The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.
Cardiovascular disease remains the number one cause of death worldwide. Traditional in vitro models rely on monolayer culture. However, the heart, specifically the heart muscle or the myocardium, is complex in its 3D anisotropy and cellular composition.
Therefore, it is critical to improve the complexity of the tissue composition within in vitro models to better mimic both the cellular constituents and the structure of the myocardium. Here in this work, we demonstrate a protocol for development of a 3D mature stem cell-derived human cardiac tissue within a novel microfluidic device. This device incorporates a 3D main tissue channel with innate elliptical microposts that induce high degrees of alignment of the surrounding hydrogel encapsulated cardiac cells.
The main channel is flanked by media channels with injection ports that enable a nutrient diffusion throughout the tissue. Due to the care required in handling the microfluidic devices, visual representation of this protocol is helpful to properly establish 3D cardiac tissue within a microfluidic device with embedded microposts for enhanced tissue alignment. To dissociate the human cardiac fibroblast, first take out the flask from the incubator.
Put inside the biosafety cabinet and begin to aspirate the spent media from the flask. Then take three MLs of PBS 1X to wash the flask. Close the cap and swirl the flask so that the bottom is properly coated in PBS.
Aspirate the PBS. Take three MLs of 37 degree trypsin and add to the flask. Then tilt the flask and swirl so that the bottom is entirely coated.
Then put in the incubator for four to six minutes. Neutralize the trypsin with FGM3 by taking three MLs and adding that to the flask. Pipette the solution up and down to dislodge any cells that remain on the back of the flask.
Collect the cell suspension in a 15 ML Falcon tube. Take 10 microliters of the cell suspension and dispense in a hemocytometer to count the cells with a microscope. Centrifuge the cell suspension at 250 G for four minutes.
After centrifugation, aspirate the supernatant being careful not to disturb the cell pellet. Resuspend the cell pellet with fresh FGM3 to make a desired 75 million cells per ML suspension. Resuspend the cells, then set the tube down with a loosely fitted cap.
To dissociate the cardiomyocytes, take the plate out of the incubator and aspirate the media. Take six MLs of PBS and wash the wells so that there's one ML of PBS in each well. Aspirate the PBS, being careful not to disturb the cells attached to the plate.
Add six MLs of warmed trypLE Express so that there's one ML in each well of a six well plate. Incubate the cells with the trypLE for 10 minutes. After 10 minutes, neutralize the trypLE with six MLs of RPMI plus B27 plus insulin so that you're adding one ML of RMPI to each well.
Use one a ML pipette to collect the solution and blast it against the backs of the wells to ensure all cells are lifted off. Collect the cell suspension in a 15 ML Falcon tube. Centrifuge the tube at 300 G for three minutes.
After centrifugation, aspirate the media and the trypLE. This step is important to wash the sensitive cardiomyocytes. This step ensures that all trypLE is gone in 3D cardiac tissue formation.
Resuspend the cell pellet in five MLs of RPMI plus B27 plus insulin. Use a one ML pipette to pipette the solution up and down to ensure a homogeneous cell suspension. Take 10 microliters of the cell suspension for counting with the hemocytometer.
Centrifuge the cells at 300 G for three minutes. Aspirate the media after centrifugation for the second time. Add the appropriate amount of RPMI plus B27 plus insulin to achieve a 75 million cells per ML suspension, then resuspend.
To prepare the collagen solution for encapsulation, have all the reagents on an icebox in the hood. Then take 75 microliters of the stock collagen. The collagen is very viscous, so slowly uptake with the pipette, then dispense in a microcentrifuge tube on ice.
Take 13.85 microliters of RPMI and add to the collagen in the Falcon tube. Then take 10 microliters of phenol red and add to the mixture on ice and resuspend. Lastly, take 1.15 microliters of one normal NaOH and add to the suspension to neutralize.
Then take a 200 microliter pipette tip and resuspend the suspension. Next step is encapsulation of the cells within the collagen metrogel. Then take an aliquot of eight microliters of CMs and add to a fresh Falcon tube on ice.
Then take two microliters of CS and add to that cell mixture. Resuspend the cell suspension and grab 5.6 microliters of the cell suspension and put in a fresh Falcon tube. Then grab the collagen that you just prepared, take four microliters of the collagen, then take 2.4 microliters of metrogel.
Pipette the mixture up and down to ensure a homogeneous distribution of the cell hydrogel suspension. Once the gel injection is prepared, you can insert into devices. Take a new tip and resuspend.
Take the Petri dish of devices, insert the tip in the injection port, and slowly and steadily inject. You'll see as the device channel fills up with the cell hydrogel suspension. Once the other port is filled, stop injection and remove the tip.
After injection, flip the devices with tweezers and place inside the larger Petri dish with water in the incubator for nine minutes. After nine minutes, take the devices out of the incubator and flip upright, then place back in the incubator for nine more minutes to complete hydrogel polymerization. Now it's time to add the media.
So, take RMPI plus B27 and insert the tip into the media port and start slowly pushing. Then do so on the opposite media port. You may find that the media is not injecting, so you can put a droplet of media on top of the port and then take the pipette and use negative pressure to pull the media through from the other side.
So you'll see as the media starts to get pulled through the channel. Once media has been added to all of the media channels, the devices will be put back in the incubator at 37 degrees. The microfluidic device is shown in the upper left-hand corner.
Days one, seven, and 14 of culture are shown this image. After 14 days of culture, the cells should condense into highly aligned structures that form around the microposts. Traces of day 14 are shown here in C of spontaneous contraction, as well as immunofluorescent images of actin stained and cardiac marker stained tissues.
What should be seen is highly aligned actin fibers that form after 14 days in culture, as well as parallel striated mature sarcomeres and localized gap junctions. During this procedure, it is critical to keep all materials at low temperatures on ice during cell hydrogel injection to prevent premature polymerization. The microfluidic devices should be handled with care, both during fabrication, injection, and extended tissue culture.
The cell injections should be a steady process to ensure the entire channel has filled while performed quickly to prevent premature polymerization or hydrogel drying out before media addition.
