Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

Our tumor-on-a-chip platform was developed to physiologically model the growth of human vascularized microtumors and allows us to answer both basic and translational research questions, questions that focus on basic tumor biology, drug efficacy, tumor responsiveness, underlying immunologic processes, potential immunotherapeutic intervention, and personalized therapy approaches. Effectively translating preclinical discoveries into potent cancer treatments hinges on faithfully replicating the disease state and model systems. This protocol outlines the establishment of the vascularized microtumor or VMT to mimic the tumor microenvironment in vitro.

The VMT faithfully reproduces key aspects of a human tumor, yielding clinically relevant findings. Our model allows us to perform physiologically relevant, translational human cancer research. Specifically our protocol in contrast to other models incorporates dynamic flow conditions, enables the integration of immune cells and immunotherapy, and further allows us for the incorporation of personalized medicine approaches.

We are employing the VMT model to propel personalized medicine initiatives forward in both preclinical and clinical arenas. This model deepens our understanding of microenvironmental factors in cell-cell interactions influencing therapeutic resistance and cancer progression. Moreover, the VMT shows promise in discovering customized patient-specific treatments for enhanced clinical care.

To begin, place the HBSS cell dissociation reagent EGM-2 and DMEM in a 37 degree Celsius water bath for 10 to 15 minutes. Thaw thrombin and laminin overnight at four degree Celsius and fibrinogen at room temperature. Add 1.5 microliters of thrombin into a 500-microliter microcentrifuge tube.

Place the UV-sterilized high-throughput plates into a desiccator for 30 minutes to remove air trapped in the microfluidics. Place the T75 flask under the microscope at 4x magnification to confirm cell confluency and transduction efficiency. Wash the cells twice with five milliliters of HBSS.

Then add one milliliter of dissociation reagent to the flask, and incubate at 37 degrees Celsius with 5%carbon dioxide for one to two minutes. Gently tap the flask using the palm, and observe under the microscope that all cells have been lifted. Wash the cells with nine milliliters of appropriate media, and collect suspension into a 15-milliliter conical tube.

Immediately remove a small aliquot, and count the cells. Centrifuge the cell suspension at 300 g and four degrees Celsius for three to five minutes. Then remove the supernatant, and resuspend the pellet in appropriate media on ice.

Using the given equation, calculate the number of cells needed. Resuspend the cells at a concentration of 1 x 10 to the 6 cells per milliliter, and calculate the volume of cells needed using the following equation. Centrifuge the required volume of cell suspension, as demonstrated.

Then in a calculated volume of fibrinogen, resuspend the pellet on ice. To begin, place the UV-sterilized high-throughput plates and thrombin aliquots into the tissue culture hood. Place the prepared cell fibrin mix on ice to slow clotting.

Using a P20 pipette. draw six microliters of the cell fibrin mix. Then gently place the pipette tip into the thrombin aliquot, and mix twice without introducing air bubbles.

Lift the high-throughput plate at an angle, and quickly insert the pipette tip into one of the loading ports. Push the pipette plunger to the first stop in a smooth and fluid motion, and inject the cell fibrin mix into the tissue chamber. Ensure the gel crosses entirely through the chamber.

Gently place the plate flat without removing the pipette tip or displacing the pipette. Remove the pipette tip using a hand from the P20 and leave it in the loading port hole. After two minutes, remove the pipette tips with gentle twisting motions from the loading ports, and place the lid on the plate.

Incubate the plate for 15 to 20 minutes at 37 degrees Celsius for gel polymerization. Place the microfluidic device on the microscope stage to observe the even distribution of cells throughout the chamber without any air bubbles. Insert the P20 pipette tip into M1 or M3, and expel four microliters of laminin slowly to coat the entire top panel.

Remove the pipette tip as demonstrated previously, and incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 10 minutes, Add 275 microliters of EGM-2 complete media to the uncoupled reservoirs of wells located in rows A and B.Insert the P200 pipette tip into the media inlet hole of the wells containing 275 microliters of EGM-2, and slowly expel 75 microliters of medium, watching the media travel through the channel and bubble up on the other side. After removing the pipette tip, push the remaining media from the tip into the media reservoir. Add 50 microliters of media to entirely cover the low side wells in rows G and H.Incubate the plates for one to two hours, as demonstrated.

Under a microscope, identify any bubbles in the media channels and remove them by reintroducing 75 microliters of media into the channels. Check medium inlets or outlets for air bubbles using the naked eye. Then insert the P200 pipette tip into the hole and pull the bubble out by lifting the plunger.

In vascularized microorgans or VMOs, endothelial cells initially distributed evenly within the tissue chamber, but by Day 2, they began to stretch out and luminize. By Day 4, the endothelial cells anastomosed with the outer microfluidic channels forming a continuous vascular network. The tight vascular barrier function was confirmed by perfusing FITC-dextran through the microvascular network with minimal leakage.

Perfusing MDA-MB-231 cancer cells into the BMOs resulted in their adherence to the endothelial lining and subsequent extravasation into the extracellular space, forming multiple micrometastases within 24 hours post-perfusion. Through time-lapse microscopic imaging, the extravasation of T-cells into the extracellular space of the VMO was observed over 45 minutes. In vascularized microtumors with fully formed vessels, T-cells rapidly adhered to the vascular wall upon perfusion.