Methods to enrich enteroendocrine cells and their subtypes in the contiguous, intestinal monolayer systems

The method of any of the above claims, wherein the thin layer of fluid at the upper surface is maintained in a range of about 0.001 mm to about 10 mm, optionally about 0.001 mm to about 1 mm, above the luminal side of the cell monolayer.

The method of any of the above claims, wherein the thin layer of fluid at the upper surface is maintained by adding hormones and/or compounds to the medium composition to induce luminal fluid liquid secretion, optionally wherein the hormones and/or compounds comprise chemicals (e.g. ionomycin), hormones (e.g. vasoactive intestinal peptide, serotonin, gastrin, etc.), cytokines, metabolites, bacteria and/or bacteria components, optionally further adding hypertonic medium to the luminal side to stimulate fluid secretion towards the luminal side to create a thick mucus layer.

The method of any of the above claims, further comprising adding chemical compounds (e.g., diterpenoid (e.g., forskolin), Methylxanthines (e.g., IBMX)), prebiotics (e.g., oligofructose and inulin-type fructans), hormone (e.g., serotonin, arginine vasopressin, angiotensin II), receptor agonists (e.g., BIMU8, Liraglutide (Saxenda), AM1638 (e.g., FFAR1 agonist), AR231453 (e.g., GPR119 agonist), and a GPBAR1 agonist), cytokines (e.g., IL-10, IL-6, TNF-α), bacteria or food metabolites (e.g., acetate, propionate and butyrate), bacteria and/or bacteria components (e.g., LPS, Akkermansia muciniphila, Bifidobacterium spp., Lactobacillus spp.) to the live cell construct to increase the density of the EEC cells.

The method of any of the above claims, further comprising adding chemical compounds (e.g., diterpenoid (e.g., forskolin), Methylxanthines (e.g., IBMX)), prebiotics (e.g., oligofructose and inulin-type fructans), hormone (e.g., serotonin, arginine vasopressin, angiotensin II), receptor agonists (e.g., BIU8, Liraglutide (Saxenda), AM1638 (e.g., FFAR1 agonist), AR231453 (e.g., GPR119 agonist), and a GPBAR1 agonist), cytokines (e.g., IL-10, IL-6, TNF-α), bacteria or food metabolites (e.g., acetate, propionate and butyrate), bacteria and/or bacteria components (e.g., LPS, Akkermansia muciniphila, Bifidobacterium spp., Lactobacillus spp.) to the live cell construct to mimic differentiation stages of EEC cells to further increase a density of the EEC cells.

The method of any of the above claims, wherein the thin layer of fluid is maintained by a microfluidic flow setup.

The method of any of the above claims, wherein high barrier integrity is maintained.

The method of any of the above claims, wherein the EEC cells secrete serotonin.

The method of any of the above claims, wherein the EEC cells secrete glucagon-like peptide-1 (GLP-1).

The method of any of the above claims, wherein the EEC cells secrete peptide YY (PYY), or other intestinal hormones including cholecystokinin, motilin, neurotensin, leptin and/or secretin.

The method of any of the above claims, wherein the EEC cells comprise L-cells.

The method of any of the above claims, wherein the method is used for screening of drug, metabolite, foodstuff or compound-induced secretion of hormones from EEC cells.

The method of any of the above claims, wherein the method is used for screening of drug, metabolite, foodstuff or compounds that block secretion of hormones from EEC cells.

The method of any of the above claims, wherein the method is used for screening of drug, metabolite, foodstuff or compounds that potentiate secretion of hormones from EEC cells.

The method of any of the above claims, wherein the gastrointestinal epithelial cells are selected from the group consisting of mammalian, avian, reptilian, amphibian, and insect cells.

The method of any of the above claims, wherein the gastrointestinal epithelial cells are human gastrointestinal epithelial cells.

The method of any of the above claims, wherein the gastrointestinal epithelial cells are selected from the group consisting of colon, small intestine, stomach, esophagus, tongue, nasopharynx, oropharynx, laryngeopharynx, and pancreatic epithelial cells.

The method of any of the above claims, further comprising adding one or more compounds in an expansion medium to prevent early lineage-fate decision of stem cells during a proliferation stage, optionally wherein the formation of EEC cells can be further enhanced by addition of the one or more compounds in the expansion medium.

A live cell construct produced by the method of any of the above claims, wherein the live cell construct comprises a substantially continuous cell monolayer comprising EEC cells and subtypes of EEC cells.

A live cell culture system, the system comprising:

• a cell support structure, the cell support structure having both an upper surface and a lower surface, the cell support structure further comprising a porous carrier on the upper surface,
• a culture vessel housing the cell support structure and providing a contained area for a culture medium, wherein the culture medium is contained below the lower surface of the cell support structure,
• wherein the cell support structure is configured to generate a live cell construct from stem cells seeded on the cell support structure, the system configured to cause the stem cells to differentiate into enteroendocrine (EEC) cells by maintaining a thin layer of fluid at the upper surface of the support structure, wherein the thin layer of fluid at the upper surface is maintained by hormones and/or compounds in the culture medium contained in the culture vessel inducing luminal fluid liquid secretion.