. H. Larsson, and G. C. Hansson. The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host–microbial interactions. Proc. Natl. Acad. Sci. 108(Supplement 1):4659, 2011. https://doi.org/10.1073/pnas.1006451107.

    Article  Google Scholar 

  33. Johansson, M. E., M. Phillipson, J. Petersson, A. Velcich, L. Holm, and G. C. Hansson. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc. Natl Acad. Sci. 105(39):15064–15069, 2008.

    Article  Google Scholar 

  34. Karam, S. M. Lineage commitment and maturation of epithelial cells in the gut. Front. Biosci. 4:D286–D298, 1999. https://doi.org/10.2741/karam.

    Article  Google Scholar 

  35. Kernéis, S., A. Bogdanova, J.-P. Kraehenbuhl, and E. Pringault. Conversion by Peyer’s patch lymphocytes of human enterocytes into M cells that transport bacteria. Science. 277(5328):949–952, 1997. https://doi.org/10.1126/science.277.5328.949.

    Article  Google Scholar 

  36. Krupa, L., B. Bajka, R. Staroń, et al. Comparing the permeability of human and porcine small intestinal mucus for particle transport studies. Sci. Rep. 10(1):20290, 2020. https://doi.org/10.1038/s41598-020-77129-4.

    Article  Google Scholar 

  37. Lai, S. K., Y.-Y. Wang, and J. Hanes. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv. Drug Deliv. Rev. 61(2):158–171, 2009. https://doi.org/10.1016/j.addr.2008.11.002.

    Article  Google Scholar 

  38. Lai, S. K., Y.-Y. Wang, D. Wirtz, and J. Hanes. Micro- and macrorheology of mucus. Adv. Drug Deliv. Rev. 61(2):86–100, 2009. https://doi.org/10.1016/j.addr.2008.09.012.

    Article  Google Scholar 

  39. Lea, T., et al. Caco-2 Cell Line. In: The Impact of Food Bioactives on Health: In Vitro and Ex Vivo Models, edited by K. Verhoeckx, P. Cotter, and I. López-Expósito, et al. Cham: Springer International Publishing, 2015, pp. 103–111.

    Google Scholar 

  40. Leal, J., H. D. C. Smyth, and D. Ghosh. Physicochemical properties of mucus and their impact on transmucosal drug delivery. Int. J. Pharm. 532(1):555–572, 2017. https://doi.org/10.1016/j.ijpharm.2017.09.018.

    Article  Google Scholar 

  41. Lesuffleur, T., N. Porchet, J.-P. Aubert, et al. Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations. J. Cell Sci. 106(3):771–783, 1993.

    Article  Google Scholar 

  42. Lock, J. Y., T. L. Carlson, and R. L. Carrier. Mucus models to evaluate the diffusion of drugs and particles. Adv. Drug Deliv. Rev. 124:34–49, 2018. https://doi.org/10.1016/j.addr.2017.11.001.

    Article  Google Scholar 

  43. Lock, J., T. Carlson, C.-M. Wang, A. Chen, and R. Carrier. Acute exposure to commonly ingested emulsifiers alters intestinal mucus structure and transport properties. Sci. Rep. 2018. https://doi.org/10.1038/s41598-018-27957-2.

    Article  Google Scholar 

  44. Lozoya-Agullo, I., F. Araújo, I. González-Álvarez, et al. Usefulness of Caco-2/HT29-MTX and Caco-2/HT29-MTX/Raji B coculture models to predict intestinal and colonic permeability compared to Caco-2 monoculture. Mol. Pharm. 14(4):1264–1270, 2017. https://doi.org/10.1021/acs.molpharmaceut.6b01165.

    Article  Google Scholar 

  45. Macierzanka, A., A. R. Mackie, and L. Krupa. Permeability of the small intestinal mucus for physiologically relevant studies: impact of mucus location and ex vivo treatment. Sci. Rep. 9(1):17516, 2019. https://doi.org/10.1038/s41598-019-53933-5.

    Article  Google Scholar 

  46. Mahler, G. J., M. L. Shuler, and R. P. Glahn. Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability. J. Nutr. Biochem. 20(7):494–502, 2009. https://doi.org/10.1016/j.jnutbio.2008.05.006.

    Article  Google Scholar 

  47. Maisel, K., M. Reddy, Q. Xu, et al. Nanoparticles coated with high molecular weight PEG penetrate mucus and provide uniform vaginal and colorectal distribution in vivo. Nanomedicine (London, England). 11(11):1337–1343, 2016. https://doi.org/10.2217/nnm-2016-0047.

    Article  Google Scholar 

  48. Mason, T. G. Estimating the viscoelastic moduli of complex fluids using the generalized Stokes-Einstein equation. Rheologica Acta. 39(4):371–378, 2000.

    Article  Google Scholar 

  49. Mason, T. G., and D. A. Weitz. Optical measurements of frequency-dependent linear viscoelastic moduli of complex fluids. Phys. Rev. Lett. 74(7):1250, 1995.

    Article  Google Scholar 

  50. Nance, E. A., G. F. Woodworth, K. A. Sailor, et al. A dense poly(ethylene glycol) coating improves penetration of large polymeric nanoparticles within brain tissue. Sci. Transl. Med. 4(149):149ra119, 2012. https://doi.org/10.1126/scitranslmed.3003594.

    Article  Google Scholar 

  51. Navabi, N., M. A. McGuckin, and S. K. Lindén. Gastrointestinal cell lines form polarized epithelia with an adherent mucus layer when cultured in semi-wet interfaces with mechanical stimulation. PLoS ONE.8(7):e68761, 2013. https://doi.org/10.1371/journal.pone.0068761.

    Article  Google Scholar 

  52. Pan, F., L. Han, Y. Zhang, Y. Yu, and J. Liu. Optimization of Caco-2 and HT29 co-culture in vitro cell models for permeability studies. Int. J. Food Sci. Nutr. 66(6):680–685, 2015. https://doi.org/10.3109/09637486.2015.1077792.

    Article  Google Scholar 

  53. Paone, P., and P. D. Cani. Mucus barrier, mucins and gut microbiota: the expected slimy partners? Gut. 69(12):2232, 2020. https://doi.org/10.1136/gutjnl-2020-322260.

    Article  Google Scholar 

  54. Pontier, C., J. Pachot, R. Botham, B. Lenfant, and P. Arnaud. HT29-MTX and Caco-2/TC7 monolayers as predictive models for human intestinal absorption: role of the mucus layer. J. Pharm. Sci. 90(10):1608–1619, 2001. https://doi.org/10.1002/jps.1111.

    Article  Google Scholar 

  55. Srinivasan, B., A. R. Kolli, M. B. Esch, H. E. Abaci, M. L. Shuler, and J. J. Hickman. TEER measurement techniques for in vitro barrier model systems. J. Lab. Autom. 20(2):107–126, 2015. https://doi.org/10.1177/2211068214561025.

    Article  Google Scholar 

  56. Swidsinski, A., Y. Dörffel, V. Loening-Baucke, et al. Reduced mass and diversity of the colonic microbiome in patients with multiple sclerosis and their improvement with ketogenic diet Original Research. Front. Microbiol. 8:225, 2017. https://doi.org/10.3389/fmicb.2017.01141.

    Article  Google Scholar 

  57. Ude, V. C., D. M. Brown, V. Stone, and H. J. Johnston. Using 3D gastrointestinal tract in vitro models with microfold cells and mucus secreting ability to assess the hazard of copper oxide nanomaterials. J. Nanobiotechnol. 17(1):70, 2019. https://doi.org/10.1186/s12951-019-0503-1.

    Article  Google Scholar 

  58. Wu, S., M. Wu, D. Tian, L. Qiu, and T. Li. Effects of polystyrene microbeads on cytotoxicity and transcriptomic profiles in human Caco-2 cells. Environ. Toxicol. 35(4):495–506, 2020. https://doi.org/10.1002/tox.22885.

    Article  Google Scholar 

  59. Yee, S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man–fact or myth. Pharm. Res. 14(6):763–766, 1997. https://doi.org/10.1023/a:1012102522787.

    Article  Google Scholar 

  60. Yildiz, H. M., C. A. McKelvey, P. J. Marsac, and R. L. Carrier. Size selectivity of intestinal mucus to diffusing particulates is dependent on surface chemistry and exposure to lipids. J Drug Target. 23(7–8):768–774, 2015. https://doi.org/10.3109/1061186X.2015.1086359.

    Article  Google Scholar 

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Acknowledgments

We would like to thank the UMD Bioworkshop for core facility usage and Michele Kaluzienski for assisting with manuscript preparation. This work was funded by the University of Maryland New Directions Fund and Faculty-Student Research Award (KM) and the AHA Predoctoral Research Fellowship (JM).

Conflict of Interest

The authors (JM, AS, and KM) have no conflict of interest to declare.

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No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

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  1. Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA

    Jacob McCright, Arnav Sinha & Katharina Maisel

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  1. Jacob McCright
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Correspondence to Katharina Maisel.

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McCright, J., Sinha, A. & Maisel, K. Generating an In Vitro Gut Model with Physiologically Relevant Biophysical Mucus Properties. Cel. Mol. Bioeng. 15, 479–491 (2022). https://doi.org/10.1007/s12195-022-00740-0

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