Effects of 3D Geometries on Cellular Gradient Sensing and Polarization

During cell migration, cells become polarized, change their shape, and move in response to various internal and external cues. Cell polarization is defined through the spatio-temporal organization of molecules such as PI3K or small GTPases, and is determined by intracellular signaling networks. It results in directional forces through actin polymerization and myosin contractions. Many existing mathematical models of cell polarization are formulated in terms of reaction-diffusion systems of interacting molecules, and are often defined in one or two spatial dimensions. In this paper, we introduce a 3D reaction-diffusion model of interacting molecules in a single cell, and find that cell geometry has an important role affecting the capability of a cell to polarize, or change polarization when an external signal changes direction. Our results suggest a geometrical argument why more roundish cells can repolarize more effectively than cells which are elongated along the direction of the original stimulus, and thus enable roundish cells to turn faster, as has been observed in experiments. On the other hand, elongated cells preferentially polarize along their main axis even when a gradient stimulus appears from another direction. Furthermore, our 3D model can accurately capture the effect of binding and unbinding of important regulators of cell polarization to and from the cell membrane. This spatial separation of membrane and cytosol, not possible to capture in 1D or 2D models, leads to marked differences of our model from comparable lower-dimensional models.Comment: 31 pages, 7 figure

A study of external pneumatic compression for the prevention of deep venous thrombosis

Thesis. 1977. Ph.D.--Massachusetts Institute of Technology. Dept. of Mechanical Engineering.MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING.Vita.Bibliography: p. 200-205.by Roger D. Kamm.Ph.D

Analysis of nanoprobe penetration through a lipid bilayer

AbstractWith the rapid development of nanotechnology and biotechnology, nanoscale structures are increasingly used in cellular biology. However, the interface between artificial materials and a biological membrane is not well understood, and the harm caused by the interaction is poorly controlled. Here, we utilize the dissipative particle dynamics simulation method to study the interface when a nanoscale probe penetrates the cell membrane, and propose that an appropriate surface architecture can reduce the harm experienced by a cell membrane. The simulation shows that a hydrophilic probe generates a hydrophilic hole around the probe while a hydrophobic probe leads to a ‘T-junction’ state as some lipid molecules move toward the two ends of the probe. Both types of probe significantly disrupt lipid bilayer organization as reflected by the large variations in free energy associated with penetration of the membrane. Considering the hydrophilic/hydrophobic nature of the lipid bilayer, three other hydrophilic/hydrophobic patterns – band pattern, axial pattern and random pattern – are discussed to reduce the damage to the lipid membrane. Both the free energy analysis and simulation studies show that the axial pattern and the random pattern can both minimize the variations in free energy with correspondingly smaller adverse effects on membrane function. These results suggest that the axial pattern or random pattern nanoprobe generates a mild interaction with the biological membrane, which should be considered when designing nondestructive nanoscale structures

In vitro models of molecular and nano-particle transport across the blood-brain barrier

The blood-brain barrier (BBB) is the tightest endothelial barrier in humans. Characterized by the presence of tight endothelial junctions and adherens junctions, the primary function of the BBB is to maintain brain homeostasis through the control of solute transit across the barrier. The specific features of this barrier make for unique modes of transport of solutes, nanoparticles, and cells across the BBB. Understanding the different routes of traffic adopted by each of these is therefore critical in the development of targeted therapies. In an attempt to move towards controlled experimental assays, multiple groups are now opting for the use of microfluidic systems. A comprehensive understanding of bio-transport processes across the BBB in microfluidic devices is therefore necessary to develop targeted and efficient therapies for a host of diseases ranging from neurological disorders to the spread of metastases in the brain

In Vitro Microfluidic Models for Neurodegenerative Disorders

Microfluidic devices enable novel means of emulating neurodegenerative disease pathophysiology in vitro. These organ-on-a-chip systems can potentially reduce animal testing and substitute (or augment) simple 2D culture systems. Reconstituting critical features of neurodegenerative diseases in a biomimetic system using microfluidics can thereby accelerate drug discovery and improve our understanding of the mechanisms of several currently incurable diseases. This review describes latest advances in modeling neurodegenerative diseases in the central nervous system and the peripheral nervous system. First, this study summarizes fundamental advantages of microfluidic devices in the creation of compartmentalized cell culture microenvironments for the co-culture of neurons, glial cells, endothelial cells, and skeletal muscle cells and in their recapitulation of spatiotemporal chemical gradients and mechanical microenvironments. Then, this reviews neurodegenerative-disease- on-a-chip models focusing on Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Finally, this study discusses about current drawbacks of these models and strategies that may overcome them. These organ-on-chip technologies can be useful to be the first line of testing line in drug development and toxicology studies, which can contribute significantly to minimize the phase of animal testing steps

3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes

The blood-brain barrier (BBB) regulates molecular trafficking, protects against pathogens, and prevents efficient drug delivery to the brain. Models to date failed to reproduce the human anatomical complexity of brain barriers, contributing to misleading results in clinical trials. To overcome these limitations, a novel 3-dimensional BBB microvascular network model was developed via vasculogenesis to accurately replicate the in vivo neurovascular organization. This microfluidic system includes human induced pluripotent stem cell-derived endothelial cells, brain pericytes, and astrocytes as self-assembled vascular networks in fibrin gel. Gene expression of membrane transporters, tight junction and extracellular matrix proteins, was consistent with computational analysis of geometrical structures and quantitative immunocytochemistry, indicating BBB maturation and microenvironment remodelling. Confocal microscopy validated microvessel-pericyte/astrocyte dynamic contact-interactions. The BBB model exhibited perfusable and selective microvasculature, with permeability lower than conventional in vitro models, and similar to in vivo measurements in rat brain. This robust and physiologically relevant BBB microvascular model offers an innovative and valuable platform for drug discovery to predict neuro-therapeutic transport efficacy in pre-clinical applications as well as recapitulate patient-specific and pathological neurovascular functions in neurodegenerative disease

In Vitro Model of Tumor Cell Extravasation

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium

3D BLOOD-BRAIN BARRIER MICROVASCULAR NETWORK MODEL INCLUDING HUMAN IPS-DERIVED ENDOTHELIAL CELLS, PERICYTES AND ASTROCYTES

The blood-brain barrier (BBB) is a selective barrier that help to maintain brain homeostasis, however it also creates an obstacle to drug delivery. For years, in vivo animal models have been widely used for BBB studies and drug evaluations. Although these techniques are considered the gold standard, 80% of drug candidates that were successful in animal models later failed in clinical trials. For that reason, a cost-effective in vitro BBB model that adequately reflects human in vivo conditions is required. Here we developed a 3D microfluidic model of the BBB by self-organized vascular network including (iPS)-derived endothelial cells, human brain pericytes, and astrocytes

Tumor-vascular interactions promote STING-driven inflammation in the tumor microenvironment

The recruitment of T cells following intratumoral administration of Stimulation of Interferon Genes (STING) agonists in the tumor microenvironment (TME) is a critical event in the STING-driven antitumor immune response, a pathway with great relevance in the context of cancer immunotherapy. We have previously demonstrated that LKB1 mutation is associated with suppression of tumor cell STING levels and reduced production of T-cell chemoattractants such as CXCL10 in KRAS-driven non-small cell lung cancer (NSCLC). Consistent with this, immunohistochemical staining of patient samples showed poor infiltration of CD3, CD4, and CD8 T cells into LKB1 negative versus LKB1 intact cancer epithelium, and instead, retention of T-cells in stroma. To examine how LKB1 alters immune cell recruitment in a STING-dependent manner, we used a 3-D microfluidic co-culture system to study interactions between vasculature and tumor spheroids derived from a KRAS/LKB1 mutated (KL) cell line with LKB1 reconstitution +/- STING deletion. To form the vasculature, we co-cultured tumor spheroids with fibroblasts and endothelial cells for 7 days, and identified changes in morphology, cytokine production, and gene expression that occur in co-culture. We first observed that co-culture induced synergistic production of multiple immune cell chemo-attractants such as CXCL10, CCL2, CCL5, and G-CSF. Interestingly, this more physiologic ex vivo tumor model of LKB1 reconstitution revealed particularly strong cooperative production of STING-dependent cytokines such as CXCL10 in the vasculature. Moreover, STING depletion in LKB1 reconstituted tumor cells did not significantly attenuate production of CXCL10 and other cytokines in co-culture, suggesting that tumor/vessel interaction may promote STING activation in the vasculature regardless of cancer cell-intrinsic STING function. Furthermore, although there was no appreciable response after treatment of KL cancer cells with cGAMP based STING agonists, treatment of isolated 3-D vascular networks with cGAMP enhanced vascular permeability and increased production of CXCL10 and CCL5, possibly contributing to defective chemokine gradients that retain T cells near the vasculature. Thus, developing these more complex models that incorporate the vasculature may elucidate important aspects of STING biology and may ultimately aid further development of effective immunotherapies targeting this signaling axi

Dynamic filopodial forces induce accumulation, damage, and plastic remodeling of 3D extracellular matrices

The mechanical properties of the extracellular matrix (ECM)–a complex, 3D, fibrillar scaffold of cells in physiological environments–modulate cell behavior and can drive tissue morphogenesis, regeneration, and disease progression. For simplicity, it is often convenient to assume these properties to be time-invariant. In living systems, however, cells dynamically remodel the ECM and create time-dependent local microenvironments. Here, we show how cell-generated contractile forces produce substantial irreversible changes to the density and architecture of physiologically relevant ECMs–collagen I and fibrin–in a matter of minutes. We measure the 3D deformation profiles of the ECM surrounding cancer and endothelial cells during stages when force generation is active or inactive. We further correlate these ECM measurements to both discrete fiber simulations that incorporate fiber crosslink unbinding kinetics and continuum-scale simulations that account for viscoplastic and damage features. Our findings further confirm that plasticity, as a mechanical law to capture remodeling in these networks, is fundamentally tied to material damage via force-driven unbinding of fiber crosslinks. These results characterize in a multiscale manner the dynamic nature of the mechanical environment of physiologically mimicking cell-in-gel systems