The blood–brain barrier is playing a critical role in controlling the influx and efflux of biological substances essential for the brain’s metabolic activity as well as neuronal function. Thus, the functional and structural integrity of the BBB is pivotal to maintain the homeostasis of the brain microenvironment. The different cells and structures contributing to developing this barrier are summarized along with the different functions that BBB plays at the brain–blood interface. We also explained the role of shear stress in maintaining BBB integrity. Furthermore, we elaborated on the clinical aspects that correlate between BBB disruption and different neurological and pathological conditions. Finally, we discussed several biomarkers that can help to assess the BBB permeability and integrity in-vitro or in-vivo and briefly explain their advantages and disadvantages.

The blood-brain barrier (BBB) is a fundamental component of the central nervous system. Its functional and structural integrity is vital in maintaining the homeostasis of the brain microenvironment. On the other hand, the BBB is also a major hindering obstacle for the delivery of effective therapies to treat disorders of the Central Nervous System (CNS). Over time, various model systems have been established to simulate the complexities of the BBB. The development of realistic in vitro BBB models that accurately mimic the physiological characteristics of the brain microcapillaries in situ is of fundamental importance not only in CNS drug discovery but also in translational research. Successful modeling of the Neurovascular Unit (NVU) would provide an invaluable tool that would aid in dissecting out the pathological factors, mechanisms of action, and corresponding targets prodromal to the onset of CNS disorders. The field of BBB in vitro modeling has seen many fundamental changes in the last few years with the introduction of novel tools and methods to improve existing models and enable new ones. The development of CNS organoids, organ-on-chip, spheroids, 3D printed microfluidics, and other innovative technologies have the potential to advance the field of BBB and NVU modeling. Therefore, in this review, summarize the advances and progress in the design and application of functional in vitro BBB platforms with a focus on rapidly advancing technologies.The BBB consists of highly specialized vascular endothelial cells (EC) lining the brain microvessels in juxtaposition with closely associated pericytes [1], extracellular matrix components, and astrocytic end-feet processes [2]. Along with other cells such as neurons and microglia, this cellular milieu modulates the BBB properties, supports its viability and functions [3]. At the brain microvascular level, the BBB functions as a highly dynamic and active interface between the systemic circulation and the CNS. The BBB maintains a stable brain environment to protect the CNS from unsolicited cells, bacteria, viruses, and potentially harmful substances (either endogenous or exogenous) apart from protecting against systemic fluctuations. The BBB also regulates the transport of essential molecules and nutrients necessary to maintain the optimal CNS environment and support neuronal activities [4]. The BBB responds to many physiological and pathological cues, including rheological changes [5], inflammatory stimuli, oxidative stress [6], diabetes, and hypercholesterolemia [7-10], acute brain injury [3], etc. The intrinsically unique and utmost complex functional interaction between the BBB endothelium and the surrounding cellular milieu (including astrocytes, pericytes, neurons, microglia, myocytes as well as specialized cellular compartments such as endothelial glycocalyx [11,12] has been termed "neurovascular unit (NVU)" [2,13]. In addition, the basement membrane, which exerts essential functions in cellular support and signal transduction,

Gelatin methacrylate (GelMA) has been gaining popularity in recent years as a photo-crosslinkable biomaterial widely used in a variety of bioprinting and tissue engineering applications. Several studies have established the effects of process-based and material-based parameters on the physical–mechanical properties and microstructure of GelMA hydrogels. However, the effect of encapsulated cells on the physical–mechanical properties and microstructure of GelMA hydrogels has not been fully understood. In this study, 3T3 fibroblasts were encapsulated at different cell densities within the GelMA hydrogels and incubated over 96 h. The effects of encapsulated cells were investigated in terms of mechanical properties (tensile modulus and strength), physical properties (swelling and degradation), and microstructure (pore size). Cell viability was also evaluated to confirm that most cells were alive during the incubation. It was found that with an increase in cell density, the mechanical properties decreased, while the degradation and the pore size increased.

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Background Understanding the pathophysiology of the blood brain–barrier (BBB) plays a critical role in diagnosis and treatment of disease conditions. Applying a sensitive and specific LC–MS/MS technique for the measurement of BBB integrity with high precision, we have recently introduced non-radioactive [13C12]sucrose as a superior marker substance. Comparison of permeability markers with different molecular weight, but otherwise similar physicochemical properties, can provide insights into the uptake mechanism at the BBB. Mannitol is a small hydrophilic, uncharged molecule that is half the size of sucrose. Previously only radioactive [3H]mannitol or [14C]mannitol has been used to measure BBB integrity. Methods We developed a UPLC–MS/MS method for simultaneous analysis of stable isotope-labeled sucrose and mannitol. The in vivo BBB permeability of [13C6]mannitol and [13C12]sucrose was measured in mice, using [13C6]sucrose as a vascular marker to correct for brain intravascular content. Moreover, a Transwell model with induced pluripotent stem cell-derived brain endothelial cells was used to measure the permeability coefficient of sucrose and mannitol in vitro both under control and compromised (in the presence of IL-1β) conditions. Results We found low permeability values for both mannitol and sucrose in vitro (permeability coefficients of 4.99 ± 0.152 × 10−7 and 3.12 ± 0.176 × 10−7 cm/s, respectively) and in vivo (PS products of 0.267 ± 0.021 and 0.126 ± 0.025 µl g−1 min−1, respectively). Further, the in vitro permeability of both markers substantially increased in the presence of IL-1β. Corrected brain concentrations (Cbr), obtained by washout vs. vascular marker correction, were not significantly different for either mannitol (0.071 ± 0.007 and 0.065 ± 0.009 percent injected dose per g) or sucrose (0.035 ± 0.003 and 0.037 ± 0.005 percent injected dose per g). These data also indicate that Cbr and PS product values of mannitol were about twice the corresponding values of sucrose. Conclusions We established a highly sensitive, specific and reproducible approach to simultaneously measure the BBB permeability of two classical low molecular weight, hydrophilic markers in a stable isotope labeled format. This method is now available as a tool to quantify BBB permeability in vitro and in vivo in different disease models, as well as for monitoring treatment outcomes.

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Microfluidics-based organ-on-a-chip technology allows for developing a new class of in-vitro blood-brain barrier (BBB) models that recapitulate many hemodynamic and architectural features of the brain microvasculature not attainable with conventional two-dimensional platforms. Herein, we describe and validate a novel microfluidic BBB model that closely mimics the one in situ. Induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (BMECs) were juxtaposed with primary human pericytes and astrocytes in a co-culture to enable BBB-specific characteristics, such as low paracellular permeability, efflux activity, and osmotic responses. The permeability coefficients of [13C12] sucrose and [13C6] mannitol were assessed using a highly sensitive LC-MS/MS procedure. The resulting BBB displayed continuous tight-junction patterns, low permeability to mannitol and sucrose, and quasi-physiological responses to hyperosmolar opening and p-glycoprotein inhibitor treatment, as demonstrated by decreased BBB integrity and increased permeability of rhodamine 123, respectively. Astrocytes and pericytes on the abluminal side of the vascular channel provided the environmental cues necessary to form a tight barrier and extend the model’s long-term viability for time-course studies. In conclusion, our novel multi-culture microfluidic platform showcased the ability to replicate a quasi-physiological brain microvascular, thus enabling the development of a highly predictive and translationally relevant BBB model.

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