The aim of this assignment is to develop and characterize a basement membrane using sprayed hydrogels. After successful fabrication, the attachment of brain endothelial cells, astrocytes and the formation of a tight cell layer (the blood-brain barrier) on this membrane will be studied.
The blood-brain barrier (BBB) is the unique transport regulator of nutrients, inflammation cytokines, disease pathogens and peripheral cell types into and out of the brain tissue, thereby preserving brain homeostasis. In Figure 1 the BBB in rat cortex is shown: a blood vessel (endothelial cells) wrapped in a basement membrane, lined by astrocytic endfeet. The key characteristic of the BBB is the formation of tight junctions between adjacent endothelial cells, which prevent paracellular transport of e.g. drug compounds. Additionally, breakdown of the BBB is involved in several neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and Multiple Sclerosis. Hence, to perform drug screening experiments and study disease characteristics, it is essential to have a realistic model of the BBB.
Figure 1. Rat cerebral cortex with astrocytic (yellow) endfeet wrapping around blood vessels (red). Cell nuclei are cyan. [Madelyn May / Rensselaer Polytechnic Institute / Troy, New York, USA].
At the BIOS group a novel in vitro model of the BBB is being developed: a microfluidic BBB-on-a-chip (Figure 2 and Figure 3). Brain endothelial cells are cultured on top of a filter membrane and astrocytes on the bottom of this membrane.
To make this BBB model more physiologically relevant, we aim at replacing the filter membrane by an engineered basement membrane consisting of crosslinked extracellular matrix molecules. It is hypothesized that the basement membrane helps the differentiation of brain endothelial cells and astrocytes to form the blood-brain barrier. Previous results at the Developmental BioEngineering group suggest that it is possible to use enzymatically crosslinked macromolecular networks of conjugates of naturally occurring polysaccharides and ECM proteins like collagen IV and laminin to form thin layers of hydrogels by spray deposition (Figure 4). For help in constructing a blood-brain barrier, the hydrogels will be composed of ECM molecules naturally occurring in the basement membrane of the blood-brain barrier.
Figure 2. Cross section of the BBB-on-a-chip including: monolayer of brain endothelial cells (EM), a filter membrane (M) and astrocytes (A). Also included are electrodes (E).
Figure 3. Assembled microfluidic BBB device. The channels and the membrane (white square) can be recognized. Electrodes are sticking out of the chip.
Figure 4. Left to right: Device used for spraying of hydrogels. SEM picture of sprayed hydrogels showing nanometer fibrils typically seen in ECM. Sprayed hydrogel with embedded cells.
In this assignment, protocols need to be developed to fabricate a functional basement membrane. To this end, functionalized hydrogels and proteins need to be sprayed and cross-linked in situ to form a thin membrane.
Next, the attachment of endothelial cells and astrocytes to this membrane needs to be examined during a period of cell culture. The properties of the so-formed BBB will be tested by functional assays.
If you are interested in this assignment, feel free to contact us:
Janneke C. Alers
Marinke van der Helm
053 489 2154
053 489 2839