(W1230-10-66) A Novel Approach to Harvest Human Mucus on RepliGut® Planar Platform for Research Related to Gastrointestinal Tract and Its Associated Disorders, along with Drug Delivery Studies

Purpose: The mucus layer that lines the entirety of the gastrointestinal (GI) tract is a highly dynamic matrix and is essential for innate immunity. Mucus serves as the first line of defense by acting as a barrier against various pathogens, toxins, and foreign particles. This barrier function makes it an important factor to consider for drug delivery studies. Mucus thickness and rheology has been shown to be affected by various gastrointestinal, metabolic, and neurological disorders, including cystic fibrosis and Crohn’s Disease ^1,2,3,4, and plays a significant role in host defense, cancer progression, and bioavailability of therapeutics. Although a key component of many health issues, the ability to study the role of mucus in these pathological and homeostatic conditions is hampered by the difficulty in isolating mucus from intestine and the confounding effects of intestinal microbiota that are vastly variable between animals and people. To overcome this obstacle, we have used the primary intestinal stem-cell derived model, RepliGut® Planar, in air-liquid interface (ALI) format to manufacture sterile human intestinal mucus that is both antibiotic and bacteria free.

Methods: Human intestinal tissue was obtained post-mortem via an established Organ Procurement Organization following consent of family under strict ethical guidelines established by the Organ Procurement Transplantation Network (OPTN). Mucus secreted from the surface of transverse colon human intestinal cells in ALI cultures was collected. Detailed characterization studies including percent solids, proteomics, macro- and microscopic rheology, and atomic force microscopy were performed.

Results: Proteomics data revealed that MUC2 and MUC5AC are the top two abundant proteins in the cell-derived human mucus along, followed byother protein components such as MUC5B and TFF3 that are essential for mucus stability and maintenance. Rheology studies indicated that the mucus viscoelasticity is similar to ex-vivo human intestinal scrapes. Atomic force microscopy revealed a compact mesh-like structure with bright protein nodes indicative of MUC2 protein crosslinking as well as linear strands indicative of MUC5AC/MUC5B. We have successfully generated approximately 20 mL per batch of mucus and have assessed variability between runs and observed consistency in the protein content.

Conclusion: We have developed a new approach to obtaining human intestinal mucus by taking advantage of an in vivo-like stem-cell derived intestinal tissue construct, enabling refined studies of this important biofluid for interactions with microbes, nutrients, and therapeutics important in GI disease manifestation.

References: 1. Strugala V, Dettmar PW, Pearson JP. Thickness and continuity of the adherent colonic mucus barrier in active and quiescent ulcerative colitis and Crohn's disease. Int J Clin Pract. 2008 May;62(5):762-9. doi: 10.1111/j.1742-1241.2007.01665.x. Epub 2008 Jan 8. PMID: 18194279.
2. Sun J, Shen X, Li Y, Guo Z, Zhu W, Zuo L, Zhao J, Gu L, Gong J, Li J. Therapeutic Potential to Modify the Mucus Barrier in Inflammatory Bowel Disease. Nutrients. 2016 Jan 14;8(1):44. doi: 10.3390/nu8010044. PMID: 26784223; PMCID: PMC4728657.
3. A. E. Dorofeyev, I. V. Vasilenko, O. A. Rassokhina, R. B. Kondratiuk, "Mucosal Barrier in Ulcerative Colitis and Crohn’s Disease", Gastroenterology Research and Practice, vol. 2013, Article ID 431231, 9 pages, 2013. https://doi.org/10.1155/2013/431231
4. Schreiber O, Petersson J, Waldén T, Ahl D, Sandler S, Phillipson M, et al. (2013) iNOS-Dependent Increase in Colonic Mucus Thickness in DSS-Colitic Rats. PLoS ONE 8(8): e71843. https://doi.org/10.1371/journal.pone.0071843