scholarly journals Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1563
Author(s):  
Cristiana L. Pires ◽  
Catarina Praça ◽  
Patrícia A. T. Martins ◽  
Ana L. M. Batista de Carvalho ◽  
Lino Ferreira ◽  
...  
Keyword(s):  
Culture Media ◽  
Lucifer Yellow ◽  
Cell Monolayer ◽  
Time Interval ◽  
Cell Monolayers ◽  
Morphological Studies ◽  
Confluent Cell ◽  
Vitro Model ◽  
Permeability Assay

Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to Papp values that are dependent on the sampling duration. The assay also leads to a small decrease in the cell monolayer TEER, which is fully recovered when cell monolayers are incubated with culture media for two full days. When this procedure is followed, the cell monolayers may be used for permeability assays on days 22, 25, and 28, triplicating the throughput of this important assay.

scholarly journals Metabolism of Selected 2-Arylbenzofurans in a Colon In Vitro Model System

Foods ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2754
Author(s):  
Ondrej Vesely ◽  
Petr Marsik ◽  
Veronika Jarosova ◽  
Ivo Doskocil ◽  
Karel Smejkal ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Biological Effects ◽  
Bacterial Fermentation ◽  
Basolateral Side ◽  
Sufficient Stability ◽  
Polar Metabolites ◽  
Fermentation Model ◽  
Vitro Model ◽  
Permeability Assay

2-arylbenzofurans represent a small group of bioactive compounds found in the plant family Moraceae. As it has not been investigated whether these substances are stable during passage through the gastrointestinal tract, their biological effects may be altered by the metabolism of intestinal microbiota or cells. The aim of the present study was to investigate and compare mulberrofuran Y (1), moracin C (2), and mulberrofuran G (3) in an in vitro model of human intestinal bacterial fermentation and in an epithelial model using the Caco-2 cell line. The analysis of compounds by LC-MS-Q-TOF showed sufficient stability in the fermentation model, with no bacterial metabolites detected. However, great differences in the quantity of permeation were observed in the permeability assay. Moreover, mulberrofuran Y (1) and moracin C (2) were observed to be transformed into polar metabolites by conjugation. Among the test compounds, mulberrofuran Y (1) was mostly stable and accumulated in endothelial cells (85.3%) compared with mulberrofuran G (3) and moracin C (2) (14% and 8.2%, respectively). Thus, only a small amount of mulberrofuran Y (1) was conjugated. Moracin C (2) and mulberrofuran G (3) were metabolized almost completely, with only traces of the unchanged molecule being found on the apical and cellular sides of the system. Only conjugates of mulberrofuran Y (1) and moracin C (2) were able to reach the basolateral side. Our results provide the basic description of bioavailability of these three compounds, which is a necessary characteristic for final evaluation of bio-efficacy.

Technical Report: Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro

2001 ◽  
Vol 13 (6) ◽  
pp. 355 ◽  
Author(s):  
P. S. P. Gupta ◽  
S. Nandi ◽  
B. M. Ravindranatha ◽  
P. V. Sarma
Keyword(s):  
Embryonic Development ◽  
Genetic Manipulation ◽  
Culture Media ◽  
Cell Monolayer ◽  
Basic Research ◽  
Cost Effective ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Vitro Fertilization

In vitro fertilization (IVF) technology provides an opportunity to produce embryos for genetic manipulation, embryo transfer and basic research in developmental physiology, and can be exploited for emerging biotechnologies such as transgenesis and cloning. In the present study, the effects of different concentrations of commercially available pregnant mare serum gonadotrophin (PMSG) (Folligon; Intervet, International B.V., Boxmeer, Holland) in oocyte culture media, on maturation, fertilization and embryonic development of buffalo oocytes in vitro were investigated. Oocytes aspirated from abattoir-derived ovaries were cultured in media containing TCM-199 + PMSG at 0, 2.5, 20, 30, 40 and 50 IU mL–1 in presence or absence of steer serum (10%) for 24 h in a CO2 incubator. The maturation rate was assessed on the basis of degree of expansion of cumulus cells. The matured oocytes were inseminated with 9–10 x 106 spermatozoa mL–1 in Brackett and Oliphant medium and the cleavage rate was recorded 40–42 h after insemination. Uncleaved oocytes were stained with aceto-orcein for evaluation of fertilization rates. The cleaved embryos were further cultured in TCM-199 + 10% steer serum on buffalo oviducal cell monolayer for 7 days. Maturation, fertilization, cleavage and embryonic development were significantly higher (P<0.05) in oocytes cultured in TCM-199 + 10% steer serum supplemented with 40 and 50 IU PMSG mL–1. It is concluded that commercially available PMSG can effectively be used in place of pure follicle-stimulating hormone for in vitro maturation of buffalo oocytes, making it cost effective for IVF studies.

scholarly journals Effect of Escherichia coli Cytotoxic Necrotizing Factor 1 on Repair of Human Bladder Cell Monolayers In Vitro

1999 ◽  
Vol 67 (7) ◽  
pp. 3657-3661 ◽  
Author(s):  
Michael D. Island ◽  
Xaioling Cui ◽  
John W. Warren
Keyword(s):  
Escherichia Coli ◽  
Bladder Epithelium ◽  
Cell Monolayers ◽  
E Coli ◽  
Cytotoxic Necrotizing Factor ◽  
Bladder Cell ◽  
Bladder Cells ◽  
Vitro Model ◽  
Cytotoxic Necrotizing Factor 1

ABSTRACT We hypothesized that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) might impair migration or proliferation of bladder cells and could potentially interfere with repair of the bladder epithelium. Using experimentally wounded human T24 bladder epithelial cell monolayers as an in vitro model, we found that both the number of T24 cells and the maximum distance they migrated into wounded regions was significantly decreased by bacterial extracts containingE. coli CNF1.

Characterization of an In Vitro Model to Study the Permeability of Human Arterial Endothelial Cell Monolayers

1988 ◽  
Vol 60 (02) ◽  
pp. 240-246 ◽  
Author(s):  
Erna G Langeler ◽  
Victor W M van Hinsbergh
Keyword(s):  
Endothelial Cell ◽  
Calcium Ions ◽  
Electrical Resistance ◽  
Serum Proteins ◽  
Cell Monolayer ◽  
Extracellular Calcium ◽  
Endothelial Cell Monolayer ◽  
Passage Rate ◽  
Cell Monolayers

SummaryA model has been developed to study the transport of fluid and macromolecules through human arterial umbilical cord endothelial cell monolayers in vitro. Cells were cultured on fibronectin- coated polycarbonate filters and formed within a few days a tight monolayer, with an electrical resistance of 17 ± 4 Ohm · cm2. The cells were connected by close cell contacts with tight junctions. The passáge-rate of horse radish peroxidase (HRP) through these filters was 20-40 fold lower than through filters without an endothelial monolayer. The continuous presence of 10% human serum was needed to maintain the electrical resistance of the monolayer and its barrier function towards macromolecules. Chelation of extracellular calcium resulted in an increased permeability and a decreased electrical resistance of the monolayer. This process was reversible by re-addition of calcium ions to the cells. The permeation rate of dextrans of various molecular weights (9-480 kD) was inversely related to the molecular mass of the molecule. No difference was measured between the passage rate of dextran of 480 kD and dextran of 2,000 kD. Incubation of the endothelial cell monolayer with 2-deoxy-D-glucose resulted in a decreased permeability but it had no effect on electrical resistance. This suggests that the passage-process is energy- dependent.Fluid permeation through the endothelial cell monolayer on filters was measured in a perfusion chamber under 20 mmHg hydrostatic pressure. It was decreased by the presence of serum proteins and responded reversibly on the chelation and readdition of extracellular calcium ions.

THE BINDING OF ANTITHROMBIN TO CAPILLARY ENDOTHELIAL CELLS GROWN IN VITRO

1987 ◽  
Author(s):  
A de Agostini ◽  
J Marcum ◽  
R Rosenberg
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Heparan Sulfate ◽  
Cell Monolayer ◽  
Whole Cells ◽  
Cell Monolayers ◽  
Bacterial Enzyme ◽  
Capillary Endothelial Cells ◽  
Binding Cell

Cloned endothelial cells from rat epididymal fat pads synthesize anticoagulantly active heparan sulfate proteoglycans containing the disaccharide, GlcA→ AMN-3,6-O-SO3, which is a marker for the antithrombin-binding domain of heparin. To demonstrate that antithrombin (AT) binds to cell surface heparan sulfate, a binding assay employing 125I-AT and cell monolayers has been developed. Post-confluent endothelial cells (7 days) were incubated with radiolabeled AT for 1 h at 4° and washed with PBS. Bound radioactivity was quantitated after solubilizing whole cells. Under these conditions, ∼1% (2174±50 cpm/5x104 cells) of the 125I-AT bound to the endothelial cell monolayer, whereas none of the radiolabeled protein bound to CHO cells or bovine smooth muscle cells. Utilization of unlabeled AT (1 μM) in experiments conducted as described above resulted in a reduction (73%) of the binding of the labeled species to endothelial cells. To assess whether heparan sulfate was responsible for AT binding, cell monolayers were incubated for 1 h at 37° with purified Flavobacterium heparinase (0.2 units). Over 90% of 125I-AT binding to these cellular elements was suppressed with the bacterial enzyme. Internalization of radiolabeled AT by endothelial cells was examined by incubating the protease inhibitor and cells at 4° and 37 . An initial rapid binding was observed at both temperatures. At 4° AT binding plateaued within 15 min, whereas at 37° binding did not plateau until 60 min and was 30% greater than that observed at 4. These data suggest that surface-associated AT can be internalized by endothelial cells. In addition, AT binding was shown to increase with the length of endothelial cell postconfluence, indicating an accumulation of heparan sulfate by these cells during quiescence. In conclusion, our studies support the hypothesis that the vascular endothelium is coated with heparan sulfate-bound AT, which is responsible for the antithrombotic properties of these natural surfaces.

Transport ofSchisandra chinensisextract and its biologically-active constituents across Caco-2 cell monolayers - an in-vitro model of intestinal transport

2008 ◽  
Vol 60 (3) ◽  
pp. 363-370 ◽  
Author(s):  
Vamsi L. M. Madgula ◽  
Bharathi Avula ◽  
Young W. Choi ◽  
Srinivas V. Pullela ◽  
Ikhlas A. Khan ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Intestinal Transport ◽  
Biologically Active ◽  
Active Constituents ◽  
Cell Monolayers ◽  
Vitro Model
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