Cell Monolayers Research Articles

Absorption and transport mechanism of colloidal nanoparticles (CNPs) in lamb soup based on Caco-2 cell

Soup is an important presence in diet, but its absorption and transport mechanism by the human body remains unclear. In this study, Caco-2 intact cell and monolayer cell models were constructed to simulate small intestine absorption on colloidal nanoparticles (CNPs) isolated from lamb soup. The intracellular localization of CNPs was viewed by laser confocal microscopy (LSCM). CNPs uptake and release pathways were explored by differences in CNPs concentrations in 5 endocytosis inhibitor models and 4 exocytosis inhibitor models. Results indicated that CNPs endocytosis by Caco-2 cells was restrained by Nystatin and Cytochalasin D, with exocytosis being inhibited by Nocodazole and Monensin. Therefore, the major absorption pathways for CNPs were caveolin-dependent endocytosis, macropinocytosis and phagocytosis. The major transport pathways were microtubule-vesicle-mediated protein transport to the membrane and transportation between the Golgi apparatus and membrane. This study may provide theoretical support for the transport mechanism of soup products in the small intestine.

Studies on absorption mechanism and pharmacokinetic properties of albendazole-bile acid conjugate: In vivo and in vitro

PurposeTo improve the oral bioavailability of albendazole (ABZ), a series of albendazole-bile acid conjugates (ABCs) were synthesized. ABC's transmembrane transport mechanism and in vivo pharmacokinetic properties were preliminarily studied. MethodsThe transmembrane transport mechanism of ABCs was studied using the Caco-2 monolayer cell model and intestinal perfusion model. The concentration of ABCs and ABZ were evaluated using High-Performance Liquid Chromatography (HPLC) and HPLC-Mass Spectrometry (HPLC-MS/MS). ResultsCompared to ABZ, better permeability was observed for different types and concentrations of ABCs using the Caco-2 monolayer cell model, with ABC-C8 showing the highest permeability. The transmembrane transport of ABCs was affected by ASBT inhibitors, indicating an ASBT-mediated active transport mechanism. Additionally, introducing cholic acid resulted in ABZ no longer being a substrate for P-gp, MRP2, and BCRP, effectively reversing ABZ efflux. In vivo unidirectional intestinal perfusion results in rats showed that ABCs altered the absorption site of ABZ from the jejunum to the ileum. The absorption efficiency of ABCs in each intestinal segment was higher than that of ABZ, and the transmembrane transport efficiency decreased with increasing concentrations of ASBT inhibitors. This further confirmed the presence of both passive diffusion and ASBT-mediated active transport mechanisms in the transport of ABCs. The solubility of ABCs in gastric juice and pharmacokinetics in rats showed that ABZ-C4 exhibited enhanced solubility. Moreover, ABCs significantly increased oral bioavailability compared to ABZ, with ABC-C4 showing an approximately 31-fold increase in bioavailability. ConclusionThe transmembrane transport mechanism of ABCs involves a combination of ASBT-mediated active transport and passive diffusion. Moreover, the incorporation of BAs successfully reverses the efflux of ABZ by efflux proteins. Among the synthesized conjugates, ABC-C4 demonstrated superior dissolution behavior both in vitro and in vivo.

C-C Chemokine Receptor 7 Promotes T-Cell Acute Lymphoblastic Leukemia Invasion of the Central Nervous System via β2-Integrins.

C-C Chemokine Receptor 7 (CCR7) mediates T-cell acute lymphoblastic leukemia (T-ALL) invasion of the central nervous system (CNS) mediated by chemotactic migration to C-C chemokine ligand 19 (CCL19). To determine if a CCL19 antagonist, CCL198-83, could inhibit CCR7-induced chemotaxis and signaling via CCL19 but not CCL21, we used transwell migration and Ca2+ mobilization signaling assays. We found that in response to CCL19, human T-ALL cells employ β2 integrins to invade human brain microvascular endothelial cell monolayers. In vivo, using an inducible mouse model of T-ALL, we found that we were able to increase the survival of the mice treated with CCL198-83 when compared to non-treated controls. Overall, our results describe a targetable cell surface receptor, CCR7, which can be inhibited to prevent β2-integrin-mediated T-ALL invasion of the CNS and potentially provides a platform for the pharmacological inhibition of T-ALL cell entry into the CNS.

Nucleocytoplasmic transport senses mechanical forces independently of cell density in cell monolayers.

Cells sense and respond to mechanical forces through mechanotransduction, which regulates processes in health and disease. In single adhesive cells, mechanotransduction involves the transmission of force from the extracellular matrix to the cell nucleus, where it affects nucleocytoplasmic transport (NCT) and the subsequent nuclear localization of transcriptional regulators, such as YAP (also known as YAP1). However, if and how NCT is mechanosensitive in multicellular systems is unclear. Here, we characterize and use a fluorescent sensor of nucleocytoplasmic transport (Sencyt) and demonstrate that NCT responds to mechanical forces but not cell density in cell monolayers. Using monolayers of both epithelial and mesenchymal phenotype, we show that NCT is altered in response both to osmotic shocks and to the inhibition of cell contractility. Furthermore, NCT correlates with the degree of nuclear deformation measured through nuclear solidity, a shape parameter related to nuclear envelope tension. In contrast, YAP is sensitive to cell density, showing that the YAP response to cell-cell contacts is not via a mere mechanical effect of NCT. Our results demonstrate the generality of the mechanical regulation of NCT.

P21-62 The copper-based nanopesticide Kocide 3000® worsens development of colitis in mice as a function of sex, and disrupts the intestinal barrier function in an enteroid-derived epithelial cell monolayer model from mouse gut organoids

P21-62 The copper-based nanopesticide Kocide 3000® worsens development of colitis in mice as a function of sex, and disrupts the intestinal barrier function in an enteroid-derived epithelial cell monolayer model from mouse gut organoids

Large extracellular vesicles from preeclampsia disrupt the integrity of brain endothelial cell monolayers

Large extracellular vesicles from preeclampsia disrupt the integrity of brain endothelial cell monolayers

Characterization of drusen formation in a primary porcine tissue culture model of dry AMD

Age-related macular degeneration (AMD) affects millions of individuals worldwide and is a leading cause of blindness in the elderly. In dry AMD, lipoproteinaceous deposits called drusen accumulate between the retinal pigment epithelium (RPE) and Bruch's membrane, leading to impairment of oxygen and nutrient trafficking to the neural retina, and degeneration of the overlying photoreceptor cells. Owing to key differences in human and animal ocular anatomy and the slowly progressing nature of the disease, AMD is not easily modeled invivo. In this study, we further characterize a "drusen-in-a-dish" primary porcine RPE model system by employing vital lipid staining to monitor sub-RPE deposition over time in monolayers of cells cultured on porous transwell membranes. We demonstrate for the first time using a semi-automated image analysis pipeline that the number and size of sub-RPE deposits increases gradually but significantly over time and confirm that sub-RPE deposits grown in culture immunostain positive for multiple known components found in human drusen. As a result, we propose that drusen-in-a-dish cell culture models represent a high-throughput and cost-scalable alternative to animal models in which to study the pathobiology of drusen accumulation and may serve as useful tools for screening novel therapeutics aimed at treating dry AMD.

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

Assessing Receptor Activation in 2D and 3D Cultured Hepatocytes: Responses to a Single Compound and a Complex Mixture.

Responding to global standards and legislative updates in Canada, including Bill S-5 (2023), toxicity testing is shifting towards more ethical, in vitro methods. Traditional two-dimensional (2D) monolayer cell cultures, limited in replicating the complex in vivo environment, have prompted the development of more relevant three-dimensional (3D) spheroidal hepatocyte cultures. This study introduces the first 3D spheroid model for McA-RH7777 cells, assessing xenobiotic receptor activation, cellular signaling, and toxicity against dexamethasone and naphthenic acid (NA)-fraction components; NAFCs. Our findings reveal that 3D McA-RH7777 spheroids demonstrate enhanced sensitivity and more uniform dose-response patterns in gene expression related to xenobiotic metabolism (AhR and PPAR) for both single compounds and complex mixtures. Specifically, 3D cultures showed significant gene expression changes upon dexamethasone exposure and exhibited varying degrees of sensitivity and resistance to the apoptotic effects induced by NAFCs, in comparison to 2D cultures. The optimization of 3D culture conditions enhances the model's physiological relevance and enables the identification of genomic signatures under varied exposures. This study highlights the potential of 3D spheroid cultures in providing a more accurate representation of the liver's microenvironment and advancing our understanding of cellular mechanisms in toxicity testing.

The Relevance of the Endothelium in Cardiopulmonary Disorders.

The endothelium is a cell monolayer that lines vessels and separates tissues from blood flow. Endothelial cells (ECs) have a multitude of functions, including regulating blood flow and systemic perfusion through changes in vessel diameter. When an injury occurs, the endothelium is affected by altering its functions and structure, which leads to endothelial dysfunction, a characteristic of many vascular diseases. Understanding the role that the endothelium plays in pulmonary vascular and cardiopulmonary diseases, and exploring new therapeutic strategies is of utmost importance to advance clinically. Currently, there are several treatments able to improve patients' quality of life, however, none are effective nor curative. This review examines the critical role of the endothelium in the pulmonary vasculature, investigating the alterations that occur in ECs and their consequences for blood vessels and potential molecular targets to regulate its alterations. Additionally, we delve into promising non-pharmacological therapeutic strategies, such as exercise and diet. The significance of the endothelium in cardiopulmonary disorders is increasingly being recognized, making ECs a relevant target for novel therapies aimed at preserving their functional and structural integrity.

Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood-Brain Barrier.

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood-brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood-brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.

Relaxation dynamics in the self-propelled Voronoi model for epithelial monolayers

Cell monolayers and epithelial tissues display slow relaxation dynamics during the reversible transition between mesenchymal and epithelial cells, a phenomenon directly relevant to embryogenesis, tumor metastases, and wound healing. In active cells, persistent motion induces collective dynamics that significantly influence relaxation behavior. To better understand the role of persistence in cell relaxations, we perform extensive simulations and employ cage-relative measures to address the previously overlooked system size effects in model active cells. We identify the glass transition at various persistence values, demonstrating conventional near-equilibrium supercooled dynamics at low persistence. However, highly persistent cells exhibit distinctive intermittent dynamics associated with intermittent local T1 transitions, where cell velocity correlates over space with a characteristic length ξ. More significantly, we formulate a universal relationship predicting the global relaxation time based on the T1 transition and the spatial velocity correlation across a wide range of persistence values. Specifically, the relaxation time demonstrates a power-law dependence on the irreversible T1 transition rate, multiplied by exp(ξ). Here, ξ vanishes at small persistence in nearly equilibrated cells, and the irreversible T1 transition rate diminishes towards the mode-coupling glass transition point. Published by the American Physical Society 2024

Scanning Electron Microscopy (SEM) Evaluation of the Ultrastructural Effects on Conjunctival Epithelial Cells of a New Multiple-Action Artificial Tear Containing Cross-Linked Hyaluronic Acid, Cationic Liposomes and Trehalose.

The authors performed an ex vivo and in vivo evaluation of the ultrastructural effects on the conjunctival epithelial cells of a new multiple-action tear substitute containing cross-linked hyaluronic acid, lipids and trehalose (Trimix®), using scanning electron microscopy (SEM) with conjunctival impression cytology. The ex vivo study highlights the persistence and distribution of the product at 5 and 60 min on a monolayer of conjunctival epithelial cells and an increase in microvilli density at the 60 min evaluation. In vivo examination was conducted on three subjects with different grades of ocular surface inflammation, treated with one drop of the product twice daily for thirty days. At the baseline (T0) and twelve hours after the last administration of the tear drop (T30), impression cytology of the upper bulbar conjunctiva for SEM evaluation of conjunctival epithelial cells was carried out. Slit lamp examination (SLE), corneal and conjunctival Fluotest, tear film break-up time (TBUT), and ocular surface disease index (OSDI) questionnaires were also performed to correlate the ultrastructural results with the clinical findings. After 30 days of treatment, a significant improvement in all clinical and symptomatic parameters and in the condition of the ocular surface was detected, with microvillar regeneration and strengthening in all the patients, and a complete restoration in 2/3 of them. The persistence and distribution of the product on the epithelial cells was also noted 12 h after the last administration. The results, therefore, suggest a marked epitheliotropic effect along with a high residence time of the tear substitute.

Sodium Tungstate Promotes Neurite Outgrowth and Confers Neuroprotection in Neuro2a and SH-SY5Y Cells.

Sodium tungstate (Na2WO4) normalizes glucose metabolism in the liver and muscle, activating the Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Because this pathway controls neuronal survival and differentiation, we investigated the effects of Na2WO4 in mouse Neuro2a and human SH-SY5Y neuroblastoma monolayer cell cultures. Na2WO4 promotes differentiation to cholinergic neurites via an increased G1/G0 cell cycle in response to the synergic activation of the Phosphatidylinositol 3-kinase (PI3K/Akt) and ERK1/2 signaling pathways. In Neuro2a cells, Na2WO4 increases protein synthesis by activating the mechanistic target of rapamycin (mTOR) and S6K kinases and GLUT3-mediated glucose uptake, providing the energy and protein synthesis needed for neurite outgrowth. Furthermore, Na2WO4 increased the expression of myocyte enhancer factor 2D (MEF2D), a member of a family of transcription factors involved in neuronal survival and plasticity, through a post-translational mechanism that increases its half-life. Site-directed mutations of residues involved in the sumoylation of the protein abrogated the positive effects of Na2WO4 on the MEF2D-dependent transcriptional activity. In addition, the neuroprotective effects of Na2WO4 were evaluated in the presence of advanced glycation end products (AGEs). AGEs diminished neurite differentiation owing to a reduction in the G1/G0 cell cycle, concomitant with lower expression of MEF2D and the GLUT3 transporter. These negative effects were corrected in both cell lines after incubation with Na2WO4. These findings support the role of Na2WO4 in neuronal plasticity, albeit further experiments using 3D cultures, and animal models will be needed to validate the therapeutic potential of the compound.

Contactless Single Cell Extraction from Monolayer Cell Culture Using A MEMS Acoustic Droplet Ejector.

To achieve a contactless and damage-less extraction of a single (or a few) human retinal pigment epithelial (RPE) cell(s) from a cell monolayer with acoustic droplet ejection. An acoustic droplet ejector based on a self-focusing acoustic transducer (SFAT) is designed, microfabricated, and placed on a precision movable stage that aligns it to the targeted cell(s) in a Petri dish. The device delivers 20.1 MHz focused ultrasound (FUS) with a focal diameter of 100 μm, which ejects droplets capable of extracting and transferring only the targeted cell(s) from a monolayer. The extraction and collection of 1-10 cells are successfully demonstrated. The number of ejected cells can be controlled by the FUS pulse width. As confirmed by fluorescence-based cell viability assays and re-culture experiments, the ejected cell(s) and remaining monolayer cells are intact without damage after cell ejection. Furthermore, real-time reverse transcription polymerase chain reaction (RT-PCR) tests for both housekeeping genes (GAPDH and β -actin) and RPE-specific genes (MITF, PEDF, and PMEL17) show no significant difference between the acoustically ejected cells and those collected manually with a micropipette. The proposed technology realizes a contactless, damage-free extraction of cells with high spatial resolution and precise control of the number of cells ejected, with a simple setup. This powerful technology not only enables efficient, high-precision cell extraction for quality check applications but also opens new avenues for other advanced biotechnologies such as bioprinting.

Nanofiber-boosted retrograded starch/pectin microparticles for targeted 5-Aminosalicylic acid delivery in inflammatory bowel disease: In vitro and in vivo non-toxicity evaluation

Incorporating 5-aminosalicylic acid (5-ASA) into a colon-specific carrier is crucial for treating inflammatory bowel diseases (IBD), as it enhances therapeutic efficacy, targets the affected regions directly, and minimizes side effects. This study evaluated the impact of incorporating cellulose nanofibers (CNF) on the in vitro and in vivo biological performance of retrograded starch/pectin (RS/P) microparticles (MPs) containing 5-ASA. Using Fourier Transform Infrared (FTIR) Spectroscopy, shifts in the spectra of retrograded samples containing CNF were observed with increasing CNF proportions, suggesting the establishment of new supramolecular interactions. Liquid absorption exhibited pH-dependent behaviors, with reduced absorption in simulated gastric fluid (∼269 %) and increased absorption in simulated colonic fluid (∼662 %). Increasing CNF concentrations enhanced mucoadhesion in porcine colonic sections, with a maximum force of 3.4 N at 50 % CNF. Caco-2 cell viability tests showed biocompatibility across all tested concentrations (0.0625–2.0000 mg/mL). Evaluation of intestinal permeability in Caco-2 cell monolayers demonstrated up to a tenfold increase in 5-ASA permeation, ranging from 29 % to 48 %. An in vivo study using Galleria mellonella larvae, with inflammation induced by LPS, showed reduction of inflammation. Given the scalability of spray-drying, these findings suggest the potential of CNF-incorporated RS/P microparticles for targeted 5-ASA delivery in IBD.

Molecular weight-mediated interaction changes for enhancing structural stability, release behavior and M cells-targeting transport efficacy of starch-based nanoparticles

Molecular weight (Mw) of ligand-mediated nanocarriers plays a pivotal role in their architecture and properties. In this study, self-assembled ovalbumin (OVA)-loaded nanoparticles were meticulously engineered by starch polyelectrolytes with different Mw. Results unveiled that, tailoring Mw of GRGDS pentapeptides-grafted carboxymethyl starch (G-CMS) displayed strong binding-affinity and transport efficiency through microfold cells (M cells) pathway in the simulated intestinal epithelial cell monolayer in which M cells were randomly located in the Caco-2 cells monolayer. Notably, nanoparticles assembled from G-CMS with relatively higher Mw exhibited more compact structures due to the stronger interactions between layers compared to that with relatively lower Mw, which rendered remarkably stable and only 19.01 % in vitro OVA leakage under conditions of the upper gastrointestinal tract. Subsequently, more intact nanoparticles reached M cells after in vitro digestion and exhibited higher transport efficiency through the M cells pathways (apparent permeability: 9.38 × 10−5 cm/s) than Caco-2 cells, attributing to specific- and non-specific binding affinity towards M cells. Therefore, optimal Mw tailoring of starch polyelectrolytes can mediate the molecular interactions among their assembled layers and the interactions with M cells to balance the structural compactness, release and transport efficacy of nanoparticles, holding promise for advancing M cells-targeting oral delivery technologies.

Pneumatic extrusion bioprinting-based high throughput fabrication of a melanoma 3D cell culture model for anti-cancer drug screening

The high incidence of malignant melanoma highlights the need forin vitromodels that accurately represent the tumour microenvironment, enabling developments in melanoma therapy and drug screening. Despite several advancements in 3D cell culture models, appropriate melanoma models for evaluating drug efficacy are still in high demand. The 3D pneumatic extrusion-based bioprinting technology offers numerous benefits, including the ability to achieve high-throughput capabilities. However, there is a lack of research that combines pneumatic extrusion-based bioprinting with analytical assays to enable efficient drug screening in 3D melanoma models. To address this gap, this study developed a simple and highly reproducible approach to fabricate a 3D A375 melanoma cell culture model using the pneumatic extrusion-based bioprinting technology. To optimise this method, the bioprinting parameters for producing 3D cell cultures in a 96-well plate were adjusted to improve reproducibility while maintaining the desired droplet size and a cell viability of 92.13 ± 6.02%. The cross-linking method was optimised by evaluating cell viability and proliferation of the 3D bioprinted cells in three different concentrations of calcium chloride. The lower concentration of 50 mM resulted in higher cell viability and increased cell proliferation after 9 d of incubation. The A375 cells exhibited a steadier proliferation rate in the 3D bioprinted cell cultures, and tended to aggregate into spheroids, whereas the 2D cell cultures generally formed monolayered cell sheets. In addition, we evaluated the drug responses of four different anti-cancer drugs on the A375 cells in both the 2D and 3D cell cultures. The 3D cell cultures exhibited higher levels of drug resistance in all four tested anti-cancer drugs. This method presents a simple and cost-effective method of producing and analysing 3D cell culture models that do not add additional complexity to current assays and shows considerable potential for advancing 3D cell culture models' drug efficacy evaluations.

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures.

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.