INCOTERMS (Normas internacionales de negociación)

To investigate whether the protocol for chemically functionalizing glass with tyra- mine groups was performed successfully, crosslinking of hyaluronic-based hydrogel was allowed directly in contact with the glass slide. It was hypothesized that direct crosslinking would result in a proper binding between both materials. For this, 20 μl hyaluronic-based hydrogel was transferred onto a treated glass slide directly after finalizing the gel mixture with H2O2 and smeared out to increase the surface area for chemical bonding. Glass slides were chemically functionalized as previouslt described in Chapter 4with tyramine-NHS concentrations of 0.5-5 mg/ml. Upon manipulating the hydrogel after gelation, the hydrogel structure was partially torn and partially remained on the glass slide treated with even the lowest tyramine- NHS concentration, as can be seen in FigureA.1a, whereas the hydrogel indirectly crosslinked on the glass slide could be moved freely without damaging the structure

(FigureA.1b). This indicates successful transfer of tyramine groups onto the glass surface.

(A) (B)

FIGUREA.1: A) Arrows indicate ruptures in hyaluronic-based hydrogel structure

upon manipulation with a pipette tip after gelation directly on tyramine coated glass slides. B) No structural damage of hydrogel that is indirectly crosslinked onthe tyramine coated glass slide after manipulation of the hydrogel. (A, B both:

tyramine-NHS=0.5 mg/ml, H2O2=0.1 wt % final concentration).

The next step was then to determine whether crosslinking of hyaluronic-based hydrogel would leave enough free reactive tyramine groups on the surface for in- direct crosslinking on the glass surface resulting in chemical bonds with the tyra- mine groups on the glass slide. To examine the possibilities in ensuring free reac- tive groups, hydrogel mixtures were finalized with reduced concentrations of H2O2 (0.05-0.12% v/v). Gelation times were roughly monitored using the vial tilting method. This was used as indicator for the time after which the gel injection set up could be dissassembled again, while simultaneously ensuring a completed crosslinking reac- tion. From this it became clear that hyaluronic-based hydrogels finalized with H2O2 at final concentrations of 0.01, 0.012 and 0.015% v/v only were sufficiently rigid for channel feature transfer using soft-lithography. Subsequently, after transferring the crosslinked hydrogels onto tyramine functionalized glass slides (again 0.5, 1.0, 2.0 and 5.0 mg/ml tyramine-NHS) with an additional droplet of the corresponding H2O2concentration, none of the conditions remained attached on the glass slide.

Appendix B

Electrode characterisation

B.1

Reconstruction K

from Linderholm et al.

(A) (B)

FIGURE B.1: Cell constants as calculated by Linderholm et al. (A) were recon-

structed in MATLABR _{(B). s and w represent the electrode spacing and width, re-}

spectively. A) Lines A, B and C correspond to Kcellfor a constant electrode spacing

(s=100μm), constant electrode width (w=20 μm) and assumption of semi-infinite

medium for constant electrode width (w=20μm), respectively. Copied from [23].

B) The blue and red line represent cell constants as determined for semi-infinite medium and channel height dependent situations, respectively (w=20μm).

B.2

Raw impedance data

(A) Electrode pair 3-4, spacing = 150μm.

(B) Electrode pair 3-5, spacing = 300μm.

(D) Electrode pair 3-9, spacing = 2600μm.

(E) Electrode pair 3-12, spacing = 3050μm.

(G) Electrode pair 3-18, spacing = 5650μm.

FIGURE B.2: Raw impedance data of 1.2% agarose prepared with

varying NaCl concentration as measured between electrode pairs 3-4, 3-5, 3-6, 3-9, 3-12, 3-15 and 3-18 in A), B), C), D), E), F) and G), respec- tively. Per graph, the specific NaCl concentrations are indicated with

B.3

COMSOL model electrode characterisation

The 2D simulation was made using COMSOL MultiphysicsR _{5.3a in the AC/DC -}

Electric current physics interface solved in a time-independent manner. The agarose gel inside the gel support was modelled with a 1 mm high and 11 mm long rect- angle with 12 platinum electrodes (0.5μm high and 10μm wide,σ= 8.9·106 S/m) placed at the bottom spaced corresponding to the actual electrode set-up. It has to be remarked that the Ag/AgCl electrodes were ommitted from the 2D simplified model based on their position that was not on the same axis in the hydrogel tunnel. Subsequently, per electrode pair, a potential difference of 5 mV was applied as in- put signal with one electrode selected as ground, whereas the remaining electrodes were selected to be floating potentials. Simulations were done per electrode pair for different conductivities of the agarose gel.

Appendix C

Future outlook

C.1

COMSOL model TEER

The 3D simulations were made using COMSOL MultiphysicsR_{5.3a in the AC/DC}

-Electric current physics interface solved in the frequency domain. The total model was comprised of 1) the hydrogel tunnel inside the gel support which was modelled with a block (1mm x 1mm x 40mm, height x width x length, respectively); 2) the glass slide which was represented with a similar block in direct contact with the the hy- drogel channel which was a semi-circular tube with a radius of 30μm; 3) a cell layer of 1μm thick lining the hydrogel channel domain; 4) 6 platinum electrodes complet- ing one electrode set (0.5μm x 50μm x 10μm, height x width x length, respectively) placed on top of the glass, positioned corresponding to the actual electrode set-up. A total overview of the structure and the material properties are shown in FigureC.1

and TableC.1, respectively. Subsequently, a potential of 50 mV was applied as input signal with one electrode selected as ground, whereas the remaining electrodes were selected to be floating potentials. Simulations were done for TEER values of 10, 100 and 1000Ωm2.

The last electrode positioned underneath the hydrogel channel was set as ground (nr. 6), and a potential was applied on the first electrode in the channel (nr.3). Mea- surements were performed at frequencies ranging from 100_-109_{Hz. The impedance} was determined at the line highlighted in FigureC.1B, which is positioned at the in- ner surface of the cell layer at the highest point. Subsequently, the found impedance data was plotted against the frequencies for each TEER values, as can be seen in FigureC.2.

TABLEC.1: An overview of the electrical properties of the materials as used in the COMSOL model.

Conductivity (S/m) Rel. permittivity (-)

Cell medium 1.7 80

Cell layer h/(TEER·A)≈2.7/TEER 100

FIGUREC.1: A) A schematic of the 3D structure used in COMSOL comprised of glass, hydrogel, a cell layer, medium and six electrodes. B). Zoomed view of the channel which is composed of cell medium and covered with a cell layer. The black line at the inner surface of the cell layer at the highest point, indicates the line used for determining the both impedance of the system as well as the voltage along the

channel.

FIGURE C.2: Impedance data over a 1 μm thick cell layer in a

semi-circular hydrogel channel (radius 30μm) as measured between

two electrodes aligned directly under the hydrogel channel, spaced 450 μm, with in between them two floating electrodes. Different

impedance data was achieved per TEER value, indicating that this method could be used for measuring TEER.

C.2

COMSOL model shear stress

3D simulations were performed in COMSOL MultiphysicsR _{5.3a in the Fluid Flow}

module using the Single-Phase, Laminar Flow physics. The channel was represented by a 2 cm long tube with semi-circular crosssection with radii of 20μm, 30μm and 50 μm, corresponding to the available channel feature dimensions on the silicon moulds. An outlet boundary was selected to one of the crosssections and set at atmospheric pressure, whereas the opposite crosssection was set at an average ve- locity (m/s), also defined as parameter. The appropriate mesh size was determined by further refining throughout computations until no difference in outcomes were noted. The channel was filled with cell medium, which has a reported fluid viscos- ity of 0.78·10-3 Pa·s, a density corresponding to regular water and was assumed to be incompressible. [37] Subsequently, the model was calculated for varying input velocities and three radii in total, from which the shear stress on the wall was calcu- lated. Final data analysis was done by plotting the modeled shear stress against the volume flow matching the channel radius and average velocity, which is shown in FigureC.3ctogether with illustrations showing the flow profile and the shear stress distribution (FigureC.3a,b).

FIGURE C.3: A) Crosssectional view of the flow profile of a semi-

circular channel with B) corresponding wall shear stress which will have the highest values on the top of the curvature and on the bot- tom along the axis. Both channels have a radius of 20 μm and are

subjected to an average velocity of 0.02 m/s. C) For the developed semi-circular channel cross sections is the model shear stress plotted against the corresponding flow. Grey dotted lines indicate the range of shear stress values reported forin vivoBBB, 0.2 and 3 Pa. [33], [34]

Appendix D

3D

view

final

designs -

injection set up

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