Descripción de los instrumentos utilizados

4.3.1 Hydrogel channel fabrication

The developed injection set-up offered a method for transferring channel features of the silicon mould into 1%, 2% and 3% agarose and 5% and 10% hyaluronic-based hydrogels without being compromised by the presence of air bubbles, as depicted in Figure4.3a,c. The 2.5% HA-ta/Dex-ta/Gel-ta appeared to lack stiffness required to obtain the channel structure. Subsequently, the created hydrogel channels could be aligned with the electrodes incorporated in the glass slide by transferring the PMMA gel support into the glass holder (Figure4.3b,d).

Occasionally, some abnormalities in hydrogel geometries were observed as de- picted in Figure 4.4. The first type of deformities were related to the AZR_40XT

channel features. The silicon moulds were re-usable up to at least twenty times. After that, some deformities were observed in hydrogel channels which could be traced back to impairments of the AZR_{40XT channel features on the silicon mould}

(Figure4.4a,b). The second type of deformations however, could not be explained that easily. They were hypothesized to arise from structural changes of dissolved hydrogel components when stored at 4◦C over time. These might affect the silane bonds of the FDTS coating in such a manner that gelation is impaired, hence the waving channel structures. For this reason, it is advised to store dissolved compo- nents at 4◦C up to seven days at most or, preferably, prepare fresh solutions prior to each experiment.

4.3.2 Channel feature transfer

Although the proposed method proved successful in fabrication of hydrogel chan- nels in general, reproducibility is questionable. The reproducibility was quantified by comparing the width of the AZR_{40XT channel features with the width of the}

successfully fabricated hydrogel channels as shown in Figure 4.5. First, it can be remarked that an exact transfer of the channel features into the hydrogel was not achieved. As an exception to the aforementioned observation, the 50 μm feature with squared crosssection appeared to be transferred 1:1 in all agarose hydrogels. Second, the coefficients of determination (R2_{) are relatively high, implying that the} trend lines are a good model for predicting the hydrogel channel width dependent on the used mould dimensions. Again, an exception in the overall trend is observed, in this case for the 3% agarose hydrogel. The latter is striking since this specific hydrogel has been used elaborately in literature [19], [26] and was therefore hypoth- esized to ensure best feature transfer. Third, standard deviations of the measured channel widths corresponding to the semi-circular cross sections (40μm and 60μm) are seemingly bigger compared to the squared cross section (50μm). Moreover, the average measured channel width for the 40μm channels are exceeding the expected values for most hydrogels, whereas the opposite holds true for the 60μm channels.

FIGURE4.3: An image of the actual injection (A) and measurement set-up (B), with zoomed view of a silicon mould with channel fea- tures (30 μm radius) and glass slide with incorporated electrodes.

Connector pins were soldered to wires that enabled connecting the electrodes to a measurement apparatus. Using both set-ups allowed for hydrogel channel fabrication (C, 50μm radius) and subsequently

alignment of the electrodes underneath the channel upon transferring the PMMA gel support in the glass holder (D, zoomed view presented

FIGURE4.4: Abnormalities in hydrogel channel structures: A) defor- mities in specific segments of hydrogel channels could be traced back to B) impaired channel features on the silicon mould; C) deformed channel geometries were observed when using hyaluronic-based hy- drogel components that had been dissolved and subsequently stored

for a period longer than seven days. Scale bars represent 100μm.

The used method for quantifying channel feature transfer has some limitations, which might explain unexpected results. First, limitations can be related to the use of the, at that time, readily available silicon moulds and their channel features. These investigated channel features differed regarding their cross sectional area, which were square (50μm) and semi-circular shaped (40 and 60μm). Both shapes might be- have differently when subjected to vertical pressure such as applied in the measure- ment set up. Upon applying vertical pressure, a square shaped channel would start collapsing while maintaining its width due to a vertical load distribution, whereas a semi-circular shaped channel would have a horizontal load distribution result- ing in variations of the measured channel width. The latter solely holds true when free movement of the hydrogel on the glass surface is allowed (even when limited), which is the case for hydrogels that are not chemically anchored to the glass slide as observed for the majority of the hydrogels. This will be further elaborated on in Section4.3.3. To assess this effect, cross sectional imaging of the channel is desired. Related to this topic, due to a lack of channel sealing on the glass, channel bound- aries might be positioned in other focus planes which would affect the measured channel width. As final shortcoming, the range in channel width was limited. For

FIGURE4.5: A,B) Measurements of the AZR_{40XT channel features}

and hydrogel channels, respectively, were performed using the LAS software. C, D) The average hydrogel channel width (channel di- mension) is plotted against the width of the channel feature (mould dimension), together with a linear line to indicate the expected be- haviour (y=x) and for each investigated hydrogel a corresponding

trend line (n=3).

a better overview it is desirable to have a broader spectrum of channel widths, for instance 20-100μm which corresponds to precapillary arteriole dimensions that are part of the BBB microvasculature together with the capillaries (≤10μm). [29]

To eliminate these limitation, one could repeat these measurements with a fresh, carefully selected set of silicon moulds. All with the same shape and height features and a broader range of diameters. Optional is repeating the measurements twice, using circular and square shaped features. Interestingly, although brain endothelial cells are typically exposed to high curvatures in the capillaries, endothelial cells’ ca- pacity to remodel square cross sectional hydrogel channels towards elliptical shapes might question the need for semi-circular hydrogel channels. [16]

FIGURE4.6: Microbeads with diameters of 2μm (C) and 10μm (A,B)

visualized hydrogel channel sealing on glass slides. Some segments were sealed sufficiently (A,C), whereas others did not (B). Channels are indicated in each subfigure with an

|{z}

. Scale bars represent: A and B = 200μm, C = 400μm.

4.3.3 Sealing hydrogel channels

Sealing of the hydrogel channels on the glass slide was visualized by flushing the channels with two solutions containing 2μm and 10μm microbeads, respectively. In general, a proper sealing of the channels could not be achieved, independent of the used hydrogel type or the concentration of the tyramine coating (Figure4.6b). Occasionally, applying pressure through the measurement set-up would suffice and microbeads could be flushed through (segments of) the hydrogel channels as shown in Figure4.6a,c. The latter is hypothesized to affect the sealing in two ways. First, upon applying pressure, the hydrogel structure is mechanically stabilized. Second, the hydrogel will absorb liquid from the PBS reservoir until it reaches a maximum swollen state as determined by the boundaries set by the gel support and the glass slide, thereby sealing the channels. Figure4.6c illustrates this observation, where the 2μm beads can be found inside the channel and underneath the PMMA gel sup- port due to a rupture elsewhere in the hydrogel (segment not shown), whereas they are not present underneath the hydrogel that is tightly pressed onto the glass slide. This implies that proper control of the hydrogel swelling behaviour offers possi- bilities in channel sealing on the glass slide. Nevertheless, although this might be sufficient for cell culture purposes, performing TEER measurements requires fully sealed channels without any liquid leakage.

To explore what prevented the hydrogel from chemically binding to the glass slide, two factors were questioned and examined: 1) the chemically functionalized glass slide and 2) the presence of free reactive tyramine groups on the hydrogel surface after crosslinking required for subsequent crosslinking with the functional- ized glass. (AppendixA.2) First, successful chemical functionalization of the glass slide with tyramine using the aforementioned protocol was confirmed by allowing hyaluronic-based hydrogel to crosslink directly on the treated glass surface, which resulted in proper attachment of the hydrogel onto the glass slides. The second factor was tested by indirect crosslinking of hyaluronic-based hydrogels comprised

of decreasing H2O2 concentrations on similarly treated glass slides. However, hy- drogels crosslinked with the lowest H2O2 concentration that would still allow for transfer of channel features, did not chemically bind to the tyramine treated glass slide upon indirect crosslinking.

Since indirect crosslinking of hydrogel on a substrate other than the same hy- drogel has not been reported to our knowledge, comparison of the obtained results could not be done. However, inspiration for sealing open hydrogel channels on a glass surface may be found in other methodologies used for creating hydrogel mi- crovasculature, based on sacrificial gel structures or applying a molecular coating of the hydrogel on the glass slide. These options will be further elaborated on in Chapter7.1.