Co-culture of Caco2 and HT-29 cells as an innovative method to mimic in vitro the morphology and permeability properties of human intestinal epithelium

For investigating the complexity of the human intestinal epithelium, a valid experimental approach is represented by co-culture. In the present study an intestinal co-culture Caco2/HT-29 (70/30) was set up starting from the parental populations of differentiated cells as previously described [1, 2]. Co-culture was harvested at 0 (T0), 6 (T6), and 14 (T14) days of post confluence after plating. Transmission electron microscopy was carried out to monitor the morphological features of cell differentiation. Alkaline Phosphatase (ALP), Aminopeptidase N (APN) and Dipeptidyl Peptidase IV (DPP IV) activity were assayed as known markers of intestinal cell differentiation. The measure of TEER and the apparent permeability of Lucifer Yellow allows to monitor the integrity of the tight junctions and the permeability of the cell layer formed. At T0 a classical monolayer is present, with a mixed population of immature absorptive elements and secretive cells. At T6 and T14, cells are progressively organized in a multilayer with a parallel growth of microvilli. At T6, co-culture demonstrates good properties of permeability and barrier components, such as mucus, representing an appropriate model for absorption study. At T14, the brush border is even more developed respect to T6 and, together with the increase of the specific activity of ALP, APN, and DPP IV, indicate co-culture as a good model for digestion study. The advantage of this co-culture described is the use of the whole cell population without particular inducers of subclones and growth supports. In conclusion, the morphological and biochemical features of co-cultured parental cells change with time, strongly supporting i) an active interaction between the two parental cell lines and ii) the versatility of this model, with more than one prevalent cell type depending on the post confluent stage

L-Carnitine Reduces Oxidative Stress and Promotes Cells Differentiation and Bone Matrix Proteins Expression in Human Osteoblast-Like Cells

Bone fragility and associated fracture risk are major problems in aging. Oxidative stress and mitochondrial dysfunction play a key role in the development of bone fragility. Mitochondrial dysfunction is closely associated with excessive production of reactive oxygen species (ROS). L-Carnitine (L-C), a fundamental cofactor in lipid metabolism, has an important antioxidant property. Several studies have shown how L-C enhances osteoblastic proliferation and activity. In the current study, we investigated the potential effects of L-C on mitochondrial activity, ROS production, and gene expression involved in osteoblastic differentiation using osteoblast-like cells (hOBs) derived from elderly patients. The effect of 5mM L-C treatment on mitochondrial activity and L-C antioxidant activity was studied by ROS production evaluation and cell-based antioxidant activity assay. The possible effects of L-C on hOBs differentiation were assessed by analyzing gene and protein expression by Real Time PCR and western blotting, respectively. L-C enhanced mitochondrial activity and improved antioxidant defense of hOBs. Furthermore, L-C increased the phosphorylation of Ca2+/calmodulin-dependent protein kinase II. Additionally, L-C induced the phosphorylation of ERK1/2 and AKT and the main kinases involved in osteoblastic differentiation and upregulated the expression of osteogenic related genes, RUNX2, osterix (OSX), bone sialoprotein (BSP), and osteopontin (OPN) as well as OPN protein synthesis, suggesting that L-C exerts a positive modulation of key osteogenic factors. In conclusion, L-C supplementation could represent a possible adjuvant in the treatment of bone fragility, counteracting oxidative phenomena and promoting bone quality maintenance

Betaine promotes cell differentiation of human osteoblasts in primary culture

BACKGROUND: Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation. METHODS: The effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes. RESULTS: Real time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content. CONCLUSIONS: Our study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality

An innovative in vitro co-culture of Caco2 and HT-29 cells for mimicking human intestinal epithelium : a morphological analysis

Intestinal epithelium represents the physiological interface between our body and nutrients with their associated molecules. The necessity to evaluate this interaction together with the difficulties encountered using in vitro primary cultures of bioptic intestinal fragments led to the development of cell lines obtained by colon adenocarcinoma and established in culture. Among these, HT-29 e Caco2 cell lines are the most used. We previously demonstrated that i) subcultured Caco2 cells form a regular monolayer with microvilli and a well-developed junctional apparatus, indicating their absorbent properties (Ferraretto et al., 2007) and ii) differentiated HT-29 cells are a heterogeneous population in which both muco-secreting, entero-endocrine, and absorptive cytotypes co-exist (Gravaghi et al., 2007). Considering that a single cell line can not represent a complete model, we decided to co-culture the two cell lines in RPMI medium and at post confluency in order to have differentiated cells mimicking as near as possible the morpho-physiological intestinal microenvironment. After plating, cells were harvested at 0, 3, 6, 10, and 14 days of post confluence (T0, T3, T6, T10, and T14, respectively) for ultrastructural analysis by transmission electron microscopy (TEM). TEM observations strongly suggest that co-cultures display original features from T0 to T14, with cells initially poorly differentiated but which progressively formed multilayers (from T6) and present: i) microvilli (from T6); ii) a complete junctional apparatus (from T6), in particular desmosomes, (T14); iii) mucus granules (from T3). In parallel, immunofluorescence analysis for the expression of occludin, E-cadherin, and desmocollin 2 is ongoing to evaluate the molecular composition of cell junctions. Our results clearly indicates that co-cultures of HT-29 and Caco2 cells may represent a valid model of intestinal human epithelium characterized by the possibility to stop the cell growth at the post confluency time more appropriate depending on the application

Influence of microbial inoculation and length of storage on fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage

Increasing the length of storage of whole-plant corn silage (WPCS) has been shown to increase ruminal in vitro starch digestibility by facilitating hydrolysis of the protein matrix surrounding starch granules. It is possible that microbial inoculants could improve fermentation, thereby enhancing proteolytic activity in the silo. Additionally, microbial inoculants have potential to reduce or prevent the growth of toxigenic fungi in silage. Therefore, the objective of this study was to determine the effects of storage length and microbial inoculation with heterofermentative and homofermentative inoculants containing Enterococcus faecium on the fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage, as well as the effect of microbial inoculation on silage mycotoxin concentrations. Whole-plant corn (333 \ub1 10 g of DM/kg as-fed) was ensiled in quintuplicate vacuum pouches untreated (CON) or after the following treatments: E. faecium at 1.5 7 105 cfu/g (EF); Lactobacillus plantarum at 1 7 105 and E. faecium at 5 7 104 cfu/g of fresh forage (LPEF); and L. buchneri and Lactococcus lactis at 1.5 7 105 cfu/g (LBLL). Silos were stored for 0, 30, 60, 90 or 120 d. Silage pH was greater with LBLL compared to the other three treatments (P = 0.005). Total acids were greater with LPEF than EF (P = 0.005). Ammonia-N (expressed as g/kg of N) was greatest with CON compared to the other treatments (P = 0.001). Concentrations of lactic acid were lower, and concentrations of acetic acid were greater with LBLL compared to the other treatments (P = 0.001). An interaction between microbial inoculation and storage length was observed for soluble CP concentrations as well as ruminal in situ starch disappearance (P = 0.001 and P = 0.012, respectively). Soluble CP (expressed as g/kg of CP) was greater with CON compared to the other treatments at d 30 and 90, but not different at d 60 and 120. Ruminal in situ starch disappearance was reduced for CON compared to the other three treatments at d 60 and 90. However, at d 120, ruminal in situ starch disappearance was similar across all treatments. Overall, the use of microbial inoculants improved fermentation profile. Microbial inoculation also increased starch disappearance in the earlier stages of fermentation but by 120 d of storage, starch disappearance was similar between inoculated silage and CON. Results from this study failed to support the hypothesis that microbial inoculants would reduce mycotoxin contamination