383100 Biomimetic Topographical Replicas of Small Intestine for Regenerative Medicine and Drug Delivery Platforms

Tuesday, November 18, 2014
Galleria Exhibit Hall (Hilton Atlanta)
Abigail Koppes1, Robert Montgomery2,3, Megha Kamath1, David Breault2,3 and Rebecca L. Carrier1, (1)Chemical Engineering, Northeastern University, Boston, MA, (2)Division of Endocrinology, Children's Hospital, Boston, MA, (3)Department of Pediatrics, Harvard Medical School, Boston, MA

Introduction: Diseases and disorders of the intestinal tract affect millions of people worldwide each year. Inflammatory bowel disease (IBD) represents the leading chronic gastrointestinal disease with increasing healthcare burdens and expenditures [1]. There are over 1.4 million persons living with IBD in the U.S. alone, and as there is no cure, this number increases yearly indicating a need for new therapeutic strategies [2]. Current in vitro models of IBD utilize 2D systems and Caco-2 epithelial cell lines with a limited ability to mimic complex tissue. Native small intestine possesses distinct structures (e.g., crypts-villi) and a heterogeneous cell population (stem, absorptive, goblet, enteroendocrine, and paneth cells). Therefore, we propose to develop functional engineered small intestine with relevant heterogeneous cell populations and spatial organization required for successful regenerative medicine and drug discovery platforms. Recent breakthroughs have enabled long-term primary intestinal culture in the form of enteroids, but size limitations and lack of access to luminal surfaces limits practical applications [3]. Recent work also suggests cell fate is impacted by 3D features and local topography/stiffness [4,5]. Therefore, we hypothesize that incorporating the 3D topography of native intestine into growth substrates with tunable mechanical and adhesion properties will enable dissociated native primary enteroid spatial organization similar to native tissue in a scalable format.

Materials and Methods: To fabricate structurally biomimetic growth substrates, fresh porcine small intestine was rinsed, fixed with 1% GTA: 1% PFA in 0.1M PBS (4C, 24hrs), sectioned (1cm^2), placed in 0.1% OsO4 in 0.1M PBS (4C, 72hrs), and agitated to remove the epithelium. Samples were rinsed, ethanol dehydrated, critically point dried, treated with silane (APTS, 12 hrs), and 10:1 v:v PDMS was solution cast for 12hrs followed by baking (60C, 2hrs). The PDMS negative mold was released from the tissue via soaking in TEA (12hrs, 25C), and rinsed in 100% ethanol (25C, 24hrs) and DIH2O (25C, 24hrs), prior to drying (75C, 2hrs). Samples were visualized with a Hitachi S-4800 UHR field emission SEM [6]. Collagen-I:10% Matrigel (v:v) hydrogels were cast into 24-well plates with the PDMS negative replicas or flat surfaces and allowed to gel (2 hrs, 37oC). Primary mouse enteroids from isolated crypts [7] were dissociated and seeded on replica hydrogels or flat controls for 5 days under standard culture conditions. On day 6, samples were incubated with EdU for 24 hrs. Samples were then fixed, stained for alpha-tubulin cytoskeleton and nuclei (Hoechst), and imaged with a Nikon A1R confocal at 20x.

Results and Discussion: Native small intestinal tissue molds were used as templates to fabricate biomimetic PDMS and hydrogel replicas with irregular, but spatially organized multi-scale topographical features. Fixed native tissue and topographical replicas show micro to nano-scale features, containing irregular, but spatially organized crypt-villus structures and smaller features of the epithelial basement membrane (not shown). Preliminary qualitative results indicate that cells positive for EdU (indicating DNA synthesis) are present in higher amounts on topographical replicas compared to flat hydrogel controls (Fig. 1). On topographical replicas, the proliferating cells appear in dense clusters with satellite EdU+ cells throughout the population, whereas on flat substrates there are sparse EdU+ cells throughout the monolayer. Further, microtubules appear to localize to the apical surface, indicating polarity, on replicas but not flat controls. Previous work in our lab has show that crypt-like structures aid Caco-2 differentiation 8,9, indicating the importance of topography in epithelial differentiation and similar effects may be apparent for primary enteroids.

Conclusions: Manipulating primary enteroid spatial organization and differentiation into functional models of native small intestine via presentation of biomimetic topographical cues may provide a better system for regenerative medicine and drug delivery and discovery applications. This work shows the ability to engineer growth substrates that mimic the native intestinal multi-scale topography, and these substrates may impact primary enteroid cell morphology and proliferation. Future work will determine the impact of physical properties (e.g., stiffness), chemical properties (e.g., substrate) and structure (e.g., intestinal topography) on cell behavior (e.g., attachment, signaling, differentiation and spatial organization) for a molecular understanding of how biomimetic topography impacts phenotypic changes.

Acknowledgements: Northeastern STEM Future Faculty Fellowship (ANK), and NIH R21EY021312 (RLK).

References: 1) Park K, Inflamm Bowel Dis. 2011;17(7):1603-1609. 2) Donohoe CL, Reynolds JV. The Surgeon. 2010;8(5):270-279. 3) Sato T, Gastroenterology. 2011;141(5):1762-1772. 4) Evans ND, Eur Cell Mater. 2009;18(1-13):13-14. 5) Reilly GC J Biomech. 2010;43(1):55-62. 6) Pfluger CA, Biomacromol. 2010;11(6):1579–1584. 7) Sato T, Nature. 2009;459(7244):262-265. 8) Wang L, Biomaterials. 2010;31(29):7586-7598. 9) Wang L, Biomaterials. Dec 2009;30(36):6825-6834.

Fig 1. Primary enterocytes, derived from dissociated mouse enteroids, exhibit increased proliferation (green) on hydrogels with topographical features (right) of small intestine compared to flat controls (left). Bar = 10 um, Max projection with orthogonal views. Blue= Hoechst nuclei, Red=alpha-tubulin, Green=EdU.

Text Box: Fig 1. Primary enterocytes, derived from dissociated mouse enteroids, exhibit increased proliferation (green) on hydrogels with topographical features (right) of small intestine compared to flat controls (left). Bar = 10 um, Max projection with orthogonal views. Blue= Hoechst nuclei, Red=alpha-tubulin, Green=EdU.


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