Confluent Cell Monolayers Research Articles

Tuning of Mechanical Properties in Photopolymerizable Gelatin-Based Hydrogels for In Vitro Cell Culture Systems.

The mechanical microenvironment plays a crucial role in the evolution of colorectal cancer, a complex disease characterized by heterogeneous tumors with varying elasticity. Toward setting up distinct scenarios, herein, we describe the preparation and characterization of gelatin methacrylamide (GelMA)-based hydrogels via two different mechanisms: free-radical photopolymerization and photo-induced thiol-ene reaction. A precise stiffness modulation of covalently crosslinked scaffolds was achieved through the application of well-defined irradiation times while keeping the intensity constant. Besides, the incorporation of thiol chemistry strongly increased stiffness with low to moderate curing times. This wide range of finely tuned mechanical properties successfully covered from healthy tissue to colorectal cancer stages. Hydrogels prepared in phosphate-buffered saline or Dulbecco's modified Eagle's medium resulted in different mechanical and swelling properties, although a similar trend was observed for both conditions: thiol-ene systems exhibited higher stiffness and, at the same time, higher swelling capacity than free-radical photopolymerized networks. In terms of biological behavior, three of the substrates showed good cell proliferation rates according to the formation of a confluent monolayer of Caco-2 cells after 14 days of cell culture. Likewise, a characteristic apical-basal polarization of cells was observed for these three hydrogels. These results demonstrate the versatility of the presented platform of biomimetic materials as in vitro cell culture scaffolds.

Active Forces in Confluent Cell Monolayers

We use a computational phase-field model together with analytical analysis to study how intercellular active forces can mediate individual cell morphology and collective motion in a confluent cell monolayer. We explore the regime where intercellular forces dominate the tissue dynamics, and polar forces are negligible. Contractile intercellular interactions lead to cell elongation, nematic ordering, and active turbulence characterized by motile topological defects. Extensile interactions result in frustration, and perpendicular cell orientations become more prevalent. Furthermore, we show that contractile behavior can change to extensile behavior if anisotropic fluctuations in cell shape are considered.

Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy

Introduction: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty. Methods: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 μm Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers. Results: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0. Conclusions: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (>60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (<2,000 cells/mm2) of the donated cornea.

Adherent Bacteria and Parasiticidal Secretion Products of Human Cervicovaginal Microbiota-Associated Lactobacillus gasseri Confer Non-Identical Cell Protection against Trichomonas vaginalis-Induced Cell Detachment.

Trichomonas vaginalis, a protozoan parasite specific to the human genital tract, is one of the most common sexually transmitted pathogens. Its pathogenicity is strongly associated with its expression of a broad array of proteases triggering cytotoxic effects in host epithelial cells. Vaginal microbiota-associated Lactobacillus, including those of L. gasseri in particular, can counteract T. vaginalis pathogenesis, but the mechanisms involved have yet to be clarified. T. vaginalis strain G3 (Tv G3) cytotoxicity was assessed by examining cell morphology, cell detachment, and fluorescent labeling of the F-actin cytoskeleton and immunolabeling of vinculin-position focal adhesions (FAs) by confocal laser scanning electron microscopy on confluent cervicovaginal epithelial HeLa cell monolayers. The inhibitory effects of bacterial cells and secreted products of L. gasseri ATCC 9857 and KS 120.1 on the Tv G3 viability and parasite deleterious effects on HeLa cells were investigated. Pre-adhering L. gasseri cells delayed but did not inhibit Tv G3-induced cell detachment, F-actin cytoskeleton disorganization and the disappearance of vinculin-positive focal FAs. L. gasseri KS 120.1 secretion products had a rapid parasiticide activity by killing time- and concentration-dependent Tv G3 parasites after direct contact. By killing Tv G3 parasites already associated with the epithelial cells, secretion products have abolished parasite-induced cell detachment. Our findings suggest that vagina microbiota-associated L. gasseri creates a physical barrier and exerts pharmacological-type mechanisms to counteract the deleterious cytotoxic effects of T. vaginalis.

Comparative Safety, Immunogenicity, and Efficacy of CEF Cell-Based and DF-1 Cell Line Adapted Infectious Bursal Disease Vaccines in Specific-Pathogen-Free Chickens.

Infectious bursal disease (IBD) is an immunosuppressive and economically important disease of young chickens caused by infectious bursal disease virus (IBDV). The National Veterinary Institute (Bishoftu, Ethiopia) produces intermediate IBDV vaccine using primary chicken embryo fibroblast (CEF) cells, a method with technical and economical cumbersome. This study assessed the safety, immunogenicity, and efficacy of DF-1 cell line-adapted IBDV LC–75 vaccine strain in reference to the CEF-based vaccine. Confluent monolayer of DF-1 cells was infected with IBDV and cells with cytopathic effects were passaged until 3rd passage. Viral growth was confirmed using a one-step RT-PCR targeting IBDV VP2 gene. Viral titer increased from 1st passage through 3rd passage. Safety was assessed in 30 specific-pathogen-free chickens (15 chickens/group) injected with 10-fold field dose of each vaccine intraocularly and monitored for 21 days. For immunogenicity and efficacy, 60 specific-pathogen-free chickens were grouped into 3 (20 chickens/group). First and 2nd group received DF-1 cell and CEF-based IBDV vaccines, respectively. The 3rd group served as unvaccinated control. Antibody response was measured using iELISA. Chickens were challenged 4 weeks postvaccination with very virulent IBDV (vvIBDV) intraocularly and followed-up for 10 days. Vaccination did not cause any adverse reactions during the 21 days of follow-up. In addition, both vaccines induced higher antibody titer 14 and 24 days-post-vaccination as compared to unvaccinated controls (p < 0.05). Moreover, DF-1 and CEF-based IBDV LC–75 vaccines rendered a complete protection against vvIBDV. Contrarily, morbidity and mortality in unvaccinated chickens was 50% and 30%, respectively. The results indicated that DF-1 and CEF cell-based IBDV vaccines are comparably immunogenic and efficacious. Therefore, DF-1 cell-line can be considered an affordable and convenient alternative to the CEF-based approach. The suitability of DF-1 cells to grow other IBDV strains and safety of these vaccines on bursa of Fabricius should further be investigated.

Polycystin-2 (TRPP2) regulates primary cilium length in LLC-PK1 renal epithelial cells.

Polycystin-2 (PC2, TRPP2) is a Ca2+ permeable nonselective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. However, little is known as to whether PC2 contributes to the primary cilium structure. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild-type LLC-PK1 renal epithelial cells. Confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated α-tubulin antibody to identify primary cilia and measure their length. Although primary cilia length measurements did not follow a Normal distribution, the data were normalized by Box-Cox transformation rendering statistical differences under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 μM) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length compared to their respective scrambled RNA transfected cells. The data indicate that conditions that regulate PC2 function or gene expression modify the length of primary cilia in renal epithelial cells. Blocking of PC2 mitigates the effects of elevated external Ca2+ concentration on primary cilia length. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

Methanol extraction revealed anticancer compounds Quinic Acid, 2(5H)‑Furanone and Phytol in Andrographispaniculata.

Andrographis paniculata (Ap) has been a part of traditional medicine for its anti-inflammatory effects, treatment of snake bites and liver abnormalities. Several investigations have revealed its bioactive components to be andrographolides. The methanolic extracts of leaves from Ap were characterized, the non-andrographolides were identified and screened for anti-proliferative activity. The extracts showed significant toxicity against numerous cancer cells including HeLa, MCF7, BT549, 293 and A549 cells. Anti-proliferative activity and effect on cancer cell invasion (metastatic potential) and cell migration were examined. The extracts revealed significant inhibition of the ability of HeLa cells in repairing the gap created by a vertical wound made on a confluent cell monolayer. Similarly, a 40% reduction in cell migration was observed in presence of the extracts. Significant anti-angiogenic activity in terms of reduced blood capillary formation was observed on the Chorioallantoic membranes of embryonated hen eggs co-inoculated with HeLa cells and the extracts. Analysis of HeLa cells treated with the extracts using flow cytometry indicated the arrest of cell cycle progression at the G2/M phase. Variation in the Bax/Bcl-2 ratio and elevated caspase-3 levels by immunoblotting confirmed cell death induction via the apoptotic pathway. Investigation of the extracts by gas chromatography-mass spectrometry displayed the predominant components to be 2(5H)-Furanone (14.73%), Quinic acid (17.32%), and Phytol (11.43%). These components have been previously known to have anticancer activity, while being studied individually in other plants. This is the first study, to the best of our knowledge, on the anti-proliferative and anti-angiogenic activity of the non-andrographolide components from Ap.

Comparison of human amniotic membrane decellularisation approaches for hESC-derived RPE cells culture

ObjectiveRecent clinical studies have shown that the transplantation of functional retinal pigment epithelium (RPE) cells can prevent the onset of RPE degeneration in age-related macular degeneration. This study aimed to...

Mechanical coupling of supracellular stress amplification and tissue fluidization during exit from quiescence

Cellular quiescence is a state of reversible cell cycle arrest that is associated with tissue dormancy. Timely regulated entry into and exit from quiescence is important for processes such as tissue homeostasis, tissue repair, stem cell maintenance, developmental processes, and immunity. However, little is known about processes that control the mechanical adaption to cell behavior changes during the transition from quiescence to proliferation. Here, we show that quiescent human keratinocyte monolayers sustain an actinomyosin-based system that facilitates global cell sheet displacements upon serum-stimulated exit from quiescence. Mechanistically, exposure of quiescent cells to serum-borne mitogens leads to rapid amplification of preexisting contractile sites, leading to a burst in monolayer tension that subsequently drives large-scale displacements of otherwise motility-restricted monolayers. The stress level after quiescence exit correlates with the level of quiescence depth at the time of activation, and a critical stress magnitude must be reached to overcome the cell sheet displacement barrier. The study shows that static quiescent cell monolayers are mechanically poised for motility, and it identifies global stress amplification as a mechanism for overcoming motility restrictions in confined confluent cell monolayers.

Valorization of a Levulinic Acid Platform through Electrospinning of Polyhydroxyalkanoate-Based Fibrous Membranes for In Vitro Modeling of Biological Barriers

In vitro models of biological barriers provide a reliable tool for investigating the physiopathological processes involved in the development of numerous diseases. Producing sustainable in vitro models obtained from solvents and biopolymers derived from industrial by-products add an important value to this underestimated source of valuable (bio)materials. This works aims at demonstrating the suitability of processing together solvents derived from levulinic acid (LA) (extracted from biomasses) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) (whose production is facilitated by LA) to produce electrospun membranes as proof-of-concept of a sustainable, engineered biological barrier fully derived from LA as the starting feedstock. The electrospinning process is initially optimized by identifying the most suitable conditions for obtaining self-supporting microporous membranes. In particular, LA-derived solvents (γ-valerolactone, 2-methyltetrahydrofuran, methyl ethyl ketone, and methyl and ethyl levulinate), PHBV concentration, and electrospinning process parameters were investigated. Self-standing and hydrophobic PHBV mats with a micropore size in the range of 1–7 μm and an average elastic modulus of 75 MPa are successfully obtained by using methyl ethyl ketone/formic acid as solvent. Preliminary cell experiments demonstrate that the developed fibrous PHBV mats promote the formation of a confluent monolayer of epithelial cells after 48 h and therefore they can potentially be used to mimic biological epithelial barriers.

Possible Therapeutic Utility of anti-Cell Adhesion Molecule 1 Antibodies for Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly aggressive malignant tumor, and the effective therapeutic drugs are limited. Thus, the establishment of novel therapeutic method is desired. Considerable proportion of MPMs are shown to express cell adhesion molecule 1 (CADM1), and to use CADM1 to bind to and proliferate on the pleural mesothelial surface, suggesting that CADM1 is a possible therapeutic target. Here, anti-CADM1 ectodomain chicken monoclonal antibodies, 3E1 and 9D2, were examined for their possible therapeutic utility. The full-length form of CADM1 was expressed in eight out of twelve human MPM cell lines. MPM cell lines were cultured on a confluent monolayer of mesothelial MeT-5A cells in the presence of 9D2, the neutralizing antibody. 9D2 suppressed the cell growth of CADM1-positive MPM cells with the loss and aggregation of CADM1 molecules on the MPM cell membrane, but not of CADM1-negative MPM cells. Co-addition of 3E1, lacking the neutralizing action, enhanced the growth-suppressive effect of 9D2. The two antibodies were tested as drug delivery vectors. 3E1 was converted into a humanized antibody (h3E1) and conjugated with monomethyl auristatin E (MMAE), a tubulin polymerization inhibitor. When the resulting h3E1–MMAE antibody-drug conjugate (ADC) was added to the standard cultures of CADM1-positive MPM cells, it suppressed the cell growth in a dose-dependent manner. Co-addition of 9D2 enhanced the growth-suppressive effect of h3E1–MMAE ADC. Anti-CADM1 ectodomain antibodies were suggested to serve as both antibody drugs and drug vectors in the treatment of MPM.

Genome-Wide Analysis and Molecular Characterization of Orf Virus Strain UPM/HSN-20 Isolated From Goat in Malaysia.

Contagious ecthyma commonly known as Orf is a globally important, highly contagious zoonotic, transboundary disease that affects domestic and wild ruminants. The disease is of great economic significance causing an immense impact on animal health, welfare, productivity, and trade. Detailed analysis of the viral genome is crucial to further elucidate the molecular mechanism of Orf virus (ORFV) pathogenesis. In the present study, a confluent monolayer of lamb testicle cells was infected with the processed scab sample obtained from an infected goat. The presence of the virus was confirmed using polymerase chain reaction and electron microscopy, while its genome was sequenced using next-generation sequencing technology. The genome sequence of Malaysian ORFV strain UPM/HSN-20 was found to contain 132,124 bp with a G + C content of 63.7%. The homology analysis indicates that UPM/HSN-20 has a high level of identity 97.3–99.0% with the other reference ORFV strain. Phylogenetic analysis revealed that ORFV strain UPM/HSN-20 is genetically more closely related to ORFV strain XY and NP from China. The availability of the genome-wide analysis of ORFV UPM/HSN-20 strain from Malaysia will serve as a good platform for further understanding of genetic diversity, ORFV infection, and strategic development for control measures.

Poly(2-Methoxyethyl Acrylate) (PMEA)-Coated Anti-Platelet Adhesive Surfaces to Mimic Native Blood Vessels through HUVECs Attachment, Migration, and Monolayer Formation

Confluent monolayers of human umbilical vein endothelial cells (HUVECs) on a poly(2-methoxyethyl acrylate) (PMEA) antithrombogenic surface play a major role in mimicking the inner surface of native blood vessels. In this study, we extensively investigated the behavior of cell–polymer and cell–cell interactions by measuring adhesion strength using single-cell force spectroscopy. In addition, the attachment and migration of HUVECs on PMEA-analogous substrates were detected, and the migration rate was estimated. Moreover, the bilateral migration of HUVECs between two adjacent surfaces was observed. Furthermore, the outer surface of HUVEC was examined using frequency-modulation atomic force microscopy (FM-AFM). Hydration was found to be an indication of a healthy glycocalyx layer. The results were compared with the hydration states of individual PMEA-analogous polymers to understand the adhesion mechanism between the cells and substrates in the interface region. HUVECs could attach and spread on the PMEA surface with stronger adhesion strength than self-adhesion strength, and migration occurred over the surface of analogue polymers. We confirmed that platelets could not adhere to HUVEC monolayers cultured on the PMEA surface. FM-AFM images revealed a hydration layer on the HUVEC surfaces, indicating the presence of components of the glycocalyx layer in the presence of intermediate water. Our findings show that PMEA can mimic original blood vessels through an antithrombogenic HUVEC monolayer and is thus suitable for the construction of artificial small-diameter blood vessels.

Extending applications of AFM to fluidic AFM in single living cell studies.

In this article, a review of a series of applications of atomic force microscopy (AFM) and fluidic Atomic Force Microscopy (fluidic AFM, hereafter fluidFM) in single-cell studies is presented. AFM applications involving single-cell and extracellular vesicle (EV) studies, colloidal force spectroscopy, and single-cell adhesion measurements are discussed. FluidFM is an offshoot of AFM that combines a microfluidic cantilever with AFM and has enabled the research community to conduct biological, pathological, and pharmacological studies on cells at the single-cell level in a liquid environment. In this review, capacities of fluidFM are discussed to illustrate (1) the speed with which sequential measurements of adhesion using coated colloid beads can be done, (2) the ability to assess lateral binding forces of endothelial or epithelial cells in a confluent cell monolayer in an appropriate physiological environment, and (3) the ease of measurement of vertical binding forces of intercellular adhesion between heterogeneous cells. Furthermore, key applications of fluidFM are reviewed regarding to EV absorption, manipulation of a single living cell by intracellular injection, sampling of cellular fluid from a single living cell, patch clamping, and mass measurements of a single living cell.

Proteomic Analysis of Control and Disrupted Endothelial Monolayers

The microvascular wall regulates normal blood‐tissue exchange, but under inflammatory conditions, elevated endothelial permeability leads to edema, poor tissue oxygenation, and disrupted tissue homeostasis. Much remains unknown about the mechanisms involved. Using an unbiased proteomic analysis of confluent human umbilical vein endothelial cell (HUVEC) monolayers that were stimulated with 0.1 U/mL thrombin, which produces a time‐dependent increase in permeability that peaks 30 min and recovers to baseline levels at 120 min, novel candidate proteins that may participate in this mechanism were identified. Cell lysates were collected, proteins digested, and the peptides were characterized using a Thermo Q‐exactive‐HF mass spectrometer coupled to a Thermo Easy nLC 1200 to get label‐free quantitation (LFQ) of relative protein expression. High‐resolution mass spectrometric data were analyzed on the MaxQuant processing suite, and the protein groups file subsequently analyzed with the Perseus processing suite. The analysis revealed identified 4627 unique proteins present in HUVEC. Ratios of the LFQ intensities of 30 min. thrombin vs. control and 120 min. thrombin vs. control were generated. Statistical significance (p &lt; 0.05) for 30 min. thrombin vs. control or 120 min. thrombin vs. control was determined using Welch’s t‐test of log(2)‐transformed LFQ intensities. In addition, a z‐score cutoff calculated as [(t test difference of individual protein) − (median t test difference of dataset)] / (standard deviation of t test difference of dataset) was used. The criteria of |z‐score| &gt; 1 and p &lt; 0.05 with Welch’s t test were utilized to filter the dataset, identifying 214 proteins to have altered expression after 30 min thrombin treatment, and 172 proteins having altered expression after 120 min thrombin. When entering the filtered datasets into String‐db, tight junction signaling was identified as enriched with thrombin treatment. Employing a more stringent filtering approach to identify candidate proteins for further study (|z‐score| &gt; 2), we found that the tight junction protein claudin‐11 (CLDN11) had significantly decreased expression after 30 min thrombin (73% decrease from control) but was not significant changed at 120 min. Expression of CLDN11 was confirmed to be decreased by thrombin at 30 min using SimpleWestern (WES) analysis. To determine whether experimental depletion of CLDN11 expression decreases endothelial barrier function, selective CLDN11 siRNA was used. However, evaluation of barrier function using electric cell‐substrate impedance sensing showed no significant changes for up to 48 h after transfection with the siRNA, when CLDN11 protein levels were significantly decreased. Immunofluorescence labeling of CLDN11 in confluent HUVEC revealed that while some CLDN11 could be found in junctions, most labeling was punctate in the cytoplasm, possibly residing in vesicles. Collectively, the results suggest that thrombin significantly alters endothelial tight junctions and reduces CLDN11 expression. However, selective reduction of CLDN11 levels alone does not appear to be sufficient to alter HUVEC monolayer barrier function, and this may be because most CLDN11 fails to localize in junctions in this cultured endothelial cell model. Future studies will be directed at evaluating CLDN11 localization in postcapillary venules and determining its potential role in microvascular barrier function using intact microvessels and in vivo.

Evolution of a confluent gut epithelium under on-chip cyclic stretching

The progress of food in the gastrointestinal (GI) tract is driven by a peristaltic motion generated by the muscle belt surrounding the GI tract. In turn, the response of the intestinal epithelial cells to the peristaltic stresses affects the dynamics of the epithelial structure. In this work, we study the effect of cyclic stretching (0.125 Hz, 10% strain) on the spatial organization of the intestinal epithelium using intestinal cells deposited on a flat elastomeric substrate to mimic the peristaltic motion in vitro. A confluent monolayer of Caco-2 cells is grown on a PDMS chip to probe the morphological and orientational response of the tissue to cyclic stretching. The PDMS chips are either covalently or noncovalently coated with laminin to recapitulate the basement membrane. We observe a significant orientational response where the cells rearrange their long axes perpendicular to the stretching direction for both coating conditions. The experiment is modeled by a vertex model where the cells store elastic energy with varying strain and effectively have a rotational diffusive motion of their long axes through rearrangements of their shapes. The model also predicts a transition between the perpendicular orientation and the orientation at an oblique angle determined by the level of the cell elastic anisotropy. It provides a general framework to study cell response and relaxation dynamics under cyclic stretching across different cell types. We also discuss potential relevance of peristalsis in determining planar cell polarity in three-dimensional architectures.

The crescendo pulse frequency of shear stress stimulates the endothelialization of bone marrow mesenchymal stem cells on the luminal surface of decellularized scaffold in the bioreactor

ABSTRACT A completely confluent endothelial cell (EC) monolayer is required to maintain proper vascular function in small diameter tissue-engineered vascular graft (TEVG). However, the most effective method for EC attachment to the luminal surface and formation of an entire endothelium layer that works in vitro remains a complicated challenge that requires urgent resolution. Although pulsatile flow has been shown to be better suited for the generation of functional endothelium, the optimal frequency setting is unknown. Several pulsatile flow frequencies were used to implant rat bone mesenchymal stem cells (MSC) into the lumen of decellularized porcine carotid arteries. The endothelium's integrity and cell activity were investigated in order to determine the best pulse frequency settings. The results showed that MSC were maximally preserved and exhibited maximal morphological changes with improved endothelialization performance in response to increased pulse stimulation frequency. Increased pulse frequency stimulation stimulates the expression of mechanoreceptor markers, cytoskeleton reorganization in the direction of blood flow, denser skeletal proteins fibronectin, and stronger intercellular connections when compared to constant pulse frequency stimulation. MSC eventually develops an intact endothelial layer with anti-thrombotic properties on the inner wall of the decellularized tubular lumen. Conclusion: The decellularized vessels retain the three-dimensional structure of the vasculature, have a surface topography suitable for MSC growth, and have good mechanical properties. By increasing the frequency of pulsed stimulation, MSC endothelialize the lumen of the decellularized vasculature. It is expected to have anti-thrombotic and anti-neointimal hyperplasia properties after implantation, ultimately improving the patency of TEVG.

Atomic Level Dissection of the Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) Homophilic Binding Interface: Implications for Endothelial Cell Barrier Function.

PECAM-1 (platelet endothelial cell adhesion molecule 1) is a 130 kDa member of the immunoglobulin (Ig) gene superfamily that is expressed on the surfaces of platelets and leukocytes and concentrated at the intercellular junctions of confluent endothelial cell monolayers. PECAM-1 Ig domains 1 and 2 (IgD1 and IgD2) engage in homophilic interactions that support a host of vascular functions, including support of leukocyte transendothelial migration and the maintenance of endothelial junctional integrity. The recently solved crystal structure of PECAM-1 IgD1 and IgD2 revealed a number of intermolecular interfaces predicted to play important roles in stabilizing PECAM-1/PECAM-1 homophilic interactions and in formation and maintenance of endothelial cell-cell contacts. We sought to determine whether the protein interfaces implicated in the crystal structure reflect physiologically important interactions. Approach and Results: We assessed the impact of single amino acid substitutions at the interfaces between opposing PECAM-1 molecules on homophilic binding and endothelial cell function. Substitution of key residues within the IgD1-IgD1 and IgD1-IgD2 interfaces but not those within the smaller IgD2-IgD2 interface, markedly disrupted PECAM-1 homophilic binding and its downstream effector functions, including the ability of PECAM-1 to localize at endothelial cell-cell borders, mediate the formation of endothelial tubes, and restore endothelial barrier integrity. Taken together, these results validate the recently described PECAM-1 IgD1/IgD2 crystal structure by demonstrating that specific residues visualized within the IgD1-IgD1 and IgD1-IgD2 interfaces of opposing molecules in the crystal are required for functionally important homophilic interactions. This information can now be exploited to modulate functions of PECAM-1 in vivo.

Construction and Evaluation of a Bio-Engineered Pump to Enable Subpulmonary Support of the Fontan Circulation: A Proof-of-Concept Study.

Our objective was to create a bio-engineered pump (BEP) for subpulmonary Fontan circulation support capable of luminal endothelialization and producing a 2-6 mmHg pressure gradient across the device without flow obstruction. To accomplish this, porcine urinary bladder submucosa was decellularized to produce a urinary bladder matrix (UBM) which produced acellular sheets of UBM. The UBM was cultured with human umbilical vein endothelial cells producing a nearly confluent monolayer of cells with the maintenance of typical histologic features demonstrating UBM to be a suitable substrate for endothelial cells. A lamination process created bilayer UBM sheets which were formed into biologic reservoirs. BEPs were constructed by securing the biologic reservoir between inlet and outlet valves and compressed with a polyurethane balloon. BEP function was evaluated in a simple flow loop representative of a modified subpulmonary Fontan circulation. A BEP with a 92-mL biologic reservoir operating at 60 cycles per minute produced pulsatile downstream flows without flow obstruction and generated a favorable pressure gradient across the device, maintaining upstream pressure of 6 mm Hg and producing downstream pressure of 13 mm Hg. The BEP represents potential long-term assistance for the Fontan circulation to relieve venous hypertension, provide pulsatile pulmonary blood flow and maintain cardiac preload.

Increased METTL3-mediated m6A methylation inhibits embryo implantation by repressing HOXA10 expression in recurrent implantation failure

BackgroundRecurrent implantation failure (RIF) is a major limitation of assisted reproductive technology, which is associated with impaired endometrial receptivity. Although N6-methyladenosine (m6A) has been demonstrated to be involved in various biological processes, its potential role in the endometrium of women with RIF has been poorly studied.MethodsGlobal m6A levels and major m6A methyltransferases/demethylases mRNA levels in mid-secretory endometrium from normal and RIF women were examined by colorimetric m6A quantification strategy and quantitative real-time PCR, respectively. The effects of METTL3-mediated m6A modification on embryo attachment were evaluated by an vitro model of a confluent monolayer of Ishikawa cells co-cultured with BeWo spheroids, and the expression levels of homeo box A10 (HOXA10, a well-characterized marker of endometrial receptivity) and its downstream targets were evaluated by quantitative real-time PCR and Western blotting in METTL3-overexpressing Ishikawa cells. The molecular mechanism for METTL3 regulating HOXA10 expression was determined by methylated RNA immunoprecipitation assay and transcription inhibition assay.ResultsGlobal m6A methylation and METTL3 expression were significantly increased in the endometrial tissues from women with RIF compared with the controls. Overexpression of METTL3 in Ishikawa cells significantly decreased the ration of BeWo spheroid attachment, and inhibited HOXA10 expression with downstream decreased β3-integrin and increased empty spiracles homeobox 2 expression. METTL3 catalyzed the m6A methylation of HOXA10 mRNA and contributed to its decay with shortened half-life. Enforced expression of HOXA10 in Ishikawa cells effectively rescued the impairment of METTL3 on the embryo attachment in vitro.ConclusionIncreased METTL3-mediated m6A modification represents an adverse impact on embryo implantation by inhibiting HOXA10 expression, contributing to the pathogenesis of RIF.