Bilayer Microfluidic Device for Combinatorial Plug Production

We at the Systems Biology for Oncology Group use a combined computational and experimental approach to unravel the intricate mechanisms shaping the tumor microenvironment. We use mathematical models based on clinical data and cancer biology to identify factors influencing individual response to therapy, with the ultimate aim of improving precision oncology. Given the heterogeneous and dynamic nature of tumor response to therapy, perturbation biology has shown the potential to overcome the limitations of genomic biomarkers.

Our approach involves subjecting receptive tumor cells to an extensive anti-cancer drug screen to identify phenotypic properties of tumors in order to determine more effective anti-cancer treatments. Microfluidics is a valuable technology which aids in the analysis of a small amount of sample, such as tumor biopsies, by distributing it into individual compartments, such as droplets or plugs. It also allows in the control of the composition of each plug, thereby creating multiple populations of chemically distinct plugs.

We present here a fully PDMS-based device where fluid flow is regulated by pressure-actuated Quake valves, which allow for the quick, controlled, and programmable production of distinct plug populations. Furthermore, since the Quake valves are also PDMS-based, they can be integrated smoothly into device fabrication. Combining data obtained from high-throughput combinatorial screening of cancer cells with mathematical models will allow us to study signaling pathways regulating tumor microenvironment behavior.

This research will pave the way for new strategies for precision oncology, providing the rationale to personalize treatment to individual patients. To begin, cut a length of PTFE tubing for every control channel of the microfluidic device. Insert the pin of a 23-gauge, 0.5-inch Luer stub at one end.

Attach the Luer stub to a male Luer. Then insert the connector into a length of polyurethane tubing. Connect the other end of the polyurethane tubing directly to a solenoid valve.

Next, connect a 23-gauge, 0.5-inch Luer stub at the end of a syringe. Fill the syringe with water. Attach the free end of the PTFE tubing to the syringe.

Inject water until approximately halfway through the tubing. Now disconnect the tubing from the syringe and insert the free end into a punched hole of the corresponding control channel. Repeat until each control channel is connected to the corresponding solenoid valve.

Next, launch the main interface program. Press the Pressurize All Control Channels"option to open the valves. This will push the fluid from the tubing into the control channels of the microfluidic device to fill it up.

For each of the aqueous reagents, cut a segment of PTFE tubing long enough to connect the pumps to the microfluidic device inlets. Connect a 23-gauge, 0.5-inch Luer stub to the end of a syringe. Fill the syringe with the required reagent.

Inject the reagent into the tubing until the tubing is full. Insert the free end of the tubing into a corresponding inlet on the microfluidic chip. With the software, apply a pressure of 400 millibars to each of the inlet aqueous reagent.

Sequentially depressurize the control channels individually using the main interface program. Press the corresponding icons on the program in the box labeled Control Channels Manual Pressurization"to actuate individual valves if necessary. After repeating the pressurization for the oil reagents, click on Depressurize All Control Channels"to simultaneously depressurize the channels.

Now click on Pressurize All Control Channels"to repressurize the control channels. Encode the composition, sequence, and replicates of each plug population in a CSV file by marking the necessary control channels with a 0"if the corresponding inlet needs to be open, or with a 1"if it needs to be closed. This will serve as input for the automatic experiment in the main interface program.

Next, click on the folder icon in the Experiment File"tab to load the CSV file. Input the relevant fields such as iterations of experiment, time of depressurization, and time of pressurization. Then choose the inlet channels corresponding to barcode production in the Barcode Inlet"section, along with the duration for which they need to be open.

Alternatively, the barcodes can be hardcoded into the input CSV file. Now reduce the pressure of the inlet oil reagents from 400 millibars to 200 millibars. Next, connect a PTFE tubing to the outlet to collect the plugs.

Use prefilled tubing to neutralize the difference in pressure at the outlet. Lastly, press Run Experiment"to start the program and plug production. The valves were able to regulate fluid flow until approximately 800 millibars of input pressure.

At 1200 millibars, the input pressure was too high for flow regulation. The flow rate of distilled water, when injected at constant pressure, dropped to zero. Upon depressurization, the flow rate recovered to the pre-actuation levels.

However, under constant flow rate, the valve actuation did not result in complete inlet closure. To demonstrate the device functionality, a quantitative combinatorial library of plugs was generated.