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Citation: Brandl, S.; Reindl, M.
Blood–Brain Barrier Breakdown in
Neuroinflammation: Current In Vitro
Models. Int. J. Mol. Sci. 2023,24,
12699. https://doi.org/10.3390/
ijms241612699
Academic Editor: Monique F. Stins
Received: 21 June 2023
Revised: 7 August 2023
Accepted: 8 August 2023
Published: 11 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
Blood–Brain Barrier Breakdown in Neuroinflammation:
Current In Vitro Models
Sarah Brandl and Markus Reindl *
Clinical Department of Neurology, Medical University of Innsbruck, 6020 Innsbruck, Austria;
sarah.brandl@i-med.ac.at
*Correspondence: markus.reindl@i-med.ac.at
Abstract:
The blood–brain barrier, which is formed by tightly interconnected microvascular endothe-
lial cells, separates the brain from the peripheral circulation. Together with other central nervous
system-resident cell types, including pericytes and astrocytes, the blood–brain barrier forms the
neurovascular unit. Upon neuroinflammation, this barrier becomes leaky, allowing molecules and
cells to enter the brain and to potentially harm the tissue of the central nervous system. Despite the
significance of animal models in research, they may not always adequately reflect human patho-
physiology. Therefore, human models are needed. This review will provide an overview of the
blood–brain barrier in terms of both health and disease. It will describe all key elements of the in vitro
models and will explore how different compositions can be utilized to effectively model a variety
of neuroinflammatory conditions. Furthermore, it will explore the existing types of models that are
used in basic research to study the respective pathologies thus far.
Keywords: blood–brain barrier; neurovascular unit; neuroinflammation; in vitro models
1. Introduction
Typically, 98% of small-molecule drugs, and close to 100% of large-molecule drugs, fail
to enter the central nervous system (CNS) through the tight barrier of endothelial cells [
1
].
Moreover, approximately 30% of all the drugs that are specifically developed for treating
CNS diseases encounter failure in penetrating the endothelial cell layer when attempting
to access the CNS [
2
]. However, under certain conditions, this physical barrier becomes
permeable. This allows both beneficial and detrimental cells, as well as substances to enter
the CNS tissue. Neuronal and glial injury arise due to the inflammatory environment
that results from the breakdown of the usually highly effective blood–brain barrier (BBB).
Studies investigating the BBB in different neurological disorders in vivo have only been partly
successful so far. These animal models did not always reflect the human (patho-) physiology,
and the results of animal studies cannot always be replicated in humans. To circumvent this
problem, but also to reduce and replace animal experiments, human tissue in vitro models are
still urgently needed. This review provides an overview of the current approaches in terms of
modeling the neuroinflammation of the BBB in different neurological and psychiatric diseases.
2. The Neurovascular Unit in Health
There are different definitions of the vascular BBB: they vary from just the brain
microvascular endothelial cells (BMECs) with pericytes in the basement membrane (BM),
astrocyte endfeet enwrapping the capillaries, and other cells signaling the formation of
barrier features, but not themselves participating in the physical barrier, to the inclusion of
the BM and glycocalyx. In contrast, the neurovascular unit (NVU) has become a collective
term for all cell types that are involved in BBB integrity, including microglia and neurons.
Int. J. Mol. Sci. 2023,24, 12699. https://doi.org/10.3390/ijms241612699 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023,24, 12699 2 of 40
2.1. Microvascular Endothelial Cells
BMECs are the key regulators of the brain microenvironment, and the most important
component of the BBB. They cover all brain microcapillaries with a total surface of between
12 and 18 m
2
in an adult human [
3
]. Moreover, BMECs express crucial factors regulating
the permeability of the BBB. Microvascular heterogeneity results from the diverse functions
that the cell layer needs to exhibit in different tissues. For example, the glomerular endothe-
lium of the kidney is fenestrated with intracellular pores for rapid filtration, absorption,
and secretion, whereas the sinusoidal endothelium of the spleen or liver is discontinuous
with larger pores for large solute exchanges at higher rates [
4
,
5
]. In contrast, BMECs exhibit
a continuous, tight layer with the help of gap-, adherens-, and tight junctions (TJs) that
bring them into proximity and limit paracellular transport across this layer [
6
,
7
]. Adherens
junctions consist of transmembrane cadherin–cadherin complexes between adjacent cells,
and they are responsible for the adhesion between cells, catenins, and scaffolding proteins
in the cytoplasm. Vascular endothelial-cadherins mediate the connection between two
BMECs, whereas N-cadherins establish the connections with pericytes [
8
]. Gap junctions
are composed of tissue-specific connexins that form hexamers at the membrane. The align-
ment of two neighboring endothelial cell–surface hemichannels allows for intercellular
communication through the exchange of ions and small molecules between BMECs [
7
,
9
].
TJs form the physical separation between the bloodstream and the CNS tissue. They
consist of the transmembrane proteins occludin, claudins (especially claudin-5), and junc-
tional adhesion molecules [
10
,
11
]. Their extracellular domains are in close interaction with
their counterparts on neighboring cells, thereby narrowing the paracellular cleft [
12
]. The
carboxyl-terminal of these transmembrane proteins interact with the actin cytoskeleton and
scaffolding proteins, such as zonula occludens-1 and -2 (ZO-1, -2). ZO-1 also establishes
interactions between adherens- and gap junction proteins [7].
Many reports from the literature state that only small lipophilic molecules with a
molecular weight below 400–600 Da and with a few (<8) hydrogen bonds, as well as gases,
can diffuse freely across the BBB. However, there is evidence that there is no distinct cutoff,
but rather a molecular weight penalty. There are certain molecules with a higher molec-
ular weight that can diffuse through the BBB by lipid solubility, the largest known being
cytokine-induced neutrophil chemoattractant-1 that has about 7 kDa. Furthermore, some
substrate classes, such as anti-helminthics, (opiate) peptides, and their analogs, can also
cross the BBB by diffusion despite higher molecular weights [
2
,
13
,
14
]. Additionally, diverse
transcellular transport systems for other molecules ensure a physiological brain pH and
metabolism [
15
]. Nutrients, such as glucose and amino acids, need solute carrier-mediated
transporters to enter the CNS. Many of them have a specific direction and substrate pre-
cision. Hormones, organic anions and cations, amines, nucleotides, vitamins, and fatty
acids are also carrier-mediated transported [
16
]. Receptors mediate the transport of pro-
tein ligands, such as amyloid-beta (A
β
), transferrin, insulin, and apolipoprotein E [
17
,
18
].
Ions are transported across the endothelium via adenosine triphosphatases, uniporters,
exchangers, and symporters [
15
]. The active efflux of drugs (or drug conjugates), xenobi-
otics, or nucleosides is mediated by adenosine triphosphate-binding cassette transporters.
The most prominent ones to name are P-glycoprotein (P-gp) and the multi-drug resistance
protein-1 [
6
]. To conclude, many proteins and other macromolecules cannot cross the BBB,
and they are held back in the bloodstream under physiological conditions.
Basolateral BMECs are embedded in the BM. This extracellular matrix (ECM) consists
primarily of structural elements, especially collagen type IV; specialized proteins such as
laminins, nidogen, fibronectin, and proteoglycans like perlecan; and agrin [
19
]. Different
α
-
and
β
-integrin receptors, which form transmembrane heterodimers, provide the functional
link between the BMEC cytoskeleton and the ECM. Additionally, the BM has an impact on
the integrity of endothelial junctions [19,20].
Toward the vascular lumen, BMECs express the glycocalyx, which is a thin layer of
a villiform substance [
21
]. Its major components are proteoglycan protein polymers and
glycosaminoglycan chains, including heparan sulfate, chondroitin sulfate, hyaluronic acid,
Int. J. Mol. Sci. 2023,24, 12699 3 of 40
and their associated binding proteins [
22
]. The glycocalyx is important for many of the
physiological functions of the BBB. Among them, it maintains the low permeability of the
BBB, prevents inflammation triggers, and the coagulation response [
21
]. Furthermore, it
was shown to sense changes in the shear force of the blood flow, subsequently inducing the
release of endogenous vasoactive mediators [
23
]. As it is negatively charged, the glycocalyx
forms an electrostatic barrier for negatively charged molecules, proteins, and plasma
cells [
24
]. During inflammation, the glycocalyx sheds off the BMECs to enable leukocyte
binding to vascular cell adhesion molecules [
21
]. Furthermore, glycocalyx degradation
decreases the physical barrier, enhances the permeability, promotes inflammation by a direct
interaction of the BMECs with plasma components or blood cells, as well as interferes with
various receptor functions, such as lipid mediators [
24
,
25
]. Alterations in the glycocalyx
have been shown to affect the BBB integrity, which could promote the development of a
broad range of neurological diseases [26].
2.2. Pericytes
Pericytes are present in most of the non-epithelial tissues around vessels. However,
they are most abundant in the CNS, especially in the retina, where they cover approximately
30% of the vessel surface with varying frequencies depending on their location [
27
,
28
].
They are embedded in the BM, which is where they are in close association with BMECs at
a distance of less than 20 nm. The membrane facing the microvessels expresses N-cadherin
and connexins, which bring the two cell types into a closer proximity. Through their
close interaction, the two cell types can exchange ions, second messengers, metabolites,
and ribonucleic acids [
8
]. The distinct functions of pericytes have been discussed rigor-
ously, with contractile abilities that resemble those of smooth muscle cells being the most
significant function [
29
]. Furthermore, they play an important role for the BBB regarding
integrity, thereby supporting angiogenesis and microvascular stability [
30
]. This becomes
especially evident in the platelet-derived growth factor receptor-
β
deficient mice, which
do not develop pericytes. These lead to microvascular reductions and microaneurysms.
The platelet-derived growth factor is secreted by BMECs, and this leads to the recruitment
of pericytes during angiogenesis and, subsequently, vessel stabilization [
8
,
31
]. Cerebral
autoregulation might also be mediated by pericytes as they have been shown to express
receptors for angiotensin I, vasopressin, or endothelin-1 [
32
–
34
]. A recent review discusses
the functions of pericytes in the heart and brain, and shows there are still uncertainties up
to today [
29
]. Injured or degenerating pericytes have been reported in various studies of
neurological diseases, such as in Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis
(ALS), stroke, or mild dementia [12].
2.3. Astrocytes
Astrocytes received their name from their primarily star-shaped morphology. As the
most prevalent cell type within the CNS, these glial cells have more functions beyond
just providing support and structure. They are involved in synaptic formation, matura-
tion, plasticity, and neurotransmitter recycling [
35
,
36
]. Furthermore, their endfeet wrap
around almost the entire outer surface of the brain capillaries, and it is there that they
are in close interaction with BMECs. They secrete essential factors for BBB maintenance
and are involved in nutrient-waste exchange. Astrocytes enhance P-gp and glucose trans-
porter protein-1 expression in BMECs, as well as in metabolic enzymes [
37
]. Specialized
transporters and pumps regulate the CNS pH, fluid homeostasis, and electrical potential.
Prominent examples are the water channel aquaporin-4 (AQP-4) and the inwardly rectify-
ing K+ channel subunit 4.1 [
38
–
40
]. Astrocytes have been shown to harbor great anti- but
also pro-inflammatory potential, which is described in further detail in Section 3.1 [
41
–
44
].
2.4. Neurons
The understanding of the importance of neurons for BBB integrity has evolved over
the last few decades. They are not involved in BBB formation during early brain de-
Int. J. Mol. Sci. 2023,24, 12699 4 of 40
velopment [
45
]. Despite their approximate distance of 20
µ
m to the microvessels, they
communicate their need, via astrocytes, for additional oxygen or nutrients [
46
,
47
]. Astro-
cytes, in conjunction with pericytes, influence the vascular tone and blood supply in the
area to restore physiological conditions. Moreover, glutamatergic neurons can modulate
BBB integrity directly by increasing levels of glutamate, as well as influence the BBB efflux
transporter gene expressions and endothelial circadian genes [48].
2.5. Microglia
Microglia are often referred to as the immune cells of the brain. In a resting state,
microglia monitor the brain microenvironment, and they are always prepared to sense
antigens via their major histocompatibility complexes [
49
]. They have important functions
in both the adult and developing brain [
50
]. The phagocytic activity of microglia is crucial
for normal brain development by their removal of defective synapses and in helping with
synapse pruning [51]. Their role after injury or infection is described below in Section 3.1.
2.6. Oligodendrocytes
Oligodendrocytes belong to the macroglia and contribute as cells of the NVU to the
BBB. Oligodendrocyte precursor cells (OPCs) have crucial functions in BBB maintenance
and vessel formation throughout life. In return, they receive trophic support [
52
]. By
producing and maintaining the myelin sheath as an insulator around axons, they contribute
to the critical transmission of nerve impulses. In addition, OPCs are known to communicate
with other cells in the BBB, such as astrocytes and pericytes. For example, pericytes
stimulate OPCs via laminin-
α
2, which provokes the differentiation of OPCs into major
oligodendrocytes at the sites of damaged, demyelinated axons [53].
3. Main Players in Neuroinflammation
3.1. Cellular Components
Both brain resident cells, such as glial cells, and peripheral immune cells contribute
to neuroinflammation. Upon activation, microglia are polarized into a pro- or anti-
inflammatory phenotype depending on the stimulus [
51
]. The so-called M1 phenotype is
involved in the damage of surrounding neuronal and glial cells by secreting neurotoxic
factors, such as pro-inflammatory cytokines and chemokines like interleukin (IL)-6, tumor
necrosis factor-alpha (TNF-
α
), C-C motif ligand (CCL)-2, superoxide, and prostaglandin-
2 [
54
]. The M1 type is activated via the classical pathway by pro-inflammatory stimuli,
such as interferon-
γ
, the lipopolysaccharide (LPS) of gram-negative bacteria, or aggregated
pathogenic proteins (A
β
,
α
-synuclein and others) [
49
,
51
,
55
]. The M2 phenotype is involved
in tissue repair and wound healing by secreting anti-inflammatory mediators, such as
arginase-1 or chitinase-3. This phenotype can be induced by IL-4 or IL-13 in the alternative
pathway, or via acquired deactivation by IL-10 or the transforming growth factor-beta
(TGF-
β
). While both phenotypes are in homeostasis during acute stimulation, the pro-
inflammatory phenotype is predominant in chronic inflammation. Therefore, excessive
microglia activation can lead to a potentiating of tissue damage through a positive feedback
loop. This is the case in many neurodegenerative diseases, such as Parkinson’s Disease
(PD), AD, or ALS [56].
Similar to microglia, activated astrocytes also exhibit neuroprotective or neurotoxic
phenotypes. Liddelow et al. [
57
] proposed that, depending on the activation trigger, as-
trocytes develop different entities that are comparable to the M1/M2-type microglia. A1
astrocytes rapidly develop after acute CNS injury, such as CNS brain trauma or neuroin-
flammation. In response to the pro-inflammatory mediators that are secreted by M1-type
microglia, they induce a secondary inflammatory response [
49
]. This A1-type astrocyte
secretes neurotoxic factors that induce the rapid death of neurons and oligodendrocytes,
thereby driving neurodegeneration and disease progression [
57
]. Moreover, it sustains a
feedback loop that promotes further M1-type microglia, as well as leads to ECM and TJ
degradation via matrix metalloprotease (MMP) and vascular endothelial growth factor
Int. J. Mol. Sci. 2023,24, 12699 5 of 40
(VEGF)-A secretion [
58
]. In contrast, in ischemia A2, astrocytes have a neuroprotective
function, aiding neuronal survival and tissue repair [
57
]. The A1/A2 nomenclature, how-
ever, has been criticized by some researchers due to the high heterogeneity of astrocytes,
which extends beyond the binary nomenclature [58–60].
Adhesion molecule expression is upregulated in BMECs during inflammatory condi-
tions so as to allow T cells to cross the BBB. P/E-selectins are involved in the initial step,
which is also called T cell rolling. The vascular cell adhesion molecule-1 and intercellular
adhesion molecule 1 lead to the arrest of CD4+ T helper (Th) cells, or other immune cells,
in the processes of capture, rolling, integrin activation, adhesion/arrest, crawling, and dia-
pedesis that occur across the BBB. In autoimmunity, it was shown that additional/added
melanoma or activated leukocyte cell adhesion molecules control T cell trafficking into
the CNS [
12
]. More detailed reviews of immune cell transmigration across the BBB can be
found elsewhere [61–64].
3.2. Soluble Components
Cytokines mediate the communication within the immune system. Depending on the
specific cytokine and the receptor on the receiving immune cell, they can signal a particular
pathway for differentiation (such as Th1, Th2, or Th17). Chemokines are released by a
variety of cells, including endothelial cells. They play a role in embryonic development
and, depending on the chemokine, they recruit specific immune cells. The role of cytokines
and chemokines in neuroinflammation and the BBB has recently been reviewed, and the
reader is referred to these excellent review articles for more details [65–68].
MMPs are a group of endopeptidases that are secreted by various cell types, including
members of the NVU [
69
]. Physiologically, they are important contributors in CNS develop-
ment, angio- and neurogenesis, as well as synaptic plasticity, learning, and memory [
69
,
70
].
Among others, MMP-2, -3, and -9 are engaged in BBB degradation and neuroinflamma-
tion [
70
,
71
]. They are activated through the proteolysis of the N-terminal pro-domain via
reactive oxygen species (ROS), IL-17, IL-1
β
, or TNF-
α
. In an active state, they degrade
ECM and TJ proteins with the subsequent activation of pro-angiogenic factors, such as
VEGF [
72
,
73
]. Additionally, they further promote a pro-inflammatory microenvironment
by cleaving more pro-MMPs and pro-forms of IL-1
β
or TNF-
α
[
71
]. Astrocytes, pericytes,
and microglia increase the expression of MMP-9 upon brain injury by increased ROS
through the albumin release or LPS [
71
,
74
,
75
]. Additional pro-inflammatory cytokines
are produced by the microglia that are activated via MMP-3 by apoptotic neurons [
76
].
The importance of different MMPs has been observed in a broad spectrum of pathological
BBB alterations, ranging from proteolysis after injuries, stroke-associated dementia and
hyperglycemia, multiple sclerosis (MS), over epilepsy, and many others [69,70,77–80].
Lipid mediators are rapidly synthesized in multistep enzymatic pathways, and they
are bioactive lipids with functions in both health and disease. Depending on their structure,
they are classified into eicosanoids, lysophospholipids, and others. After synthesis, they
are released into the extracellular space, where they stimulate BBB cells and modulate their
functions via G-protein-coupled receptors. Through the activation of intracellular signaling
pathways, the paracellular permeability is ultimately increased [81].
ROS comprise a group of chemically reactive molecules that are involved with oxygen
(such as superoxide anions, hydrogen peroxide, and hydroxyl radicals), and they are
produced by several endo- and exogenous processes. They have the capability of oxidizing
or damaging biological molecules, and they have a dual role in the NVU [
82
–
84
]. Low
levels of ROS, together with reactive nitrogen species, act in signaling transduction for
normal cell function maintenance [
85
]. ROS also contribute to the regulation of blood
flow by vasodilation, the clearance of damaged cells, and tissue repair. Maintaining ROS
level balance underlies a complex interplay with cellular antioxidant systems, such as the
enzymes superoxide dismutase, catalase, and glutathione peroxidase [
86
]. Imbalances
in this regulation cause higher ROS levels, increased blood flow resistance, decreased
nitric oxide bioavailability, as well as increased apoptosis and immune response [
83
].
Int. J. Mol. Sci. 2023,24, 12699 6 of 40
ROS overproduction has also been associated with several neurodegenerative diseases,
especially AD. ROS are involved in several aspects of disease development, including BBB
breakdown (which interferes with brain energy supply and homeostasis),and increasing A
β
peptide deposition in vascular walls [
86
]. ROS promote a senescence-associated secretory
phenotype, which involves the secretion of several pro-inflammatory cytokines, MMPs,
and insoluble proteins/ECM [84].
4. In Vitro Modeling
Not every model is suitable for each research approach. Before starting to develop a
BBB in vitro model, the following questions should be considered:
• What is the research question and the purpose of the experiment?
• What basic biological requirements does the model need to fulfill?
• What cells and what ECM are needed for the assays that are to be performed?
• What time can be invested in each approach?
• Does it need to be applicable for high-throughput screening?
• Are there additional devices that are needed for the model?
• How long are the cells cultured, and when is the endpoint?
An additional overview of the factors that need to be considered for in vitro modeling
is illustrated in Figure 1[87].
Figure 1.
Considerations for BBB/NVU model development. Adapted from Cameron et al. [
87
].
Created with BioRender.com.
4.1. Cell Types and Sources
The choice of cell types for each in vitro model is critical and depends on the re-
searcher’s needs and resources. Cells can be derived from animals (especially rodents,
but bovine and porcine tissues are also utilized) or humans. In general, they can be
Int. J. Mol. Sci. 2023,24, 12699 7 of 40
distinguished between immortalized cell lines, primary cells, and stem cells. Each cellular
model has certain pros and cons. Primary cells are isolated from postmortem brain tissue
(or from donor tissue that was collected during a surgical procedure), and they are then
frozen in a “low passage”. Thus, postmortem tissues are easier to acquire, but the material
isolated from surgeries often serves a better yield [
88
]. They exhibit characteristics close to
the in vivo NVU. However, they depend on a relatively high amount of cell-type-specific
growth factors, and they can only divide a limited number of passages before reaching
senescence, which affects cellular morphology and functionality [
89
]. Furthermore, isola-
tion of these cells raises ethical problems and requires high-level technical skills; moreover,
purchasing primary cells can be expensive [
90
]. Therefore, the immortalized cell lines of
primary cells were established to tackle this drawback. They possess most cell-type-specific
functions, are cost-effective, applicable for long-term culture, and can be expanded rapidly.
Unfortunately, immortalization can interfere with morphology and barrier formation. Thus,
different cell lines from the same cell type can have aberrant characteristics, which makes it
crucial to validate their features (such as cell-type-specific gene/protein expression and
barrier integrity before performing experiments [91,92]).
Over the last few years, an increasing number of in vitro BBB/NVU models have been
based on stem cells. The opportunity to differentiate them into various cell types with
healthy or pathological phenotypes has facilitated new perspectives for investigating the
NVU in health and disease [
93
]. There are several approaches for the use of stem cells as
neural stem cells have a low potential for self-renewal and show immune incompatibility
upon transplantation [
94
]. Therefore, they have been used to model diseases by inserting
certain gene mutations, and they are then re-injected into animal models rather than being
used for BBB models [
95
]. Mesenchymal stem cells are isolated from bone marrow. Due to
the secretion of growth factors that contribute to neuroregeneration and remyelination, they
have already been used to treat CNS diseases. Furthermore, since mesenchymal stem cells
express similar phenotypic markers as pericytes, they have successfully been implemented
in in vitro models as substitutes for primary pericytes [
96
–
98
]. However, they might be not
applicable for neuroinflammatory BBB in vitro models because they have been shown to
act immunosuppressively, and they might have AQP-4-regulating functions [
96
,
99
]. Finally,
induced pluripotent stem cells (iPSCs) are somatic cells that have been reprogrammed to a
state of pluripotency by utilizing an overexpression of specific transcription factors [
100
].
To achieve in-vivo-like paracellular barrier properties, iPSC-based human BBB models are
the models of choice; this is because they have a higher transendothelial electrical resistance
(TEER) than can be achieved in models utilizing other BMECs [
101
]. Furthermore, gene-
editing techniques, such as CRISPR/Cas9, enable for disease modeling through using
iPSCs [
102
]. Lippmann et al. [
103
] provided a detailed and widely used protocol for the
differentiation of iPSCs into induced brain microvascular endothelial cell-like cells (iBMEC)
for the purpose of BBB modeling. This protocol has been modified and further optimized
over the last decade [
104
–
106
]. However, attention needs to be paid to the cell identity,
as a recent paper revealed that these differentiation protocols produce a homogeneous
epithelial cell population instead of an endothelial one, despite exhibiting a high TEER.
Fortunately, this phenotype can be rescued by the overexpression of appropriate BMEC-
specific ETS transcription factors (such as ETV2,ERG, and FLI1). Although rescuing
the phenotype, Lu et al. [
107
] stated that, for reliable BBB-forming BMECs, more work
needs to be conducted, including thorough characterizations with the latest technologies.
Moreover, the narrow experimental window caused by de-differentiating iPSCs under
in vitro conditions results in high costs for culturing, and time-intensive procedures pose
limitations in iPSC models [
90
]. However, as an aspect of personalized medicine, patient-
derived iPSCs are becoming increasingly important. In addition to being used for modeling
diseases, these cells can also be helpful for the individual assessments of putative drug
responses [
93
,
100
,
102
]. For example, in monogenetic diseases, such as Huntington’s disease
(HD), the mutations are already present in patient cells and do not need to be created
additionally [108,109].
Int. J. Mol. Sci. 2023,24, 12699 8 of 40
Undoubtedly, BMECs are the most fundamental compartment of the BBB. Therefore,
culturing them as a monoculture can be utilized as a BBB model. However, as mentioned
above, other cell types crucially contribute to BBB integrity [
104
]. To date, due to their sim-
plicity, there are still monoculture models in use. These cultures are utilized in basic research
for toxicity and proliferation evaluations, transport experiments, and the characterization
of the secretion or immune response, especially when the response of only BMECs is aimed
at to be studied without the interference of other NVU cell types [
87
]. Additionally, it needs
to be noted that BMECs have a regional heterogeneity and an arterial-capillary-venous
zonation in vivo, which could lead to varying results in in vitro experiments depending on
the utilized BMEC source [
110
]. Very apparent differences in morphology and function are
visible in astrocytes and BMECs between gray and white matter, such as the expression of
specific receptors and transporters. The spatial density and orientation of the BMECs vary
depending on the location, and astrocytes show fibrous morphology with long processes in
the white matter while being more star-shaped in gray matter. Furthermore, oligodendro-
cytes are more abundant in white matter compared to the gray matter. Pericyte coverage
is also heterogeneous along the vessels. Differences have been reviewed in more detail
by Villabona-Rueda et al. [
111
]. Depending on the pathology that is to be modeled, these
differences need to be considered. For example, MS patients show different types of lesions
and BBB dysfunctions in their gray and white matter, and neurodegeneration in PD and
AD primarily affects gray matter [111,112].
4.2. Three-Dimensional ECM
In a 2D perspective, the ECM is set up solely by a layer of glial and/or neuronal cells
that are seeded onto a BM-mimicking substance. The currently used BM substitutes are
primarily collagen type I/IV and/or fibronectin [
87
]. To take ECM modeling to the third
dimension, a functional scaffold that supports appropriate cell growth and differentiation is
necessary. It must fulfill certain requirements, including ECM-comparable bio-physiological
properties (low stiffness, good hydrophilicity, elasticity, and degradation) and a low toxicity
for the cells [
113
]. Hydrogels are the medium of choice for these demands. They consist
of a biocompatible network of cross-linked polymer chains of natural or synthetic origin.
Natural polymers are either polysaccharide-based, such as hyaluronic acid or chitosan,
or protein-based, such as collagen, laminin, fibrin, or fibronectin [
114
]. Common substances
incorporate several proteins, such as gelatin, Geltrex
™
, or Matrigel
®
. The latter ones
have a thick structure and weak cross-linking, which makes them beneficial for 3D BBB
self-assembled models [
115
]. There are also plant-based materials available, such as,
for example, alginate, which is a polysaccharide that is derived from brown algae [
116
].
Although natural polymers reflect the physiological composition of the ECM, they have
several disadvantages, such as batch-to-batch variability, an (often) animal origin, and weak
mechanical properties, thereby leading to more rapid disintegration [
117
]. In contrast,
synthetic polymers are chemically defined and highly versatile, but they also poorly reflect
the physiological ECM composition. The primary synthetic substance that has been used
for BBB modeling is polyethylene glycol. Natural polymers combined with synthetic
polymers ensure a balance between mechanical strength and biocompatibility. These
hybrid hydrogels can have similarities with different CNS tissue characteristics, since ratios
between the constituent polymers, as well as functional groups or cross-linking agents,
can be modified. Consequently, their properties, such as degradation rate or stiffness, are
tunable, and they can be used to model healthy or pathological neural tissues. Examples of
often-used hydrogels are polyethylene glycol-hyaluronic acid, polyethylene glycol-collagen,
or gelatin-methacryloyl [
118
]. All hydrogels need some kind of cross-linking. This can
be achieved physically in the case of natural polymers, and this happens without the
addition of exogenous agents, which occur solely by the change in temperature or pH.
Hydrogels with synthetic compartments need chemical cross-linking, which is typically
photo-polymerization by an external substance that induces gelation [119].
Int. J. Mol. Sci. 2023,24, 12699 9 of 40
4.3. Transwell
With the first model being established in 1953, the transwell model setup has not
changed substantially since [
120
]. This approach is still widely used due to its cost-
effectiveness and high-throughput ability. In general, transwells consist of a polystyrene
multi-well carrier plate, which is available in several sizes, and its respective inserts.
The bottom of the inserts consists of a porous membrane that allows for the exchange
of nutrients and other molecules between the apical and basolateral compartments of
the insert. The pore size and density can vary depending on the application. Usually,
between 0.2 and 3
µ
m pores are used for transport studies and tissue engineering [
121
–
123
].
Thus, interactions between different cell types can be studied in co-culture models while
maintaining a physical separation. Smaller pore sizes are also recommended for the proper
formation of BMEC monolayers for barrier function assays [
121
]. When the chemotaxis
or invasion of cells is studied, membranes with larger pores of up to 12
µ
m are used [
124
].
The material of the membrane should be chosen individually depending on the application.
Polycarbonate membranes are made of a translucent thermoplastic polymer, and they are
employed in many cell culture studies due to their ease of use and low cost. However,
they are not fully transparent, and visualizing apically seeded cells under the microscope
requires cell labeling with fluorescent or chromogenic dyes. Furthermore, they have high
protein binding capacities, which is unsuitable for certain types of assays. Polyethylene
terephthalate membranes consist of polyester and exhibit low protein binding. They are
clear and offer good cell visibility. However, they have limited chemical resistance, which
is unfavorable for some applications. Polyvinylidene fluoride membranes have uniform
or asymmetric pores, low protein binding, as well as high chemical and thermal stability.
They are suitable for a wide range of applications, but are also more expensive. Coating the
membranes with BM components, such as collagen type IV or fibronectin, is recommended
for better cell attachments and morphologies [
123
]. Recent models used the co-cultures of
human immortalized cell lines [
123
,
125
], primary cells [
122
], or iPSCs [
126
–
129
]. Some of
them incorporated hydrogels on one side of the insert with glial cells to obtain a more phys-
iologically relevant ECM setup [
129
–
131
]. Transwell co-cultures are often criticized for their
lack of physiological relevance due to the absence of contact between different cell types.
The thickness of the membrane, which is typically at least 10
µ
m thick, restricts the cell–cell
interactions across the membrane. This problem was addressed by Zakharova et al. [
132
],
who developed polydimethylsiloxane (PDMS) membranes with tunable thicknesses and
pore sizes. They fabricated an optically transparent, 2
µ
m thin membrane that enabled for
a lower permeability in BMECs when co-cultured with astrocytes when compared to other
membrane substances.
4.4. 3D Models
Three-dimensional models have made some advances compared to the classical tran-
swell models. First, a 3D ECM provides a more in-vivo-like environment than the planar
cell layers of most transwell models. Second, aside from membrane-based microfluidic
models, the different cell types are in direct contact with each other, which facilitates the
interaction and exchange of secreted growth factors or other regulatory molecules. Third,
a medium flow mimicking the shear stress of the blood circulation can be implemented.
The neuroinflammatory response profile to TNF-
α
stimulations in 2D vs. 3D models
was investigated by Herland et al. [
133
] in mono- and co-culture models by utilizing tran-
swell and microfluidic BBB-on-a-chip setups. Co-culturing primary BMECs with astrocytes
and pericytes under flow conditions resulted in significantly higher cytokine secretion (i.e.,
of IL-6, the granulocyte colony-stimulating factor) in the 3D model. Cucullo et al. [
134
]
showed, in 2011, the importance of shear stress on BMECs, as it altered the gene expression
patterns of junctional proteins, CYP450 proteins, ion channels, drug transporters, adhesion
molecules, and integrins. Notably, evidence has arisen of the notion that shear stress
does not affect cell morphology, but rather it tightens the barrier itself by an increase in
adherens junction and TJ protein expression [
87
]. The diameter of the vessel (channel,
Int. J. Mol. Sci. 2023,24, 12699 10 of 40
tubing), the dynamic viscosity of the fluid (medium), as well as the flow rate need to be
adjusted to get close to the physiological amount of shear stress that is needed in the model.
The in vivo shear stress is approximately 0.3–2 Pa (4 to 30 dyn/cm
2
), and this is with a
dynamic viscosity, which is dependent on the rheology of blood and is usually calculated
as 3.5–5.5~cP [
87
,
135
,
136
]. The calculation of the shear stress adjustment is described in
detail elsewhere [
87
,
137
,
138
]. For medium-flow generation in microfluidic devices, syringe
and peristaltic pumps are widely used in several other methods [
139
]. However, there
are new advances in pumping systems that passively pump media in a non-mechanical
manner [
140
]. It is important to note that this is a continuous rather than pulsatile flow, as
is keeping the shear stress on a moderate, physiological level [
141
]. Faley et al. [
142
] created
a 3D microfluidic model with a tubular structure filled with the monolayers of endothelial
cells. The authors compared different endothelial cell lines, ranging from human umbilical
vein endothelial cells to human dermal microvascular endothelial cells, as well as to two
iPSC-derived BMEC lines. The iBMECs showed a 10–100 times lower permeability to
different-sized markers compared to the other cell lines. Moreover, the model maintained
the barrier function and efflux transporter activity for up to 21 days under perfusion condi-
tions. It is worth mentioning that Faley et al. saw the best results with a subphysiological
wall shear of 0.3 dyn/cm
2
. Elevated flow rates led to an increase in permeability and
angiogenic sprouting.
One common problem regarding microfluidics is the formation of bubbles, which are
generated when plugging or removing pipes, exchanging reagents, or if the systems are
not fully tight. These bubbles tend to accumulate at right angles in the microfluidic chips,
particularly at the device inlets. Moreover, these bubbles cause blockages in the channels
known as “dead zones.” As such, they lead to flow interference, which increases the
pressure and causes cell membrane damage. To address this problem, active (for example
lasers or acoustic generators) and passive (which do not require additional equipment)
de-bubblers can be used [143].
4.4.1. Microfluidic Models
The first microfluidic models were described in the late 1990s, and they consisted
of a hollow fiber cartridge system that was composed of a bundle of porous polypropy-
lene fibers. Through the fibers, there was a medium flow applied by a variable-speed
pulsatile pump. Endothelial and glial cells could be seeded through separate loading
ports. The tubing was made of gas-permeable silicon [
144
,
145
]. Continuing up to the
present day, this original setup has been further developed and optimized, and the research
on novel microfluidic devices is still intensive. However, the major components are still
present: (1) a carrier in which the cells are incorporated, (2) an ECM compartment, (3) a
BMEC monolayer, and (4) medium flow. The material of microfluidic chips has mainly
been PDMS since it fulfills most needs. It is biocompatible, easy to fabricate, cost-efficient,
and transparent. However, one limitation of the material is its tendency in the absorption
of hydrophobic molecules. This problem is minimized by conducting protein modifications
at its surface [146].
However, there are versatile possibilities for the chip appearance, and there are four
major designs [
137
]. The sandwich design comprises a PDMS chip with two superposed
channels, which are separated by a polycarbonate or PDMS membrane. Glial cells or
neurons are seeded into the upper channel, which mimics the CNS matrix. The lower
channel reflects the capillary with the BMECs being seeded onto the membrane. Similar to
this setting is the parallel design. Cells are seeded not in vertically but in horizontally sepa-
rated channels. These channels have small interconnecting pipes with a diameter of 3
µ
m
to enable communication between the cell types [
147
]. As a further development of the
parallel design, chips with two channels for BMECs and an additional channel in between
with a CNS matrix replicate have been established. This design is membrane-based as well,
but incorporates a hydrogel-aided compartment for the ECM [
148
]. The communication
between cells can be further enhanced by 3D tubular structure designs. Without the need
Int. J. Mol. Sci. 2023,24, 12699 11 of 40
for a membrane, the cells are in direct proximity. Cylindrical channels lined with BMECs
are surrounded by a cell-containing hydrogel, thus reflecting the ECM. The microvessel
structure can either be achieved artificially or with self-assembled microvessels in hydro-
gels [
149
]. Campisi et al. [
150
] combined an iBMEC network with human brain pericytes
and astrocytes within a single fibrin hydrogel. After vasculogenesis, the model exhibited
physiologically relevant structures and functionality. Organs-on-a-chip microfluidic models
in various setups can be purchased as well. Different companies have launched chips for
all kinds of research areas. The reviews of Nikolakopoulou and Jagtiani [
151
,
152
] sum up
the currently available products.
4.4.2. 3D Bioprinting
Over the last few decades, 3D bioprinting has pushed the precision of BBB model
fabrication to a new level. Templates are designed by computer-aided design software and
then printed by specialized 3D printers. Depending on the printing technology, precise
structures with a resolution down to 100
µ
m (extrusion-based bioprinting) and even 10
µ
m
(laser-assisted bioprinting) are possible [
113
]. There are different fabrication techniques,
each of which imply strengths but also limitations [
119
]. Inkjet bioprinting offers a high
speed and resolution with low cost. It is favorable for printing cells as it ensures a high cell
viability of up to 90% [
153
]. A printer head connected to a cartilage creates droplets of bioink
by a thermal or piezoelectric actuator [
154
]. To prevent the printer head from clogging,
printing high cell densities or viscous material need to be avoided [
119
]. Microextrusion
bioprinting allows for the printing of high-viscous material in a continuous strand through
a syringe via a screw plunger or through air pressure. Compared to other bioprinting
methods, it is relatively cheap and simple [
154
]. However, it compromises cell viability
by exposing them to high mechanical stress. Therefore, extrusion-based bioprinting is
used more for scaffold and sacrificial structure printing [
155
]. In laser-assisted bioprinting,
the bioink is suspended at the bottom of a donor layer, which has a ribbon structure on
top. The printing of droplets is achieved by an absorbing layer that is stimulated by a
laser pulse. This pulse leads to an evaporation of the donor layer, creating a bubble at
the interface of the bioink layer, which is pushed onto the substrate. There, the bioink
droplet is then cross-linked. This allows for the printing of very precise structures with a
broad range of bioink viscosity. Due to the contact-free printing, cells do not experience
mechanical stress. Although cell viability is high, the technique’s high costs restrict its use in
research [
119
,
154
]. Different kinds of light-based bioprinting (stereo-, soft-, and two-photon
lithography) are rapid and highly precise methods, and they are conducted by a hydrogel
layer being exposed to a light pattern (usually UV or near-UV wavelengths); through this,
the illuminated area is cross-linked. This process is repeated vertically until the whole
construct is built. Scaffolds can be printed in such precision that Mariano et al. [
156
], for the
first time, developed a 1:1 scale biomimetic BBB model. Two years later, the same group
published a model with even thinner capillary walls of only 2
µ
m instead of 10
µ
m, as
was the case in the prior model [
157
]. Despite the high cell viability and fast fabrication,
a drawback of these methods is the limitation to single-material structures [119,154].
The printable material in 3D bioprinting is highly diverse. It needs to be distinguished
between substances that incorporate cells and those that are utilized for frameworks or
for dissolving support. Bioinks with cells are hydrogels, as described in Section 4.2. The
frameworks for the chips themselves or scaffolds, where cell-laden hydrogels are added in
between, are usually made by highly viscous substances that retain their structure. Resins
or PDMS are the materials of choice [
157
,
158
]. Sacrificial inks can be used to print hollow
structures into matrices since they dissolve under specific circumstances, like when there
are temperature changes. Examples are Pluronic (F-127) or polyvinyl alcohol [
158
]. Even the
chips themselves can be printed, which is achieved by utilizing transparent polymers like
transparent poly methyl methacrylate) or duralumin [
158
]. Wang et al. [
159
] developed an
Objet VeroClear photopolymer-based chip with a perylene-C coating to ensure transparent
appearances, chemical resistance, and biocompatibility.
Int. J. Mol. Sci. 2023,24, 12699 12 of 40
4.4.3. Cell Aggregates (Spheroids and Organoids)
Spheroids and organoids are both self-assembled cell aggregates in a static three-
dimensional environment. However, they differ in composition, complexity, and size.
Spheroids are more simple cell structures that are primarily generated by cell lines, primary
cells, or tumor cells/tissues in suspension or non-adherent conditions. They do not require
an ECM, and they usually do not exhibit a defined tissue architecture. Organoids are
derived from culturing iPSCs, embyronic, or neural stem cells, and they can recapitulate
many aspects of tissue and organ function through which to assess neurotoxicity and to
develop new drugs. However, cell differentiation requires an ECM and a growth factor
cocktail [
160
]. The first approaches were established a decade ago and are progressing
rapidly [
161
,
162
]. For example, Nzou et al. [
163
] studied the suitability of their human
cortex organoid model for drug development by assessing the BBB impairment under
hypoxic and neuroinflammatory conditions, as well as the impact of ROS and inflammation-
reducing reagents. One major advantage of organoids is the possibility of modeling
different brain regions. Thus, organoids resembling the human midbrain can be developed,
including astrocytes, oligodendrocytes, and dopaminergic neurons that are myelinated and
exhibit synaptic connections [
162
]. Furthermore, the self-assembled structures can be set up
based on patient iPSCs, which allows for disease- and patient-specific modeling. In using
this approach, the potential neurotoxicity for the patient can be screened and might also be
helpful in therapy development in the future [
163
]. A disadvantage is that organoids are
limited in size (around one millimeter) due to the lack of nutrient availability in their core.
This often leads to cellular stress within the CNS-mimicking organoid center, resulting in
necrotic cells with fragmented nuclei. This issue could be solved by vascularization within
the organoid. However, this is still a major challenge for researchers in this field [
164
]. A
study by Wörsdörfer et al. [
165
] suggested the implementation of mesodermal progenitor
cells into organoid models in order to tackle the lack of vessels within the 3D structure. Their
tumor organoid model incorporated blood vessels, as well as BM and cell–cell junctions.
Additionally, the mesodermal progenitor cells also differentiated into microglia-like cells
that could potentially be used for tumor modeling but also neuroinflammation in general.
Ham et al. [
166
] developed vascularized cerebral organoids by adding VEGF from the
beginning of the differentiation of embryonic stem cells, thus creating open-circle vascular
tubes with a two-layer structure and BBB characteristics without disturbing neurogenesis.
However, in long-term cultures, the density of blood vessels decreased and four-month
old organoids had distorted open-circle morphologies, which was possibly caused by the
missing blood pressure. Therefore, the importance of shear stress within, as well as on, the
outer surface of cerebral organoids is still a matter of research [89,166].
4.5. Model Validation
4.5.1. Cell Identity and Viability
The validation of the cell-type identity is not only for the iPSCs of special impor-
tance. Although manufacturers promise the expression of their commercially immortalized
and primary cell lines, they still might change their functions and morphology due to
immortalization or cell culture conditions, as well as due to the number of passages. There-
fore, cell-type-specific markers should be assessed via immunofluorescence, Western blot,
qPCR, or flow cytometry before the performing experiments [
119
]. BMEC identity is
usually assessed by a combination of general endothelial cell markers—such as vascular
endothelial-cadherin (CD31), specific markers for functionality, and TJ protein expres-
sion—which is a topic that will be covered in the next section. Astrocyte markers are
primarily GFAP and AQP-4. While GFAP can also be found on other glial cells in the devel-
opment of natural tissues, it is expressed only by astrocytes in a differentiated neuronal
culture [
167
]. AQP-4 is most abundantly expressed by astrocytic endfeet, but other ependy-
mal cells show low expressions as well [
168
]. Both markers can be instrumental in disease
modeling as their expression enhances or decreases in the course of pathogenic changes
or through the reactivity of astrocytes [
167
]. Pericyte identification can be challenging if
Int. J. Mol. Sci. 2023,24, 12699 13 of 40
they are isolated from tissues with vascular smooth muscle cells, but this can be neglected
in the microvasculature, as this cell type is not usually found there. The platelet-derived
growth factor receptor-
β
and neural/glial antigen-2 are accepted markers for pericyte iden-
tity [
169
]. The oligodendrocyte mature markers are oligodendrocyte transcription factors 1
and 2, or myelin-associated proteins, including myelin basic protein, 2
0
,3
0
-cyclic nucleotide-
3
0
-phosphodiesterase, as well as myelin oligodendrocyte glycoprotein (MOG) [
119
,
170
].
The latter is of special importance for the investigation of inflammatory demyelinating
diseases. Depending on their reactive state, specific microglial markers can be used. Resting
microglia express CD11b and CD45. When entering a pro-inflammatory phenotype through
external stimuli, M1 microglia express CD86 and inducible nitric oxide synthase, whereas
anti-inflammatory M2 microglia upregulate their arginase-1 and CD206 expression [
171
].
Furthermore, the development, maturation, and functionality of neural cells can be investi-
gated in 3D fabrications. For neural differentiation, nestin is often used as a progenitor and
as a neural stem cell marker [
172
]. The neuron-specific class III
β
-tubulin and its antibody
TUJ are markers for premature or undifferentiated neural cells. Major neurons can be
identified by microtubule-associated protein 2. Mature neuronal subtypes can be further
divided by the markers gamma-aminobutyric acid (GABA) and its enzymes GAD65/67 for
interneurons; tyrosine hydroxylase for dopaminergic neurons; tryptophan hydroxylase for
serotonergic neurons; and choline acetyltransferase for cholinergic neurons [119].
Assessing the cellular viability is particularly important in 3D models since nutrient
and oxygen availability is not always ensured; moreover, it is dependent on the thickness
and porosity of the matrix and the chip itself. Furthermore, extensive UV cross-linking
during fabrication is compromised with cell viability. Besides checking cell morphology
and the counting tools that are available for confocal microscopes from 3D stacked image
sequences (such as ImageJ plugin 3D Objects Counter), several reagents can be used for
viability evaluation [
173
]. Tetrazolium salts (like MTT) are reduced by living cells into
formazan dyes, which can then be quantified via visible light absorbance measurements.
Likewise, resazurin-based substances, such as Alamar Blue, can be used for estimating the
living cell population, whereby the number of living cells is estimated through reduction
reactions that produce the red fluorescent resorufin. The advantage of tetrazolium- and
resazurin-based chemicals is their simple application to the culture medium, which is
incubated for a defined time and can be measured immediately afterward. However, these
methods do not reflect the dead-cell compartments, and reductions in a 3D ECM occur only
slowly [
174
]. For the evaluation of both viable and dead cells, a mixture of Calcein-AM
(which can easily enter living cells) and ethidium homodimers (which only enter dead
or damaged cells) is the combination of choice, if it is applied with different fluorescent
tags [
119
]. Apoptosis can be evaluated in assays with markers like caspase-3 and -9, as well
as Bcl-2 and Bax, or with terminal deoxynucleotidyl transferase dUTP nick end labeling
assays. Since apoptosis is detectable in diverse pathological conditions, such as traumatic
brain injury (TBI) or ALS, assays should be also included in the in vitro modeling of these
diseases [175,176].
4.5.2. TJ Formation and Permeability
BBB permeability is most frequently tested by TEER measurements. The presuppo-
sition is that the barrier has a setup that allows for measurements on the basolateral and
apical side. As such, an electrical field is created at both sides of the barrier, and the resis-
tance is measured by electrodes. The higher the resistance, the stronger the barrier function
and the more tightly the cell junctions are connected. The advantage of this method is the
non-invasive and rapid manner in which the barrier can be assessed.
TEER =R∗A
where Rdepicts the electrical resistance in
Ω
(which is equal to the measured resistance
minus the resistance of the blank) and Adescribes the growth area in cm
2
. The average
physiological electrical resistance in vivo is around 1800
Ω
cm
2
, but this can reach values as
Int. J. Mol. Sci. 2023,24, 12699 14 of 40
high as 8000
Ω
cm
2
[
177
,
178
]. However, only very few current in vitro platforms can reach
values as high as this [
159
,
179
,
180
]. Although TEER measurement is the gold standard
for permeability assessments in transwell and microfluidic model systems, the technique
lacks consistency in the results. A critical review from Vigh et al. [
181
] highlights the
range of values that depend on the device used for measurements. The authors stated
that the correct interpretation of values, as well as the comparison between the models,
is only possible with an explicit description of the technical parameters and the setup.
Palma-Florez et al. [
182
] tried to address this limitation by developing a BBB microfluidic
chip with an integrated micro-TEER device that was in a close proximity to the barrier. To
improve TEER, certain substances can be added. For instance, synthetic glucocorticoids
like dexamethasone or hydrocortisone (cortisol) have been found to increase TEER values
in vitro. However, they may impact experimental results, particularly in studies that are
related to inflammation as glucocorticoids have anti-inflammatory properties [183,184].
Permeability can also be assessed by specific markers that are non-toxic and do not
bind, or through those that become internalized by the cells within the model. Furthermore,
they should be metabolically inert, available in different molecular sizes, quantifiable,
and reliable [
12
]. Detailed descriptions of how permeability needs to be calculated in 2D
and 3D setups were published by, for example, Hajal et al. [
185
] and Wong et al. [
186
].
Kadry et al. [
12
] provided an overview of the different reagents that can be used to test
permeability within the context of various approaches.Since none of the substances that
are currently available fulfill all the aforementioned requirements, the choice of marker
needs to be made based on the specific research question. Both small (for examining small
changes) and large molecules (to assess BBB integrity loss) can be used as markers for
BBB dysfunction. Since none of the substances currently available fulfill all the necessary
requirements, the choice of marker for BBB dysfunction must be made based on the specific
research question. Small molecules are suitable for studying small changes, while large
molecules can be utilized to evaluate BBB integrity loss. As such, a combination of different
markers is the most reliable approach through which to evaluate BBB integrity
[12,187].
TJ
proteins can either be directly evaluated via immunofluorescence, qPCR, or via Western
blotting. The most commonly accepted markers for the BBB are claudin-5, ZO-1, and oc-
cludin. Additionally, the markers for efflux transporters, such as P-gp, BCRP, Mrp, or solute
carriers can be utilized to ensure BMEC functionality and polarity [
187
]. A recent study
by Nakayama-Kitamura et al. [
123
] set up evaluation parameters for the in vitro human
BBB likeness that is needed for drug development. The parameters comprised TJ markers
(claudin-5 and ZO-1), TEER, the endothelial cell marker CD31, transporters (P-gp, glucose
transporter protein-1, and BCRP), and receptor-mediated transcytosis (transferrin receptor).
Additionally, they checked the permeability of caffeine (which is usually BBB-permeable)
and Lucifer Yellow (which is impermeable if the BBB is functional).
5. Disease-Specific Modeling of Neuroinflammation
The following section will discuss the various conditions that involve neuroinflamma-
tion. However, since there are more than 600 neurological diseases known today, it will
only cover a fraction of the most prominent examples that come with BBB impairment [
188
].
Key features of the discussed neurological diseases are summarized in Table 1.
Int. J. Mol. Sci. 2023,24, 12699 15 of 40
Table 1. Key features of the neurological diseases with BBB impairment.
Disease Key Features References
CNS Autoimmune Diseases
• Complex interplay of genetic and environmental factors
• Disease onset and severity are driven by triggers, such as infections or tumors
• Autoantibodies and reactive T cells
[189,190]
Multiple Sclerosis
•
Chronic inflammatory demyelinating disease with autoimmune aspects. Th1 and Th17 CD4+ T cells might be autoreactive,
thus producing interferon-
γ
(by Th1 cells), IL-17, and IL-22 (by Th17 cells), which dysregulate TJ proteins and upregulate
adhesion molecules
• Human leukocyte antigen gene polymorphisms and viral infections (Epstein-Barr Virus)
•
Inflammatory processes are provoked by neurotoxic astrocytes and microglia, which attract immune cells and lead to
progressive or relapsing axonal demyelination with oligodendrocyte apoptosis, astrogliosis, axonal loss, and secondary
neurodegeneration
[16,191–193]
NMDARE • NMDA receptor autoantibodies lead to an internalization of the receptor
• Autoimmune Encephalitis with psychiatric symptoms, autonomic fluctuations, and seizures [190,194]
NMOSD
•
The majority of patients are seropositive for the antibodies that are against AQP-4 on the endfeet of astrocytes that induce
complement-dependent cytotoxicity
• Association with acute optic neuritis, myelitis, area postrema syndrome, and distinct MRI lesions
[189,195,196]
MOGAD
•
Patients have autoantibodies against MOG on the oligodendrocytes that can activate FcR-mediated antibody-dependent
cellular cytotoxicity and which complement-dependent cytotoxicity
•
Association with acute disseminated encephalomyelitis, optic neuritis, transverse myelitis, brain stem syndrome, and encephalitis
[189,197]
CNS Infections
Bacterial Infections
•
The endotoxin LPS of gram-negative and lipoteichoic acid from gram-positive bacteria leads to BBB disruption via toll-like
receptor-4, thereby inducing pro-inflammatory cytokines for the innate immune response
• The endotoxin hypothesis suggests that gut-bacterial endotoxin causes/contributes to neurodegeneration
[198,199]
Viral Infections
• Entry via transcytosis, and the infection of BMECs or CNS-invading immune cells occurs as a Trojan horse
• HIV-1, SARS-CoV-2, and others reduce TJs and upregulate adhesion molecules/cytokines
• Can cause/trigger other neurological disorders, such as dementia, epilepsy, autoimmune diseases, etc.
[200,201]
Int. J. Mol. Sci. 2023,24, 12699 16 of 40
Table 1. Cont.
Disease Key Features References
Acute CNS Injuries
Stroke
•
Ischemia (in 85% of cases) with nutrient and oxygen deprivation in the hyperacute phase (<6 h), whereby the TJ opening
occurs due to hypoxia-induced ROS and transcription factors (hypoxia-inducible factor-1
α
). This is followed by cytotoxic
(ionic) edema due to the cell swelling that occurs because of ion and water homeostasis imbalance
•
A refractory period with increasing BBB openings (hours or days) leads to plasma protein entry in the CNS, possibly resulting
in a vasogenic edema by fluid increase due to BBB openings and the extravasation of plasma proteins
• Potential hemorrhagic conversion with a loss of microvessel integrity and the entry of blood components into the CNS
[47,81,202–204]
Traumatic Brain Injury
•
Inflammation by microglia and astrocyte activation (via albumin/DAMPs) and by neutrophilic infiltration; chronic inflamma-
tion might be linked to enhanced white matter-/neurodegeneration and encephalopathy
•
Elevated neuronal stimulation and toxicity through the overproduction of glutamate receptors and GABA-receptor internal-
ization
•
Further BBB breakdown and dysregulated metabolism by ROS and other pro-inflammatory molecules, as well as in the
sub-acute stages of TBI that is enhanced by NOX and activated by MMPs
[75,203,205,206]
Epilepsy
• Recurrent seizures whose frequency has been shown to correlate positively with BBB breakdown
•
Higher K+ influx to the CNS due to leukocyte–BMEC interactions. Astrocytes and microglia produce pro-inflammatory
mediators that lower TJ protein expression and BBB tightness. The extravasation of serum proteins and immune cells augment
the inflammatory responses
•
The downregulation of the K+ channel subunit 4.1 (via albumin binding to TGF-
β
receptor) and excitatory amino acid
transporter-2 (via TNF-α) in astrocytes cause an increase in the K+ and glutamate that enhance neuronal hyperactivity
[203,207]
CNS Tumors
Primary Tumors • Glioblastoma multiforme (from astrocytes) is the most aggressive one, but does not metastasize
• Cancer cells release exosomes with VEGF-A and pro-inflammatory cytokines, which lead to a TJ decrease [12,208]
Brain Metastases • A total of 30% of brain cancers, often derived from lung cancers, breast cancers, or melanomas
•
Microvessels have heterogeneous permeability with their active efflux of molecules, enhanced angiogenesis, and inflammation
[152,203]
Int. J. Mol. Sci. 2023,24, 12699 17 of 40
Table 1. Cont.
Disease Key Features References
Neurodegeneration
Alzheimer’s Disease
•
The most common cause of progressive cognitive impairment worldwide (increasing with the rising percentage of aged
people) that has genetic and environmental risk factors
• Genetic forms (in total 1%) are caused by mutations in the genes APP,PSEN1, or PSEN2
• Neuroinflammation and degeneration, Aβplaques, and neurofibrillary tangles (hyperphosphorylated tau)
[16,209]
Amyotrophic Lateral Sclerosis
• Muscle weakness and atrophy with a progressive decrease in motor functions through motor neuron degeneration
•
The 43 insoluble TAR-DNA-binding protein aggregations in the soma of motor neurons exhibit neurotoxic properties. This
also occurs in other misfolded protein aggregates (with mutations in SOD1 or C9orf72) in familial forms (10%)
• Glutamate-mediated excitotoxicity, increased oxidative stress, mitochondrial dysfunction, and neuroinflammation
[210–212]
Parkinson’s Disease
• Age-associated neurodegenerative disease with dopaminergic neuronal loss in the substantia nigra
• Neuroinflammation mediated by Lewy body depositions (the aggregations of α-synuclein oligomers or fibrils)
• Genetic factors, environmental toxins, and mitochondrial dysfunction play a role
[213–216]
Huntington’s Disease
• Glutamine (CAG) repeat expansion mutations in the HTT gene
• Affected areas are the basal ganglia (i.e., the dorsal striatum and cortical compartments)
• Reactive microglia and astrocytes play major roles in neuroinflammation generation
[108,217–219]
Abbreviations:
A
β
, amyloid-beta; BBB, blood–brain barrier; CNS, central nervous system; DAMPs, damage-associated molecular patterns; GABA, gamma-aminobutyric acid; HIV-1,
human immunodeficiency virus-1; IL, interleukin; LPS, lipopolysaccharide; MOG, myelin oligodendrocyte glycoprotein; MOGAD, MOG-associated antibody disease; NMDARE,
N-methyl-D-aspartate receptor encephalitis; NMOSD, neuromyelitis optica spectrum disorders; NOX, nicotinamide adenine dinucleotide phosphate oxidase; ROS, reactive oxygen
species; TGF-
β
, transforming growth factor-beta; Th, T helper; TJ, tight junction; TNF-
α
, tumor necrosis factor-alpha; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2;
and VEGF-A, vascular endothelial growth factor-A.
Int. J. Mol. Sci. 2023,24, 12699 18 of 40
5.1. Neurological Autoimmune Diseases
BBB dysfunction is an early event in MS, which is followed by immune cell infiltration
and the extravasation of plasma proteins. How the BBB contributes to MS pathogenesis and
progression is discussed elsewhere [
46
,
220
–
223
]. Nishihara et al. [
224
] showed that different
Th subsets had comparable migration rates through the BBB in a transwell setup under
inflammatory conditions (mediated by TNF-
α
and interferon-
γ
). However, it was observed
that the Th1 cells preferentially migrated through the barrier in non-inflammatory condi-
tions. The authors also investigated the movement through the blood-cerebrospinal fluid
barrier and saw a 10- to 20-fold higher migration compared to the BBB model, especially
regarding Th17 cells. Based on these findings different Th cell subsets might use different
anatomical routes to enter the CNS. The Wnt/
β
-catenin pathway is involved in numerous
crucial regulations of embryonic development and adult tissue homeostasis. Among other
functions, it is important for maintaining the BBB’s integrity and restoring it in disease,
including in the case of neurodegenerative diseases like AD, PD, and ALS [
225
,
226
]. In MS,
in vivo and in vitro studies have revealed that abnormalities in Wnt/
β
-catenin signaling
contribute to re-myelination failure. OPCs cannot detach from the vasculature and are
unable to migrate to the demyelinated region. Furthermore, un-detached OPCs lead to
perivascular clustering and the secretion of Wif1, which reduces Wnt and TJ integrity [
227
].
Derada Troletti et al. [
228
] showed that endothelial to mesenchymal transition, which
they provoked by TGF-
β
and IL-1
β
additions, mediated the inflammation-induced BMEC
dysfunction, and they found that this might also play a role in MS pathophysiology. The
main transcription factor of endothelial de-differentiation, ETS1, has also been shown to be
associated with BBB breakdown [
229
]. Cerutti et al. [
230
] developed an in vitro technique
for studying the interaction between BMECs with human leukocytes in a microfluidic
model, which was coupled with live-cell imaging that could also be advantageous for
leukocyte extravasation into the CNS in MS in vitro studies. It appears to be impossible
to create a universal in vitro model for neuroinflammation in MS due to the reliance of
adaptive and innate immune cells on the disease’s development, as well as due to the
specific region of the brain that is affected [192].
For drug development, BBB in vitro models are urgently needed to investigate the
effects of treatments for retained integrity or barrier restoration. In the CNS, the differ-
ent pathogenic autoantibodies have different modes of action. Without proper treatment,
CNS autoimmunity can have severe effects in patients, leading to cognitive impairment,
seizures, or even comas in NMDA-receptor encephalitis (NMDARE), or MS-like relapses
in AQP-4 antibody-positive neuromyelitis optica spectrum disorders (NMOSD). Acute
relapses are typically treated with general immunosuppression via the use of corticos-
teroids. Although corticosteroids decrease the permeability of the BBB and the produc-
tion of pro-inflammatory cytokines, chemokines, and cell adhesion molecules, they can
also have severe side effects. Therefore, more targeted therapeutics are needed [
231
].
Takeshita et al. [
232
] developed a tri-culture transwell model, whereby they applied the
antibodies from NMOSD patients who were with or without the IL-6 receptor blocking
therapeutic antibody satralizumab. The addition of satralizumab led to higher TEER val-
ues and decreased the transmigration of peripheral blood mononuclear cells through the
barrier. When studying the recombinant antibodies from NMOSD patients’ cerebrospinal
fluid, Shimizu et al. [
233
] observed a strong binding in one of their antibodies against
glucose-regulated protein 78, which is an endoplasmic reticulum chaperone that has also
been found on the surface of BMECs in vitro. Therefore, the hypothesis that arose was that
other non-disease-defining autoantibody targets are relevant for the BBB breakdown in
these diseases, and that they could either be possible therapeutic targets or even be em-
ployed for the enhanced accessibility of immunotherapeutics into the brain. To investigate
this hypothesis, Li et al. [
194
] analyzed the human monoclonal antibodies from patients
with NMDARE and GABA
B
-receptor encephalitis for non-disease-defining antibody target
identification. Antibodies that showed vascular binding in mouse brain sections were
applied to a BMEC monoculture transwell system to evaluate if there were effects on
Int. J. Mol. Sci. 2023,24, 12699 19 of 40
their microvasculature.One of their monoclonal antibodies decreased TEER and occludin
expression, which was also confirmed in vivo. Finally, myosin-X was identified as a novel
target epitope.
5.2. CNS Infections
Many bacterial species are able to infect the CNS with intact, but also especially im-
paired, BBBs via diverse mechanisms [
234
]. Brown et al. [
235
] investigated, in their 3D
microfluidic NVU dual chamber model, the impact of inflammatory stimulation on the
BBB, and this was induced by LPS (mimicking a systemic bacterial infection) or via pro-
inflammatory cytokines (which could result in a local or systemic inflammation). By apply-
ing either 100 µg/mL of LPS or a 100 ng/mL cytokine mix (of IL-1, TNF-α, and monocyte
chemoattractant protein-1,2) to the vascular compartment, inflammation was induced,
and the metabolic profile on both sides was investigated over 24 h of exposure. An activa-
tion of pro-inflammatory cytokines was found in both vascular and brain compartments,
whereas, at later time points, this activation was only seen in a subset in both compartments.
This could suggest the activation of anti-inflammatory cytokines at later time points within
the brain section, whereas the vascular compartment remained as more pro-inflammatory.
The bacterial infection of the CNS that causes bacterial meningitis has been modeled with
different pathogens, and this was achieved with several BMEC cell lines in the transwell
models [
236
]. Streptococcus pneumoniae (among others) was shown to upregulate VEGF via
hypoxia-inducible factor-1αinduction.
Consequently, BBB permeability was elevated, thus enabling more bacterial transmi-
grations into the CNS [
237
]. VEGF secretion was also observed by Caporarello et al. [
238
]
when infecting their BMEC-pericyte co-culture BBB model with Haemophilus influenzae type
a. The authors demonstrated that different adenosine receptors on both cell types were
activated upon infection and that VEGF, was released by the cells, which can cause pericyte
detachment, BMEC proliferation, and BBB breakdown. More recently, the impact of the gut
microbiome on the CNS and BBB has been extensively investigated and reviewed [
239
–
242
].
In line with the “endotoxin hypothesis,” developing multi-organs-on-a-chip platforms
could assist in filling in the knowledge gap concerning how the microbiome may contribute
to neurodegeneration [243–245].
Besides bacteria, fungi can penetrate the BBB, with Cryptococcus neoformans being the
most common one to cause meningitis. A promising NVU-on-a-chip was established by
Kim et al. [
246
], which was used to analyze the penetration of the BBB by Cryptococcus
neoformans. The pump-free model comprised a unilateral medium flow, human neural
stem cells, BMECs, and pericytes. The authors observed an elevation of inflammatory and
angiogenesis-related cytokines, such as IL-8 and thrombospondin-1, but there was no BBB
impairment. Therefore, a transcytosis-mediated entry of the pathogen into the CNS was
proposed, and the authors concluded that including additional tissues in the model to
create a multi-organ-on-chip model would be beneficial. This could help in examining the
gut–brain axis and in studying fungal penetration from the gut through the BBB, as well as
in verifying its neurotropism.
Over the last few years, the COVID-19 epidemic has led to comprehensive research on
severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Since neurological
symptoms were reported early on during the acute phase, but also in long-term COVID-19,
the transmigration through the BBB and the effects of a severe viral infection of the CNS
were extensively studied. Bipolar neuron-to-neuron spread in the olfactory epithelium,
transport via the vagal nerve to the brain stem, or transmigration through the blood-
cerebrospinal fluid barrier or BBB (directly or on leukocytes) were all under discussion
as entry routes for SARS-CoV-2 [
247
]. Kase et al. [
248
] observed that the pseudo-typed
lentivirus particles of major SARS-CoV-2 strains were able to infect microglia, whereas they
rarely infected other CNS cell types such as iPSC-derived neurons and astrocytes. Another
study by Andrews et al. [
249
] produced contradicting results in their stem-cell-derived
organoids, with astrocytes being the major target of SARS-CoV-2. Rhea et al. [
250
] tested the
Int. J. Mol. Sci. 2023,24, 12699 20 of 40
radiolabeled monomeric SARS-CoV-2 spike protein subunit S1 on its ability to cross the BBB
in vivo and in vitro. The authors suggested an adsorptive transcytotic manner with the par-
ticipation of angiotensin-converting enzyme 2, which is the main endogenous receptor of
the virus, in a murine brain and lung. Moreover, they noticed a more effective uptake of S1
in all brain regions when it was taken up across the BBB compared to the nasal route. This
uptake of S1 was only slightly affected by LPS-induced inflammation, whereas LPS inflam-
mation altered the S1 clearance from blood and its uptake by peripheral tissues. However,
these transwell experiments with human iBMECs showed a limited permeability for S1,
probably due to technical reasons. Buzhdygan et al. [
251
] used a hydrogel-based BBB model,
which they exposed to the SARS-CoV-2 spike protein subunits S1 and S2. Although the
spike protein induced a BBB integrity loss, it did not affect BMEC viability; rather, it en-
hanced the vascular cell/intercellular adhesion molecule-1, the pro-inflammatory response
(i.e., IL-1
β
, IL-6, C-X-C motif ligand-10, and CCL5), and MMP (especially MMP-3/12)
secretion. A study by DeOre et al. [
252
], who employed a SARS-CoV-2 spike protein in a
hydrogel-based microfluidic BBB model, revealed that the angiotensin-converting enzyme
2 expression was altered by the addition of the S1 spike protein subunit when it was paired
with fluid shear stress. Furthermore, RhoA was identified as a major regulator of the
BBB breakdown through the spike protein. Zhang et al. [
253
] found an impairment of
the BM but not TJs in their co-culture BBB transwell model with BMECs and astrocytes
after SARS-CoV-2 infection. The virus was able to replicate in the BMECs; furthermore, it
passed the barrier, probably via transcytosis, and degraded the BM (by upregulated MMP-
9). Manosso et al. [
254
] investigated the microbiota–gut–brain communication during
SARS-CoV-2 infection and suggested that the SARS-CoV-2-induced cytokine storm leads to
microglial activation, astrocyte reactivity, and neuronal degeneration, which then promote
the development of psychiatric and neurological symptoms. In a study by Ju et al. [
255
],
it was suggested that the envelope protein is responsible for the breakdown of the BBB
during SARS-CoV-2 infection. The transwell model used showed a decrease in cell viability
and an increase in inflammatory mediators (major histocompatibility complex-I, IL-1
β
,
and—particularly—IL-6) when the envelope protein was added. Furthermore, the ZO-1
mRNA levels were decreased after SARS-CoV-2 envelope protein administration.
Human immunodeficiency viruses (HIVs), different flaviviruses, and new-world al-
phaviruses have also been studied for BBB penetration and CNS infection in vitro [
201
,
256
].
Several studies showed the ability of the Zika virus and other flaviviruses in terms of
infecting and activating BMECs [
257
–
262
]. Moreover, the virus crossed the BBB via tran-
scytosis without significantly increasing permeability [
258
–
260
]. However, controversial
results from other studies have suggested that TJ breaks down in a virus strain-dependent
manner, thereby enabling for a CNS entry via a paracellular route [263]. For HIV-1, it was
demonstrated that TJ proteins are disrupted upon infection [
201
]. Exposure to a HIV-1
transactivator of transcription proteins led to ZO-1 downregulation, which was mediated
by BMEC autophagy induction in vitro [
264
,
265
]. Furthermore, the transactivator of tran-
scription was able to cross the BBB in a bidirectional manner [
266
]. Investigating the impact
of this protein on the BBB is of special importance since it affects most cell types in the CNS,
thereby contributing to neurotoxicity in HIV-1 associated neurocognitive disorders [
267
].
A recent review of Swingler et al. [
268
] highlights the applicability of the iPSC-derived
organoids of different brain regions for various neurotropic viruses. By organoid modeling,
the molecular regulation of neurotrophic viral infections, as well as cellular responses
become more accessible. However, the known limitations of organoids, such as size and
necrotic center formation, apply here as well.
5.3. Acute CNS Injuries
5.3.1. Stroke
Functional and structural BBB alterations have been observed in vivo and in vitro in
several stages of stroke. Due to the vast knowledge about the impact on the consequences
of BBB breakdown in ischemic stroke, the current BBB in vitro models focus on replicating
Int. J. Mol. Sci. 2023,24, 12699 21 of 40
neuroinflammation through oxygen-glucose deprivation (OGD). The goal of these models
is to restore the tightness of the BBB or to prevent its impairment, with the ultimate aim of
facilitating drug development [
269
–
273
]. Cell death due to ischemia and peripheral immune
cells causes more ROS, miRNAs, and damage-associated molecular patterns (DAMPs) to
be generated, which activate glial cells. Microglial and astrocyte activation lead to pro-
inflammatory mediator release (especially TNF-
α
and IL-1
β
), as well as the upregulation of
pro-inflammatory genes in BMECs [
12
]. MMPs, which are primarily secreted by neutrophils,
further digest TJ proteins [
274
]. Fattakhov et al. [
269
] and Kadir et al. [
270
] provided
protocols for the establishment of triple-culture models in transwell setups. Lyu et al. [
271
]
evaluated the restorative potential of stem cell therapies with their microfluidic NVU-on-a-
chip. After the functional response to OGD, the authors tracked the infiltration of candidate
stem cells through the BBB and observed that different types of stem cells exerted unique
neurorestorative effects on the structural and functional integrity of the NVU rather than
on the direct replacement of the neurons. A 3D microfluidic model by Cho et al. [
147
]
with rat brain endothelial cells was developed to screen BBB-targeting drugs. The authors
induced ischemic conditions by OGD, as well as showed the protective functions of the
antioxidant edaravone and Rho kinase-inhibitor Y-27632. Wevers et al. [
273
] modeled stroke
by introducing hypoglycemic conditions in a glucose-free medium, hypoxia by 10
µ
M of
antimycin-A (an inhibitor of complex III of the electron transport chain), and disrupted the
perfusion by moving the chips from a rocking platform to static conditions in the incubator.
Due to the relatively high-throughput BBB chip, with 40 chips in parallel, this model was
proposed as a tool for the drug screening of anti-inflammatory and free radical scavengers.
Similar drugs were utilized in the NVU organoid model of Nzou et al. [
272
] when they
were investigating their neuroprotective impacts. After inducing hypoxia by exposing the
organoids to 0.1% O
2
for 24 h, BBB breakdown, as well as pro-inflammatory cytokine and
ROS production was observed.
5.3.2. Traumatic Brain Injury
BBB impairment has been observed in all stages of traumatic brain injury (TBI), rang-
ing from mild to severe. The disruption of the BBB occurs within the first hours after TBI
due to neuroinflammation, and this may remain for years [
275
,
276
]. Despite decades of
investigations through using in vivo models, there is no efficient neuroprotective treatment
for TBI that has yet passed clinical trials. This is partly caused by the incomplete knowledge
about molecular mechanisms in the complex pathophysiology of TBI. Therefore, in vitro
models could be helpful in investigating the underlying structural and functional alter-
ations at the cellular level. Different methods have been employed to induce neurotrauma
in vitro since TBI itself can also result from different impacts, such as blunt-force, blast,
or compression [
277
]. These injury models are induced statically or dynamically by me-
chanical or chemical forces. Static mechanical injuries are impact-based methods such
as weight drop. Dynamic methods include microfluidic compartmentalization or scratch
assays, as well as stretch-induced traumas that are caused by culture monolayer or axonal
stretching. Chemical injuries are used for the microenvironment investigations of post-TBI
tissues, and these include altering culture conditions with nutrient or oxygen deprivation,
or treatments with chemicals [
278
]. Schlotterose et al. [
279
] developed a 3D-printed device
for standard and non-standard cell culture applications to induce hydrostatic pressure
on cells, resulting in their perturbation. As such, they were able to provoke TBI-typical
hallmarks, such as cell death, decreased neuronal functionality, neural axon swelling, and a
decrease in BBB tightness. Uni- and biaxial cell stretch models were set up by Rosas-
Hernandez et al. [
280
,
281
] to assess cell viability and impact on the BBB after mild TBI.
The model was based on rat BMECs on a silicone membrane, which was stretched utilizing
a custom-made stretching device. They observed a deformation-dependent increase in
cell death and apoptosis after high magnitudes of stretch, whereas the metabolic activity
in the stretched BMECs was already decreased after a lower magnitude of deformation.
Interestingly, low-magnitude stretching enhanced TJ protein expression, indicating its po-
Int. J. Mol. Sci. 2023,24, 12699 22 of 40
tentially protective role in BBB integrity. However, the authors also argued that experiments
should be repeated with human BMECs since their results were shown to be contrary to the
results of other studies that used mouse BMECs instead, thus indicating species-to-species
differences. Salvador et al. [
282
] provided a protocol through which to perform a stretch-
and OGD-induced in vitro model that could be used to mimic the impact of TBI on mouse
BMECs. This approach can also be performed with human BMECs so as to serve a more
physiologically relevant model. A systemic review by Wu et al. [
283
] summarized the
current, at the time, in vitro models for TBI up until 2021. Only a very small proportion
included investigations of the BBB alterations during trauma. Therefore, future studies are
needed to reveal the impact of BBB dysregulation in the different forms of TBI. Furthermore,
other cell types than solely BMECs, astrocytes, or neurons should be included in the setups.
For example, pericytes were shown to rapidly detach from BMECs after TBI in a mouse
model, but they were significantly enhanced again after 5 days, which indicates a biphasic
pericyte regulation in acute TBI [284].
5.3.3. Epilepsy
For decades, it has been discussed if BBB leakage is the cause or consequence for
seizure development and aggravation [
203
,
285
]. A major problem in the treatment of
epilepsy is the development of drug resistance in a third of patients. This treatment
failure could be caused by an excessive upregulation of efflux transporters, such as P-gp or
the molecular target that are changes caused by anti-epileptic drugs themselves [
47
,
286
].
To study the interactions of epileptic tissue and BBB, several in vitro models have been
developed. However, although being labeled as in vitro models, many of these models have
been of organotypic brain slices or whole brains that were isolated from rodents [
287
–
289
].
Seizure-like events can be induced by low magnesium concentrations, multiple doses of
kainic acid, or 4-aminopyridine (a Ca
2+
channel blocker) [
290
]. To achieve personalized
drug development for patients, the organotypic cultures of tissue obtained from epileptic
patients can be isolated and used in co-culture contact or in non-contact transwell models
along with BMECs, as well as in brain tissues that exhibit seizure-like activities [
291
].
This allows one to display TJ and adhesion protein expressions in the monolayer, as well
as to investigate the bidirectional interactions of BMECs with epileptic tissue, especially
after the application of different anti-epileptic drugs [
286
]. So far, there are no in vitro
models of epilepsy that incorporate more BMECs and astrocytes/isolated brain tissue [
200
].
Furthermore, working with isolated human brain tissue is limited due to high complexity
and low availability [
292
]. Yamanaka et al. [
293
] reviewed the neuroinflammatory role
of pericytes and their important impact in epilepsy pathogenesis. Pericytes undergo
redistribution and remodeling during epileptic events, thereby causing BBB alterations due
to responses to pro-inflammatory cytokines, as well as pericyte-glial scarring at permeable
microvessels. Therefore, including pericytes in epilepsy in vitro co-culture models would
possibly provide more physiologically reliable results for human drug discovery [
122
,
294
].
5.4. CNS Tumors
Brain cancers display significant heterogeneity, arising either from the CNS itself or
due to spreading from the periphery as metastases through a permeable BBB. The BBB ex-
periences several alterations during tumor cell invasion. This is also often referred to as the
blood–brain tumor barrier, as microvessels have highly heterogeneous permeability follow-
ing the active efflux of molecules, enhanced angiogenesis, and inflammation when they are
combined with less blood flow due to tumor growth. The highest permeability is frequently
found at lesion cores [
200
]. Novel-formed microvessels within the tumor tissue exhibit
neither sufficient intercellular junctions nor transport systems [
47
]. With tumor progression,
astrocytes lose their connection to the vasculature and interact with tumor cells, thereby
regulating proliferation, immune cell invasion, and drug responses [
295
]. Furthermore,
edemas due to AQP-4 upregulation is regularly observed in brain tumors [
12
]. Otherwise,
BBB permeability in brain tumors is a two-edged sword. Although the barrier is leaky,
Int. J. Mol. Sci. 2023,24, 12699 23 of 40
chemotherapeutics either cannot enter, because the breakdown of the BBB is unequally
distributed, or they are removed again from the CNS by active efflux mechanisms [
296
].
Researchers have already attempted to exploit neuroinflammation in order to open the
BBB. Blethen et al. [
296
] provide an overview of methods, such as low-intensity-focused
ultrasound, which can be employed to modulate the blood–brain tumor barrier in order to
enhance drug uptake. These methods induce a secondary inflammation process that leads
to the transient intercellular opening of BMECs and is mediated by the elevated release of
DAMPs, TNF-
α
, IL-8, and heat shock proteins into the brain parenchyma. Although this
approach was already proven to be successful in some preclinical studies, there is more
research needed for the fine-tuning of low-intensity-focused ultrasound. Tumor modeling
benefits greatly from 3D in vitro modeling as the tumor microenvironment can be mimicked
for chemotherapeutic screening and metastasis studies. Experimental setups include multi-
ple cell types in 3D scaffolds, thereby adding tumor spheroids in the brain parenchyma
or vessel compartment [
297
–
300
]. Seo et al. [
301
] established a glioblastoma multiforme
spheroid, which they placed in a vascularized 3D hydrogel to study chemosensitivity and
the drug delivery associated with the BBB in this tumor. They added TNF-
α
and saw
enhanced BBB permeability and monocytic THP-1 cell adhesion to BMECs due to higher
adhesion molecule expression. The authors also highlighted the importance of other NVU
cell types, such as astrocytes for BBB integrity, but also their prohibitive role in cancer metas-
tasis. A recent study by Zhang et al. [
302
] suggested the enhanced adhesion of circulating
breast cancer cells with metastasis potential to the brain endothelium. The microfluidic
model included varying shear stress, selecting those cells with the highest adhesion to
BMECs. As such, the authors observed not only elevated brain metastasis gene expressions
in circulating breast cancer cells, but also increased transmigrations through the BBB in
a transwell model. Moreover, their adhesion and proliferation within polyacrylamide
gels (0.6 kPa) (which were coated with collagen I) was better than the one of wild-type
cancer cells, which the research group associated with a better survival in the soft brain
microenvironment. Finally, immunocompetent glioblastoma organoids could bring about
a breakthrough in personalized medicine when co-cultured with patient blood/tumor-
derived immune cells for the development of immunotherapies [
303
]. One patient-specific
glioblastoma model was established by Cui et al. [
304
], who aimed to optimize patient-
specific responses to programmed cell death protein-1 checkpoint immunotherapy by
dissecting the immunosuppressive tumor microenvironment heterogeneity.
5.5. Neurodegeneration
5.5.1. Alzheimer’s Disease
Independently of whether it is the cause or consequence, the involvement of BBB
disruption in AD has been proven to a large extent [
305
]. Evidence for the BBB breakdown
in AD patients also comes from plasma proteins like fibrin(ogen), thrombin, albumin, and
the immunoglobulins that are found co-localized with A
β
plaques [
16
,
306
–
308
]. In AD, glial
cells are primarily engaged in neuroinflammation. Studies have shown the activated astro-
cytes and microglia around plaques, which release pro-inflammatory cytokines and trigger
further inflammatory processes. Astrocyte alterations besides astrogliosis in AD comprise
an impairment in glucose metabolism, a decreased expression of glutamate transporters,
and an imbalance in potassium [
309
]. A recent small nuclear RNA study by Xu et al. [
310
] of
human postmortem brains revealed the transcriptomic changes in astrocytes and microglia
in pathogenic conditions. Although astrocytic changes had common features in AD and
PD, the regional differences were provocative toward one of the pathologies. In contrast,
the microglia showed unique disorder gene transcriptomic patterns. In vitro BBB models
of AD incorporate inflammatory regulation by adding pro-inflammatory cytokines and A
β
.
Spampinato et al. [
305
] investigated the peripheral blood mononuclear cell transmigration
across the barrier under A
β
exposure and pro-inflammatory conditions, as well as provided
a protocol for a transwell model setup. Schreiner et al. [
311
] summarized the current AD
BBB in vitro models in great detail.
Int. J. Mol. Sci. 2023,24, 12699 24 of 40
5.5.2. Amyotrophic Lateral Sclerosis
Blood-derived components like the thrombin, immunoglobulin G, or hemoglobin that
have been found in ALS patient postmortem brains indicate BBB breakdown. Furthermore,
a decrease in TJ protein expression, astrogliosis with detached astrocyte endfeet, and severe
pericyte degradation have also been observed [
16
,
312
]. In ALS progression, adaptive
immunity may contribute to the disease severity, and recent studies have highlighted the
CNS infiltration of T cells, as well as an overall shift toward pro-inflammatory T cell subsets
and reduced CD4+ T cells [
313
]. For example, CD4+ Tregs are reduced during disease
progression. Their suppressive capability is also diminished in vitro by isolating patient T
cells. Interestingly, removing Tregs from their environment could restore this characteristic
feature, thererby displaying a possible therapeutic target for the autologous transplantation
of T cells [
314
]. Most studies regarding BBB breakdown in ALS have been performed in
SOD1 mutant rodent models or in postmortem human tissues [
315
,
316
]. In vitro, not many
models have been established until recently. The majority of models have been of the
cell cultures of motor neuron cell lines with the abovementioned mutations, or of patient-
derived motor neurons. Therefore, the focus has been to understand cellular mechanisms
more specifically. In recent years, there has been a significant focus on using iPSCs to better
understand the causes of ALS and to develop effective treatment approaches [
317
]. A recent
review by Arjmand et al. [
318
] provides an input about how an ALS organ-on-a-chip can
be approached. Embryonic or neural stem cells or iPSCs can be derived from patient
fibroblasts. The microenvironment should involve an excess of glutamic acid to mimic the
glutamate-induced excitotoxicity.
5.5.3. Parkinson’s Disease
α
-Synuclein acts in a pro-inflammatory manner via different mechanisms in PD [
81
].
On the one hand, it binds to microglia and astrocytes via toll-like receptor-2 and -4, thus
leading to the release of inflammatory mediators [
319
,
320
]. On the other hand, monomeric
α
-synuclein was shown to induce the release of pro-inflammatory cytokines/chemokines
by pericytes in a transwell model, thus leading to reduced BMEC integrity [
321
]. En-
hanced BBB impairment was also seen in PD patients through using quantitative MRI
imaging [
322
]. However, although pre-formed
α
-synuclein fibrils lead to a downregu-
lation of TJ proteins, BBB breakdown seems to be dependent on multiple inflammatory
events since the functionality of the BBB by the addition of
α
-synuclein alone was not
altered significantly in vitro [
323
]. Therefore, including multiple cell types to model PD
is crucial for reflecting the pathology, and for performing reliable experiments. Pediadi-
takis et al. [
324
] established a “human Substantia Nigra Brain-Chip” consisting of a PDMS
chip with two perfusable microchannels and an ECM component mixture-coated PDMS
membrane in between. Thus, they introduced iPSC-derived dopaminergic neurons, human
primary astrocytes, pericytes, and microglia into one of the two channels, and iPSC-derived
BMECS on the surface of the opposite channel. Continuous exposure of
α
-synuclein fibrils
in the “brain” channel showed an accumulation of pSer129-
α
Syn fibrils, mitochondrial
impairment, and an enhanced BBB permeability after a few days.
5.5.4. Huntington’s Disease
A study by Vignone et al. [
109
] compared the iBMECs of HD patients with those of
the unaffected controls. They aimed to investigate whether changes in the transcriptomic
profiles indicated if the BMECs themselves were functionally compromised when pro-
moting BBB dysfunction. The results indicated alterations in the BBB properties, and in
functions such as receptor-mediated transcytosis. A gene expression analysis of iBMECs
by Linville et al. [
325
] showed similar results regarding TJ proteins, while they could not
observe the impaired paracellular permeability in their transwell setup. Furthermore,
the authors saw differences in their immune cell adhesion and immune activation tran-
scripts between the juvenile HD iBMECs and adult postmortem HD BMECs. Therefore,
they suggested BBB gene expression changes due to the elevated CAG repeat expansion
Int. J. Mol. Sci. 2023,24, 12699 25 of 40
during HD progression. Besides these studies using iBMEC monocultures, not many in-
vestigations have been conducted on the differentiation of other HD-relevant cell types.
However, this could be due to the difficulty of replicating differentiation protocols (for in-
stance, striatal medium spiny neurons [
326
]). There are still many tasks that need to be
completed in order to accurately model the BBB for HD. One key area that needs develop-
ment is the creation of an in vitro co-culture model that includes other CNS resident cells
to better determine the BBB phenotype.
6. Discussion
The barrier function of the BBB is strictly regulated by various cell types in the CNS.
While in the past BMECs have primarily been employed for in vitro modeling, investiga-
tions on the surrounding astrocytes, pericytes, microglia, neurons, and oligodendrocytes
have shed new light on the importance of these CNS-resident cells. In neuroinflammation,
both central and peripheral cells produce pro-inflammatory mediators that lead to BBB
impairment. Inflammation-inducing factors vary across the spectrum of neurological dis-
eases, such as the production of autoantibodies, the release of ROS and DAMPS due to
ischemia and trauma, and the influence of metastases on the microvascular efflux pumps
and permeability. While a huge variety of different models exist for some neuropathologies,
there is a devastating lack of in vitro models for others. Some disease models could bene-
fit from the already-established setups of other diseases. While monoculture transwells
may not be suitable for more complex physiological research questions or personalized
approaches, they still offer advantages in conducting basic high-throughput toxicity or
permeability investigations. Models that incorporate multiple cell types better mimic
human (patho)physiology, even more so when applied to a 3D model setup with an ECM
substitute and shear stress. The tunability of the incorporated cells can be established by
using patient-derived iPSCs to enable more personalized research regarding drug tolerance,
toxicity, and efficacy. It is worth noting that as the model becomes more complex, there are
more potential sources of error that can accumulate. To validate the model, the cell-type
identity and viability need to be confirmed. Permeability can be measured by TEER, or
via size-defined particles that can pass a leaky barrier. Appropriate barrier formation is
evaluated via TJ protein detection. For the future, a robust and reliable platform mimicking
different neurological diseases that are as close to in vivo conditions as possible is desirable
in order to reduce lab-to-lab aberrancies. Although in vitro models will never fully reflect
human physiology, they are great tools that allow for increasing reduction and replacement
of animal models.
7. Conclusions
There are several key considerations for BBB in vitro modeling that have been ad-
dressed in this review. First, considerations need to be made regarding the “why” of the
experiment. The model setup can completely differ depending on whether the transport
of substances across a BMEC monolayer is examined, or how the tumor microenviron-
ment in glioblastoma organoids changes due to different immunotherapy approaches.
Each pathology brings different characteristics and biological requirements that need to
be adapted accordingly in the BBB model. For example, the majority of patients with
NMOSD exhibit AQP-4 antibody seropositivity. To investigate the pathomechanism of
these antibodies against astrocytes, it is essential to evaluate the AQP-4 expression of these
cells before conducting experiments. Investigations of AD or PD need the presence of A
β
or
α
-synuclein fibrils, respectively, on the “brain” side. Acute CNS injuries need to be induced
before the investigations, such as OGD in ischemia models or those in the differently
provoked injuries in TBI. Additionally, when modeling neuroinflammation, it is important
to choose a method for inducing inflammation. Most models aim for stimulation with
LPS or TNF-
α
, but the addition of other pro-inflammatory substances can be considered as
well. Moreover, the cell and ECM types, as well as the general setup (simple BBB vs. full
NVU), need to be considered. For experiments on the function of the different transporters
Int. J. Mol. Sci. 2023,24, 12699 26 of 40
or receptors of BMECs, a BMEC monoculture model that shows the respective protein
expression can be sufficient. However, investigations of the different cell types’ interactions
might require more complex setups. For transwell models, the multi-well format, pore size,
and membrane material, as well as a BM substitute need to be defined. For hydrogel-based
models, the hydrogel composition and cross-linking substance need to be evaluated; in
addition, some additives might be required for enhanced cell viability. Organoids need a
cocktail of growth factors and an ECM for proper differentiation. Furthermore, the time
frame of each experimental approach may vary. Utilizing iPSCs for the model can be
time-consuming since the differentiation protocols require several weeks to complete. Then,
there is a short time window where the differentiated cells can be used for the model
before de-differentiation. Primary cells can be commercially purchased or isolated directly
(if ethically applicable), but they can only be used for a few passages, which limits the
number of attempts and cells for the model establishment. In contrast, immortalized cell
lines can be used over many passages in higher quantities. Moreover, in particular for
drug safety studies in pharmaceutical production, a high-throughput platform is essential.
Often, the quantity of the model compromises its quality or, in this case, its complexity.
Hence, high-throughput models most likely will not require time-intensive fabrication.
For some models, additional devices are needed. For the introduction of shear stress, a per-
fusion system is required. This can involve different pumping systems or an incubator-safe
rocking platform. BBB/NVU-on-a-chip models need specially fabricated chips made of
PDMS or similar materials. It should be noted that there are various printing techniques
used for 3D bioprinted models that require different devices based on the printing material
and complexity of the model. To measure the TEER, specialized TEER meters need to be
purchased. Immunofluorescence staining requires fluorescence microscopes. If experiments
are conducted in 3D setups, a laser confocal microscope can be beneficial for creating a
3D representation of the staining. Finally, the culture time is dependent on the cells used
and the experimental procedure performed. Experiments are usually started when BMECs
have been cultured long enough to develop a tight monolayer. The formation of TJs and
low permeability need to be assessed by TJ marker immunofluorescence staining, TEER
measurements, or permeability assays. The enhancement of TJs can be reached by the
addition of glucocorticoids, but this should be avoided in inflammation-related studies.
Additional incubation time might be necessary, if inflammation needs to be provoked or
if the effects of different drugs, cytokines, ROS, antibodies, or immune cells on the BBB are
being studied.
Ultimately, the selection of a suitable model setup and its complexity depend on the
specific research question at hand. As the British statistician George E. P. Box once said
(which is also applicable for biological models): “All models are wrong, but some are
useful” [327].
Funding: Supported by the intramural funding program of the Medical University Innsbruck Ph.D.
Research Training Groups, Project 2022-1-2 “CONNECT”.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
AβAmyloid-beta
AD Alzheimer’s disease
ALS Amyotrophic lateral sclerosis
AQP-4 Aquaporin-4
BBB Blood–brain barrier
Int. J. Mol. Sci. 2023,24, 12699 27 of 40
BM Basement membrane
BMEC Brain microvascular endothelial cell
CCL C-C motif ligand
CNS Central nervous system
DAMP Danger-associated molecular pattern
ECM Extracellular matrix
GABA Gamma-aminobutyric acid
HIV Human immunodeficiency virus
IL Interleukin
iBMEC Induced brain microvascular endothelial cell-like cell
iPSC Induced pluripotent stem cell
LPS Lipopolysaccharide
MMP Matrix metalloproteinase
MOG Myelin oligodendrocyte glycoprotein
MOGAD MOG-associated antibody disease
MS Multiple sclerosis
NMDARE N-methyl-D-aspartate receptor encephalitis
NMOSD Neuromyelitis optica spectrum disorders
NVU Neurovascular unit
OGD Oxygen-glucose deprivation
OPC Oligodendrocyte progenitor cell
P-gp P-glycoprotein
PD Parkinson’s disease
PDMS Polydimethylsiloxane
ROS Reactive oxygen species
SARS-CoV-2 Severe acute respiratory syndrome coronavirus type 2
TBI Traumatic brain injury
TEER Trans-endothelial electrical resistance
TGF-βTransforming growth factor-beta
Th T helper
TJ Tight junction
TNF-αTumor necrosis factor-alpha
VEGF Vascular endothelial growth factor
ZO-1 Zonula occludens-1
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