4. M ICROFLUIDIC C ULTURE P LATFORMS WITH P ERMEABLE AND
4.3 Microfluidic Platforms with a Semi-permeable Barrier
4.3.2 Demonstration of ligand traps by two fluorescent proteins
In the last chapter, the microfluidic platforms use an etched coverslip as the base. The two chambers communicate with each other through the gap between the etched surface and the bottom of the PDMS barrier, whose height is defined primarily by the etching depth. In fabricating the co-culture platforms with semi-permeable valve barriers, however, we used a two-layer fabrication technique similar to that shown in Fig. 2.2; but the fabricated platform is similar to the one in Chapter 3, i.e., with a continuous gap for cell-cell interactions. In the fabrication process, instead of an array of lines, a long rectangular SU-8 layer was first patterned on the silicon wafer as shown in Fig. 4.4. A second layer, defining two PDMS valve barriers and two larg- er cell culture chambers and a narrow middle gel channel, was then created. The schematic of the mold to construct the first PDMS layer is shown in Fig. 4.4(b). The second PDMS layer is produced in the same way as those in previous chapters. This new fabrication process avoids the glass etching process involving hazardous hydrof- luoric acid (HF). In the platform, the height of the gap is defined by the thickness of the first rectangular SU-8 block, which is also easy to be controlled by the selection of
the type of SU-8 and the spinning speed. In this dissertation, we fabricate microfluidic platforms having a 20 µm gap between the two chambers of 100 µm in height.
Figure 4.4: Schematics of the process to fabricate the mold for the first PDMS layer.
(a) A rectangular SU-8 layer is first patterned on a silicon wafer, whose thickness de- fines the gap height between the two chambers. (b) Another SU-8 layer is aligned and patterned onto the first SU-8 layer to define the channels and chambers.
As mentioned in section 4.2.2, the coverslip was treated by ethylene glycol to enhance the attachment of the gel and the PDMS surface was coated with APTES to facilitate the releasing of the PDMS barrier. Biotin coated polystyrene particles (0.7 – 0.9 µm in diameter; Spherotech, Lake Forest, IL) were chosen as the ligand trap. In- stead of agarose, collagen was used as the scaffold for the ligand traps because imple- ment of agarose involves high temperature which will damage the receptor proteins.
The gel was prepared by adding 10 µl 10× PBS and 0.38 µl NaOH (Sigma-Aldrich, Atlanta, GA) into 73 µl polystyrene particles solution. Vortex mixer was used to dis- perse the particles. Subsequently, 16.6 µl collagen (BD Biosciences, San Jose, CA)
mg/ml and a particle concentration of 2.59×1010 particle/ml. After the valve was acti- vated by injecting DI water in the control chamber, the platform was placed on ice.
Subsequently, the pre-gel mixture was filled into the mid-channel in a manner similar to that shown in Fig. 4.1. After loading, the whole platform was placed into a humidi- fied incubator at 37oC for 30 min to polymerize the matrix.
Figure 4.5: Demonstration of the semi-permeable barrier with two fluorescent pro- teins. Avidin-Alexa Fluor 488 (green) is trapped by the biotin coated nanoparticles inside the barrier and therefore cannot transport from one chamber to the other. How- ever, ovalbumin-Alexa Fluor 647 (red) can pass through the barrier and perfuse into the other chamber. (The width of the barrier is 200 µm)
After gelation, the semi-permeable valve barrier was formed by releasing the DI water from the control chamber. Two fluorescent proteins, avidin-Alexa Fluor 488 and ovalbumin-Alexa Fluor 647 (both from Invitrogen, Carlsbad, CA), were added into one chamber. Avidin was known to bind to biotin with a high degree of affinity.
The dissociation constant of biotin and avidin was measured as 10-15 M (Green, 1963).
Therefore, we expected avidin-Alexa Fluor 488 to be trapped by the immobile biotin coated particles inside the barrier, while the ovalbumin-Alexa Fluor 647 could pass the barrier and perfuse into the other chamber.
The experimental result is shown in Fig. 4.5, which clearly indicates that the avidin (green) binds to the immobile biotin inside the barrier and therefore is trapped by the biotin coated polystyrene particles. But the ovalbumin (red) can pass through the barrier and perfuse into the opposite chamber. As a result, avidin was effectively blocked from the molecular exchange between the two chambers.
In the fluorescent imaging based experiment, however, a high concentration for each fluorescent protein had to be used to obtain sufficient fluorescent signals for demonstration. The concentration added to the chamber was 0.48 µM/ml for avidin and 1.47 µM/ml for ovalbumin. According to the collagen matrix recipe described above, the ligand traps’ binding ability to avidin is calculated as 1.69 µM per ml gel.
The experiment shows that the gel quickly becomes saturated with avidin, es- pecially if there exists a transverse flow, which seems a problem. In real biological systems, however, the amount of ligands secreted by cells is orders of magnitude low- er in concentration than the fluorescent proteins used here for demonstration. For ex-
measured ranging from 5.5 ng/106 cells/day to 191.2 ng/106 cells/day (Ozawa, et al., 2004). Suppose there are 10,000 myoblast clones in one chamber, which will secrete 2.4 – 83.3 × 10-6 nM VEGF in 10 days. This is a very tiny fraction, about 0.0023 – 0.082%, of the total binding ability of the ligand traps in the barrier (Suppose the gel’s binding ability to VEGF is 1.69 µM/ml and the total volume of the gel in the barrier is 0.06 µl). Therefore, we expect that saturation would be very rare in real bioassays.
To guide the design of the semi-permeable barrier, an analytical model of the barrier’s performance has been developed and is presented in the next section.