The influence of astrocytes and their culturing environment on blood-brain barrier modeling in a microfluidic chip

Abstract: The blood-brain barrier (BBB) is a highly selective barrier separating the brain from the circulatory system. The physical barrier is built up by a monolayer of endothelial cells, which is the main component used for in vitro modeling. To create more physiologically relevant in vitro models, the field of organs-on-chips has recently been introduced, combining microfluidics and microengineering to create culture environments for cells. To mimic the BBB, a continuous monolayer of brain endothelial cells is cultured inside these organ-on-chip devices. A step towards more in vivo like models is the introduction of co-cultured astrocytes, a brain-specific cell type in close contact with the blood-brain barrier in vivo. Astrocytes are reported to have a function in inducing and maintaining BBB-specific properties of the endothelial cells. During injury to the brain, astrocytes become reactive, changing their function, morphology and molecular expression. In vitro it has been shown that the state of astrocytes is influenced by their culture environment. In this study, a method to incorporate hydrogels seeded with astrocytes in a microfluidic channel is presented, along with an investigation on the effect of different hydrogel compositions on astrocyte reactivity and morphology. Firstly, using a silane-based surface treatment a collagen type I hydrogel seeded with astrocytes was successfully incorporated and fixated in a microfluidic channel inside a polydimethylsiloxane device. Secondly, by varying collagen type I and hyaluronic acid concentrations, the lowest astrocyte reactivity was found in the hydrogels containing only collagen type I and in a mixed gel of collagen type I and hyaluronic acid with a low total gel concentration. A mixture of collagen type I and hyaluronic acid resulted in the most star-shaped astrocytes, characteristic morphology for astrocytes in vivo. Because of the small sample size, further investigation is recommended before conclusions can be drawn on the best hydrogel composition. The results from these two experiments were used to establish a co-culture system in a blood-brain barrier-chip. Unfortunately, as no continuous monolayer of endothelial cells was formed inside the chip and air bubbles inside the hydrogel structures affected the astrocyte culture, the effect of the presence of astrocytes cultured in a three dimensional environment could not be quantified. In order to achieve a reliable co-culture system optimization of chip design and protocol for incorporation of hydrogel is recommended.