When sealing of the hydrogel channels on a glass substrate is achieved, the hydrogel- based microfluidic chip allows for cell culture of endothelial cells, lining the channel wall. Elevated TEER values have been reported forin vitro cultured hCMEC/D3 cells exposed to a shear stress[36], which might implicate an important role for shear stress in regulating the barrier function through endothelial cells. To be more pre- cise, brain endothelial cells maintain a cobblestone-like morphology when exposed to shear stress or a highly curved substrate, whereas endothelial cells from other organs tend to elongate and axially align in response to shear stress. To further re- search the impact of the specific shear stress values on barrier function, a controllable shear stress variable would be desirable. However, as observed in the preliminary cell culture study on the hyaluronic-based hydrogel (Chapter5), remodeling of the matrix is likely to occur, which will affect the cross sectional area of the hydrogel channels and thereby the shear stress on the walls. For optimal implementation of shear stress in this proposed hydrogel-based BBB on chip, monitoring crosssectional channel geometries is required. Although mathematical equations to determine wall shear stress are known for both squared and circular channel crosssections, the more complex semi-circular crosssections might preferably be resolved by using finite ele- ment modeling. This has already been performed for the used semi-circular channel geometries. (Detailed description in AppendixC.2.) For further optimization, the cross section can be adapted based on confocal images over time to compensate for remodeled channel geometries, which will most likely occur during the first days of cell culture.
Chapter 8
Conclusion
It was aimed to fabricate a hydrogel-based 3D semi-circular microvascular BBB-on- chip with integrated sensors for barrier function assessment through TEER mea- surements. During the scope of this thesis, first steps were made in achieving this goal. We have developed a combination of devices that allowed for channel fea- ture transfer in hyaluronic-based hydrogels with dimensions down to 40μm that subsequently could be aligned with electrodes integrated in a glass substrate. The hydrogel proved suitable for conventional endothelial cell culture purposes, which was concluded from the characteristic cobblestone morphology and active remodel- ing capacity of the hCMEC/D3 cell line. Subsequently, preliminary tests were per- formed for characterization of the proposed electrode configuration and showed to potentially be suited for impedance spectroscopy measurements in the developed device. Future research on channel sealing is vital for completing the device such that it facilitates cell culture in the hydrogel-based microfluidic chip.
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Appendix A
Hydrogel chip fabrication
A.1
Silicon mould fabrication
TABLEA.1: An overview of spincoating details for fabrication of sili- con chips with channel topography features at various heights. (Stan-
dard protocol available at clean room University of Twente.)
AZ40XT film thickness Spincoating steps 20μm Step 1: 800 rpm, 100 r/s 20s Step 2: 4000 rpm, 400 r/s, 30s Step 3: 4500 rpm, 1200 r/s, 5s 30μm Step 1: 800 rpm, 100 r/s, 15s Step 2: 2500 rpm, 400 r/s, 30s Step 3: 3500 rpm, 1200 r/s, 1s 50μm Step 1: 800 rpm, 100 r/s, 15s Step 2: 1500 rpm, 400 r/s, 30s Step 3: 3000 rpm, 1200 r/s, 1s