Central Nervous System

A dynamic in vitro BBB model for the study of immune cell trafficking into the central-nervous system

By Luca Cucullo, Nicola Marchi, Mohammed Hossain, and Damir Janigro

Although there is significant evidence correlating overreacting or perhaps misguided immune cells and the blood–brain barrier (BBB) with the pathogenesis of neuroinflammatory diseases, the mechanisms by which they enter the brain are largely unknown. For this purpose, we revised our humanized dynamic in vitro BBB model (DIV-BBBr) to incorporate modified hollow fibers that now feature transmural microholes (2 to 4 lm +) allowing for the transendothelial trafficking of immune cells. As with the original model, this new DIV-BBBr reproduces most of the physiological characteristics of the BBB in vivo. Measurements of transendothelial electrical resistance (TEER), sucrose permeability, and BBB integrity during reversible osmotic disruption with mannitol (1.6 mol/L) showed that the microholes do not hamper the formation of a tight functional barrier. The in vivo rank permeability order of sucrose, phenytoin, and diazepam was successfully reproduced in vitro. Flow cessation followed by reperfusion (Fc/Rp) in the presence of circulating monocytes caused a biphasic BBB opening paralleled by a significant increase of proinflammatory cytokines and activated matrix metalloproteinases. We also observed abluminal extravasation of monocytes but only when the BBB was breached. In conclusion, the DIV-BBBr represents the most realistic in vitro system to study the immune cell trafficking across the BBB.

Brain Barrier

A new dynamic in vitro model for the multidimensional study of astrocyte–endothelial cell interactions at the blood–brain barrier

By Luca Cucullo , Mark S. McAllister, Kelly Kight, Ljiljana Krizanac-Bengez,Matteo Marroni, Marc R. Mayberg, Kathe A. Stanness, Damir Janigro

Blood–brain barrier endothelial cells are characterized by the presence of tight intercellular junctions, the absence of fenestrations, and a paucity of pinocytotic vesicles. The in vitro study of the BBB has progressed rapidly over the past several years as new cell culture techniques and improved technologies to monitor BBB function became available. Studies carried out on viable in vitro models are set to accelerate the design of drugs that selectively and aggressively can target the CNS. Several systems in vitro attempt to reproduce the physical and biochemical behavior of intact BBB, but most fail to reproduce the three-dimensional nature of the in vivo barrier and do not allow concomitant exposure of endothelial cells to abluminal (glia) and lumenal (flow) influences. For this purpose, we have developed a new dynamic in vitro BBB model (NDIV-BBB) designed to allow for extensive pharmacological, morphological and physiological studies. Bovine aortic endothelial cells (BAEC) developed robust growth and differentiation when co-cultured alone. In the presence of glial cells, BAEC developed elevated Trans-Endothelial Electrical Resistance (TEER). Excision of individual capillaries proportionally decreased TEER; the remaining bundles were populated with healthy cells. Flow played an essential role in EC differentiation by decreasing cell division. In conclusion, this new dynamic model of the BBB allows for longitudinal studies of the effects of low and co-culture in a controlled and fully recyclable environment that also permits visual inspection of the abluminal compartment and manipulation of individual capillaries.

Brain Barrier

In vitro Models of the Blood–Brain Barrier: Tools in Translational Mediciney

By Alberto Williams-Medina, Michael Deblock, and Damir Janigro

Medical progress has historically depended on scientific discoveries. Until recently, science was driven by technological advancements that, once translated to the clinic, fostered new treatments and interventions. More recently, technology-driven medical progress has often outpaced laboratory research. For example, intravascular devices, pacemakers for the heart and brain, spinal cord stimulators, and surgical robots are used routinely to treat a variety of diseases. The rapid expansion of science into ever more advanced molecular and genetic mechanisms of disease has often distanced laboratory-based research from day-to-day clinical realities that remain based on evidence and outcomes. A recognized reason for this hiatus is the lack of laboratory tools that recapitulate the clinical reality faced by physicians and surgeons. To overcome this, the NIH and FDA have in the recent past joined forces to support the development of a “human-on-a-chip” that will allow research scientists to perform experiments on a realistic replica when testing the effectiveness of novel experimental therapies. The development of a “human-on-a-chip” rests on the capacity to grow in vitro various organs-on-a-chip, connected with appropriate vascular supplies and nerves, and our ability to measure and perform experiments on these virtually invisible organs. One of the tissue structures to be scaled down on a chip is the human blood–brain barrier. This review gives a historical perspective on in vitro models of the BBB and summarizes the most recent 3D models that attempt to fill the gap between research modeling and patient care. We also present a summary of how these in vitro models of the BBB can be applied to study human brain diseases and their treatments. We have chosen NeuroAIDS, COVID-19, multiple sclerosis, and Alzheimer’s disease as examples of in vitro model application to neurological disorders. Major insight pertaining to these illnesses as a consequence of more profound understanding of the BBB can reveal new avenues for the development of diagnostics, more efficient therapies, and definitive clarity of disease etiology and pathological progression.

Brain Barrier

Morphological and functional characterization of an in vitro blood–brain barrier model

Kathe A. Stanness, Lesnick E. Westrum, Eleonora Fornaciari, Patrizia Mascagni, Jay A. Nelson, Stephan G. Stenglein, Tim Myers, Damir Janigro

Cell culture models have been extensively used for studies of blood–brain barrier BBB.function. However, several in vitro models fail to reproduce some, if not most, of the physiological and morphological properties of in situ brain microvascular endothelial cells. We have recently developed a dynamic, tridimensional BBB model where endothelial cells exposed to intraluminal flow form a barrier to ions and proteins following prolonged co-culturing with glia. We have further characterized this cell culture model to determine whether these barrier properties were due to expression of a BBB phenotype. Endothelial cells of human, bovine or rodent origin were used. When co-cultured with glia, intraluminally grown endothelial cells developed features similar to in vivo endothelial cells, including tight junctional contacts at interdigitating processes and a high transendothelial resistance. This in vitro BBB was characterized by the expression of an abluminal, ouabain-sensitive NarK pump, and thus favored passage of potassium ions towards the lumen while preventing Kq extravasation. Similarly, the in vitro BBB prevented the passage of blood–brain barrier-impermeant drugs such as morphine, sucrose and mannitol. while allowing extraluminal accumulation of lipophylic substances such as theophylline. Finally, expression of stereo-selective transporters for Aspartate was revealed by tracer studies. We conclude that the in vitro dynamic BBB model may become an useful tool for the studies of BBB-function and for the testing of drug passage across the brain endothelial monolayer. q 1997 Elsevier Science B.V.

Brain Barrier

Side by side comparison between dynamic versus static models of blood–brain barrier in vitro: A permeability study

By Stefano Santaguida, Damir Janigro, Mohammed Hossain, Emily Oby, Edward Rapp, Luca Cucullo,

Endothelial cells in vivo are continuously exposed to shear stress, a tangential force generated by the flow of blood across their apical surfaces that affects endothelial cell structure and function. By contrast, the Transwell apparatus cannot reproduce the presence of intraluminal blood flow that is essential for the formation and differentiation of the BBB. In contrast, the dynamic in vitro model of the BBB (DIV-BBB) mimics both functionally and anatomically the brain microvasculature, creating quasi-physiological conditions for co-culturing human and non-human endothelial cells and astrocytes in a capillary-like structure. We used intraluminal bovine aortic endothelial cells (BAEC) co-cultured with extraluminal glial cells (C6) to obtain elevated trans-endothelial electrical resistance (TEER) and selective permeability to sucrose and phenytoin. The experiments were performed in parallel using Transwell systems DIV-BBB models and data were then cross compared. By contrast with Transwell, C6 and BAEC co-cultured in the DIV-BBB demonstrated predominantly aerobic metabolism evidenced by a robust increase in glucose consumption that was paralleled by a similar change in lactate production. BAEC exposed to glia under dynamic conditions grow in a monolayer fashion and developed a more stringent barrier as demonstrated by high TEER values and a selective permeability to [14C] phenytoin and the well-known paracellular marker [3H] sucrose. In conclusion, these data demonstrate that the exposure to intraluminal flow plays an essential role in promoting endothelial cell differentiation and increasing BBB tightness, thus making the use of the DIV-BBB well suited for pharmacological studies.

Brain Barrier

A new dynamic in vitro modular capillaries-venules modular system: Cerebrovascular physiology in a box

By Luca Cucullo, Mohammed Hossain, William Tierney, and Damir Janigro

Background: The study of the cerebrovascular physiology is crucial to understand the pathogenesis of neurological disease and the pharmacokinetic of drugs. Appropriate models in vitro often fail to represent in vivo physiology. To address these issues we propose the use of a novel artificial vascular system that closely mimics capillary and venous segments of human cerebrovasculature while also allowing for an extensive control of the experimental variables and their manipulation. Results: Using hollow fiber technology, we modified an existing dynamic artificial model of the blood–brain barrier (BBB) (DIV-capillary) to encompass the distal post-capillary (DIV-venules) segments of the brain circulatory system. This artificial brain vascular system is comprised of a BBB module serially connected to a venule segment. A pump generates a pulsatile flow with arterial pressure feeding the system. The perfusate of the capillary module achieves levels of shear stress, pressure, and flow rate comparable to what observed in situ. Endothelial cell exposure to flow and abluminal astrocytic stimuli allowed for the formation of a highly selective capillary BBB with a trans-endothelial electrical resistance (TEER; >700 ohm cm2) and sucrose permeability (< 1X10-u cm/sec) comparable to in vivo. The venule module, which attempted to reproduce features of the hemodynamic microenvironment of venules, was perfused by media resulting in shear stress and intraluminal pressure levels lower than those found in capillaries. Because of altered cellular and hemodynamic factors, venule segments present a less stringent vascular bed (TEER <250 Ohm cm2;Psucrose > 1X10-4 cm/sec) than that of the BBB. Abluminal human brain vascular smooth muscle cells were used to reproduce the venular abluminal cell composition. Conclusion: The unique characteristics afforded by the DIV-BBB in combination with a venule segment will realistically expand our ability to dissect and study the physiological and functional behavior of distinct segments of the human cerebrovascular network.