Physiology and pathophysiology of human gut: intestine-on-chip
Inventors
Apostolou, Athanasia • Varone, Antonio • Kasendra, Magdalena • Luc, Raymond
Assignees
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Publication Number
US-12018284-B2
Publication Date
2024-06-25
Expiration Date
Abstract
An in vitro microfluidic intestine on-chip is described herein that mimics the structure and at least one function of specific areas of the gastrointestinal system in vivo. In particular, a multicellular, layered, microfluidic intestinal cell culture, which is some embodiments is derived from patient's enteroids-derived cells, is described comprising L cells, allowing for interactions between L cells and gastrointestinal epithelial cells, endothelial cells and immune cells. This in vitro microfluidic system can be used for modeling inflammatory gastrointestinal autoimmune tissue, e.g., diabetes, obesity, intestinal insufficiency and other inflammatory gastrointestinal disorders. These multicellular-layered microfluidic intestine on-chips further allow for comparisons between types of gastrointestinal tissues, e.g., small intestinal duodenum, small intestinal jejunum, small intestinal ileum, large intestinal colon, etc., and between disease states of gastrointestinal tissue, i.e. healthy, pre-disease and diseased areas. Additionally, these microfluidic gut-on-chips allow identification of cells and cellular derived factors driving disease states and drug testing for reducing inflammation.
Core Innovation
The invention relates to patient-derived, multicellular, layered microfluidic intestine/gut-on-chip platforms that include intestinal epithelial cells and endothelial cells, with immune-cell interaction capabilities. The platform models healthy versus diseased states and supports comparisons across intestinal regions, and it enables intestinal epithelial differentiation into multiple intestinal cell types within a microfluidic culture device having a membrane with opposing first and second surfaces.
The platform uses intestinal enteroids or colonoids comprising human primary intestinal epithelial cells that are disrupted into enteroid or colonoid fragments, seeded on a first membrane surface to create seeded primary intestinal epithelial cells, expanded to form an epithelial monolayer, and differentiated into two or more different differentiated intestinal cell types, including L-cells. The document also characterizes ileal and duodenum Intestine-Chips containing human microvasculature endothelial cells and reports major intestinal epithelial lineages, including absorptive enterocytes, goblet cells, enteroendocrine subtypes such as L-cells and enterochromaffin cells, and Paneth cells.
The system further establishes seeded endothelial cells on a second membrane surface and enables immune recruitment through intestine-specific microvascular endothelial cells, with assays described for immune-cell adhesion and recruitment under flow. The document reports barrier function stability, physiological cell-type ratios, improved maturation of absorptive and enteroendocrine markers, reduced stemness/proliferation signals, and improved epithelial barrier formation and morphology under dynamic flow and cyclic stretch.
Claims Coverage
The partial claims provided include 1 independent claim. The independent claim includes 4 inventive features centered on a membrane-based microfluidic device seeded with intestinal enteroid/colonoid fragments and endothelial cells, followed by monolayer expansion and differentiation into multiple intestinal cell types including L-cells.
Membrane-based microfluidic co-culture of intestinal epithelial monolayer and endothelial cells
A method of culturing intestinal cells in vitro using a microfluidic culture device with a cell growth region comprising a membrane having a first surface and a second surface on opposing sides, wherein intestinal enteroid or colonoid fragments are seeded on the first surface and endothelial cells are seeded on the second surface.
Enteoid/colonoid fragment seeding to form seeded primary intestinal epithelial cells
Disrupting an intestinal enteroid or colonoid comprising human primary intestinal epithelial cells into enteroid or colonoid fragments and seeding the fragments on the first surface to create seeded primary intestinal epithelial cells.
Epithelial monolayer expansion and differentiation into multiple intestinal cell types including L-cells
Expanding the seeded primary intestinal epithelial cells to create a monolayer of epithelial cells and differentiating the monolayer of epithelial cells to create two or more different differentiated intestinal cell types, wherein one of the two or more different differentiated intestinal cell types comprises L-cells.
Delayed endothelial cell seeding on opposing membrane surface
Seeding endothelial cells on the second surface so as to create seeded endothelial cells, wherein the endothelial cells are seeded 1-6 days after seeding the primary intestinal epithelial cells.
The independent claim covers a membrane-based microfluidic co-culture method in which intestinal enteroid/colonoid fragments are seeded to form and differentiate an epithelial monolayer into multiple intestinal cell types including L-cells, while endothelial cells are seeded on an opposing membrane surface after a defined delay.
Stated Advantages
Improved epithelial barrier formation and morphology under dynamic flow and cyclic stretch.
Improved longevity using a multi-step seeding strategy.
Improved maturation of absorptive and enteroendocrine markers on-chip compared with 3D ileal enteroids.
Reduced stemness/proliferation signals on-chip, including reduced LGR5 and Ki67.
Stable barrier function, including stability of 3 kDa dextran impermeability across multiple donors.
Physiological cell-type ratios by day-8.
Strong barrier and physiological ratios in duodenum Intestine-Chips.
Expression and localization of drug transporters and CYP3A4 and nuclear receptor (PXR/VDR) responses in duodenum Intestine-Chips.
Accelerated barrier formation upon inclusion of endothelial cells in Colon Intestine-Chip characterization.
Documented Applications
Drug testing on a multicellular intestinal gut-on-chip platform.
Inflammation modeling using the patient-derived intestinal gut-on-chip system.
Modeling healthy versus diseased states, including diabetes/obesity/IBD.
Comparisons across intestinal regions including duodenum, jejunum, ileum, and colon.
Tissue-tissue interface modeling using a duodenum Intestine-Chip, including epithelium morphology improvement with flow and flow plus stretch.
Modeling intestinal barrier function and barrier stability using a permeability assay with 3 kDa dextran across multiple donors.
Assessment of drug transporters and drug metabolism-related markers in duodenum Intestine-Chips, including drug transporters and CYP3A4 with nuclear receptors (PXR/VDR).
Comparative evaluation of ileal Intestine-Chip versus 3D ileal enteroids for epithelial lineage presence, abundance, and maturation kinetics using microscopy and mRNA/qPCR.
Colon Intestine-Chip characterization including accelerated barrier formation upon inclusion of endothelial cells and time-dependent maturation across donors.
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