Bioengineered intestinal tubules as a tool to test intestinal biological efficacy of lettuce species.

Lettuce (Lactuca sativa) is one of the most consumed and cultivated vegetables globally. Its breeding is focused on the improvement of yield and disease resistance. However, potential detrimental or beneficial health effects for the consumer are often not targeted in the breeding programs. Here, a bioengineered intestinal tubule was used to assess the intestinal efficacy of extracts from five plant accessions belonging to four Lactuca species. These four species include the domesticated L. sativa, closely related wild species L. serriola, and phylogenetically more distant wild relatives L. saligna and L. virosa. We assessed the epithelial barrier integrity, cell viability, cell attachment, brush border enzyme activity, and immune markers. Extracts from L. sativa cv. Salinas decreased cell attachment and brush border enzyme activity. However, extracts from the non-edible wild species L. saligna and L. virosa reduced the epithelial barrier functions, cell attachment, cell viability, and brush border enzyme activity. Since wild species represent a valuable germplasm pool, the bioengineered intestinal tubules could open ways to evaluate the safety and nutritional properties of the lettuce breeding material originating from crosses with wild Lactuca species.

A combined microphysiological-computational omics approach in dietary protein evaluation.

Food security is under increased pressure due to the ever-growing world population. To tackle this, alternative protein sources need to be evaluated for nutritional value, which requires information on digesta peptide composition in comparison to established protein sources and coupling to biological parameters. Here, a combined experimental and computational approach is presented, which compared seventeen protein sources with cow's whey protein concentrate (WPC) as the benchmark. In vitro digestion of proteins was followed by proteomics analysis and statistical model-based clustering. Information on digesta peptide composition resulted in 3 cluster groups, primarily driven by the peptide overlap with the benchmark protein WPC. Functional protein data was then incorporated in the computational model after evaluating the effects of eighteen protein digests on intestinal barrier integrity, viability, brush border enzyme activity, and immune parameters using a bioengineered intestine as microphysiological gut system. This resulted in 6 cluster groups. Biological clustering was driven by viability, brush border enzyme activity, and significant differences in immune parameters. Finally, a combination of proteomic and biological efficacy data resulted in 5 clusters groups, driven by a combination of digesta peptide composition and biological effects. The key finding of our holistic approach is that protein source (animal, plant or alternative derived) is not a driving force behind the delivery of bioactive peptides and their biological efficacy.

Novel Dietary Proteins Selectively Affect Intestinal Health In Vitro after Clostridium difficile-Secreted Toxin A Exposure.

Bacterial gastroenteritis forms a burden on a global scale, both socially and economically. The Gram-positive bacterium Clostridium difficile is an inducer of gastrointestinal bacterial infections, often triggered following disruption of the microbiota by broad-spectrum antibiotics to treat other conditions. The clinical manifestatiaons, e.g., diarrhea, are driven by its toxins secretion, toxin A (TcdA) and toxin B (TcdB). Current therapies are focused on discontinuing patient medication, including antibiotics. However, relapse rates upon therapy are high (20-25%). Here, eighteen dietary proteins were evaluated for their capacity to restore gut health upon C. difficile-derived TcdA exposure. We used bioengineered intestinal tubules to assess proteins for their beneficial effects by examining the epithelial barrier, cell viability, brush-border enzyme activity, IL-6 secretion, IL-8 secretion and nitric oxide (NO) levels upon TcdA challenge. TcdA effectively disrupted the epithelial barrier, increased mitochondrial activity, but did not affect alkaline phosphatase activity, IL-6, IL-8 and NO levels. Intervention with dietary proteins did not show a protective effect on epithelial barrier integrity or mitochondrial activity. However, bovine plasma and potato protein increased alkaline phosphatase activity, egg-white protein increased IL-6 and IL-8 release and wheat, lesser mealworm and yeast protein increased NO levels after TcdA exposure. Hence, dietary proteins can influence parameters involved in intestinal physiology and immune activation suggesting that supplementation with specific dietary proteins may be of benefit during C. difficile infections.

Development and validation of bioengineered intestinal tubules for translational research aimed at safety and efficacy testing of drugs and nutrients.

Currently used intestinal cell models have limited translational value, therefore, development of novel in vitro intestinal models that recapitulate the human in vivo setting more closely are of interest. Here, an advanced intestinal model was developed by the incorporation of physiological parameters, such as extracellular matrix (ECM) elements and shear stress, to cultured Caco-2 cells in a 3-dimensional environment. Caco-2 cells grown on ECM-coated hollow fiber membranes (HFM) under physiological shear stress show an improved phenotype, as demonstrated by the presence of enterocytes, goblet, Paneth, enteroendocrine and stem cells. Additionally, this model showed signs of an improved morphology due to the appearance of villi-like structures. Similar to epithelial cells grown on Transwells™, the current model remains easy to use, cost efficient and allows apical and basolateral access. The bioengineered intestinal tubule was validated by exposure to Clostridium difficile toxin A, the leading cause of healthcare-associated diarrhea. The loss of the tight junction network was supported by an increase in inulin-FITC leakage and the number of goblet cells increased, in agreement with clinical findings. In addition to toxicity screening, the bioengineered intestinal tubules are considered useful for drug and nutrient safety and efficacy testing.

Bioengineered bile ducts recapitulate key cholangiocyte functions.

Investigation of diseases of the bile duct system and identification of potential therapeutic targets are hampered by the lack of tractable in vitro systems to model cholangiocyte biology. Here, we show a step-wise method for the differentiation of murine Lgr5+ liver stem cells (organoids) into cholangiocyte-like cells (CLCs) using a combination of growth factors and extracellular matrix components. Organoid-derived CLCs display key properties of primary cholangiocytes, such as expressing cholangiocyte markers, forming primary cilia, transporting small molecules and responding to farnesoid X receptor agonist. Integration of organoid-derived cholangiocytes with collagen-coated polyethersulfone hollow fiber membranes yielded bioengineered bile ducts that morphologically resembled native bile ducts and possessed polarized bile acid transport activity. As such, we present a novel in vitro model for studying and therapeutically modulating cholangiocyte function.

Evaluating Human Intestinal Cell Lines for Studying Dietary Protein Absorption.

With the global population rising, the need for sustainable and resource-efficiently produced proteins with nutritional and health promoting qualities has become urgent. Proteins are important macronutrients and are involved in most, if not all, biological processes in the human body. This review discusses these absorption mechanisms in the small intestine. To study intestinal transport and predict bioavailability, cell lines are widely applied as screening models and often concern Caco-2, HT-29, HT-29/MTX and T84 cells. Here, we provide an overview of the presence and activities of peptide- and amino acid transporters in these cell models. Further, inter-laboratory differences are discussed as well as the culture micro-environment, both of which may influence cell culture phenotype and performance. Finally, the value of new developments in the field, including culturing cells in 3-dimensional systems under shear stress (i.e., gut-on-chips), is highlighted. In particular, their suitability in screening novel food proteins and prediction of the nutritional quality needed for inclusion in the human diet of the future is addressed.