Recent in vitro Models for the Blood-Brain Barrier

Joao Basso,^ab* Maria Mendes,^ac* Maria Ferreira,³ Joao Sousa,^ab Alberto Pais^b and Carla Vitorino^{ab c}

^aFaculty of Pharmacy, University of Coimbra, Polo das Ciencias da Saiide, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal ^bCoimbra Chemistry Centre, Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal

^cCentre for Neurosciences and Cell Biology (CNC), University of Coimbra, Faculty of Medicine, Rua Larga, Polo 1,1st floor, 3004-504 Coimbra, Portugal This email address is being protected from spam bots, you need Javascript enabled to view it

The blood-brain barrier (BBB) is essential for the regulation and maintenance of the neurological function and homeostasis of the brain, being a complex cellular system which strictly controls the transport of substances between the blood circulation and brain tissue. Several central nervous system (CNS) disorders may, however, lead to impairment in the BBB integrity. As such, further insights on the brain pathophysiology are required. An understanding of the brain microenvironment and underlying response mechanisms is

*These authors contributed equally to this chapter.

Nanoparticles for Brain Drug Delivery

Edited by Carla Vitorino, Andreia Jorge and Alberto Pais

Copyright © 2021 Jenny Stanford Publishing Pte. Ltd.

ISBN 978-981-4877-31-2 (Hardcover), 978-1-003-11932-6 (eBook) w w w. j e n ny sta nf о rd. со m crucial for the development of several in vitro models which mimic in vivo conditions, designed for diverse purposes and studies. Through the correlation between BBB function and the pathogenicity of the target disease, in vitro models enable both the analysis of the BBB influence in the CNS disorders and the exploration of the BBB as a promising target for pharmacological modulation.

This chapter provides a comprehensive and detailed description of the currently existing in vitro models of the BBB, addressing the advantages and drawbacks of each mentioned model (monolayer cultures, Transwell cultures and microfluidic systems), on a comparative perspective.

Introduction to Blood-Brain Barrier Models

The concept of the blood-brain barrier (BBB) is not new. In fact, it arose between the late ends of the 19th and the beginning of the 20th century, after the intravenous or intrathecal administration of dyes resulted in selective compartmental organ staining [1]. The BBB is a physical and chemical barrier which plays a vital role in maintaining the homeostasis of the central nervous system (CNS) by controlling the transport of substances and drugs between the systemic blood circulation and the brain tissue. Therefore, not only the BBB mediates the transport of nutrients and metabolites to the brain, it also prevents the crossover of toxic substances and pathogens and their action within the CNS [2]. The dysfunction or disruption of this barrier is associated to several neurological conditions, including Alzheimer's disease, brain infections, brain tumours, epilepsy, ischaemic stroke, multiple and amyotrophic lateral sclerosis and traumatic brain injuries [3, 4].

The anatomical and physiological structure of the BBB includes the relationship between endothelial cells, pericytes, neurons, astrocytes and the extracellular matrix (ECM), thus establishing a neurovascular unit [2]. Cerebral capillaries are composed of series of endothelial cells which completely surround the lumen and are connected by junctional complexes which include tight, adherens and gap junctions [5].

Studies on the structure and function of the BBB are extremely importance, as neurological disorders are considered the second leading cause of death worldwide [6].

Clarifying the details of the BBB transport mechanism is a very important step towards a successful drug delivery to the brain and understanding what occurs within this organ. As a result, various strategies are being developed to accurately reproduce the microenvironmental complex and predominant extracellular conditions of the brain. These models also aim at easing the measurement of biological phenomena with high resolution and high throughput.

Hence, BBB models are a powerful tool for the pharmaceutical industry and academia to identify novel and commercialised drugs and compounds which easily pass the BBB and are effective at treating CNS disorders. Ideally, and to approximate in vitro assays to in vivo conditions, a BBB-on-chip model should be able to simulate the organisation and unique characteristics of the BBB, thus reflecting its physiological complexity and endothelium with barrier function. These models should not only possess the same cell types, distribution (3D vessel-like structure of endothelial cells), cell-cell junctions and a polarised expression of influential proteins, but also reproduce the selective permeability, low fluid phase activity and transport processes and pathways of different substances by expressing the same enzymes, receptors, transporters and efflux transporters as those expressed in vivo [7]. The interaction between different cells is also an important factor, as it plays an important role in preserving tissue function, regeneration and repairing [8]. High transepithelial electrical resistance (TEER) values, high selective permeability to different molecules and shear stress are also envisioned, as these are characteristic of this barrier [7].

Several experimental BBB models have been developed during the last decades to study its function and anatomy, which may be grouped in in vivo and in vitro models. The first use consists on experimentation in living animals to closely mimic the complexity of the BBB microenvironment, since the experiments usually occur under natural conditions and with an intact membrane. The use of animal models by the industry, as tools for pharmacokinetic and pharmacodynamicstudies,isstillarequirementbefore the translation to human clinical trials. Nonetheless, there are some drawbacks to the use of these models, including the animal acquisition and the experimentation, which are both expensive and time consuming. Ethical concerns on the use and protection of laboratory animals for scientific purposes should also be considered, as well as the four Rs policy: replacement, responsibility, refinement and reduction [9]. Finally, the data obtained from animal experimentation is obtained from a different species, thus being considerate an approximation of the human environment. Therefore, there is commonly a lack of correlation between animal and human studies, which may be ascribed to differences in protein expression and physiology (cross-species differences) and poorly designed studies [10].

In vitro cellular BBB models include monolayer cultures, Transwell (со-)cultures, spheroids and microfluidic systems [11]. These approaches are based on cell culturing and provide some advantages over animal models, including a reduced cost and labour. Importantly, they may be developed using human cell cultures.

The use of endothelial cells alone or in combination with other components of the neurovascular units, in a controlled environment, is a frequently adopted strategy to yield a significant amount of data, with these models presenting a good reproducibility and high-throughput screening performance. To reproduce the physical barrier between the endothelial lumen and the brain tissue, cells are cultured over a microporous membrane, which separates the upper (luminal) compartment from the lower (abluminal) compartment. These are the Transwell models, the most common approach for BBB modelling. The simpler models rely on the use of endothelial cells over the membrane. However, these models do not take into consideration the communication between endothelial cells and other types of cells. A more trustworthy approach includes the co-culture of different types of cells, thus better reproducing the respective physiological interaction. While endothelial cells are cultured on the upper compartment, pericytes, astrocytes and/or neurons are cultured on the lower compartment [11].

Transwell BBB models are static systems, that is, they do not contemplate the free flow of fluids and the shear stress forces on the endothelial cells. In addition, most of the described models in the literature are incomplete co-culture models of the neurovascular unit. Altogether, these approaches do not fully replicate the microenvironment and physiological behaviour of the BBB. The use of microfluidic systems attempts to overtake these current limitations of the in vitro Transwell models. A brief comparison between in vivo and in vitro BBB modelling is presented in Table 12.1.

Table 12.1 Brief comparison between in vitro and in vivo BBB models

Source: Adapted with permission from Ref. [7], Copyright (2018), with permission from Elsevier.

BBB, blood-brain barrier; PK, pharmacokinetic; PD, pharmacodynamic.