Cell Monolayer Deformation Microscopy reveals mechanical fragility of cell monolayers following EMT

Amy A. Sutton ◽  
Clayton W. Molter ◽  
Ali Amini ◽  
Johanan Idicula ◽  
Max Furman ◽  
K. Chien ◽  
R. Van de Velde ◽  
I.P. Shintaku ◽  
A.F. Sassoon

Immunoelectron microscopy of neoplastic lymphoma cells is valuable for precise localization of surface antigens and identification of cell types. We have developed a new approach in which the immunohistochemical staining can be evaluated prior to embedding for EM and desired area subsequently selected for ultrathin sectioning.A freshly prepared lymphoma cell suspension is spun onto polylysine hydrobromide- coated glass slides by cytocentrifugation and immediately fixed without air drying in polylysine paraformaldehyde (PLP) fixative. After rinsing in PBS, slides are stained by a 3-step immunoperoxidase method. Cell monolayer is then fixed in buffered 3% glutaraldehyde prior to DAB reaction. After the DAB reaction step, wet monolayers can be examined under LM for presence of brown reaction product and selected monolayers then processed by routine methods for EM and embedded with the Chien Re-embedding Mold. After the polymerization, the epoxy blocks are easily separated from the glass slides by heatingon a 100°C hot plate for 20 seconds.

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 313-327
H. Honda ◽  
R. Kodama ◽  
T. Takeuchi ◽  
H. Yamanaka ◽  
K. Watanabe ◽  

Cell monolayers on culture dishes were divided into two groups: tensile monolayers and non-tensile ones. In the development of an epithelium, a non-tensile cell monolayer turns into a tightly bound tensile one. Detection of these states was carried out by using the boundary shortening procedure, a computer-based geometrical method to show how much the polygonal cell boundary contracts. Non-tensile monolayers were divided further into two groups according to their motility: a fluctuating monolayer in which cells move laterally, and a stable monolayer in which cells are immobilized. Quantitative determination of cell motility was performed by analysing time-lapse cellular patterns. These computer-based geometrical analyses enabled us to divide monolayers into three groups: tensile stable monolayers, non-tensile stable monolayers and fluctuating monolayers, and this study therefore gives an insight into the way in which changing conformations of cells may be assayed.

2004 ◽  
Vol 287 (2) ◽  
pp. F319-F328 ◽  
Nicolas Markadieu ◽  
Daniel Blero ◽  
Alain Boom ◽  
Christophe Erneux ◽  
Renaud Beauwens

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP3 and PI( 3 , 4 )P2 but not PI( 4 , 5 )P2 stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP3 levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP3 was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP3 also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP3 and PI( 3 , 4 )P2 mediate the effect of insulin on sodium transport across A6 cell monolayers.

Parasitology ◽  
2016 ◽  
Vol 143 (13) ◽  
pp. 1713-1722 ◽  
A. CIAN ◽  

SUMMARYBlastocystisspp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects.In silicoanalysis of the whole genome sequence ofBlastocystissubtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified inBlastocystissubtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to eitherBlastocystisculture supernatants or each recombinant protease. BothBlastocystisculture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis ofBlastocystisby increasing intestinal cell permeability.

2002 ◽  
Vol 96 (4) ◽  
pp. 913-920 ◽  
Christoph Wandel ◽  
Richard Kim ◽  
Margaret Wood ◽  
Alastair Wood

Background The efflux transporter P-glycoprotein, a member of the adenosine triphosphate-binding cassette superfamily, is a major determinant of the pharmacokinetics and pharmacodynamics of the opioid loperamide, a well-recognized antidiarrheal agent. Animal studies indicate that P-glycoprotein limits morphine entry into the brain. In this study, the authors examined whether other opioids of importance to anesthesiologists such as fentanyl, sufentanil, and alfentanil, and also morphine-6-glucuronide and morphine-3-glucuronide, are P-glycoprotein substrates and whether, in turn, these opioids act also as P-glycoprotein inhibitors. Methods The transcellular movement of the various opioids, including loperamide and morphine, was assessed in L-MDR1 (expressing P-glycoprotein) and LLC-PK1 cell monolayers (P-glycoprotein expression absent). A preferential basal-to-apical versus apical-to-basal transport in the L-MDR cells but not the LLC-PK1 cells is seen for P-glycoprotein substrates. In addition, the effect of the various opioids on the transcellular movement of the prototypical P-glycoprotein substrate digoxin was examined in Caco-2 cell monolayers. IC50 values were calculated according to the Hill equation. Results Loperamide was a substrate showing high dependence on P-glycoprotein in that basal-apical transport was nearly 10-fold greater than in the apical-basal direction in L-MDRI cells. Morphine also showed a basal-to-apical gradient in the L-MDR1 cell monolayer, indicating that it too is a P-glycoprotein substrate, but with less dependence than loperamide in that only 1.5-fold greater basal-apical directional transport was observed. Fentanyl, sufentanil, and alfentanil did not behave as P-glycoprotein substrates, whereas the morphine glucuronides did not cross the cell monolayers at all, whether P-glycoprotein was present or not. Loperamide, sufentanil, fentanyl, and alfentanil inhibited P-glycoprotein-mediated digoxin transport in Caco-2 cells with IC50 values of 2.5, 4.5, 6.5, and 112 microm, respectively. Morphine and its glucuronides (20 microm) did not inhibit digoxin (5 microm) transport in Caco-2 cells, and therefore IC50 values were not determined. Conclusions Opioids have a wide spectrum of P-glycoprotein activity, acting as both substrates and inhibitors, which might contribute to their varying central nervous system-related effects.

1988 ◽  
Vol 60 (02) ◽  
pp. 240-246 ◽  
Erna G Langeler ◽  
Victor W M van Hinsbergh

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.

1987 ◽  
A de Agostini ◽  
J Marcum ◽  
R Rosenberg

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.

Pharmaceutics ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 176 ◽  
Laura Hellinen ◽  
Heidi Hongisto ◽  
Eva Ramsay ◽  
Kai Kaarniranta ◽  
Kati-Sisko Vellonen ◽  

The retinal pigment epithelial (RPE) cell monolayer forms the outer blood–retinal barrier and has a crucial role in ocular pharmacokinetics. Although several RPE cell models are available, there have been no systematic comparisons of their barrier properties with respect to drug permeability. We compared the barrier properties of several RPE secondary cell lines (ARPE19, ARPE19mel, and LEPI) and both primary (hfRPE) and stem-cell derived RPE (hESC-RPE) cells by investigating the permeability of nine drugs (aztreonam, ciprofloxacin, dexamethasone, fluconazole, ganciclovir, ketorolac, methotrexate, voriconazole, and quinidine) across cell monolayers. ARPE19, ARPE19mel, and hfRPE cells displayed a narrow Papp value range, with relatively high permeation rates (5.2–26 × 10−6 cm/s. In contrast, hESC-RPE and LEPI cells efficiently restricted the drug flux, and displayed even lower Papp values than those reported for bovine RPE-choroid, with the range of 0.4–32 cm−6/s (hESC-RPE cells) and 0.4–29 × 10−6 cm/s, (LEPI cells). Therefore, ARPE19, ARPE19mel, and hfRPE cells failed to form a tight barrier, whereas hESC-RPE and LEPI cells restricted the drug flux to a similar extent as bovine RPE-choroid. Therefore, LEPI and hESC-RPE cells are valuable tools in ocular drug discovery.

Kai Chien

Untreated glass is the ideal supporting substrate for cell cul¬ture growth since it is extremely flat and smooth, transparent and insoluable in organic solvent. However, difficulties have been encountered in removing polymerized epoxy resin from a glass surface following in situ cell monolayer embedment. Vari¬ous techniques have been made to grow cell cultures on either coated glass surfaces or plastic substrates. The purpose of this abstract is to describe a heat separation technique which when used together with a newly designed embedding mold allows cell monolayers to be transferred from untreated coverglass or glass slide to pre-shaped tissue blocks. The resulting tissue block can be easily separated and used directly for orientation light microscopy prior to ultramicrotomy.

2009 ◽  
Vol 297 (2) ◽  
pp. C440-C450 ◽  
Shannon A. Bainbridge ◽  
James M. Roberts ◽  
Frauke von Versen-Höynck ◽  
Jessa Koch ◽  
Lia Edmunds ◽  

Hyperuricemia develops as early as 10 wk of gestation in women who later develop preeclampsia. At this time the invasive trophoblast cells are actively remodeling the uterine spiral arterioles, integrating into and finally replacing the vascular endothelial lining. In the nonpregnant population uric acid has several pathogenic effects on vascular endothelium. We therefore sought to examine the effects of uric acid (0–7 mg/dl) on trophoblast cell invasion through an extracellular matrix using an in vitro Matrigel invasion assay. We also assessed trophoblast integration into a uterine microvascular endothelial cell monolayer in a trophoblast-endothelial cell coculture model. Additionally, we addressed the importance of redox signaling and trophoblast-induced endothelial cell apoptosis. Uric acid elicited a concentration-dependent attenuation of trophoblast invasion and integration into a uterine microvascular endothelial cell monolayer. The attenuated trophoblast integration appeared to be the result of reduced trophoblast-induced endothelial cell apoptosis, likely through the intracellular antioxidant actions of uric acid. In a test of relevance, pooled serum (5% vol/vol) from preeclamptic women attenuated the ability of trophoblast cells to integrate into the endothelial cell monolayers compared with pooled serum from healthy pregnant controls, and this response was partially rescued when endogenous uric acid was previously removed with uricase. Taken together these data support the hypothesis that elevations in circulating uric acid in preeclamptic women contribute to the pathogenesis of the disorder, in part, through attenuation of normal trophoblast invasion and spiral artery vascular remodeling.

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