An exploration of the reflow technique for the fabrication of an in vitro microvascular system to study occlusive clots

Abstract

Embolic ischemia and pulmonary embolism are health emergencies that arise when a particle such as a blood clot occludes a smaller blood vessel in the brain or the lungs, and restricts flow of blood downstream of the vessel. In this work, the reflow technique (Wang et al. Biomed. Microdevices 2007, 9, 657) was adapted to produce a microchannel network that mimics the occlusion process. The technique was first revisited and a simple geometrical model was developed to quantitatively explain the shapes of the resulting microchannels for different reflow parameters. A critical modification was introduced to the reflow protocol to fabricate nearly circular microchannels of different diameters from the same master, which is not possible with the traditional reflow technique. To simulate the phenomenon of occlusion by clots, a microchannel network with three generations of branches with different diameters and branching angles was fabricated, into which fibrin clots were introduced. At low constant pressure drop (ΔP), a clot blocked a branch entrance only partially, while at higher ΔP, the branch was completely blocked. Instances of simultaneous blocking of multiple channels by clots, and the consequent changes in the flow rates in the unblocked branches of the network, were also monitored. This work provides the framework for a systematic study of the distribution of clots in a network, and the rate of dissolution of embolic clots upon the introduction of a thrombolytic drug into the network.

Appendices

Appendix A : Scaling analysis

To reduce the empiricism of the reflow procedure, it is useful to understand the physical factors that govern this process. The capillary time scale t _c (Leal 2007), defined as μH/γ, where μ and γ are the viscosity and surface tension of the polymer at the temperature T ₀ , represents the time required by the photoresist-air interface to relax to a circular arc. The viscosity of AZ40XT is a complicated function of the temperature and the time, for which the polymer is maintained above the softening point. But with typical values of the height and surface tension, H = 100 μm and γ = 10mN/m, respectively, to get a capillary relaxation time comparable to the experimental reflow time of 100 s, the viscosity needs to be 10,000 Pa-s, which is extremely large. Besides, we have never observed any partially melted structures in our experiments. Therefore, in our experiments, the capillary relaxation time is expected to be much smaller than the reflow time of 100 s employed in the experiments. This implies that for any given cross-sectional area and contact width of the photoresist, during the reflow process, the air-photoresist interface relaxes rapidly to a circular arc while maintaining the contact width with the substrate. Note that gravity plays a minimal role in changing the contact width and therefore the spreading of the photoresist. This can be shown by examining the Bond number $\mathrm{Bo}=\raisebox{1ex}{$\rho {gH}^2$}\!\left/ \!\raisebox{-1ex}{$\gamma $}\right.,$ which is a ratio of the gravitational force relative to the stabilizing surface tension force (Leal 2007). Here ρ is the density of the polymer and g is the acceleration due to gravity. For typical values of the parameters, ρ = 1000 kg/m³, H = 100 μm, and γ = 10 mN/m, the Bond number is on the order of 10⁻², suggesting that gravity will play a weak role.

Appendix B: Profile of the AZ40XT photoresist covering SU-8 insert prior to reflow

The developed SU-8 features on the silicon wafer with various heights (H ₀) were covered by AZ40XT photoresist via spin-coating (800 rpm for 30 s and 2500 rpm for 2 s), followed by a soft bake, UV exposure by aligning the photomask with the already formed SU-8 structure (Fig. 4(c)), a post bake and a development step as described in section 2.4. The profile of the developed AZ40XT with SU-8 insert was characterized by optical profilometer (Veeco NT1100) at 10X magnification (3X scanning speed in z direction). An optical image (Fig. 18(a)) of the branched region between 1st and 2nd generation microchannel indicates that a good alignment was achieved to place the AZ40XT on the center top of SU-8 insert. A profilometer image (Fig. 18(b)) and its height profiles along two axes (Fig. 18(c) and (d)) show the highest portion of the hybrid-photoresist feature is 78.4 μm, higher than both the SU-8 structure (H ₀ = 66.2 μm) and regions without SU-8 insert (H ₂ = 47.4 μm, as shown in yellow-green region in Fig. 18(b)). Note that the profilometer cannot measure the height in regions where the height is not a constant. In subfigures (c) and (d), there is a range of abscissae connecting the hybrid-photoresist and non-SU-8-insert regions, where no height was reported by the profilometer. This suggests a gradual variation in the height in this region, instead of a sharp or step change.

Fig. 18

Profile of microstructure of AZ40XT photoresist covering SU-8 insert (H ₀ = 66.2 μm) characterized by optical profilometer. (a) Optical and (b) profilometer image of the bifurcation region between 1st and 2nd generation of the microchannel. The blue and red lines indicate the X and Y axes, respectively. The height profile along respective axis is plotted in (c) and (d), respectively. Scale bar is 100 μm in (a) and (b)