Comparative study of four immortalised human brain capillary endothelial cell lines, hCMEC/D3, hBMED, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies

BACKGROUND: Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. METHODS: Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (C(CL)) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. RESULTS: The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. CONCLUSIONS: Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products

Establishment of an in Vitro Human Blood-Brain Barrier Model Derived from Induced Pluripotent Stem Cells and Comparison to a Porcine Cell-Based System

The blood-brain barrier (BBB) is responsible for the homeostasis between the cerebral vasculature and the brain and it has a key role in regulating the influx and efflux of substances, in healthy and diseased states. Stem cell technology offers the opportunity to use human brain-specific cells to establish in vitro BBB models. Here, we describe the establishment of a human BBB model in a two-dimensional monolayer culture, derived from human induced pluripotent stem cells (hiPSCs). This model was characterized by a transendothelial electrical resistance (TEER) higher than 2000 Ω∙cm2 and associated with negligible paracellular transport. The hiPSC-derived BBB model maintained the functionality of major endothelial transporter proteins and receptors. Some proprietary molecules from our central nervous system (CNS) programs were evaluated revealing comparable permeability in the human model and in the model from primary porcine brain endothelial cells (PBECs)

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier

Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function and shuttling of drugs and antibodies

The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cell-derived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype.

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/β-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion

Neuregulin-1 attenuates experimental cerebral malaria (ECM) pathogenesis by regulating ErbB4/AKT/STAT3 signaling.

BACKGROUND:Human cerebral malaria (HCM) is a severe form of malaria characterized by sequestration of infected erythrocytes (IRBCs) in brain microvessels, increased levels of circulating free heme and pro-inflammatory cytokines and chemokines, brain swelling, vascular dysfunction, coma, and increased mortality. Neuregulin-1β (NRG-1) encoded by the gene NRG1, is a member of a family of polypeptide growth factors required for normal development of the nervous system and the heart. Utilizing an experimental cerebral malaria (ECM) model (Plasmodium berghei ANKA in C57BL/6), we reported that NRG-1 played a cytoprotective role in ECM and that circulating levels were inversely correlated with ECM severity. Intravenous infusion of NRG-1 reduced ECM mortality in mice by promoting a robust anti-inflammatory response coupled with reduction in accumulation of IRBCs in microvessels and reduced tissue damage. METHODS:In the current study, we examined how NRG-1 treatment attenuates pathogenesis and mortality associated with ECM. We examined whether NRG-1 protects against CXCL10- and heme-induced apoptosis using human brain microvascular endothelial (hCMEC/D3) cells and M059K neuroglial cells. hCMEC/D3 cells grown in a monolayer and a co-culture system with 30 μM heme and NRG-1 (100 ng/ml) were used to examine the role of NRG-1 on blood brain barrier (BBB) integrity. Using the in vivo ECM model, we examined whether the reduction of mortality was associated with the activation of ErbB4 and AKT and inactivation of STAT3 signaling pathways. For data analysis, unpaired t test or one-way ANOVA with Dunnett's or Bonferroni's post test was applied. RESULTS:We determined that NRG-1 protects against cell death/apoptosis of human brain microvascular endothelial cells and neroglial cells, the two major components of BBB. NRG-1 treatment improved heme-induced disruption of the in vitro BBB model consisting of hCMEC/D3 and human M059K cells. In the ECM murine model, NRG-1 treatment stimulated ErbB4 phosphorylation (pErbB4) followed by activation of AKT and inactivation of STAT3, which attenuated ECM mortality. CONCLUSIONS:Our results indicate a potential pathway by which NRG-1 treatment maintains BBB integrity in vitro, attenuates ECM-induced tissue injury, and reduces mortality. Furthermore, we postulate that augmenting NRG-1 during ECM therapy may be an effective adjunctive therapy to reduce CNS tissue injury and potentially increase the effectiveness of current anti-malaria therapy against human cerebral malaria (HCM)

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

Generation of an hiPSC-Derived Co-Culture System to Assess the Effects of Neuroinflammation on Blood-Brain Barrier Integrity

The blood-brain barrier (BBB) regulates the interaction between the highly vulnerable central nervous system (CNS) and the peripheral parts of the body. Disruption of the BBB has been associated with multiple neurological disorders, in which immune pathways in microglia are suggested to play a key role. Currently, many in vitro BBB model systems lack a physiologically relevant microglia component in order to address questions related to the mechanism of BBB integrity or the transport of molecules between the periphery and the CNS. To bridge this gap, we redefined a serum-free medium in order to allow for the successful co-culturing of human inducible pluripotent stem cell (hiPSC)-derived microglia and hiPSC-derived brain microvascular endothelial-like cells (BMECs) without influencing barrier properties as assessed by electrical resistance. We demonstrate that hiPSC-derived microglia exposed to lipopolysaccharide (LPS) weaken the barrier integrity, which is associated with the secretion of several cytokines relevant in neuroinflammation. Consequently, here we provide a simplistic humanised BBB model of neuroinflammation that can be further extended (e.g., by addition of other cell types in a more complex 3D architecture) and applied for mechanistic studies and therapeutic compound profiling

Development of a Direct-Contact Triculture Model of the Human Blood Brain Barrier and Potential Applications as a Preclinical Drug Screening Tool

Given the high cost and emerging ethical and public concerns associated with the use of animal models, the use of in vitro techniques is gaining considerable attention in research programs related to CNS drug discovery and development. More specifically, various in vitro cell culture models for studying and predicting drug transport across the Blood-Brain Barrier (BBB) were developed as preclinical research tools for neurological drug discovery and development. The main goal of the present studies has been to improve conventional in vitro methods used in mimicking the BBB physiology and functions by allowing physiologically complex interactions of the human BBB cells (astrocytes, pericytes, and Brain Microvessel Endothelial cells or the BMECs) to occur in vitro. This goal was achieved by carrying out a systematic seeding of layers corresponding to all the principal cellular components of a human BBB, such that a confluent and continuous layer of BMECs would form on a basement of human brain astrocytes and pericytes. Therefore, it was hypothesized that if complex interactions among these BBB cells were allowed to occur in vitro, then this would lead to an improved multicellular culture model of the BBB system, showing better paracellular tightening in comparison to the conventional BBB monoculture models. Results presented in this dissertation indicate that a confluent and continuous layer of hCMEC/D3 cells (an immortalized BBB cell line) was formed directly on a basal coculture made of astrocytes and pericytes layers to make a physiological configuration resembling that of the human BBB in vivo. The resulting direct-contact triculture model was more restrictive to the permeation of various paracellular markers (mannitol, sucrose, PEG-4000 and inulin) than its corresponding monoculture of hCMEC/D3 cells. In order to compare the transcellular absorption between mono-and tri-culture models, digoxin (an FDA recommended probe substrate for efflux transporters) and elacridar (an FDA recommended potent inhibitor of efflux transporters) were used to compare efflux activity between the mono-and tri-culture models. While, a statistically significant effect of elacridar on the absorption of digoxin was observed in the hCMEC/D3 monocultures, there was no statistically significant effect of elacridar on the absorption of digoxin in the triculture model. The observed difficulty to increase the absorption of digoxin using a potent Pgp inhibitor (elacridar) in the triculture model is in agreement with previously published research showing that it is difficult to improve brain drug exposure even when efflux transporters at the human BBB are inhibited. In addition, the triculture model showed a valid ranking order of transcellular BBB permeants; L-histidine (a non-Pgp substrate) showed higher apparent permeability, followed by thiamine (a Pgpsubstrate, but essential for brain function), followed by propranolol (a weak Pgp substrate), then paclitaxel, and the lowest permeability corresponded to verapamil which is a strong Pgp substrate. The final chapter of this dissertation provides possible strategies on how the triculture methodology can be further improved and applied in an industrial setting as a screening tool for studying the effect of pharmaceutical drugs and drug products on the BBB physiology and functions. Ultimately, it is hoped that the triculture will be useful in the creation of IVIVC models to predict clinical pharmacokinetic and pharmacodynamic parameters

Model of Nanoparticles Transport across the human Blood-Brain-Barrier Microvasculature

8siThe blood-brain-barrier (BBB) represents a near-impenetrable hurdle against the delivery of therapeutic to the central nervous system. Since only small compounds can cross the BBB, this reduces the treatments available for neurodegenerative diseases and cancer1. Polymer nanoparticles (NPs) have emerged as a potential solution for delivering therapeutics across the BBB to brain targets. The development of in vitro methods for quantifying NP transport behavior represents an invaluable tool for assessing therapeutic delivery capabilities2. In this work, we modelled NP transport across a previously established 3D in vitro microfluidic model of the human BBB, where a self-assembled microvasculature from human induced-pluripotent stem cell-derived endothelial cells, brain pericytes and astrocytes are supported within an extracellular matrix and fibrin gel3. Differences in NP transport were observed between commercially available polystyrene and in-house produced polyurethane NPs. The platform was also capable of elucidating the effect of surface-grafted human holo-transferrin, an attractive brain-associated ligand, on NP transport across the BBB. Importantly, a pre-clinical model and protocol are presented for reliably testing the transport capabilities of nanocarriers, with the aim to optimize their design for therapeutic delivery across the human BBB.openopenSharon WL Lee, Marco Campisi, Tatsuya Osaki, Luca Possenti, Giulia Adriani, Clara Mattu, Roger D. Kamm, Valeria ChionoWL Lee, Sharon; Campisi, Marco; Osaki, Tatsuya; Possenti, Luca; Adriani, Giulia; Mattu, Clara; Kamm, Roger D.; Chiono, Valeri