Intestinal Cell Culture Research Articles

Intestinal Organoids: New Frontiers in the Study of Intestinal Disease and Physiology.

The development of sustainable intestinal organoid cell culture has emerged as a new modality for the study of intestinal function and cellular processes. Organoid culture is providing a new testbed for therapeutic research and development. Intestinal organoids, self-renewing 3-dimensional structures comprised intestinal stem cells and their differentiated epithelial progeny allow for more facile and robust exploration of cellular activity, cell organization and structure, genetic manipulation, and vastly more physiologic modeling of intestinal response to stimuli as compared to traditional 2-dimensional cell line cultures. Intestinal organoids are affecting a wide variety of research into gastrointestinal pathology. The purpose of this review is to discuss the current state-of-the-art and future effect of research using enteroids and colonoids (organoids grown from the small and large intestines, respectively).

Intestinal Crypt Organoid: Isolation of Intestinal Stem Cells, In Vitro Culture, and Optical Observation.

The isolation and culture of intestinal stem cells (ISCs) was first demonstrated in the very recent decade with the identification of ISC marker Lgr5. The growth of ISCs into crypt organoids provides an in vitro model for studying the mucosal physiology, intestinal cancer tumorigenesis, and intestinal regeneration. Here, we describe two different isolation protocols and demonstrate a fixation method that aids in the confocal observation of the organoids.

An Integrated Caco-2TC7cells/biosensors Device for the Real Time Monitoring of Intestinal Glucose and Polyphenols Absorption and Hypoglycemic Effect of Phytochemicals

An integrated device, for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is shown here. The SB was made of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor located in the basolateral compartment (BC) of a cell culture plate. This system was able to monitor the glucose absorption and the hypoglycemic effect induced by different polyphenols and could be proposed to provide an effective strategy to manage postprandial hyperglycemia with natural compounds.

Designer matrices for intestinal stem cell and organoid culture.

Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.

High Iron-Sequestrating Bifidobacteria Inhibit Enteropathogen Growth and Adhesion to Intestinal Epithelial Cells In vitro

The gut microbiota plays an important role in host health, in particular by its barrier effect and competition with exogenous pathogenic bacteria. In the present study, the competition of Bifidobacterium pseudolongum PV8-2 (Bp PV8-2) and Bifidobacterium kashiwanohense PV20-2 (Bk PV20-2), isolated from anemic infant gut microbiota and selected for their high iron sequestration properties, was investigated against Salmonella Typhimurium (S. Typhi) and Escherichia coli O157:H45 (EHEC) by using co-culture tests and assays with intestinal cell lines. Single and co-cultures were carried out anaerobically in chemically semi-defined low iron (1.5 μM Fe) medium (CSDLIM) without and with added ferrous iron (30 μM Fe). Surface properties of the tested strains were measured by bacterial adhesion to solvent xylene, chloroform, ethyl acetate, and to extracellular matrix molecules, mucus II, collagen I, fibrinogen, fibronectin. HT29-MTX mucus-secreting intestinal cell cultures were used to study bifidobacteria competition, inhibition and displacement of the enteropathogens. During co-cultures in CSDLIM we observed strain-dependent inhibition of bifidobacterial strains on enteropathogens, independent of pH, organic acid production and supplemented iron. Bp PV8-2 significantly (P < 0.05) inhibited S. Typhi N15 and EHEC after 24 h compared to single culture growth. In contrast Bk PV20-2 showed less inhibition on S. Typhi N15 than Bp PV8-2, and no inhibition on EHEC. Affinity for intestinal cell surface glycoproteins was strain-specific, with high affinity of Bp PV8-2 for mucin and Bk PV20-2 for fibronectin. Bk PV20-2 showed high adhesion potential (15.6 ± 6.0%) to HT29-MTX cell layer compared to Bp PV8-2 (1.4 ± 0.4%). In competition, inhibition and displacement tests, Bp PV8-2 significantly (P < 0.05) reduced S. Typhi N15 and EHEC adhesion, while Bk PV20-2 was only active on S. Typhi N15 adhesion. To conclude, bifidobacterial strains selected for their high iron binding properties inhibited S. Typhi N15 and EHEC in co-culture experiments and efficiently competed with the enteropathogens on mucus-producing HT29-MTX cell lines. Further studies in complex gut ecosystems should explore host protection effects of Bp PV8-2 and Bk PV20-2 mediated by nutritional immunity mechanism associated with iron-binding.

The dietary biogenic amines tyramine and histamine show synergistic toxicity towards intestinal cells in culture

Tyramine and histamine are the biogenic amines (BA) most commonly found at high concentrations in food; they may even appear together at toxic concentrations. The present work examines, via real-time cell analysis, whether histamine and tyramine show synergistic toxicity towards intestinal cell cultures. Employing a constant equipotency ratio, their interaction was examined via the combination index (CI) method of Chou & Talalay. Co-treatment with tyramine and histamine was associated with a stronger cytotoxic effect than was treatment with either BA or on its own. Indeed, a synergistic interaction (CI<1) was observed in the range of concentrations found in foods. The results also show that histamine, at concentrations below the legal limit, increases the cytotoxicity of tyramine at concentrations frequently reached in some foods. The synergistic cytotoxicity of tyramine and histamine should be taken into account when establishing legal limits designed to ensure consumer safety.

Establishment of primary bovine intestinal epithelial cell culture and clone method.

The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

Real-time monitoring of glucose and phenols intestinal absorption through an integrated Caco-2TC7cells/biosensors telemetric device: Hypoglycemic effect of fruit phytochemicals

An integrated device for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is described in this study. The SB is composed of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor, all located in the basolateral compartment (BC) of a cell culture plate. Caco-2TC7 cells, differentiated on culture inserts, separated the apical compartment that simulates the intestinal lumen, from the BC which represented the bloodstream. The system recorded currents relative to glucose (1mM) absorption, obtaining bioavailability values (5.1%) comparable to HPLC analysis (4.8%). Phloridzin and phloretin, specific phenolic inhibitors of SGLT1 and GLUT2 glucose transporters, reduced the glucose transport of almost 10 times. They were minimally absorbed in the BC with a bioavailability of 0.13% and 0.49% respectively. The hypoglycemic potential of blueberry and pomegranate juices was also studied. In particular, the amount of glucose absorbed through the Caco-2TC7 monolayer was 8‰ for pomegranate and 1.7‰ for blueberry, demonstrating the potential hypoglycemic effect of the juices. Polyphenols absorption was also monitored by the SB and an increase was recorded during the first 50min in presence of both blueberry and pomegranate juices, then a constant decrease occurred. The proposed device has been developed as innovative tool for the dynamic monitoring of natural compounds effects on glucose absorption, in order to manage postprandial hyperglycemia.

Infant digestion physiology and the relevance of in vitro biochemical models to test infant formula lipid digestion.

Lipids play an important role in the diet of preterm and term infants providing a key energy source and essential lipid components for development. While a lot is known about adult lipid digestion, our understanding of infant digestion physiology is still incomplete, the greatest gap being on the biochemistry of the small intestine, particularly the activity and relative importance of the various lipases active in the intestine. The literature has been reviewed to identify the characteristics of lipid digestion of preterm and term infants, but also to better understand the physiology of the infant gastrointestinal tract compared to adults that impacts the absorption of lipids. The main differences are a higher gastric pH, submicellar bile salt concentration, a far more important role of gastric lipases as well as differences at the level of the intestinal barrier. Importantly, the consequences of improper in vitro replication of gastric digestions conditions (pH and lipase specificity) are demonstrated using examples from the most recent of studies. It is true that some animal models could be adapted to study infant lipid digestion physiology, however the ethical relevance of such models is questionable, hence the development of accurate in vitro models is a must. In vitro models that combine up to date knowledge of digestion biochemistry with intestinal cells in culture are the best choice to replicate digestion and absorption in infant population, this would allow the adaptation of infant formula for a better digestion and absorption of dietary lipids by preterm and term infants.

Hydrocortisone-induced anti-inflammatory effects in immature human enterocytes depend on the timing of exposure.

The immature human gut has a propensity to exaggerated inflammatory responses that are thought to play a role in the pathogenesis of necrotizing enterocolitis (NEC). Prenatal exposure to corticosteroids has been reported to reduce the risk of NEC, while postnatal dexamethasone treatment is associated with adverse neurodevelopmental outcomes in preterm infants. The aim of this study was to investigate the direct role of hydrocortisone in gene expression patterns and inflammatory responses in immature human enterocytes. Time-dependent hydrocortisone effects in nontransformed primary human fetal intestinal epithelial cell line H4 were investigated by cDNA microarray. Fetal intestinal organ culture and cell culture experiments were conducted. Inflammatory responses were induced by stimulation with IL-1β and TNF-α with and without hydrocortisone. IL-8 and IL-6 expression and secretion were measured as functional readout. Here we report time-dependent hydrocortisone-induced changes in gene expression patterns detected by cDNA microarray. Hydrocortisone significantly attenuated IL-1β-induced inflammatory responses in the immature human gut when administered at the time of the proinflammatory insult: IL-1β-induced IL-8 and IL-6 secretion in the fetal ileum as well as H4 cells were significantly reduced. Hydrocortisone also inhibited IL-8 secretion in response to TNF-α. In contrast, TNF-α-induced IL-8 secretion was not reduced in cells treated with hydrocortisone for 48 h before stimulation. Our observations provide a physiological basis for understanding the differential clinical effects of corticosteroids in the immature human gut depending on the timing of treatment.

Individual and combined effects of roquefortine C and mycophenolic acid on human monocytic and intestinal cells

Roquefortine C (ROC) and mycophenolic acid (MPA) are secondary metabolites produced by various fungal species. It is known that both ROC and MPA may co-occur in raw materials or food. However, to date there is a lack of information regarding their toxicity. In this study, ROC and/or MPA cytotoxicity was evaluated on human intestinal (Caco-2) and monocytic cell cultures (THP-1 and CD14+). After 48 h single mycotoxin exposure, viability tests showed that monocytes (THP-1 and CD14+) were more sensitive to ROC (inhibitory concentration 50% (IC50)=55 and 45 μM, respectively) than to MPA (IC50&gt;780 μM). IC50 values determined from ROC and MPA mono-exposure experiments on Caco-2 cells were &gt;100 and &gt;780 μM, respectively. Caco-2 cell viability was significantly reduced after 48 h co-exposure at high ROC/MPA concentrations. A synergistic effect was observed at 10/78, 25/780 and 50/780 μM ROC/MPA concentrations, while an additive effect was seen at 100/780 μM. THP-1 apoptosis rate increased after 3 and/or 6 h single ROC (from 10 to 100 μM) and MPA (780 μM) exposures in a dose-dependent manner. Co-exposure to 100/780 μM of ROC/MPA led to an increase in the THP-1 apoptotic cell population. No apoptosis mechanism was observed on Caco-2 cells. This is the first time that combined ROC and MPA cytotoxic effects, as well as the associated mechanisms are investigated.

Fully defined matrices for the culture and expansion of intestinal stem cells and organoids

Fully defined matrices for the culture and expansion of intestinal stem cells and organoids

Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells.

In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.

Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract Supplement

Background: Tight junctions are found in epithelial cells and function as selective gatekeepers to regulate absorption. PT-gliadin is the gluten protein segment that is known to impair the functioning of tight junctions. This study aimed to examine the effects of a lignite extract dietary supplement (RESTORE) on tight junction function in small intestine (IEC-6) and colon (Caco-2) epithelial cells. The study also evaluated the biologic safety of the same supplement as established by the rates of apoptosis in the intestinal and proximal renal tubule cell cultures treated with the supplement. Methods: IEC-6 and Caco-2 cells were incubated until a stable trans-epithelial electrical resistance (TEER) was measured. The dietary supplement at 20% concentration or a control were placed on the cells and left overnight. These cells were then treated with and without PT-gliadin. Tight junction expression was determined by immunofluorescent microscopy. The rate of apoptosis was established in cell culture with the lignite extract at 20% concentration in order to assess a toxic concentration in normal cell lines: IEC-6, Caco-2, and human renal proximal tubule cell (RPTC) lines. Results: The lignite extract supplement increased the TEER in IEC-6 (58%) and Caco-2 (15%) compared to control. PT-gliadin dramatically decreased the TEER in both control IEC-6 (49%) and control Caco-2 (27%) membranes. The lignite supplement prevented PT-gliadin-mediated decrease in TEER. The supplement reduced apoptosis in RPTC (44%), IEC-6 (13%), and Caco-2 (24%) cell cultures. Conclusion: The lignite supplement blocked a PT-gliadin dependent decrease in TEER in small intestine and colon cell line membranes. The lignite extract was not toxic on intestinal or renal cells at high concentration, and demonstrated a statistically significant reduction in apoptosis in RPTCs. Human clinical trials are needed to evaluate the use of RESTORE to support health in gluten-sensitive individuals.

Uptake of manganese from manganese-lysine complex in the primary rat intestinal epithelial cells.

This study was conducted to compare the differences of the uptake of Mn from Mn-lysine complex (MnLys) and MnSO4 and to determine the potential mechanisms of the uptake of Mn from MnLys. We first established the primary rat intestinal epithelial cell culture model and used it to determine the uptake of Mn from different Mn sources. The MnLys increased (p<0.001) Mn uptake when compared to MnSO4 . The uptake of Mn decreased (p<0.05) with added Fe concentration increasing in the medium regardless of Mn source. The MnLys decreased (p<0.01) Mn2+ efflux transporter ferroportin 1 (FPN1) mRNA level, but did not influence (p>0.06) Mn2+ influx transporter DMT1 mRNA expression when compared to MnSO4 . The results above indicated that the increase of Mn accumulation for MnLys at least partly was due to the decrease of Mn efflux by reduced FPN1 expression. The N-ethylmaleimide, as an l-lysine transport system y+ inhibitor, decreased (p<0.001) the uptake of Mn from MnLys, but did not affect (p>0.10) the uptake of Mn from MnSO4 . The cycloheximide, as an l-lysine transport system b0,+ activator, increased (p<0.001) the uptake of Mn from MnLys, whereas also did not influence the uptake of Mn from MnSO4 . The MnLys increased (p<0.01) the system y+ member cationic amino acid transporter (CAT) 1, and system b0,+ components rBAT and b0,+ AT mRNA expression when compared to MnSO4 . These results suggested that the uptake of Mn from MnLys complex might be transported by CAT1 and system b0,+ , which was different from ionized Mn2+ uptake pathway. In conclusion, the uptake of MnLys complex not only might be absorbed as Mn2+ , but also appeared to be transported through CAT1 and system b0,+ in the primary rat intestinal epithelial cells.

Insights into the protective role of solid lipid nanoparticles on rosmarinic acid bioactivity during exposure to simulated gastrointestinal conditions

The evaluation of the digestion effects on bioactive solid lipid nanoparticles (SLN) was performed. For this purpose, witepsol and carnauba SLN loaded with rosmarinic acid (RA) were exposed to the simulated gastrointestinal tract (GIT) conditions prevailing in stomach and small intestine. The simulation of intestinal epithelium was made with a dialysis bag and intestinal cell culture lines. Changes on SLN physical properties, RA release and absorption profiles were followed at each step. Combination of digestion pH and enzymes showed a significant effect upon SLN physical properties. Zeta potential values increased at stomach conditions and decreased at small intestine simulation. Also, at intestine, SLN increased their sizes and released 40–60% of RA, maintaining its initial antioxidant activity values. Sustained release of 40% of RA from SLN was also observed in dialysis tube. At CaCo-2 cell line, both types of SLN showed similar absorbed RA % (ca. 30%). Nevertheless, in CaCo-2/HT29x mix cell lines, for carnauba SLN a lower adsorption RA % was observed than for witepsol SLN. Solid lipid nanoparticles protected RA bioactivity (in terms of antioxidant activity) until reaching the intestine. A controlled release of RA from SLN was achieved and a significant absorption was observed at intestinal cells. Overall, SLN produced with witepsol showed a higher stability than carnauba SLN.

Early tranexamic acid administration: A protective effect on gut barrier function following ischemia/reperfusion injury.

The mucus barrier is a critical component of the gut barrier and may be disrupted by pancreatic enzymes following trauma/hemorrhagic shock (T/HS). Luminal strategies against pancreatic enzyme activation or contact with the intestine are protective of the mucus layer and gut barrier integrity following T/HS. There is increasing evidence the use of tranexamic acid (TA) attenuates inflammatory responses in cardiac surgery and is readily absorbed from the gut. We therefore postulated that systemic administration of TA would attenuate mucus degradation and gut barrier failure following T/HS. This was studied in an in vitro model. Confluent monolayers of HT29-MTX (mucus-producing clone) and Caco-2 cocultures were exposed to 90 minutes of hypoxia followed by reoxygenation (H/R), luminal trypsin (5 μM), or both treatment groups. In a subset of experiments, TA (40 μM or 150 μM) was added to the basal chamber (systemic side) of intestinal cell cultures immediately following the hypoxic period. Mucus barrier function was indexed by rheologic measurement of both mucus thickness and viscosity (G', dyne/cm) and oxidant stress. Intestinal cell barrier integrity was indexed by transepithelial electrical resistance, permeability to fluorescein isothiocyanate-dextran, and apoptosis by flow cytometry. Exposure to both trypsin and H/R of Caco-2/HT29-MTX cocultures led to the most severe effect on mucus barrier function. Administration of TA immediately following hypoxia abrogated the effects noted on mucus barrier function. The epithelial barrier was also most severely impacted by both trypsin and H/R. Addition of TA after the hypoxic event was shown to be protective. Intestinal mucus physiochemical properties and intestinal barrier function were most severely impacted by exposure to both trypsin (concentration related) and H/R. The "systemic" administration of TA immediately after the hypoxic period was protective and suggests an additional role for early administration of TA in trauma patients in shock.

Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine

Tyramine and histamine, the most toxic biogenic amines (BA), are often found in high concentrations in certain foods. Prompted by the limited knowledge of BA toxicity, and increasing awareness of the risks associated with high intakes of dietary BA, the in vitro cytotoxicity of tyramine and histamine was investigated. Tyramine and histamine were toxic for HT29 intestinal cell cultures at concentrations commonly found in BA-rich food, as determined by real-time cell analysis. Surprisingly, tyramine had a stronger and more rapid cytotoxic effect than histamine. Their mode of action was also different, while tyramine caused cell necrosis, histamine induced apoptosis. To avoid health risks, the BA content of foods should be reduced and legal limits established for tyramine.

The evaluate and compare the effects of the Tacrolimus and Sirolimus on the intestinal system using an intestinal cell culture model

Tacrolimus (TAC) and Sirolimus (SRL) are produced by Streptomyces sp and effective immunosuppressive drugs commonly used in organ transplantation. Therefore, strategies for minimizing the toxicity of immunosuppressant molecules are our interest. This study was conducted to evaluate the interactive effects and the possible underlying mechanism of TAC and SRL on HCT116 cells. It was found that TAC and SRL alone inhibited cell viability. Also, it induced reactive oxygen species (ROS) formation, loss of mitochondrial membrane potential (Δψm), and able to increase DNA fragmentation in a concentration-dependent manner. The use of combined SRL and TAC showed a reservation in all toxicity observed with the two immunosuppressive drugs separately. Our result demonstrated that the mechanisms of TAC and SRL at high concentration are closely connected with oxidative stress. Furthermore, SRL at low concentration plays a protective effect against TAC (IC50) which induced cytotoxicity and genotoxicity. However, using the combination of the SRL/TAC at high concentrations (IC30) appears as an antagonist response.

Effect of Combining Pulsed Electric Fields with Maceration Time on Merlot Grapes in Protecting Caco-2 Cells from Oxidative Stress

The objective of this study was to investigate the effect of different maceration times (0, 2, 4, 8 and 14 days) after pulsed electric field (PEF) processing (1.5 kV/cm, either at 15 or 70 kJ/kg) on the release of anthocyanins from Merlot grapes (Vitis vinifera) and the bioprotective potentials of grape juice against H2O2-induced oxidative stress using human intestinal Caco-2 cell culture model. Cell viability, lactate dehydrogenase membrane leakage and nitric oxide production in Caco-2 cells were used as biomarkers to indicate general cellular health and integrity that ultimately represent the bioprotective potentials of grape juice. The results showed that PEF at 70 kJ/kg was the most effective in accelerating the release of anthocyanins from grape skin. Furthermore, the amount and the type of anthocyanins being released varied as a result of combined effect of PEF processing intensity and maceration time. Linear correlations between anthocyanins and the bioprotective capacity markers (R 2 = 0.4–0.8) were found. Compared to the untreated Merlot, PEF treatment either at 15 or 70 kJ/kg reduced the maceration time from 14 to 8 and 2 days, respectively, to achieve similar degree of bioprotective effect. This implies the possibility of using combined PEF and maceration time to tailor the profile of bioactive compounds with the same bioprotective potentials.