BARRIER BOOK ONE DISCOVERIES (THE BARRIER 1)

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Matrigel forms thick, loosely cross-linked gels which can promote 3D tissue organization.

1. Introduction

By contrast, collagens and laminins have an inherent capacity to polymerize and form a 3D gel spontaneously resulting in tighter packing and marked resistance to proteolysis [ 78 ]. On the contrary, culturing endothelial cells in these 3D microenvironments is quite a complex and challenging process. First, oxygen, nutrient and soluble growth factors availability which influences cellular differentiation is not uniform across the culture layers and gradients arise as the medium diffuses through the gel.

Second, imaging and western blot techniques to assess cell function and protein distribution become significantly more challenging due to: Finally, the addition of a third dimension amplifies the heterogeneities of the cultures, thus affecting the study reproducibility and data comparison [ 67 ]. Natural hydrogels also present some shortcomings such as batch-to-batch variability in composition due to their isolation from animal-derived sources. Also, not all natural hydrogels can be used uniformly in all models and one has to be careful to choose a gel best suited to the purpose of the experiment.

Alternatively, synthetic hydrogels are comprised of purely non-natural molecules that are biologically inert and are capable of creating defined 3D microenvironment. Such inert gels are simple to manufacture, adapts to the mechanical forces conveniently and are highly reproducible. However, in future there will be a need for ECM with combined properties of natural and synthetic hydrogels as it seems likely that different 3D matrices will induce different cellular responses which will in turn foster the development of more sophisticated 3D models.

However, its application in cerebrovascular research is limited due to the inherent complexities of the NVU and inability of current technology to create such multifaceted environments. While there has been a wide spread use of animal derived rodent, bovine, and porcine in vitro BBB models, inevitable species differences hinder their translatability to clinical neuroscience and drug development [ 84 , 85 , 86 , 87 , 88 ].

The Blood–Brain Barrier

On the other hand, the limited scalability, phenotypic drift, and high costs of isolated primary human brain endothelial cells BMEC hamper the development of widely usable human BBB in vitro models [ 89 , 90 , 91 ]. Therefore, development of robust and physiologically representative in vitro human BBB model of high fidelity remains a critical objective in the field of neurovascular research. Recent scientific advances in stem cell research have opened new avenues for studying various human diseases and promote drug development in a dish [ , , , , ].

The inherent potential of human induced iPSC or embryonic ESC pluripotent and progenitor stem cells have been exploited to develop highly functional and physiologically relevant in vitro human BBB models [ 84 , , ]. Here, we provide details on the design and applications of current stem cell BBB modeling see Table 4 including model-specific limitations. Similarly, hematopoietic stem cell-derived ECs when co-cultured with pericytes display a mature and functionally responsive BBB EC phenotype with sustained barrier tightness for many days and the ability to predict human brain drug distribution [ ].

New experimental models of the blood-brain barrier for CNS drug discovery

Thus, the model stability in vitro is outweighed by its relatively poor barrier tightness, compared to other in vitro BBB platforms [ , , ]. Additionally, given the tedious procedures and difficulties to isolate pure primary neurons or astrocytes, embryonic or adult brain neuronal progenitor cells NPC have served as potential alternative for robust BBB modeling in vitro [ 10 , 84 , , ]. Mainly, NPCs are shown to proliferate extensively and differentiate into neuronal and astrocyte lineages that are critically involved in BBB development and maturation in vivo [ ].

The differentiation protocol was subsequently optimized to improve the model quality and robustness, as tested by previously established BBB-specific gene panel [ 10 ]. Importantly, this model could have the potential for translatability to a fully humanized and functional BBB model but requires standardization to improve the barrier tightness thus mimicking the in vivo conditions.

Extensive characterization of the purified BMECs show abundant expression of TJ proteins enriched along the cell-cell contours and polarized expression of diverse array of functionally active BBB nutrient and efflux transporters [ ]. The basic differentiation protocol was extensively optimized in subsequent studies to further enhance the differentiation efficiency of hiPSC cell line and BMEC functional responses to achieve high barrier tightness. These TEER values are unprecedented in vitro and uncertainty prevails regarding the stability of the model in prolonged culture for long-term assessments.

Publication details

Nevertheless, the biochemical phenotype of BMECs differentiated from other cell lines under similar conditions also exhibited a potential improvement of barrier integrity [ ]. Interestingly, it was also demonstrated that hiPSC derived BMECs co-cultured with astrocytes in microfluidic platforms can achieve significantly high and sustained TEER values without the necessity for shear stress [ ].

In addition to retinoic acid supplementation, the source of matrigel used for early maintenance of stem cells [ ] and initial hPSC seeding density before differentiation [ ] could significantly affect BMEC yield and BBB tightness.

Importantly, the singularized-cell seeding approach described by the latter study could enhance the scalability and reproducibility of hPSC-derived BBB models. Given a large dynamic range of permeability values diazepam to sucrose , hPSC derived BBB models could have potential applications as reliable drug screening tools [ , ], although more functional assays with a diverse array of compounds are required to test the predictive strength of the model [ ].

Importantly, stem cells can be integrated with microfluidic approaches towards development of robust BBB-on-chip platforms for high throughput studies [ ]. Despite high reproducibility of cord blood stem cell-derived BBB models, their relatively low barrier tightness could be a potential limitation [ , , ]. Nevertheless, stem cell based BBB modeling is receiving significant interest as a potential alternative to current in vitro platforms. The BBB phenotype displays unique characteristics anatomically and functionally.

Novel biotechnological advancements and better understanding of the processes governing the barriergenesis and barrier functions under normal and pathological conditions have driven the development of more sophisticated and realistic BBB models for studying the pathophysiology of CNS diseases, more effective CNS drugs and helped reduce the needs for inherently complicated and highly variable in vivo studies. The ability to modulate stem cells differentiation into BBB phenotypic cells will further boost the development of clinically relevant NVU models for basic and translational studies and drug development.

Most of the studies currently available revolve around the use of very cost-effective and user friendly ECs monocultures, which however, offer a scant image of the BBB in vivo and fail to reproduce the cellular and environmental milieu of the NVU. In some instances, improved BBB tightness and selectivity have been observed but the data are not consistent and of limited reproducibility across platforms. Despite these issues, the use of in vitro models as companion tools to further basic and translational research hold great promise in us much the ability to selectively control and manipulate the biological environment although in its simplest version allow for testing and experiment conditions that are not easily reproducible in vivo.

Use of primary cell cultures when available is still advantageous at least from a data reproducibility point of view but not very cost effective. Stem cell technology although still not fully mature could indeed provide a breakthrough in BBB modeling and beyond allowing for the development of the desired primary cultures within the platform itself, thus reducing the setup cost of the platform and the dedifferentiation issues that originate from having to passage the primary cells multiple times [ ].

New experimental models of the blood-brain barrier for CNS drug discovery

Further, the ability to strictly control the biological environment inside these platforms could indirect benefit stem cell technology by allowing to study and dissect out the optimal conditions necessary to differentiate and stabilize the cells into their final mature form. Among the various approaches to reproduce the BBB in vitro , microfluidic models have significant advantages over static and previous flow-based platforms including improved experimental flexibility, biomaterial integration, cost effectiveness and possibly high throughput.

There is a growing trend in the microfluidics community to develop techniques that can be readily translated from engineering-centric labs to life science laboratories. This is achieved by limiting instrumentation requirements including external pumps [ ], developing modular approaches [ ], and simplifying experimental workflows [ ]. Better accessibility to microfluidic tools for life science studies could help significantly advance BBB research. However, these platforms are still confined to a limited number of laboratories as working prototypes and access to these systems outside the lab of origin remains limited.

The high level of technical proficiency required to use these models is also a restrictive factor.

CELLS OF THE BBB

From a manufacturing stand point, lots of progresses have been made through the use of bioprinting technology. This process allows to generate spatially-controlled cell patterns where cell function and viability are preserved within the printed construct. At current stage however, the technology is not yet mature for the generation of multipart biological environments where several elements including materials, cell types, growth and differentiation factors pose technical challenges related to the sensitivities of living cells and the construction of more complex tissue structures such as the NVU.

Further, lack of high-throughput 3D-bioprinted tissue models for research makes this technology not yet suitable for drug discovery and toxicology studies. To achieve these goals, the integration of multiple fields of research including engineering, biomaterials science, cell biology, physics and medicine will be necessary. Concerning the use of stem cells in BBB modeling, although noteworthy progresses have been made toward the differentiation of these cells into viable NVU components there are a number of issues that remain unresolved.

Starting with the short term stability of the differentiated cells which at the current technological stage cannot be maintained for more than few days before dedifferentiation occurs. Also there are huge variations in TEER and permeability values reported across the various stem cells-based BBB models developed so far. For example, retinoic acid addition to the differentiated endothelial cell co-cultured with pericytes or differentiating NPC population could elevate the TEER by many-fold. This is attributed to the increased expression of TJ proteins, nutrient and drug transporters.

Thus, it should be noted that development of BBB phenotype is stem cell source dependent. Another limitation of the stem cell based models is the lack of comparative quantification studies for the expression of BBB-specific proteins with existing models including stability. Thus microcapillary like structures forms within the 3D matrix. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. National Center for Biotechnology Information , U. Expert Opin Drug Discov. Author manuscript; available in PMC Jan 1. Kaisar , 1 Ravi K. Sajja , 1 Shikha Prasad , 1 Vinay V. Author information Copyright and License information Disclaimer.

The publisher's final edited version of this article is available at Expert Opin Drug Discov. See other articles in PMC that cite the published article. Abstract Introduction The blood-brain barrier BBB is a dynamic biological interface which actively controls the passage of substances between the blood and the central nervous system CNS. Area covered This article reviews recent technological improvements and innovation in the field of BBB modeling including static and dynamic cell-based platforms, microfluidic systems and the use of stem cells and 3D printing technologies.

Expert opinion Development of effective CNS drugs has been hindered by the lack of reliable strategies to mimic the BBB and cerebrovascular impairments in vitro. Open in a separate window. Schematic illustration of BBB anatomy A cross-section of brain microcapillary representing luminal compartment composed of basal lamina, endothelial cells and pericytes tightly ensheathed by the astrocytic end-feet. Table 1 Transport systems at the BBB. Transport systems Substrates 1.

Mono carboxylic acid Lactate 5. Neutral amino acids Phenylamine 6. Basic amino acids Arginine 7. Modeling the BBB in vitro: Schematic diagram of currently available in vitro BBB models simulating in vivo NVU milieu based on two distinct principles- static vs dynamic culture Static models include transwell and 3D ECM platform while dynamic models utilize hollow fiber based apparatus or micro fluidic devices. Cell culture-based in vitro BBB models: Mono, Co- and Triple culture platforms The simplest and most feasible in vitro BBB model consists of a monolayes of brain capillary endothelial cells seeded on a permeable support under static culture conditions.

Table 3 BBB Models based immortalized endothelial cell lines. Primary cultures Since mice are extensively used in preclinical brain cancer research [ 11 ] as well as many CNS neurodegenerative and neuroinflammatory disorders, mouse BBB models have been developed in parallel to improve data reproducibility when transitioning from in vitro to in vivo. Immortalized endothelial cell lines To reduce cost and labor associated with the procurement of primary ECs, several immortalized endothelial cell lines from diverse origin have been developed. Shear stress and cell differentiation Various types of hemodynamic forces regulate the blood vessel function and tone including the regional tissue-blood barrier homeostasis [ 28 ].

Microfluidic devices Microfluidic tissue-on-a-chip approaches have emerged as promising techniques to establish in vitro barrier tissues as potential tools in discovery biology and to study BBB endothelial responses to shear stress [ 46 , 47 ]. Technology advancements in BBB modeling 6. Stem cell based BBB models While there has been a wide spread use of animal derived rodent, bovine, and porcine in vitro BBB models, inevitable species differences hinder their translatability to clinical neuroscience and drug development [ 84 , 85 , 86 , 87 , 88 ].

Conclusion The BBB phenotype displays unique characteristics anatomically and functionally. Expert Opinion Most of the studies currently available revolve around the use of very cost-effective and user friendly ECs monocultures, which however, offer a scant image of the BBB in vivo and fail to reproduce the cellular and environmental milieu of the NVU.

Blood-brain barrier models are to be considered companion tools designed to facilitate basic and translational studies in the field of CNS drug discovery and cerebrovascular diseases. Complex co-culture systems may prove effective in the development of quasi-physiological system at the expenses of increased complexity and higher cost. Microfluidic tissue-on-a-chip approaches have emerged as promising tools to establish in vitro BBB models but the technology is not yet available mainstream.

The use of multiple in vitro systems featuring complementary characteristics could help reducing the shortcomings of each platform as standalone systems. Footnotes Declaration of interest: Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures.

Pro-regenerative properties of cytokine-activated astrocytes. Drug metabolism and pharmacokinetics, the blood-brain barrier, and central nervous system drug discovery. Traumatic injury elicits JNK-mediated human astrocyte retraction in vitro. The role of shear stress in Blood-Brain Barrier endothelial physiology. This article details the effect of shear stress on BBB endothelial physiology and function.

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Autocrine VEGF signaling is required for vascular homeostasis. Analysis of the expression of nine secreted matrix metalloproteinases and their endogenous inhibitors in the brain of mice subjected to ischaemic stroke.

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