In the current application, a collaborative, multidisciplinary research project is proposed that has the potential to create a new paradigm for the study of human physiology in health and disease. A state-of-the-art microfabricated platform will be developed to create a functional, in vitro replica, i.e. simulacrum, of the human colonic epithelium and its associated microbiome. This new technology will be used to perform novel studies and hypothesis testing of intestinal physiology that cannot currently be performed. Furthermore, the technology and protocols developed here will establish the basis for creating organ simulacra from normal and diseased primary human tissues that will change the manner in which studies of tissue function, microbiome influence, and drug effect are performed. Recent progress in organotypic culture of colonic epithelial stem cells has made it possible to create long-lived spheroids in a gelatinous matrix. However, the absence of chemical gradients as well as patterned physical support results in a disorganized and chaotic conformation that poorly mimics the structure and function of the colon, and furthermore is not amenable to microbiome co-culture. We propose to develop a novel microfabricated system that will enable ex vivo culture of human colonic epithelium and overlying microbiota that recapitulates the 3D structure and environment of the colonic mucosa in a setting which lies between an unpolarized cell culture system and the complexity of the intact human organism. Recent technical advances from our labs in sustained monolayer culture of colonic epithelial stem cells will be integrated with microfabricated scaffoldings and devices to create the colonic simulacrum. A number of innovations will be incorporated into the microengineered system with the goal of recapitulating the colonic stem-cell niche, the differentiated intestinal mucosa, the microbiota, and the dynamic information flow between these compartments. The platform will permit tight control of the luminal and basal crypt environments by providing independent fluidic and gaseous access to these compartments. The platform will support formation of mitogen, morphogen, differentiation-factor, dietary-compound and gaseous gradients to enable unprecedented investigations into colonic physiology. A number of hypotheses related to the interplay of dietary factors, the microbiome and the colonic epithelium will be tested to demonstrate the power and broad applicability of this transformative technology.

Public Health Relevance

The goal of this project is to develop a platform that enables a transformative approach to the study of the colon in health and disease. Assays of dietary and other factors in the setting of human tissue and intestinal bacteria will enable study of stem-cell self-renewal, differentiation and colon physiology using cells derived from human tissue. This technology will establish a new paradigm to understand the influence of diet and bacteria on intestinal and digestive diseases enhancing our understanding of the growth and development of the colon.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-A (51))
Program Officer
Carrington, Jill L
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University of North Carolina Chapel Hill
Schools of Arts and Sciences
Chapel Hill
United States
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Wang, Yuli; Kim, Raehyun; Gunasekara, Dulan B et al. (2018) Formation of Human Colonic Crypt Array by Application of Chemical Gradients Across a Shaped Epithelial Monolayer. Cell Mol Gastroenterol Hepatol 5:113-130
Bhatt, Aadra P; Gunasekara, Dulan B; Speer, Jennifer et al. (2018) Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells. ACS Infect Dis 4:46-52
Wang, Yuli; Kim, Raehyun; Hinman, Samuel S et al. (2018) Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche. Cell Mol Gastroenterol Hepatol 5:440-453.e1
Dutton, Johanna S; Hinman, Samuel S; Kim, Raehyun et al. (2018) Primary Cell-Derived Intestinal Models: Recapitulating Physiology. Trends Biotechnol :
Gunasekara, Dulan B; Speer, Jennifer; Wang, Yuli et al. (2018) A Monolayer of Primary Colonic Epithelium Generated on a Scaffold with a Gradient of Stiffness for Drug Transport Studies. Anal Chem 90:13331-13340
Kim, Raehyun; Wang, Yuli; Hwang, Shee-Hwan J et al. (2018) Formation of arrays of planar, murine, intestinal crypts possessing a stem/proliferative cell compartment and differentiated cell zone. Lab Chip 18:2202-2213
Williamson, Ian A; Arnold, Jason W; Samsa, Leigh Ann et al. (2018) A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology. Cell Mol Gastroenterol Hepatol 6:301-319
Gunasekara, Dulan B; DiSalvo, Matthew; Wang, Yuli et al. (2018) Development of Arrayed Colonic Organoids for Screening of Secretagogues Associated with Enterotoxins. Anal Chem 90:1941-1950
Wang, Yuli; Kim, Raehyun; Hwang, Shee-Hwan J et al. (2018) Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform. Anal Chem 90:11523-11530
Wang, Yuli; DiSalvo, Matthew; Gunasekara, Dulan B et al. (2017) Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells. Cell Mol Gastroenterol Hepatol 4:165-182.e7

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