Confluent Monolayers Research Articles

MESENCHYMAL STEM CELLS INFLUENCE TROPHOBLAST AND ENDOTHELIAL CELL FUNCTIONALITY IMPORTANT FOR PREVENTION OF PRE-ECLAMPSIA VIA A NOVEL ANTI-ANGIOGENIC PROTEIN, FKBPL

Objective: Pre-eclampsia is a disorder affecting 5–6% of all pregnancies globally. It is characterised by the new onset of hypertension and proteinuria post 20 weeks gestation. Pre-eclampsia is a leading cause of morbidity and mortality in both mothers and children. Although the pathogenesis of pre-eclampsia is poorly understood, aberrant angiogenesis and inadequate trophoblast cell function have both been implicated. Mesenchymal Stem Cell (MSC)-based therapies have shown benefits in animal models of pre-eclampsia however, the underlying mechanisms are not well understood. FKBPL is a novel anti-angiogenic protein which has a critical role in developmental, physiological and pathological angiogenesis. In this study, our objective was to evaluate the effects of MSC–conditioned medium (MSC-CM) on migration and differentiation of trophoblast and endothelial cell lines under both normoxic and hypoxic conditions. The role of FKBPL signalling in these processes was also investigated. Design and method: Human trophoblast (BeWo and Jar) and endothelial cells (HUVEC) were incubated in the presence of human bone marrow- derived MSC-CM, normal or serum free medium under normoxia (21% oxygen) or hypoxia (1% oxygen). The confluent cell monolayer was wounded and the percentage of wound closure assessed at 24 h. HUVEC were stained with Calcein prior to 6-hour incubation in the presence of MSC-CM, normal or serum free medium under normoxia or hypoxia. Tubule formation was assessed using Image J. FKBPL protein expression was evaluated using western blot analysis where cells were lysed in RIPA buffer before being subjected to western blotting and probed for FKBPL and GAPDH. Results: MSC-CM promoted cell migration significantly in all three cell lines under both normoxia and hypoxia compared to normal medium (n = 6; p < 0.001). MSC-CM also significantly increased the formation of HUVEC tubule networks under both normoxia and hypoxia compared to normal medium. Furthermore, concomitant reduction in FKBPL protein expression was observed, demonstrating potential FKBPL's involvement in MSC-driven effects on trophoblast and endothelial cell functionality. Conclusions: Our findings suggest that MSCs could be explored as potential preventative therapy for pre-eclampsia by ameliorating trophoblast and endothelial cell functionality, and that the mechanism is likely to involve a novel anti-angiogenic protein, FKBPL.

Programmed Shape‐Morphing Scaffolds Enabling Facile 3D Endothelialization

AbstractRapid formation of a confluent endothelial monolayer is the key to the success of small‐diameter vascular grafts, which is significantly important for treating dangerous and even sometimes deadly vascular disorders. However, the difficulty to homogenously locate endothelial cells onto the lumen of small‐diameter tubular scaffolds makes 3D endothelialization greatly challenging. Here, novel shape‐morphing scaffolds enabling programmed deformation from planar shapes to small‐diameter tubular shapes are designed and developed by combining biocompatible shape memory polymer and electrospun nanofibrous membrane. Endothelial cells can be conveniently seeded and attached on the 2D surface of the scaffolds and subsequently self‐rolled into 3D organization at physiological temperature. Endothelial cell responses and functions are varied on the shape‐morphing scaffolds with different nanofibrous electrospun membranes as the inner layer, arisen from the inducement of scaffolds with different morphological, physical, and biochemical characteristics. Owing to excellent properties of the nanofibrous membrane fabricated by the coelectrospinning of poly‐ε‐caprolactone (PCL) and gelatin methacrylate (GelMA), the shape‐morphing scaffolds with a nanofibrous PCL/GelMA inner layer support desirable homogeneous endothelial cell attachment as well as the rapid formation of biomimetic cell–scaffold interaction and cell–cell interaction under the 3D cell culture condition, therefore offering a visible approach for facile 3D endothelialization.

Deciphering the relative contribution of vascular inflammation and blood rheology in metastatic spreading.

Vascular adhesion of circulating tumor cells (CTCs) is a key step in cancer spreading. If inflammation is recognized to favor the formation of vascular "metastatic niches," little is known about the contribution of blood rheology to CTC deposition. Herein, a microfluidic chip, covered by a confluent monolayer of endothelial cells, is used for analyzing the adhesion and rolling of colorectal (HCT-15) and breast (MDA-MB-231) cancer cells under different biophysical conditions. These include the analysis of cell transport in a physiological solution and whole blood over a healthy and a TNF-α inflamed endothelium with a flow rate of 50 and 100 nl/min. Upon stimulation of the endothelial monolayer with TNF-α (25 ng/ml), CTC adhesion increases from 2 to 4 times whilst cell rolling velocity only slightly reduces. Notably, whole blood also enhances cancer cell deposition from 2 to 3 times, but only on the unstimulated vasculature. For all tested conditions, no statistically significant difference is observed between the two cancer cell types. Finally, a computational model for CTC transport demonstrates that a rigid cell approximation reasonably predicts rolling velocities while cell deformability is needed to model adhesion. These results would suggest that, within microvascular networks, blood rheology and inflammation contribute similarly to CTC deposition, thereby facilitating the formation of metastatic niches along the entire network, including the healthy endothelium. In microfluidic-based assays, neglecting blood rheology would significantly underestimate the metastatic potential of cancer cells.

Stem cell product-based blockage of cytokine-induced apoptosis: implications for diabetes

A feature of Type 1 diabetes mellitus (T1DM) is the cytokine-induced apoptosis of insulin-secretingBeta cells (β-cells). The cytokines are secreted from macrophages after infiltration into the pancreatic islets of Langerhans. Daily insulin supplementation is the only widely available treatment option for this disease, but this can lead to serious complications including diabetic ketoacidosis or cardiovascular disease. The utility of mesenchymal stem cells as a therapeutic and novel drug discovery tool is being widely explored. The aim of this study was to explore the therapeutic effectiveness of mesenchymal stem cells (MSC) on cytokine-induced apoptosis in β-cells. BRIN BD11,an insulin-secreting β-cell line was exposed to pro-inflammatory cytokines and endotoxin (IFN-γ,TNF-α,IL-1β and LPS) to induce β-cells apoptosis. This was performed with and without MSC serum-free conditioned media (SF-CM). SF-CM was conditioned for 24 hours on 70% confluent MSC monolayers. Cellular viability and apoptosis were determined with MTT and TUNEL assays respectively. IFN-γ (1μg/ml), TNF-α (100ng/ml),IL-1β (100ng/ml) AND LPS (500 μg/ml) all reduced BRIN BD11 viability to approximately 50% at the concentrations indicated (Figure1A). This effect was noted with and without serum where the latter resulted in decreased sensitivity to TNF-α and LPS. The TUNEL assay confirmed that this reduction in cellular viability resulted from programmed cell death (apoptosis) in all instances (Figure1C). However, the addition of SF-CM significantly decreased the sensitivity of BRIN BD11 cells to all cytokines (P≤0.05) and endotoxin (P≤0.05)in the presence and absence of serum.

A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY/MASS SPECTROMETRY METHOD DEVELOPMENT FOR THE QUANTITATIVE DETERMINATION OF BISOPROLOL FROM CACO-2 CELL MONOLAYERS

Objective: A simple, rapid high-performance liquid chromatography–mass spectrometry (MS)/MS method was developed for the determination of bisoprolol from confluent Caco-2 monolayers and from aqueous solution.Methods: Chromatography was achieved on discovery C 18, 50 mm×2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A [acetonitrile:water:formic acid, 5:95:0.1 v/v] and eluent B [acetonitrile:formic acid, 100:0.1 v/v]). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 ml/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.Results: Under these conditions, bisoprolol was eluted at 1.49 min. According to the Caco-2 test results, bisoprolol appeared to have moderate to high permeability. It should be noted that the recovery value for bisoprolol is 97.69%. Caco-2 permeability values for bisoprolol are in agreement with BCS Class I classification for these drugs and their known high bioavailability in humans.Conclusion: From results of analysis, it can be concluded that developed method is simple and rapid for the determination of bisoprolol from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for the examination of bisoprolol from Caco-2 cell monolayers.

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Analysis of ZO‐1, ARHGEF11 and Actin Cytoskeleton at the Apical Junctional Complex

BackgroundZonula occludens (ZO‐1) is a tight junction cytosolic scaffolding protein known to bind the actin cytoskeleton in epithelial cells. ARHGEF11 is a Rho guanine nucleotide exchange factor known to activate the Rho family of small GTPases which are regulators of the actin cytoskeleton of the cell. ARHGEF11 associates with ZO‐1 by binding to the ZU5 domain in the C‐terminal tail. We are interested in understanding the interplay between ZO‐1, ARHGEF11 and the actin cytoskeleton in confluent MDCK II cells.MethodsConfluent monolayers stably transfected with EGFP‐ZO‐1 or EGFP‐ZO‐1Δ ZU5 were treated with latrunculin A to observe the actin depolymerization protection capacity of ZO‐1 with and without the ZU5 domain. Additionally, studies were performed to investigate the localization of EGFP‐ZO‐1 or EGFP‐ZO‐1ΔZU5 proteins following recovery from latrunculin A exposure. Colocalization studies were performed in mCherry‐ARHGEF11/EGFP‐ZO‐1 co‐transfected cells. Pharmacological inhibition of Rho‐kinase was used to target the ARHGEF11 pathway. Electrophysiological studies were conducted to evaluate junctional permeability.ResultsFull‐length EGFP‐ZO‐1 protects against latrunculin A induced actin depolymerization more robustly than EGFP‐ZO‐1ΔZU5. In the actin polymerization recovery paradigm EGFP‐ZO‐1ΔZU5 exhibited enhanced recovery rate when compared to full‐length EGFP‐ZO‐1 following latrunculin A exposure. Enhanced actin networks are observed in mCherry‐ARHGEF11 positive cells in both wildtype and ZO‐1 knockdown MDCK II cells.ConclusionsThe increased recovery rate exhibited by EGFP‐ZO‐1ΔZU5 in the latrunculin A recovery assay coupled with the greater actin protection capacity of full‐length ZO‐1 suggests that truncated ZO‐1 is more mobile. ARHGEF11 elicits actin cytoskeleton reorganization in both wildtype and ZO‐1 knockdown cells that was partially reversed by inhibition of Rho kinase. There was limited accumulation of ARHGEF11 at the apical junctional complex in confluent monolayers.Support or Funding InformationSan Antonio Medical Foundation, Trinity University Biology Department, Mach Research AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evidence that Zinc Deficiency Impairs Gut Epithelial Barrier and intestinal Immunity

There is growing interest in dietary factors, in particular micronutrients, from the perspective of disease pathogenesis and potential for treatment. Diarrheal disease in infant results in abnormally low concentrations of serum zinc. Zinc has been tested for its ability to treat and prevent diarrheal diseases in many large field trials over a period of over 4 decades and has generally been found effective. The mechanism by which zinc deficiency causes diarrhea is not known. The present study investigated the impact of zinc deficiency on the host, particularly of intestinal tight junction (TJ) integrity and proteins that are involved in intestinal absorption and secretion along with the pathogenesis of enteric infection. We have used human colonic T84 cells to study barrier function and Shigella to test susceptibility of intestinal infection due to zinc deficiency. T84 grown onto transwell inserts in zinc deficient media showed a low transepithelial electrical resistance (TEER) compare to zinc sufficient media (1014 ± 165 Ω.cm2 vs 3663 ± 293 Ω.cm2) in confluent T84 monolayers. We furthermore analysed the correlation between TEER and paracellular ionic conductance. We found that zinc deficiency altered paracellular ionic conductance. Bacterial transmigration studies showed a significant increase in apico‐basolateral transmigration of Shigella (6.58 ± 0.13 vs 7.98 ± 0.23 log10 CFU/ml) which peaked at 6hrs post infection. Transmission electron microscopy data showed widened TJ complex due to few membrane fusion proteins and distorted TJ morphology, whereas the desmosomes were still intact. Electrophysiological studies with Ussing chamber and qPCR quantification demonstrated reduced cAMP dependent electrogenic Cl− secretion (34.5 ± 1.25 μA/cm2 vs 17.75 ± 2.3 μA/cm2) along with reduced expression of CFTR in zinc deficient cells, resulting dehydrated lumen thus aiding in bacterial colonization. Our results suggest that zinc deficiency caused (1) perturbed barrier function and integrity (2) altered Cl− secretion leading to susceptibility of infection. We conclude that adequate zinc is required to maintain intestinal barrier health to avoid risk against intestinal infection.Support or Funding InformationGovt of India National Funding AgencyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bicarbonate is required for proliferation of pulmonary endothelial cells

RationaleThe second messenger cAMP is widely known to regulate proliferation in numerous cell types. Many of these studies implicate cAMP generated by membrane‐bound cyclases (AC1‐9). While these membrane‐bound ACs generate cAMP in the near membrane compartment, cAMP can also be generated in unique subcellular compartments by the soluble adenylyl cyclase (sAC or AC10). Soluble AC localizes to the cytosol as well as organelles, such as the mitochondria, and is uniquely stimulated by bicarbonate to generate cAMP. Recently, AC10 was observed to be overexpressed in prostate carcinoma, suggesting a role for AC10 in cellular proliferation; however, the independent role of bicarbonate in proliferation has not been investigated. Thus, we tested the hypothesis that bicarbonate is required for proliferation of pulmonary endothelial cells.MethodsPulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs) were seeded at 100, 000 cells per well in media with and without bicarbonate. The cells were counted for seven consecutive days to generate growth curves and blood gas analysis performed to determine pH, pO2, pCO2, and HCO3− concentrations of the media. Additionally, a separate set of PMVECs were seeded at 100, 000 cells per well in bicarbonate free media and bicarbonate added back to the media after 24, 48, 72, 96, 120, 144, and 168 hours and counted for five days following bicarbonate add back.ResultsGrowth curves revealed that PMVECs and PAECs seeded in the bicarbonate‐containing media entered into log phase growth followed by a plateau phase at cell confluence; however, PMVECs and PAECs seeded in the bicarbonate free media did not enter log phase growth nor reach confluence. When bicarbonate was added back to PMVECs initially grown in a bicarbonate free environment, the cells proliferated to numbers comparable to PMVECs seeded in bicarbonate containing media throughout the growth curve; however, if PMVECs were incubated in bicarbonate free media for more than 96 hours, these cells did not enter into log phase growth and did not form a confluent monolayer.ConclusionThese data reveal a requirement for bicarbonate for proliferation of pulmonary endothelial cells, since neither PAECs nor PMVECs formed confluent monolayers in the absence of bicarbonate.Support or Funding InformationNIH 121513This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ZEB1 REGULATES MULTIPLE ASPECTS OF ENDOTHELIAL QUIESCENCE DURING VASCULAR REMODELLING

Generation of new blood vessels is essential for vascular remodeling in exercise to provide greater blood flow to developing muscles. As blood vessels grow, the sprouting endothelial tips need to recognise each other, and move from a motile, angiogenic phenotype to a maturing, quiescent phenotype,, form tubes and allow blood flow to occur and regulate their development. They grow in response to cytokine signalling such as VEGF and other angiogenic factors, but they mature as a consequence of cell contact and flow. As they do so, they change their metabolic state from growing, glycolytic, to quiescent oxidative metabolism. The mechanisms behind the transcriptional control of this phenotype are unknown, but recent work has demonstrated the dynamic interplay between ‘quiescence’ factors (such as FOXO1 expression) and expression of glycolytic enzymes such as PFKFB3 through c‐MYC. We investigated potential drivers of this process and identified Zeb1 (a transcription factor with known functions in cancer progression) to be expressed by quiescent (non‐angiogenic) endothelial cells and reduced in angiogenic endothelial cells in the developing murine retina. In vitro analyses demonstrates that Zeb1 is expressed by confluent monolayers of Human Vein Umbilical Cord Endothelial cells (HUVECs) but is downregulated in actively growing cells. Zeb1 knockdown promotes an angiogenic EC phenotype: including enhanced PFKFB3, c‐myc and VEGFR2 and reduced FOXO1, Tie2 and VE‐cadherin expression. Consistently, Zeb1 knockdown EC were metabolically more active (both through oxidative and glycolytic pathways). Furthermore, loss of Zeb1 promoted EC migration and proliferation in vitro. In vivo shRNA knockdown of Zeb1 led to altered vessel formation and hypercellularity. In conclusion, Zeb1 appears to be linked to key quiescence regulating signaling pathways and furthermore regulates aspects of these pathways. Zeb1 expression appears to be a critical component for endothelial cells to generate functional vasculatures that can support enhanced muscle blood flow, by its control of FOXO1, PFKFB3 and Myc.Support or Funding InformationThis work was supported by the British Heart Foundation (grant PG/11/67/29067), the University of Nottingham Cancer Research Priority Area, and the Medical Research Council Newton Fund (MR/N01247X/1)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of cannabinoids on trophoblast cell growth and syncytialization

BackgroundRecent statistics indicates a ~65% increase in pregnancy‐associated marijuana abuse in the US affecting ~4% of all pregnant women. Low birth weight, pre‐term birth and other fetal abnormalities have been linked to marijuana abuse during pregnancy. Cannabinoids (CB) are the active constituents of marijuana in which delta‐9‐tetrahydrocannabinol (Δ9‐THC), cannabidiol (CBD) and cannabinol (CBN) are most active and interact with cannabinoid receptors (CNR) such as CNR1 and CNR2.Placenta is a transient organ which develops during pregnancy. Cytotrophoblast (CT) are predominant cell type of the placenta, which syncytialize/fuse to form multinucleated syncytiotrophoblast (ST) at the maternal‐fetal interface. Any interruption in this process can adversely affect placental development and fetal growth. Human placenta and the model human trophoblast cell line, BeWo, highly express CNR1 and CNR2 (Fig 1C). However, a clear mechanistic understanding of the adverse effects of CB on pregnancy is lacking.We hypothesize that CB compromise CT syncitialization and ST formation, thus affecting placental and fetal development and leading to adverse pregnancy outcomes. We tested this hypothesis using the Forskolin (FK)‐induced BeWo cell syncitialization model.MethodsEffects of Δ9‐THC, CBD and CBN at 0.2, 2 and 20 μM on BeWo cell viability in the presence and absence of 25 μM FK were evaluated using the MTT assay. Non‐cytotoxic CB concentration of 2 μM was used to treat a confluent monolayer of BeWo cells in the presence or absence of FK for 72 h to evaluate the effects of Δ9‐THC, CBD or CBN on syncytialization. Changes in mRNA levels of syncitialization marker genes Dysferlin (DYSF) and Syncytin‐1 (SYNC‐1) were determined using real‐time PCR. ELISA was used to quantify changes in hCG secretion by syncytializing BeWo cells using the above conditions. E‐cadherin immunostaining was used to assess changes in syncytialization upon CB treatment. Short‐hairpin RNA (shRNA) was used to knock‐down of specific CNRs in BeWo cells.Results and DiscussionΔ9‐THC, CBD and CBN all decreased BeWo cell viability with IC50 values of ~10 μM. FK induced syncytialization as evidenced by induction of DYSF and SYNC‐1 mRNA (Fig 1A, B), appearance of multinuclear syncytia and increased secretion of hCG all of which were significantly decreased by 2 μM of Δ9‐THC, CBD or CBN treatment. Δ9‐THC treatment decreased phosphorylation of protein kinase A (PKA) substrates (Fig 1C). PKA signaling pathway plays a pivotal role in mediating trophoblast syncytialization. It is possible that CB potentially exert their toxicity by altering PKA‐mediated signaling in placental trophoblasts. Additionally, Δ9‐THC at 20 μM significantly increased cellular ceramide production (Fig 1D). We are currently evaluating if the effects of CB on BeWo cell proliferation and syncytialization are mediated through CNR by knocking down CNR individually or in combination. These studies will provide the first mechanistic insights into the adverse effects of marijuana abuse on pregnancy that are important for developing intervention strategies to address CB toxicity to the placenta and fetus.Support or Funding InformationNational Institutes of Health (NIH) Institute on Drug Abuse [Grant DA032507], University of Washington‐National Institute of Environmental Health Sciences (NIEHS)‐sponsored Interdisciplinary Center for Exposures, Diseases, Genomics, and Environment [Grant ES007033]. University of Washington, Alcohol and Drug Abuse Institute‐Small Grant Award.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract QS31: Micropatterned Microsphere Scaffolds: Optimizing the Performance of Engineered Dermal Substitutes

PURPOSE: Current dermal replacement products perform sub-optimally in complex wound beds, such as those that have been irradiated or those with exposed hardware, mostly as a result of insufficient cell invasion and vascularization. We have previously observed more robust endothelial cell invasion of scaffolds containing a micropatterned matrix of differential collagen stiffness in a murine model when compared to collagen controls. Herein we compare the performance of our micropatterned microsphere hydrogels (MSS) to a widely utilized commercially available dermal replacement product in vitro and in vivo. METHODS: Microspheres composed of 1% type I collagen 50-150um in diameter were created and encased in a 0.3% type I collagen bulk. For our in vitro study, polydimethylsiloxane (PDMS) wells of 4mm diameter and 2mm height were filled with the microsphere scaffolds. 3x2mm Integra® disks were placed inside PDMS wells. Non-microsphere containing 1% and 0.3% collagen scaffolds served as controls. A monolayer of endothelial cells was seeded onto this three-dimensional platform, activated for invasion with 1uM sphingosine-1-phosphate, and cultured for 3 days. The collagen hydrogels were then analyzed using confocal microscopy to quantify cell invasion. For our in vivo study, 8x2mm MSS disks were created, along with 1% and 0.3% collagen controls. 8mm Integra® disks were created, and the unilateral silicone layer was removed. A disk of each type was then implanted subcutaneously in the dorsum of 8-week old wild-type mice. The scaffolds were removed at 7 and 14 days, imaged, and analyzed with ImageJ. RESULTS: Cells formed a confluent monolayer on the surface of the collagen disks, and migrated at a significantly higher rate into the MSS scaffold during 3 days of culture compared to control cultures as well as Integra® (142um MSS vs 45um Integra®, p<0.0001). Furthermore, in our in vivo study, MSS and Integra® both demonstrated robust cellular invasion spanning the depth of the scaffold at 7 and 14 days. Integra® had a lower cell density within the bulk at all depths with 705 cells per mm2 compared to 1,454 cells per mm2 in MSS at 14 days. Control collagen disks demonstrated minimal cell invasion and notable volumetric contraction. CONCLUSION: Micropatterned differential stiffness microsphere hydrogels (MSS) promote significantly more cellular invasion both in vivo and in vitro when compared to a commercially available dermal substitute. This enhanced cellular invasion and accelerated neovascularization, which results solely from the unique architecture of the scaffolds, indicates superior efficacy in the rate of integration into the host wound bed and may result in decreased length of time between its application and definitive wound closure. Further, the significantly more robust cellular and vascular invasion and integration of these scaffolds indicates their applicability in the treatment of suboptimal wound beds, which is beyond the capability of currently available dermal substitutes. K. Celie: None. Y. Toyoda: None. J.S. Buro: None. A.J. Lin: None. J. Xu: None. A. Miller: None. J. Morgan: None. J.A. Spector:; Dr. Spector is an equity holder in Fesarius Therapeutics which has licensed the microsphere scaffold technology from Cornell University.; Fesarius Therapeutics.

Monolayer microgel composite membranes with tunable permeability

Membranes with tunable gating functionality can be produced by modification with thermo-responsive polymers and colloids. We propose a straightforward method to gain this responsiveness by adsorption of microgels to the membrane surface. To be able to predict the filtration properties of the resulting composite membrane, a comprehensive understanding of the behavior of adsorbed microgels on porous substrates under convective flow is required. Previous studies focus on constant pressure experiments at various temperatures neglecting changes in viscosity and flux. In this study, we investigate the permeability and selectivity of poly-n-isopropyl-acrylamide (PNIPAM) monolayer microgel membranes under convective flow in cross-flow mode applying a constant flux. We use in-situ spectroscopic ellipsometry on a porous substrate to prove a reproducible and confluent monolayer microgel deposition. Supporting scanning electron micrographs and confocal laser scanning micrographs confirm the high reliability of the ellipsometry data set.The monolayer microgel membranes show distinct switching between 25 and 50 °C. When increasing the temperature above the microgel's volume phase transition temperature, the molecular weight cut-off (MWCO) of the functionalized membrane shifts to lower molar masses controlling the dextran retention between 30% and 70% only when the pore size of the support is in an appropriate range. The confluent monolayers are also mechanically stable when backwashed. Our data give insight into the responsive behavior of the PNIPAM network under convective flow – a key issue for custom-made filtration tasks.

Origami-based self-folding of co-cultured NIH/3T3 and HepG2 cells into 3D microstructures

This paper describes an origami-inspired self-folding method to form three-dimensional (3D) microstructures of co-cultured cells. After a confluent monolayer of fibroblasts (NIH/3T3 cells) with loaded hepatocytes (HepG2 cells) was cultured onto two-dimensional (2D) microplates, degradation of the alginate sacrificial layer in the system by addition of alginate lyase triggered NIH/3T3 cells to self-fold the microplates around HepG2 cells, and then 3D cell co-culture microstructures were spontaneously formed. Using this method, we can create a large number of 3D cell co-culture microstructures swiftly with ease in the same time. We find that HepG2 cells confined in the 3D cell co-culture microstructures have an ability to enhance the secreted albumin compared to 2D system in a long culture period. The result indicates that the origami-based cell self-folding technique presented here is useful in regenerative medicine and the preclinical stage of drug development.

Using the scratch assay to study cell migration in an inquiry-based cell biology lab

Cell migration, a fundamental process in development, wound healing, and immune function, is a common topic in undergraduate cell biology courses. We developed laboratory exercises with an inquiry-based learning (IBL) approach in which cell migration could be examined with the scratch assay, adapted from the primary literature. A narrow scratch was created in a confluent monolayer of cells growing on the bottom of a cell culture dish. Migration into the resultant cell-free zone from both sides of the scratch was measured after one day using the scale bar function of a digital camera. The Chinese hamster ovary cell line was used, but any adherent cell type could be examined. Students used the scratch assay to formulate hypotheses and design experiments in which variables affecting cell migration could be investigated. For example, the effect of cytoskeletal disruption was evaluated by adding the microtubule- and microfilament-disrupting drugs, colcemid and phallacidin, respectively, to the growth medium when the scratch was made. Optimal drug concentration parameters were determined for students to reference. Low drug concentrations inhibited cell migration, while higher concentrations killed the cells. This study demonstrated that the scratch assay is an accessible IBL method for studying cell migration.

The effects of zinc supplementation on primary human retinal pigment epithelium

Population-based and interventional studies have shown that elevated zinc levels can reduce the progression to advanced age-related macular degeneration. The objective of this study was to assess whether elevated extracellular zinc has a direct effect on retinal pigment epithelial cells (RPE), by examining the phenotype and molecular characteristics of increased extracellular zinc on human primary RPE cells. Monolayers of human foetal primary RPE cells were grown on culture inserts and maintained in medium supplemented with increasing total concentrations of zinc (0, 75, 100, 125 and 150 μM) for up to 4 weeks. Changes in cell viability and differentiation as well as expression and secretion of proteins were investigated. RPE cells developed a confluent monolayer with cobblestone morphology and transepithelial resistance (TER) >200 Ω*cm2 within 4 weeks. There was a zinc concentration-dependent increase in TER and pigmentation, with the largest effects being achieved by the addition of 125 μM zinc to the culture medium, corresponding to 3.4 nM available (free) zinc levels. The cells responded to addition of zinc by significantly increasing the expression of Retinoid Isomerohydrolase (RPE65) gene; cell pigmentation; Premelanosome Protein (PMEL17) immunoreactivity; and secretion of proteins including Apolipoprotein E (APOE), Complement Factor H (CFH), and High-Temperature Requirement A Serine Peptidase 1 (HTRA1) without an effect on cell viability. This study shows that elevated extracellular zinc levels have a significant and direct effect on differentiation and function of the RPE cells in culture, which may explain, at least in part, the positive effects seen in clinical settings. The results also highlight that determining and controlling of available, as opposed to total added, zinc will be essential to be able to compare results obtained in different laboratories.

Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids

An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cytotoxicity of purified methionine γ- lyase produced by Pseudomonas putida on several cell lines

Many human malignant cell lines and primary tumours have absolute requirements for L-methionine (1). Under normal circumstances methionine comes from dietary proteins, most normal tissues can also synthesize methionine from either homocysteine or methylthioadenosine (2). Upon L-methionine depletion, L-methionine-dependent cancer cells are not able to divide and became arrested in the late S/ G2 phase of the cell cycle (3).Methionine cleaving enzyme (methionine γ- lyase) has been found to be an effective antitumour agent in vitro as well as in vivo (4). Methionine γ- lyase is a pyridoxal 5-phosphate dependent enzyme that catalyzes the α, γ- elimination of L-methionine to α-ketobutyrate, methanethiol and ammonia (5). Rhabdomyosarcoma (RD) cell line at passage 65, AhmedMohammed-Nahi-2003(AMN3) cell line at passage 180, AMGM (Ahmed – Majeed-Glioblastoma-Multiform) cell line at passage 65 and normal Rat Embryo Fibroblast (REF) cell line at passage 56, kindly provided by Iraqi center for cancer and medical genetics research (ICCMGR) were cultured in PRMI 1640 medium supplement with 10% heat inactivated fetal calf serum and incubated at 37 ºC with 5% CO2. Examination of cytotoxicity of methionine γ- lyase Cytotoxicity was determined with the crystal violet stain as previously described (7). In briefly , 1x105 cell/ml was seeded onto 96-well culture plates in 200 µl RPMI 1640 medium and incubated until the cell reached confluent monolayer(vary according to the kind of cell-line). After incubation period the medium was removed and 200 μl of various concentrations (1000, 500,250 and 125 μg/ml) from methionine γ- lyase were added to the plate by using 3- well replicates for each concentration. The plates were incubated at 37ºC for 96 hr. The control (cancer cell lines without treatment with enzyme) (3-well replicates) was treated with 200 μl of serum free medium and incubated at 37ºC for 96 hr. At the end of incubation period, the enzyme and medium was removed from plate and washed with PBS to remove unattached (dead) cells. Two hundred μl of crystal violet was added to each well and left for 20 min at 37ºC.The stain was removed by washing with tap water several times, and the plates were left to dry. After drying, each plate was read by using ELISA microplate spectrophotometer at 492nm wave length. The percentages of Inhibitory Rate (IR %) were estimated (8).