Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

This microfluidic cancer on-chip model which we call the evolution accelerator, provides a controllable platform for long term, real time quantitative studies of cancer dynamics within well defined environmental conditions at a single cell level. The technology allows us to probe the evolutionary dynamics of cancer under a tumor-like stress landscape. Providing information including morphology, population, motility, and migration over time on the cellular level.

With the ability to control and monitor the behavior of mixed tumor populations under well controlled stress gradients our technology may present a more physiologically relevant model for preclinical drug development. The presented pressure sealing packaging method can also be implemented in other microfluidic systems that requires sophisticated adjustment of ambient gas composition as well as the downstream experiment capacity. The keys to successfully perform a long term experiment is to secure a physically and biochemically stable environment.

Factors including temperature, humidity, and gas composition should be monitored at all times. To begin, fabricate a plate holder that is sufficient to hold simultaneous gas permeable culture dishes on a 3D printing machine. Unscrew the components of the customized three well sample plate with screw driver.

Disinfect the components via UV exposure for at least one hour and leave in a sterile environment. To start cell line seeding add five milliliters of trypsin into each PC3-Epi or PC3-EMT cell culture in a 10 centimeter culture dish and run the trypsinization for five minutes in an incubator at 37 degrees celsius. Transfer the cells from the culture dishes into 15 milliliter centrifuge tubes and then centrifuge at 150 times g for three minutes.

Discard the supernatant. Re-suspend each of the cell pellets in the 15 milliliter centrifuge tubes and count the cells of each PC3-MP or PC3-EMT using a hemocytometer. Isolate a total of 2.5 times 10 to the four of each cell type.

Mix the two isolated cell types and add in two milliliters of culture media. Seed two milliliters of the cell culture into each of the three gas permeable culture dishes. Leave the gas permeable in the incubator at 37 degrees celsius over night for the cells to attach.

Disinfect the PDMS devices via UV exposure for at least one hour and leave in a sterile environment. Now set up a gas supply system that consists of both carbon dioxide and oxygen control units, a gas pump, a gas mixing chamber, a humidifier or bubbler, and three separate sets of gas valves, and pressure gauges. Alternatively use a gas supply system that provides normoxia condition.

Make sure the relative humidity in the mixing chamber is increased to above 85%Set up an onstage incubation thermal control unit at 37 degrees celsius with separate heating sub-units for the lid and the bottom plate. The next day take out the gas permeable culture dishes from the incubator and identify that the detailed components are in order, Every well possesses a gas permeable culture dish holder, a PDMS chip holder, a glass window holder, and a pair of 35 milliliter wide glass windows. Use the fitted screws to assemble these components and clamp the gas permeable culture dish.

Then transfer the pre-warmed culture medium to a vacuum chamber to de-gas for 20 minutes. Treat the PDMS chip in an oxygen plasma system for 30 seconds in order to maintain hydrophilicity. Next load two syringes slowly with 10 milliliters of de-gassed growth media and the other two syringes with 10 milliliters of de-gassed desired reagent of interest.

For example media and media with drug. Connect each individual syringe to a 50 centimeter tubing by a 23 gauge dispensing needle into one hollow steel pin. Insert a hollow steel pin into the other end of the tubing.

Push the media into the tubing to prime the tubing and insert the steel pin into each PDMS chip through the capping layer. Fill up the reservoir layer and wet the PDMS pattern layer with media. The reservoir layer works as on-chip bubble trap to prevent air bubbles from getting into the microfluidic pattern.

Then load a one milliliter syringe with culture media. Connect the syringe to a five centimeter tubing by a 23 gauge dispensing needle into one hollow steel pin. Insert a hollow steel pin into the other end of the tubing and prime the tubing.

Insert the hollow steel pin into the center hole of the chip for excessive media to be extracted later. Load two 10 milliliter syringes per chip in the forward deck and place the other two syringes in the withdrawal deck of the syringe system. In order to avoid entrapping microbubbles in the microfluidic pattern, dispense one milliliter of the pre-warmed and de-gassed media into the 35 milliliter deep gas permeable culture dish.

Position the chips directly on top of the gas permeable culture membranes, where the chip approaches the liquid surface with a 15 degree tilt angle. Clamp the PDMS chip with the PDMS chip holder, pushing the PDMS device downward. Tape a sheet of a sealer on top of the PDMS device and clamp with PDMS chip holder, in order to prevent the chip from drying out.

Set media flow rate around the array to be 20 microliters per hour. Position the entire plate in the onstage incubator on the motorized stage of an inverted microscope. Connect the gas supply system to the gas channels through gas tubing and maintain the gauge pressure at 0.2 PSI.

This pressurizes the gas permeable culture membrane against the installed PDMS chip to ensure sealing of the device. Slowly extract excessive media in the chip using the one milliliter syringe from the center hole. Observe the chip under the microscope while extracting media and then stop extracting when the chip is sealed.

Connect the temperature sensing unit of the onstage incubator to the three well plate and set to 37 degrees celsius. In this study PC3 cultured in an evolution accelerator without the presence of external stress had growth curves while aligned. Suggesting the identicality of the cell proliferation profile as a function of position across the chip.

The initial slope of the curves reveals the proliferation rate. The doubling time for cells in the device is around 24 hours. GFP expressing PC3-EMT and mCherry expressing PC3-Epi cell lined were co-cultured in the evolution accelerator.

Starting from 40%confluence the coculture grew 40%confluent within three days. While the growth curve of total confluence is in the form of logistic growth model. The population of PC3-EMT continued to expand and PC3-Epi was suppressed in the asymptotic phase.

PC3-Epi and PC3-EMT cells were densely seated in the center of the device and were allowed to freely migrate toward the empty region. The normalized histogram of the speed of the speed of both phenotypes shows the velocity of PC3-EMT was significantly higher than PC3-Epi by a factor of 1.8 times. It is very important to avoid creating microbubbles at all cost while the culture media should be pre-warmed and de-gassed.

The PDMS chip should be wetted properly and handled gently. Due to the soft re-sealable nature of our pressure sealing method many downstream experiments can be preformed. Such as subclinical population extraction, local metabolite analysis, and spatially resolved immunofluorescence and sequencing.

Our technology demonstrates a way to reproduce key components and interactions in a complex super-micro environment in a comprehensive manner. Yet simple enough to provide quantitative, reliable, and reproducible data.