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 in 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 micro-organs or VMOs, endothelial cells initially distributed evenly within the tissue chamber but by day two, they began to stretch out and luminize.
By day four, 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 VMOs 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 VMOs was observed over 45 minutes. In vascularized micro-tumors with fully-formed vessels, T-cells rapidly adhered to the vascular wall upon perfusion.
