The great demand for new in vitro models of the blood-brain barrier has led to the development of various interesting approaches and a remarkable progress in recent years. Microfluidic systems which are able to realistically simulate the smallest functional units of organs have the potential to revolutionize basic research as well as the development of new pharmaceutical drugs and carrier systems. This study introduces a new microfluidic in vitro model of the blood-brain barrier.
The TransBBB chip offers a platform with ten parallel culture chambers in microwell format which allows the seamless integration into existing cell culture workflows. Since it is made of COP, it can be produced by injection molding which makes it easy to manufacture on a large scale . Compared to the frequently used PDMS, it also has the advantage of not exhibiting any adsorbing effects for hydrophobic substances . This is particularly important considering the fact that most CNS drug are relatively hydrophobic . It does not use an artificial membrane as culture area and instead relies on a hydrogel that mimics the physiological extracellular matrix. 3D Life biomimetic hydrogels from Cellendes® have been shown to successfully mimic extracellular matrices [29–31]. Including Matrigel as one of the gel components promotes cell adhesion and differentiation [32, 33]. When filled into the chips with a small micropipette, it forms smooth surfaces between the pillars, offering physiological culture areas.
Formation of air bubbles poses a significant issue for microfluidic systems as they can impede perfusion and also impair cell growth . It is important to know that the solubility of gases in liquids decreases with increasing temperatures. Therefore, cell culture medium used in this study was always equilibrated for at least 24 h in the incubator at 37 °C.
Perfusion is an important advantage of microfluidic systems compared to static models as it has been shown that the resulting shear stress on endothelial cells is a relevant factor for cell differentiation and the expression of tight junctions and transporters [35–37]. The TransBBB chip can be perfused by gravity driven flow induced by a programmable rocker without the use of complex tubing systems. This also allows perfusion with significantly lower medium volumes. The resulting shear stress on endothelial cells was calculated to be in a range from 0,05–0,1 Pa, which is lower than shear rates found in vivo (0,3–2 Pa) [37, 38].
The comparison of cell morphology under perfusion with large and small volumes suggests the crucial role of the ratio of total volume to culture area. The relevance of the amount of cell culture medium used in microfluidic systems in general was already discussed by Walker et al.  and Sung et al. . So far there is no conclusive explanation for the finding in our experiment but it will be subject of further investigations. However, the results of this experiment underline that it is an important aspect that should be taken into account when developing new microfluidic in vitro models.
Cells with the greatest predictive value for blood-brain barrier models would be primary human brain capillary endothelial cells. Due to the difficulty of obtaining primary human cells, PBCECs were used for this study instead. It has already been shown that these have a greater relevance than immortalized cell lines in terms of barrier tightness and protein expression [40–43]. The field of induced pluripotent stem cells (iPSCs) has made a remarkable progress in recent years. iPSCs have the potential to show similar properties compared to primary cells while being readily available . The use of iPSCs in the presented chip offers a promising option for the future.
PBCECs grew preferentially on the gel surface where they had a flat and stretched morphology leading to a dense layer that separated luminal and abluminal channel. The detection of ZO1 with an immunostaining demonstrates the formation of tight junctions. ZO-1 was also detected around the cell nuclei which could indicate a premature stage of cell development .
The permeation assay with the commonly used FITC dextran 20 kDa as fluorescence marker enables the assessment of confluency and barrier integrity. The performed determination of the ratio of abluminal and luminal fluorescence intensity allows the evaluation of permeation rates .
Moreover, the TransBBB system offers integrated electrodes for in situ measurements of trans-endothelial electrical resistances (TEER). Up to now, no significant TEER values could be measured with PBCECs cultivated in the chip. It has been reported that even minimal gaps in cell layers could lead to drastically reduced TEER values . Since one culture channel of the TransBBB chip contains up to 21 hydrogel surfaces, the probability is quite high that there could be a small gap in the cellular barrier on one of these surfaces. Therefore, the length of the individual channels and thus the number of cultivation areas per channel will be reduced in further developments. This will also allow even more culture chambers to be incorporated into one chip, which would enable greater throughput.
3-D Life biomimetic hydrogel contains peptide sequences which are cleavable by matrix-metalloproteases allowing cellular migration. The use of other cell types of the NVU like peri- or astrocytes is thus possible in order to further improve the relevance of the model.