The overall goal of this procedure is to use a microfluidic platform to construct a perfusable vascular network in a multicellular aggregate, or spheroid. This method can help address key questions in the alternate shape of regenerative medicine fields, such as how is a vascular network important in vivo and in vitro tissue models. The main advantage of this technique is that the route of drug and supplement administration can be simulated in vitro via delivery of regions of interest directly into the spheroids.
Generally, individuals new to this method will struggle because of the difficulty in introducing the cells into the target area in the microfluidic device. After casting the PDMS prepolymer, degas the material in a vacuum chamber for two hours, followed by overnight curing at 80 degrees Celsius in an air-vented oven. The next morning, peel the PDMS from the silicon wafer, and use a two-millimeter-diameter hole punch to create holes in the material at the indicated positions.
Use a one-millimeter-diameter punch to create the spheroid well, and use adhesive tape to clean the PDMS slab and a 24-by-24 millimeter glass cover slip. Treat the clean slab with air plasma for 40 seconds, and bond the PDMS slab onto the cover slip. Then, cure the PDMS at 80 degrees Celsius for at least 12 hours.
Two to three days before seeding the microfluidic device, add three times 10 to the fifth freshly thawed hLF, RFP-HUVECs, and GFP-HUVECs to 10 milliliters of fresh endothelial medium in a 10-millimeter dish. When the hLFs and RFP-HUVECs reach sub-confluency, suspend the hLFs and RFP-HUVECs in the endothelial medium to final concentrations at 1.0 times 10 to the fifth and 2.5 times 10 to the fourth cells per millimeter, respectively. After two days in suspension culture, in a biosafety cabinet, add a 99-microliter droplet of freshly prepared master mix solution into the center of a 35-millimeter Petri dish on ice.
To load the microfluidic device, use a modified 100-microliter micropipette tip to transfer a spheroid and 100 microliters of medium into a second, room-temperature Petri dish. Then, aspirate the spheroid in a minimum amount of medium, and turn the pipette upright so that the spheroid moves to the bottom of the pipette tip by gravity. Touch the pipette tip to the meniscus of the master mix droplet to eject the spheroid into the solution without depressing the pipette plunger.
Next, quickly add one microliter of freshly prepared thrombin to the droplet, and gently mix the thrombin into the solution. With the pipette set to seven microliters, slowly transfer the spheroid into the spheroid well of the PDMS slab. The most critical step of the procedure is injecting the spheroid with enough gel to allow the spheroid to settle at the bottom of the device.
After loading, remove the pipette tip gently to prevent leakage between the microposts. Incubate the spheroid at 37 degrees Celsius for 15 minutes. When the fibrin has solidified, slowly inject 20 to 30 microliters of endothelial medium into holes one A and three A to load channels one and three, respectively.
Then, transfer the device onto a wet lab wipe in a 100-millimeter dish, and incubate the device at 37 degrees Celsius and 5%CO2 for 24 hours to facilitate the removal of any bubbles at the interface between the medium and the fibrin. The next day, add two milliliters of 0.05%trypsin-EDTA to the sub-confluent GFP-HUVECs, and stop the trypsin reaction with four milliliters of complete medium when the cells have completely detached. Collect the endothelial cells by centrifugation and resuspend the pellet in fresh endothelial medium at a five times 10 to the sixth cells per milliliter concentration.
Next, inject 20 microliters of HUVECs into hole one B to load channel one, and place the device at 37 degrees Celsius for 30 minutes, tilted at a 90 degree angle to ensure that the HUVECs adhere to the fibrin in channel two. After loading channel three in the same manner, place the device in a new 100-millimeter dish containing a damp lab wipe in the cell culture incubator for seven to 14 days, replacing half of the medium in channels one and three daily. These representative images taken on day zero of HUVEC loading, show fibrin gel loaded into channel two only, without any leakage into channels one or three and with the HUVECs successfully attached to the sidewall of the fibrin gel.
The spheroid can also be observed to be properly settled at the bottom of the device. Angiogenic sprouts are observed beginning on day one, with the longest sprout reaching the spheroid on day three in this experiment, and with most of the angiogenic sprouts reaching the spheroid by day seven. Optimally, after four days in the device, flow can be observed through the vascular lumens with the vascular angle defined as the direction of the vascular tip and the center of the spheroid from the vascular root.
The vascular angles decrease in a time-dependent manner indicating migration of the angiogenic sprouts toward the spheroid. RFP and GFP-HUVECs can coordinately form a single vascular lumen, clearly indicating how angiogenic sprouts from channels one and three anastomose to RFP-HUVECs in the spheroid to form a continuous vascular network. Further, FITC-dextran injected into channel one flows into the constructed vascular network and interior of the spheroid, eventually reaching channel three, implying that the integrated vascular network could supply nutrients to and remove waste products from the spheroid.
After its development, this technique paved the way for researchers in the organic electric field to explore the efficacy of drugs or supplements of interest in in vitro tissue models.
