BUILDING BARRIERS: ENGINEERING A NOVEL IN VITRO MODEL OF THE BLOOD-BRAIN BARRIER

Jun 29, 2022, 12:00 PM
10m
Room: S3 A

Room: S3 A

Speaker

Schofield, Christina (Centre for the Cellular Microenvironment, University of Glasgow)

Description

"Introduction: The Blood-Brain Barrier (BBB) is a dynamic interface which regulates the movement of solutes. The physical barrier consists of endothelial cells (ECs) with extrinsic barrier properties induced by interactions with the neurovascular unit (NVU). Neither static nor dynamic in vitro BBB models fully capture in vivo-like conditions, and while coculturing EC monolayers with other NVU cell types has improved barrier properties, the complexity of these culture conditions detracts from their usefulness for high-throughput drug discovery, testing, and disease modelling [1]. The model proposed here utilises material-driven fibronectin (Fn) fibrillogensis by poly(ethyl acrylate), which is biologically compatible with many cell types including ECs, to present a EC monolayer with growth factors (GFs) in synergy with integrin binding sites on Fn [2]. Radically, we hypothesise that an effective in vitro model can be created using GFs and EC monolayer grown on a PEA-Fn coated electrospun membrane scaffold. PLLA electrospun fibres additionally provide a physiological-like scaffold in comparison to commercial polycarbonate or polyethylene-terephthalate semi-porous membranes.

Experimental Methods: Electrospun membranes are produced from poly L-lactic acid (PLLA) 8% solution in hexafluoro-2-propanol and coated with plasma-polymerized PEA (pPEA). Membranes were characterised using scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. hCMEC/D3, were grown on Fn-coated membranes with Fn coating with the addition of GFs at small concentrations (100 ng/ml) on the membrane. Cell/barrier characterisation includes TEER, FITC-dextran permeability and tight junction (TJ) immunofluorescence. Inserts to hold membranes with inbuilt TEER electrodes for continuous resistance measurement in cell culture were designed using AutoDesk Fusion360 and printed on a Prusa 3D printer in PLLA.

Results: hCMEC/D3 cells have low expression of TJs on membranes and their overall TEER values are considered very low. However, we were able to show high level of ZO-1 staining in specific pPEA-Fn-GF conditions, including low permeability to 10/40kDa dextran. Barrier characteristics were further improved by combining different GFs, such as FGF-2 and BDNF, and ECM proteins, such as collagen IV and laminin, and exploration of arranged electrospun fibres in differing geometries. In the investigation of key barrier-inducing GFs, novel GFs which induce higher levels of permeability than VEGF have been discovered.

Conclusion: We demonstrate that this versatile and tuneable design can induce barrier characteristics in immortalised hCMEC/D3 cells. With continued investigation into the changes the cells undergo and optimisation for increased BBB-characteristics, this is primed to be a powerful BBB model. There is room for further optimisation, particularly for the electrospun scaffold and growth factor choice and their combinations, although the model proves promising even at this early stage. This system offers a promising platform, with prospects for the study of BBB physiology and pathology, as well as for high-throughput BBB drug permeability testing.

  1. Williams-Medina, A., M. Deblock, and D. Janigro, In vitro Models of the Blood-Brain Barrier: Tools in Translational Medicine. Front Med Technol, 2020. 2: p. 623950.
  2. Vanterpool, F.A., et al., A material-based platform to modulate fibronectin activity and focal adhesion assembly. Biores Open Access, 2014. 3(6): p. 286-96."

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