Assessment of Electrospun Poly(ε-caprolactone) and Poly(lactic acid) Fiber Scaffolds to Generate 3D In Vitro Models of Colorectal Adenocarcinoma: A Preliminary Study.

Three-dimensional scaffold-based culture has been increasingly gaining influence in oncology as a therapeutic strategy for tumors with a high relapse percentage. This study aims to evaluate electrospun poly(ε-caprolactone) (PCL) and poly(lactic acid) (PLA) scaffolds to create a 3D model of colorectal adenocarcinoma. Specifically, the physico-mechanical and morphological properties of PCL and PLA electrospun fiber meshes collected at different drum velocities, i.e., 500 rpm, 1000 rpm and 2500 rpm, were assessed. Fiber size, mesh porosity, pore size distribution, water contact angle and tensile mechanical properties were investigated. Caco-2 cells were cultured on the produced PCL and PLA scaffolds for 7 days, demonstrating good cell viability and metabolic activity in all the scaffolds. A cross-analysis of the cell-scaffold interactions with morphological, mechanical and surface characterizations of the different electrospun fiber meshes was carried out, showing an opposite trend of cell metabolic activity in PLA and PCL scaffolds regardless of the fiber alignment, which increased in PLA and decreased in PCL. The best samples for Caco-2 cell culture were PCL500 (randomly oriented fibers) and PLA2500 (aligned fibers). Caco-2 cells had the highest metabolic activity in these scaffolds, with Young's moduli in the range of 8.6-21.9 MPa. PCL500 showed Young's modulus and strain at break close to those of the large intestine. Advancements in 3D in vitro models of colorectal adenocarcinoma could move forward the development of therapies for this cancer.

In Vitro Models of the Small Intestine: Engineering Challenges and Engineering Solutions.

Pathologies affecting the small intestine contribute significantly to the disease burden of both the developing and the developed world, which has motivated investigation into the disease mechanisms through in vitro models. Although existing in vitro models recapitulate selected features of the intestine, various important aspects have often been isolated or omitted due to the anatomical and physiological complexity. The small intestine's intricate microanatomy, heterogeneous cell populations, steep oxygen gradients, microbiota, and intestinal wall contractions are often not included in in vitro experimental models of the small intestine, despite their importance in both intestinal biology and pathology. Known and unknown interdependencies between various physiological aspects necessitate more complex in vitro models. Microfluidic technology has made it possible to mimic the dynamic mechanical environment, signaling gradients, and other important aspects of small intestinal biology. This review presents an overview of the complexity of small intestinal anatomy and bioengineered models that recapitulate some of these physiological aspects.

Toxicity of food contact paper evaluated by combined biological and chemical methods.

The study was focused on assessment of potential health risks of paper-based food contact materials (FCMs) in a step-wise approach using three toxicological bioassays in vitro and chemical analyses of migrating contaminants. 3T3 NRU cytotoxicity test showed high sensitivity to detect basal toxicity of FCMs extracts and served as a first-line test for selection of samples for further testing. The reconstructed human intestine model EpiIntestinal showed more realistic tissue response than cell culture monolayer and higher resistance despite prolonged exposure to the selected 6 samples, i.e. negligible decrease of viability and intestinal penetration, nevertheless an increase of IL-8 after exposure to black printed sample extract. Yeast based assays identified weak agonistic/antagonostic activity to human androgen receptor of the black printed sample. In accordance with the biological effects, the targeted LC and GC analytical methods confirmed the presence of high amounts of phthalates, photoinitiators and PAHs that could justify the hazard of the black printed sample. Heavily printed uncoated FCMs are recognized not to be suitable for direct contact with food. The selected bioassays and chemical analyses might be useful tools to detect targeted biological effects of xenobiotics suspected to contribute to human exposure from food.

µEvaluation of microcystin-LR absorption using an in vivo intestine model and its effect on zebrafish intestine.

Microcystin-LR (MC-LR) is regarded as one of the most toxic microcystins (MCs) isoforms. Microcystins could cause multiple organs dysfunction, and more attention has been drawn to the toxic effects on the gastrointestinal disorder. By using ex vivo everted gut sac model in 6 fish (Carassius auratus, Megalobrama amblycephala, Hypophthalmichthys molitrix, Aristichthys nobilis, Ctenopharyngodon idellus and Cyprinus carpio) and determining the accumulation of MC-LR in zebrafish intestine, we found a dose-dependent manner in the absorption and accumulation of MC-LR. Until now, little studies have been reported concerning the gut microbiota composition caused by different MC-LR exposure. The present study is the first time characterized the phylogenetic composition and taxonomic of the bacterial communities growth in the intestines of zebrafish treated with MC-LR using 16S rRNA pyrosequencing. After 30 days of treatment with 0, 1, 5 or 20 µg/L MC-LR, the alpha and beta diversity did not generate significant differences, indicating the existence of a core microbiota. However, db-RDA analysis showed that treatment with 20 µg/L MC-LR changed the characteristics of high abundances microbiota. The expression of Oatp2b1, stress related enzyme activities in gut and their associations with gut microbiota were also determined. The identified phylotypes including Actinobacteria, Lactobacillus and some opportunistic pathogens highlight the increasing risks of pathogen invasion and recovery tendency via potential probiotics resistance in zebrafish exposed to MC-LR.

Effect of peristaltic-like movement on bioengineered intestinal tube.

The intestine is a highly heterogeneous hollow organ with biological, mechanical and chemical differences between lumen and wall. A functional human intestine model able to recreate the in vivo dynamic nature as well as the native tissue morphology is demanded for disease research and â€‹drug discovery. Here, we present a system, which combines an engineered three-dimensional (3D) tubular-shaped intestine model (3D In-tube) with a custom-made microbioreactor to impart the key aspects of the in vivo microenvironment of the human intestine, mimicking the rhythmic peristaltic movement. We adapted a previously established bottom-up tissue engineering approach, to produce the 3D tubular-shaped lamina propria and designed a glass microbioreactor to induce the air-liquid interface â€‹condition and peristaltic-like motion. Our results demonstrate the production of a villi-like protrusion and a correct spatial differentiation of the intestinal epithelial cells in enterocyte-like as well as mucus-producing-like cells on the lumen side of the 3D In-tube. This dynamic platform offers a proof-of-concept model of the human intestine.

Protein Hydrolysates' Absorption Characteristics in the Dynamic Small Intestine In Vivo.

Background: Dietary proteins are known for their wide range of nutritional, functional and biological properties. Although the total amount of proteins may be obtained from mixtures, its "availability" for absorption in the gut is in many cases quite uncertain or even varies for the same food depending on processing conditions, the presence of other components, and so on. Methods: To obtain accurate protein hydrolysate absorption data, we have developed a small intestine model (SIM) to test them. Results: The results indicated that the protein hydrolysates were absorbed rapidly during the first 15 min, and then decreased to 90 min, then they were absorbed again from 90 min to the endpoint. The protein absorption was also affected by the protein processing method used. The Enzyme + Ultrasound (EU) processing method group had a higher absorption rate than the Enzyme (E) processing method group, and the absorption of the Enzyme + Artificial gastric juice processing method (EH) and Enzyme + Ultrasound + Artificial gastric juice processing method (EUH) groups was reduced compared to the E group alone. The amino acid analysis results showed that the amino acids were reduced and absorbed by our SIM in almost all groups except for cysteine and methionine. In general, the Pearson relation value of the amino acid contents between before SIM and after SIM was 0.887, which indicated that single amino acid absorption was mainly related to its content in the whole amino acids. The single amino acid absorption ratio among different groups also displayed differences, which ranged from 31% to 46% (E group from 39% to 42%; EU group from 40% to 47%; EH group from 31% to 39%; EUH group from 35% to 41%). CONCLUSIONS: The protein hydrolysates' varied from startpoint to endpoint, and the protein absorption was affected by processing method.

3D stromal tissue equivalent affects intestinal epithelium morphogenesis in vitro.

Current in vitro models of human intestine commonly fail to mimic the complex intestinal functions and features required for drug development and disease research. Here, we deeply investigate the interaction existing between epithelium and the underneath stroma, and its role in the epithelium morphogenesis. We cultured human intestinal subepithelial myofibroblasts (ISEMFs) in two different 3D configurations: 3D-collagen gel equivalent (3D-CGE) and 3D cell-synthetized stromal equivalent (3D-CSSE). The 3D-CGEs were obtained by means of the traditional collagen-based cell technique and the 3D-CSSE were obtained by bottom-up tissue engineering strategy. The biophysical properties of both 3D models with regard to cell growth and composition (via histological analysis, immunofluorescence, and multiphoton imaging) were assessed. Then, human colorectal adenocarcinoma cell line (CaCo-2) was cultured on both the 3D constructs in order to produce the intestinal model. We identified higher levels of matrix-associated proteins from ISEMFs cultured in 3D-CSSE compared to 3D-CGE. Furthermore, multiphoton investigation revealed differences in the collagen network architecture in both models. At last, the more physiologically relevant stromal environment of the 3D-CSSE drove the CaCo-2 cell differentiation toward the four different type of intestinal epithelial cells (absorptive, mucus-secretory, enteroendocrine, and Paneth) phenotype and promotes, in contrast to the 3D-CGE, the production of the basement membrane. Taken together, these results highlight a fundamental role of the 3D stromal environment in addressing a correct epithelium morphogenesis as well as epithelial-stromal interface establishment.

Chip-based human liver-intestine and liver-skin co-cultures--A first step toward systemic repeated dose substance testing in vitro.

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called "human-on-a-chip" concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air-liquid interface for the skin model during their co-culture with the liver equivalents respectively at (1)/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver-skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance - troglitazone - to the chip-based co-cultures.