SBIR-STTR Award

Increasing evidence indicates that systemic inflammation and the blood-brain barrier (BBB), which becomes the target of overreacting or misguided immune cells that determine BBB failure and immune extravasations into the brain parenchyma, are involved in the pathogenesis of neurological diseases such as meningitis, inflammation, Alzheimer's disease, and multiple sclerosis. Therefore understanding the mechanisms of leukocyte trafficking into the brain might provide insights into how to modulate pathologic immune responses or enhance host protective mechanisms in neuroinflammatory diseases. Essential for the success of this critical issue and for the development of novel pharmacological treatments is the use of artificial systems capable to reproduce in detail the physiology of the BBB and its functional response to the inflammatory processes. To date, we have developed a flow-based artificial co-culture system (DIV-BBB) based on microporous hollow fiber technology that is capable to reproduce a quasi-physiological environment where endothelial cells and astrocyte establish a functional BBB. This BBB model has been shown to closely mimic the characteristics and functional properties of in vivo. However, a significant body of evidence from this and other laboratories suggests that the main limitation of this model to study the role of the BBB in neurological diseases is lack of transendothelial cell trafficking due to the small diameter of the transcapillary pores (0.2-0.55m). Moreover, because the dynamic in vitro BBB model (DIV-BBB) more accurately reflects the properties of capillaries comprising the BBB;it is not entirely clear whether this system is appropriate for studying leukocyte extravasation in the brain, which is likely to occur at the post-capillary segment (venules). Therefore, to address this critical issue we also propose to prototype and validate a post-capillary (DIV-Venules) interface, which will be added to the DIV-BBB to develop the first in vitro capillary-venules model of the brain cerebrovasculature. To this end, the aims of this Phase 1 STTR proposal are the following: Specific Aim 1: To prototype a new dynamic in vitro capillary-venules model of the brain cerebrovasculature that is permissive for the extravasation of white blood cells (WBC) from vascular into the parenchimal (brain) side of the system. To this end, we will investigate three methods of manufacturing large diameter holes (2-4 5m) in the artificial hollow fibers that provide the structural support for vascular cell growth and we will determine the most cost effective way to mass-produce these modified artificial capillaries. Specific Aim 2: To evaluate the dynamic in vitro capillary-venules model and validate the system against parallel Transwell models. This will be assessed by: a) Measuring the pharmacokinetic (e.g., paracellular permeability to high polar molecules), cell viability, and other distinctive vascular properties of the DIV-BBB and the DIV-Venules modules;b) By assessing the extravasation of THP-1 cells (human monocytic cell line) in the brain compartments of the capillaries and venules modules in response to abluminal chemokines and to determine the patterns of extravasation (capillary versus venules) of THP-1 migration. The physiological response of this new in vitro brain capillary-venules model will be compared against parallel Transwell systems, which are generally considered the gold standard in cerebrovascular research. , ,

Public Health Relevance:
Understanding human neurological diseases requires simultaneous studies of various cell types (e.g., neurons, endothelium, astrocytes, white blood cells, etc.) as well as fluid phase factors (adhesion molecules, cytokines, pro-inflammatory factors, intravascular shearing forces, etc). The blood-brain barrier (BBB) exemplifies the importance of this approach to neuroscience. Loss of BBB function plays a pivotal role in the pathogenesis of many diseases of the central nervous system (CNS). In this project, we will design and prototype a new dynamic in vitro blood-brain barrier model (nDIV-BBB) with a pore size that allows white blood cells (WBC) to extravasate from the basal surface of the endothelial cells into the extracellular space of the cartridge. In addition, we will investigate three methods of manufacturing holes in the hollow fiber to determine the most cost effective way to mass-produce the cartridges. We have shown that the DIV-BBB model more accurately represents the characteristics of an in vivo blood- brain barrier than the two-dimensional (flat plate) models. However, the current pore size of the DIV-BBB model does not allow for inflammation studies that require monocytes to extravasate the barrier. In addition, we will validate the feasibility of these improved DIV-BBB models by assessing the cell growth and BBB viability as well as the formation of a tight barrier with low paracellular permeability.

Thesaurus Terms:
2-Dimensional;Address;Adhesion Molecule;Adventitial Cell;Alzheimer;Alzheimer Disease;Alzheimer Sclerosis;Alzheimer Syndrome;Alzheimer's;Alzheimer's Disease;Alzheimers Dementia;Alzheimers Disease;Architecture;Astrocytes;Astrocytus;Astroglia;Au Element;Blast Transformation;Blastogenesis;Blood - Brain Barrier Anatomy;Blood Vessels;Blood Capillaries;Blood Leukocyte;Blood Monocyte;Blood-Brain Barrier;Brain;Cns Diseases;Cns Disorder;Caliber;Capillaries;Capillary;Capillary, Unspecified;Cell Adhesion Molecules;Cell Communication And Signaling;Cell Line;Cell Lines, Strains;Cell Locomotion;Cell Migration;Cell Movement;Cell Signaling;Cell Survival;Cell Viability;Cellline;Cells;Cellular Expansion;Cellular Growth;Cellular Migration;Central Nervous System;Central Nervous System Diseases;Central Nervous System Disorders;Cerebrovascular System;Characteristics;Chemoattractants;Chemotactic Factors;Chemotaxins;Co-Culture;Cocultivation;Coculture;Coculture Techniques;Cytokines, Chemotactic;Data;Dementia, Alzheimer Type;Dementia, Primary Senile Degenerative;Dementia, Senile;Development;Diameter;Disease;Disorder;Drug Kinetics;Encephalon;Encephalons;Endothelial Cells;Endothelium;Engineering / Architecture;Environment;Extracellular Space;Extravasation;Flr;Failure (Biologic Function);Fiber;Future;Gold;Hemato-Encephalic Barrier;Homologous Chemotactic Cytokines;Human;Human, General;Inflm;Immune;Immune Response;In Vitro;Inflammation;Inflammatory;Intercellular Space;Intercrines;Intracellular Communication And Signaling;Laboratories;Leakage;Leukocyte Trafficking;Leukocytes;Liquid Substance;Lymphoblast Transformation;Lymphocyte Activation;Lymphocyte Stimulation;Lymphocyte Transformation;Ms (Multiple Sclerosis);Man (Taxonomy);Man, Modern;Marrow Leukocyte;Marrow Monocyte;Measures;Meningitis;Methods;Modeling;Molecular;Motility;Motility, Cellular;Multiple Sclerosis;Muscle, Smooth, Vascular;Nerve Cells;Nerve Unit;Nervous System Diseases;Nervous System, Brain;Nervous System, Cns;Neural Cell;Neuraxis;Neurocyte;Neurologic Disorders;Neurological Disorders;Neurons;Neurosciences;Pathogenesis;Pathologic;Pattern;Perfusion;Pericapillary Cell;Pericytes;Perivascular Cell;Permeability;Pharmacokinetics;Pharmacological Treatment;Phase;Phenotype;Physiologic;Physiological;Physiology;Play;Primary Senile Degenerative Dementia;Process;Property;Property, Loinc Axis 2;Research;Reticuloendothelial System, Leukocytes;Role;Rouget Cells;Sis Cytokines;Sttr;Sclerosis, Disseminated;Side;Signal Transduction;Signal Transduction Systems;Signaling;Small Business Technology Transfer Research;Spillage;Structure;Structure Of Venule;Surface;System;System, Loinc Axis 4;Technology;Validation;Vascular Endothelium;Venous;Venules;White Blood Cells;White Cell;Base;Biological Signal Transduction;Capillary;Cell Adhesion Protein;Cell Growth;Cell Motility;Cell Type;Cerebrovascular;Chemoattractant Cytokine;Chemokine;Complement Chemotactic Factor;Cost Effective;Cultured Cell Line;Cytokine;Dementia Of The Alzheimer Type;Design;Designing;Disease/Disorder;Failure;Fluid;Hemodynamics;Host Response;Immunoresponse;Improved;In Vivo;Insight;Insular Sclerosis;Liquid;Manufacturing Process;Migration;Monocyte;Nervous System Disorder;Neurological Disease;Neuronal;Novel;Primary Degenerative Dementia;Prototype;Public Health Relevance;Response;Senile Dementia Of The Alzheimer Type;Shear Stress;Social Role;Success;Trafficking;Two-Dimensional;Vascular;Venule;White Blood Cell;White Blood Corpuscle

Reproducing in vitro the physiological characteristics of brain vascular segments represents a critical issue. Adequate modeling of the cerebrovasculature could significantly help understand the mechanisms and improve the pharmacology of disease where a role for leukocytes migrating across the cerebrovasculature is demonstrated. Thanks to the previous support (Phase I) we were able to prototype and test drive a new dynamic in vitro model of the BBB (DIV- BBB) permissive for leukocyte extravasation. Our initial effort aimed at piercing hollow fibers in a reproducible manner;the results were recently published. We found that manually perforated hollow fibers allow leukocytes passage across the BBB in response to pro-inflammatory stimuli and hemodynamic changes. We were also able to produce a capillary-venule segment by varying the rheological parameters (e.g., changing the shear stress) of the system. In addition we recently developed a system to mechanically ""stretch"" the hollow fibers increasing the pore size up to the physiologically relevant size of ~5 [unreadable]m. Controlled traction was applied to the end of the fibers and scanning electron microscopy showed enlarged pores within the stretched fiber. To further the commercial opportunity afforded by this new BBB model, we propose the following Phase II Specific Aims: To optimize the performance of a dynamic in vitro capillary-venules model of the brain cerebrovasculature permissive for leukocyte extravasation. To determine the pattern of leukocytes extravasation in control and diseased capillary-venules segments composed of fibers with different transmural permeability properties (from Aim 1). To compare the results obtained using these DIV- BBB models to other state-of-art in vitro BBB models. We will initially tailor the use of the DIV capillary-venules system to multiple sclerosis and epilepsy research and drug development. We have assembled a multi-disciplinary team of investigators and experts in the field of leukocyte migration across the cerebrovasculature. Additional clinically relevant venues are detailed in the Commercialization Plan. Given our preliminary results and the confirmatory progress report described in detail in this application, and given the fact that i the meantime development of new drugs has remained a major issue in the treatment of neurological diseases, we believe that the combination of a strong record of accomplishment and sound experimental design aimed at improving drug development are fundamental aspects of this Phase 2 proposal.

Public Health Relevance:
Leukocyte migration into the brain parenchyma is a hallmark of various neuro-inflammatory brain diseases. In order to develop therapeutics targeting brain inflammatory process it is imperative to reproduce in vitro the modality by which leukocytes cross the blood-brain barrier (BBB) to reach the brain parenchyma. We now plan to optimize the performance of a dynamic in vitro capillary-venules model of the brain cerebrovasculature permissive for leukocyte extravasation. We will also determine the pattern of leukocytes extravasation in control and diseased (multiple sclerosis and epilepsy) capillary-venules segments composed of fibers with different permeability properties.