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Review
. 2024 Mar 12;12(3):626.
doi: 10.3390/biomedicines12030626.

Co-Culture Models: Key Players in In Vitro Neurotoxicity, Neurodegeneration and BBB Modeling Studies

Ana Rita Monteiro  1   2 , Daniel José Barbosa  3   4   5 , Fernando Remião  1   2 , Renata Silva  1   2
Affiliations
Review

Co-Culture Models: Key Players in In Vitro Neurotoxicity, Neurodegeneration and BBB Modeling Studies

Ana Rita Monteiro et al. Biomedicines. .

Abstract

The biological barriers existing in the human body separate the blood circulation from the interstitial fluid in tissues. The blood-brain barrier (BBB) isolates the central nervous system from the bloodstream, presenting a dual role: the protection of the human brain against potentially toxic/harmful substances coming from the blood, while providing nutrients to the brain and removing metabolites. In terms of architectural features, the presence of junctional proteins (that restrict the paracellular transport) and the existence of efflux transporters at the BBB are the two major in vivo characteristics that increase the difficulty in creating an ideal in vitro model for drug permeability studies and neurotoxicity assessments. The purpose of this work is to provide an up-to-date literature review on the current in vitro models used for BBB studies, focusing on the characteristics, advantages, and disadvantages of both primary cultures and immortalized cell lines. An accurate analysis of the more recent and emerging techniques implemented to optimize the in vitro models is also provided, based on the need of recreating as closely as possible the BBB microenvironment. In fact, the acceptance that the BBB phenotype is much more than endothelial cells in a monolayer has led to the shift from single-cell to multicellular models. Thus, in vitro co-culture models have narrowed the gap between recreating as faithfully as possible the human BBB phenotype. This is relevant for permeability and neurotoxicity assays, and for studies related to neurodegenerative diseases. Several studies with these purposes will be also presented and discussed.

Keywords: blood–brain barrier; co-culture models; in vitro; neurodegeneration; neurotoxicity; neurovascular unit.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematic representation of the blood–brain barrier (BBB) development in the embryonic phase (A) and adult life (B). (A) The BBB development showing the interaction between immature endothelial cells, neural progenitors, pericytes, and radial glia. The angiogenesis from the perineural vascular plexus starts with migration towards the neuroepithelium. Newly forming blood vessels recruit pericytes for stabilization. In parallel, originating from neuroepithelium and using radial glial cells as a guidance structure, neural progenitor cells migrate and begin their differentiation. (B) Adult BBB representation showing a more elaborate structure. Endothelial cells, astrocytes, microglia, pericytes, and neurons constitute the neurovascular unit. Created with Biorender.com.
Figure 2
Figure 2
Schematic representation of the characteristics of the endothelial cells of the blood–brain barrier: tight junctions, adherens junctions, and influx and efflux transporters. Endothelial cells (surrounded by a pericyte) are connected by the inter-endothelial junctions. The tight junctions comprise the proteins claudin, occludin, and junctional adhesion molecule (JAM). Endothelial cells are also connected by catenins, vascular endothelial cadherin (VE-cadherin), and platelet endothelial cell adhesion molecules (PECAMs), which constitute the adherens junctions. The figure also illustrates efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs), as well as some of the main influx carriers such as the glucose transporter (GLUT-1), large amino acid transporter (LAT-1), monocarboxylate transporter (MCT-1), and organic anion transporting polypeptide (OATP-A). Created with Biorender.com.
Figure 3
Figure 3
Illustration of the transport pathways at the blood–brain barrier. (A) Passive diffusion through which lipid-soluble compounds can cross, given the large surface area and the lipophilic nature of the membrane of the endothelium; (B) Paracellular transport, which allows water-soluble compounds to cross; (C) Transcellular transport that comprises: (1) carrier-mediated transport, including transport proteins for glucose, amino acids, purine bases, and other substances; (2) receptor-mediated transport, reserved for certain proteins as transferrin and insulin; (3) adsorptive-mediated transport, allowing to albumin and other plasma proteins enter the CNS. Created with Biorender.com.
Figure 4
Figure 4
Representative scheme of the static model employing the Transwell systems. The Transwell inserts can be used for culturing brain endothelial cells, in monoculture models, for drug permeability analyses. The endothelial cells are seeded in the upper compartment, which represents the “blood side”, while the lower compartment is reserved to represent the “brain part”. On the other hand, the inserts can be used for co-culture models, either in contact or non-contact. The other cells that can be used (astrocytes, pericytes, microglia, and neurons) can improve the BBB phenotype. Created with Biorender.com.
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Grants and funding

This work was funded by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. Daniel José Barbosa is supported by an FCT Junior Researcher position (DL57/2016/CP1355/CT0007).

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