The brain is a vital organ and it has been protected from any damage by nature in several ways,
and the brain skull is the simplest one. There are many other protective membranes and fluids
present to prevent the brain from injury and toxins.
The blood-brain barrier(BBB) is another line of defense, and it was discovered in the 19 th century
by a German physician Paul Ehrich. As the name indicates it consists of a barrier between the
blood vessels and cells or tissues of the brain. Other parts of the brain like skull and meninges
protect it from physical injury while the blood-brain barrier protects the brain from toxins and
disease-causing pathogens.
Looking into the structure, the BBB is composed of specialized human brain microvascular
endothelial cells (HBMECs), perivascular cells (pericytes), and astrocytes. Neurons and
microglia also part of the maintenance of the BBB and make the neurovascular unit(NVU).
Animal model vs in-vitro cell model
Animal models reserve the natural matrix structure and efficiently able to explore the
physiological framework. But in the case of BBB, there is some discrepancy between animal and
human compatibility in course of matrix composition which makes it less efficient for human
BBB response and failed the drug clinical trial for indicative as a highly toxic and low efficient
drug.
On the other hand, in vitro cell models culture cells from a human origin in a definite
environment to mimic tissue and organ functions. Numerous human brain endothelial cells have
been effectively used to model aspects of the BBB.
Blood-brain barrier model
BB models serve as an important tool for understanding the biological and pathological
mechanisms and processes of drug transportation to the brain. Invitro animal models for BBB
has been developed using a variety of species including rat, mouse, pig and bovine. The
establishment of the human BBB model is very crucial for clear differentiation between different
species. Several kinds of cells have been used for the in vitro BBB model including Primary
human brain endothelial cells(hBECs) and immortalized human cells. But there are some issues
with the use of these cells,
1. Restrictions in the procurement of human tissues
2. loss of hBECs phenotype
3. Low transendothelial electrical resistance (TEER) in human cell line
After that induced pluripotent cells((IPSCc) were differentiated from primary human brain
endothelial cells it has been used for the BBB model but there was still some doubt underlying as
permeability and transendothelial electrical resistance was low. There were some more issues
relating to the use of iPSCs arises which may be,

? Reproducibility
? Type and history of iPSCs used to drive hBECs
? Suitability of the model
Another human in vitro BBB model based on the co-culture of cord blood-derived ECs with
astrocytes can also be used. This BBB model may present low TEER values but with relatively
high permeability.
A simple and easy human BBB model can be generated using the cord blood-derived
hematopoietic stem cells, which can be obtained non-invasively. It has an advantage over the
previous model as it is high in reproducibility and sustainability.
According to the cell types used BBB model is divided into different types, the purpose of these
in vitro cells is to reproduce BBB functionality and predict the passage of active compounds
through the barrier.
Monolayer Models
A monolayer of endothelial cells fledged in the Transwell is used as a simple in vitro BBB
model. The insert copies the blood (luminal) side, while the well in which the insert resides
mimics the parenchymal (abluminal) side.
Co-culture models
To better mimic the anatomic structure of the in vivo BBB, BMECs are cocultured with other
cell types that directly contribute to the barrier properties of BBB. Co-culture models can be
based on the following cell types
BMECs-astrocytes coculture model
BMEC–pericyte coculture model
BMEC–pericyte–astrocyte triple coculture model
Neurons and other cells have also been used with BMECs for the coculture model.

In vitro blood-brain barrier (BBB) models. 1-Brain microvascular endothelial cell (BMEC)
monolayer model. 2,3- BMEC–astrocyte coculture models.4-BMEC–pericyte–astrocyte triple-
culture model.
Limitation of Transwell model
Above mentioned methods used for mimicking the Blood-Brain Barrier (BBB) are based on the
incubation chamber(Transwell) which is separated from the filter and coated matrix to
understand and investigate the barrier permeability. But these protocols have some limitations,
1. They do not reproduce critical microenvironmental parameters, mainly anatomical size or
hemodynamic shear stress
2. They do not have real-time visualization capability
3. They require many consumables.

SynVivo BBB model
It is a cell-based physiological microfluidic platform that offers a realistic approach to the
microenvironment essential for studying the cellular behavior, drug delivery mechanism, and
anatomical studies in real-time. This microenvironment is created by emulating a histological
slice of brain tissue cells in communication with endothelial cells across the blood-brain barrier.
3D tissue and organ on chip model are used widely to recreate and mimics the natural
microenvironment investigating several parameters with side by side architecture that make it
possible for real-time visualization. Several advantages related to this model which make it
superior to other models is
? It is Accurate in in-vivo hemodynamic shear stress
? it has Real-time visualization of cellular and barrier functionality
?  low cost and time friendly
?  Robust and easy to use protocols
3D BBB model
There are several techniques and materials used for the 3D BBB model which rely on the
embedding of cells into hydrogels to induce capillary angiogenesis. BBB-on-a-chip models use
biomimetic materials to culture BMECs which may be 2D, 2.5D, 3D to expand endothelial
barrier development and maintenance. culturing of cells within a 3D matrix offers cell-matrix
interactions across the entire cell surface, cell migration in any direction, and cell-cell
interactions in 3D. 3D BBB-on-a-chip models mostly depend on the injection of hydrogels into
a central compartment surrounded by two external fluidic channels. ECs are seeded in these

external channels and to form an adhesive link with the hydrogel to induce vascularization of the
matrix. Most of the 3D BBB model uses the natural polymer-based hydrogels which include
commercially available collagen-1, and fibrin. Other synthetic materials like polyethylene
glycol(PEG) can also be used for 3D modeling.