Before discussing the in vitro modeling of the human brain (CNS), it is important to pinpoint the main challenges that such a multiscale complex system represent. Overall, the structural complexity of the brain includes neurons, glia cells (such as astrocytes, microglia and oligodendrocytes) and pericytes, forming the neurovascular unit (NVU), immune cells and brain vascular endothelial lineage cells [23, 24], (Fig. 2A). It is estimated that the human brain contains 100 billion neurons that form the neuronal network with glial cells, transmitting information via synapses, in a process known as neurotransmission [9]. This creates a dynamic connection, stronger or weaker depending on the frequency of the synapses, between neurons that change overtime in a process known as neural plasticity. Besides that, it is estimated that neurons alone count more than 500 subtypes with specific cell-cell interactions. Moreover, the brain contains more than 250 different regions holding their unique functionality, microenvironment, cellular composition and architecture, which is very challenge to recapitulate in vitro [3]. Furthermore, the brain is connected to multiple brain subunit systems, namely brain endocrine system, choroid plexus, glymphatic system, vasculature system and barriers (i.e., BBB, blood-CSF barrier, blood-spinal cord barrier and arachnoid barrier), which are crucial for the functionality and homeostasis [3, 25]. In a recent perspective paper published by Maoz, B.M., 2021 [3], the sole-author reflects on the main challenges that should be overcome to generate an in vitro model representative of the human brain. Among the abovementioned complexity of the brain microenvironment, structure, connectivity, mechanical forces and functionality (Fig. 2B), the author also highlights a particularity of the human brain that is still mostly not understood – how neuronal electrical activity between brain cells can be translated in higher-order functions, such as self-awareness or consciousness. All these challenges show the necessity to develop in vitro bioplatforms able to close replicate parts or totally the human brain, surpassing one of the most technological and engineering challenge that we current face – the puzzling of the human brain. Overall, its achievement can shed light not just on our understanding of the brain and neuronal diseases, but ultimately on us, as humans and our evolution as specie.

(A) Illustration of the structure of the brain, particularly the brain-blood brain barrier microenvironment and cell-cell interactions; (B) The main challenges for the accurate reproduction of the brain as an in vitro model

The concept of an OoC – a biomimetic microsystem that comprises organ models with microfluidic devices, was first introduced in 2010 by Huh et al., in the group led by Donald E. Ingber [26], where the authors published a novel microfluidic device compressing a human alveolar-capillary interface to recapitulate the physiological and functionality of a breading lung. Since then, a variety of OoCs have been developed to mimic different human physiological conditions and single organs, such bone, brain, eye, heart, liver, lung, skin, vascular systems, among others (topic reviews can be found elsewhere [10, 22, 27, 28]); and progressing to the development of multi-organ models and human-on-a-chip (HoC) [28]. Beside the ability that OoCs show to recapitulate vascular perfusion in a dynamic microenvironment, these biomimetic microsystems have additional advantages over traditional animal-based models and cell-based models, namely the advantage to recreate tissue-tissue interfaces, organ-relevant complexity and functionality, allowing the on-demand application of physical, mechanical and biochemical stimuli found in the human body [3, 10, 28, 29]. Thus, comparing with animal models, OoC have the added benefits to enhance the prediction results, increase the test duration, improve reproducibility, as well as to reduce fabrication cost and operation complexity [10]. For these reasons, OoC provide a modeling bioplatform for CNS and screening of novel drugs and NFs with direct benefits that are not achieved by conventional in vitro platforms, such as the shear forces found in the brain and brain barriers, allowing to test different concentrations and timepoints [3], and evaluate the drug/NF ability to cross the BBB and target brain diseased cells in representative models of CNS.

Table 1 gathers the main advantages that BoCs have to surpass the CNS modeling challenges in comparison with the in vitro gold standard methodologies.

To achieve a significant neuromimetic platform that exceeds the capabilities of traditional in vitro methodologies, the BoC device must address some of the unique CNS modeling challenges described in Table 1 [3]. One of the most significant advantages over conventional cell cultures, is the inclusion of microfluidic-based platforms that provides the opportunity to recreate the shear stress on the BBB, enhancing the BBB properties over static models. Due to the crucial role that BBB presents as a brain’s gatekeeper, static models present limitations to emulate BBB, such as their ability to predict drug effects. Although BoCs show several advantages in mimicking of brain models and interconnectivity to brain sub-units compared to static models, the complex brain multi-functionality is still not fully represented with today’s technology. As stated by Maoz, 2021 [3], in in vitro systems, the emulation of brain capability to monitor homeostasis, process sensory inputs and outputs, present self-awareness, consciousness and cognition goes beyond any currently “platform-on-a-chip”. Some of these aspects are being studied using advanced neuronal platforms for computer software [41, 42] and controlling flight simulations [43]. But most of the BoC devices used a simplified definition of brain’s functionality targeting neurons and measuring their electrophysiological activity [44]. By so, in Sect. Brain-on-a-chip (including BBB), the state-of-the-art of BoC platforms will be discussed in more detail, including some technological limitations, as the incorporation of physiological brain aspects and sub-units, including immune system.

Due to its vital importance and cell activity, the brain has evolved with an extra protection system of blood vessels (i.e., BBB), which prevents toxins and other harmful substances from reaching it. This protective BBB, a highly selective membrane with low permeability, is also the main reason for the difficulty in creating effective drugs that are able to cross this barrier and target brain cells [9]. The efficiency of the BBB is so high, that it is estimated that 100% of large-molecule drugs do not cross the BBB, and just 2% of small-molecule drugs with mass below 500 Da are able to cross it [45]. It is generally accepted that only substances with a low molecular mass and lipophilic behavior can bypass the BBB freely (Fig. 3A) [45, 46]. However, most drugs have a higher molecular mass, which in general demands an endogenous transport system for the molecules to move across the BBB. Examples are transport-mediated transcytosis, receptor-mediated transcytosis, cell-mediated transcytosis, and absorptive transcytosis [47, 48], (Fig. 3A). Briefly, transport-mediated transcytosis or protein-mediated transport, is based on proteins that are responsible for carrying specific molecules. Among those proteins are glucose transporter type 1 (GLUT-1) and large neutral amino acid transporters (LAT), playing a crucial role in the delivery of several molecules to the brain [49]. Receptor-mediated transcytosis uses the activation of brain endothelial cell to transport endogenic molecules and is considered a promising approach to delivery drugs into the CNS [49]. Examples of this transcytosis are transferrin receptor (TfR) [50], low-density lipoprotein receptor (LDLR) [51], insulin and insulin like growth factor receptor [52], albumin receptor [53], lactoferrin receptor [54] and low-density lipoprotein-receptor related protein 1 and 2 (LRP1 and LRP2) [55]. Another important via to cross the BBB is the adsorptive-mediated transcytosis that is based on the electrostatic interaction between the negatively charged membranes of the brain endothelial cells and the positively charged molecules (usually polycationic proteins) [56]. Overall, transcytosis is being used as key-strategy to get nanomaterials through the BBB into the CNS and to enhance the efficiency of drug delivery.

(A) Representation of the distinct mechanism that molecules can use to cross BBB. (B) Illustration of the cells that compose the BBB (3D representation)

Anatomically, the BBB is composed of endothelial cells, pericytes embedded in basal lamina and astrocytes end-feet, touching the abluminal side of the brain vessels (Fig. 3B). Endothelial cells are the core structure of the cerebral blood vessels that interact with other CNS’s cells. Their morphology and function differ from peripheral endothelial cells, by presenting tight and adherent junctions, no fenestration but small transcellular pores, which restrict free diffusion and rapid exchange of molecules between the blood and brain. Also, they present specific transporters that regulates the flow of specific substrates, creating a protective barrier to molecules that reaches the brain [47]. Pericytes are vascular mural cells embedded in basal lamina, wrapping around endothelial cells and creating a close communication and regulation between them. Their main function is to help to regulate the BBB permeability, cerebral blood flow, clearance of toxic metabolites, neuroinflammation and stem cell activity [57]. Astrocytes, also known as star cells, are the most numerous glial cells (which also includes microglia) and have several functions in CNS, including dynamic signaling for clear waste, regulation of the vascular function, hemostasis, balance of neuroimmune response, brain blood flow and support to the BBB [47]. Although there is still discussion about the exact role of astrocytes in BBB, it is consensual that the BBB is formed through the coordination between endothelial cells, pericytes and astrocytes. Microglia are also a type of glia cell that acts has primary innate immune cell, i.e., specialized population of macrophages, and is found in the brain after colonize it in the early stage of the brain’s development [58]. Their main function is the immune surveillance of the CNS, synaptic pruning and phagocytosis of cellular debris, dead neurons and pathogens [59]. For years, studies on the BBB were focused on the contribution of endothelial cells, especially when using 2D cell culture and animal models [60]. Undeniably, these early studies have contributed to the understanding of cell lineage [61], expression of endothelial markers [62], thigh junctions [63, 64], efflux transporters [65], receptor systems [66], among others scientific discoveries. Just recently, the importance of the other cell types located in the brain tissues was acknowledged and added to the models [67, 68], where the BoC and its development have a great impact. Thus, to preserve the interaction between vascular endothelial and neuronal cells, fully replicating the NVU establishes a new benchmark for developing innovative in vitro BBB-BoC models.

As abovementioned, BoC devices take advantage of the OoC technological approach, which has the potential to create an accurate and simple-to-use preclinical model tool, by decoupling a complex organ, such as the brain, into different cellular structures while maintaining their interconnections. This approach allows for the precise assessment of drug molecules and/or drug nanocarriers along the different tissues, unveiling new interactions between them that are essential for the development of new therapeutic strategies for neurological diseases. Also, the possibility of integrating biosensors into it could extend its monitoring and workability for longer periods (more details in Sect. Integration of biosensing systems in BoCs). Most of the recent developments in BoC follow one of two main categories, depending on their high-throughput abilities: (i) BoC that mimics the 3D brain tissue environment (i.e., material, cell types and physiological stimulation) [32, 37], or (ii) BoC that simulates cell-to-cell or organ-to-organ interactions with interconnected multichip systems [38]. Some of these studies have also been dedicated to create BoC devices that are useful for mimicking the BBB structure, addressing the issue of transport across the endothelial layer with a porous membrane and allowing communication with brain cells [8].

An example of this advanced BBB-BoC was achieved by Brown and co-workers, 2018 [69], where a human BBB microfluidic model (named as µHuB) was developed using human cerebral microvascular endothelial cells (hCMEC/D3) and human astrocytes, using a commercial microfluidic platform, Fig. 4A. Wherein, the authors verified relevant shear stresses with expression of phenotypical tight junction markers, such as Claudin-5 and Zonula occludens protein 1 (ZO-1), with size-selective permeability close to BBB models (10 and 70 KDa). In another study, Pediaditakis et al., 2022 [32], reported the development of a BBB-brain human organotypic microphysiological system containing endothelial, pericytes, glia and cortical neurons to recreate critical aspects of neuroinflammation, serving as brain-chip model able to study novel therapeutics for brain diseases, and to understand cell-cell interactions and BBB function during neuroinflammation. In this study, the researchers report similar transcriptomic profiling to human adult cortex by using next-generation sequencing data and databases of signature genes, reporting identical proinflammatory cytokines, and compromised BBB permeability when exposed to tumor necrosis factor alpha (TNF-a), Fig. 4B.

Examples of different designs of microphysiological systems developed to recapitulate the human brain. A. (a) Co-culture of hCMEC/D3 and human astrocytes using a commercial microfluidic device (µHuB) containing an apical compartment seeded with hCMEC/D3 (green) and a central compartment containing human astrocytes (red), (b) Zoomed-in of the pore membrane (3 × 3 × 50 μm, w × h × d) that connect apical and central compartments. Scale bar = 20 μm. Reproduced with permission [69]. Copyright 2018, Wiley. B. Illustration of the Brain-Chip device designed as a two-channel microengineered chip incorporating brain endothelial-like cells (BBB) cultured on the bottom channel and separated by a porous membrane from neurons (MAP2, green), primary human brain astrocytes (GFAP, magenta; IBA1, yellow), pericytes (aSMA, red), and microglia (top channel representing the brain model). Scale bar = 50 μm. Reproduced with permission [32]. Copyright 2022, iScience. (C) Schematic illustration of the microengineered human BBB model containing human brain microvascular endothelial cells (HBMECs) (top red channel) seeded on top channel separated by a porous membrane from the human astrocytes (HAs) and human brain vascular pericytes (HBVPs) (bottom blue channel) (scale bar = 100 μm). Reproduced with permission [70]. Copyright 2020, Nature. Open access. (D) In vitro 3D BBB triculture model assembled in hydrogel. Illustration of the timeline for the fabrication of the human BBB-device, comprising endothelial cells, pericytes and astrocytes. Reproduced with permission [71]. Copyright 2022, Wiley. Open access. (E) Self-assembled in vitro 3D neuro vascular unit (NVU) platform. Endothelial cells (ECs) are enclosed within fibrin gel, facilitating the development of vascular networks within the matrix. Neurons and astrocytes are then seeded in the neighboring channel alongside the vascular networks. Reproduced with permission [37]. Copyright 2017, Nature. Open access

Indeed, the lack of efficient drugs that can cross the BBB and treat NDs is a main concern in neurosciences and medicine. In this regard, nanomedicine, which enables the design of NFs that can be engineered as active targeting drug nanocarriers, allowing for targeting, loading and controlled delivery of a high range of active substances to specific organ/tissues of the body, has received great attention in recent times, especially to enhance the targeting and efficiency of drug delivery into the CNS [4]. However, their clinical translation has been scarce, specially due to the lack of robust in vitro CNS models able to screen at the development phase some strategies related to their BBB-crossing, clinical safety, toxicity and neuroprotection [8]. In a study published by Ahn et al., 2020 [70], a microphysiological platform was designed to recapitulate key-structures and functions of the human BBB to 3D mapping the distributions of NFs in the vascular and perivascular regions (Fig. 4C). The authors reported a mimicking BBB-brain model that besides similar structure and function, key gene expressions, low permeability, and 3D astrocytic network with reduced reactive gliosis and polarized aquaporin-4 distribution, revealed a precise capture of NPs distributions with distinct cellular uptakes and BBB penetrations through receptor-mediated transcytosis. The authors pinpoint the advantage of the developed bioplatform to serve as NFs screening tool in comparison with animal models, particularly: (i) the BBB-brain model allowed time-lapse sampling and end-point fluorescence-activated cell sorting (FACS) analysis to quantify 3D nanoparticle (NP) distribution at the BBB; (ii) the compartmentalized structure of the BBB chip allowed the separated measuring of molecules in each space and BBB penetration; (iii) evaluation of the targeting efficacy of NPs at cellular levels; as well as (iv) depth mechanistic understanding of the interactions between the BBB and NPs at cellular levels [70]. This study is an example of the capableness that microphysiological platforms to screen novel NFs in an early stage of development and optimization prior to clinical trials, fostering the engineering of brain-targeted delivery systems for neurological disorders.

In the given examples, BBB is typically represented as a 2D endothelial vascular monolayer separated from the brain model through a porous membrane or pillars, and the structural design of the device assembled as vertical or “sandwich-like” (Fig. 4B-C) or planar parallel (Fig. 4A) design. In these approaches, endothelial cells may be placed onto the porous membrane (with or without glia cells) or grown in a distinct compartment to establish monolayers featuring a vascular system. Yet, blood vessels can also be formed in 3D architecture within hydrogels, as a 3D-tubular design (Fig. 4D), either with or without perivascular cells integrated into the hydrogel matrix. An example of this methodology is the work performed by Seo et al. [71], showing the fabrication of a human BBB model by coculturing BBB-composing cells within a 3D hydrogel matrix (Fig. 4D). The authors first validated the BBB model through the analysis of the expression of BBB-specific markers, BBB permeability with and without administration of inflammatory cytokines and BBB-opening agents. After the BBB validation, the authors extended their research using this 3D BBB-model as a BBB-glioblastoma-platform to study drug delivery and BBB-associated chemosensitivity. Another example for 3D BBB assembling strategy is the work developed by Bang et al. [37], presenting a self-assembled in vitro neurovascular unit (NVU) platform with a 3D model of the BBB (Fig. 4E). This framework can accurately replicate the in vivo BBB microenvironment, complete with ECM. Leveraging the intrinsic processes of vasculogenesis and angiogenesis, endothelial cells can autonomously organize to establish vasculature. In this work the authors report that this methodology mirrors natural vascular development in vivo, resulting in enhanced BBB functionalities with potential application in the screening of medicines that targets the brain for NDs.

Although these latest 3D-BBB in vitro models resemble with more accuracy the in vivo human brain, most of these studies lack in the representation of the peripheral immune system (i.e., immune cell migration and interaction across the BBB) in response to severe injury and diseases. Indeed, the immune system has a synergetic and preponderant role in the regulation of the BBB, and vice-versa, which affects the CNS during health and disease [72]. Some of these BBB-immune interactions include: (1) the transport of cytokines and substances with neuroinflammatory properties; (2) traverse of immune cells through the BBB by tightly regulated process of diapedesis; and (3) inflammatory conditions, trauma injury and AD, which increases immune cell entry into CNS [73]. The process of immune cells moving out of the bloodstream, known as diapedesis or extravasation, involves multiple steps, namely: (i) tethering and rolling of the immune cells along endothelial cell surface, (ii) activation by recognizing chemokines immobilized on proteoglycans on the surface of endothelial cells, (iii) firm arrest of the immune cells on the luminal surface of the endothelial cells, (iv) polarization and crawling to find endothelial junctions, and (v) diapedesis across the endothelial barrier [74]. This complex process is characterized by the sequential interactions between adhesion molecules and signaling molecules present on both the vascular endothelial cells and the immune cells [72]. Thus, the in vitro modelling of immune cell trafficking across BBB, requires reliable culture systems that faithfully replicate the unique characteristics of the BBB, including the continuous interaction with components of the NVU. Also, it has been shown that the presence of shear flow in in vitro BBB models emulates unique T-cell crawling behavior observed in in vivo imaging studies [75]. So, advanced BBB in vitro models should be combined with sophisticated microfluidics and live cell imaging [72]. The development of BoC as a highly tunable in vitro system integrated with immune systems will be greatly beneficial for the advancement in the understanding of brain diseases and development of novel drugs/NFs.