Chapter 10. Mission Possible: Ghost Heart Protocol

Patrik D’haeseleer, Matthew Harbowy, Ahnon Milham, and Maria Chavez

This year for Halloween, we decellularized a pig heart. We got a nice bleeding heart, hooked it up to some plumbing, stripped out all its cells with enzymes and detergents, and then bottled the thing in Everclear. Because every mad scientist’s den deserves to have some mad science on display.

Ghost heart in a jar
Figure 10-1. Ghost heart in a jar

For those of you interested in trying this yourself, we’ve posted a detailed Instructable. Don’t be put off by the complexity of the procedure—when it comes down to it, the hardest part of this project is the plumbing. Actually, this would be a great project for a parent to do with a kid who’s really into bioscience and wants to go a little farther than the dissections in AP biology at school.

So why would you want to decellularize a heart? This is a technique being developed that may eventually produce organs for transplantation, composed of a patient’s own cells. The idea is to take a donor organ and strip it of all its cells, leaving nothing but the extracellular matrix that held the cells in place. This scaffold of connective tissue—called a "ghost organ" for its pale and almost translucent appearance—can then be reseeded with a patient's own cells, with the goal of regenerating an organ that can be transplanted into the patient without fear of tissue rejection.

Amazingly, mouse and rat ghost hearts reseeded with cardiac precursor cells will actually start beating autonomously (great videos here and here). Some of those techniques seem daunting, but the Pelling Lab at the University of Ottawa has already illustrated how easy and eminently DIY-able the decellularization process is; in the case of a mouse heart or even of a whole steak, it could be as simple as soaking in detergent for a while.

Having spent a good amount of time thinking about related tissue engineering techniques for the BioPrinting community project at BioCurious, it seemed only natural to try our hand at decellularization as a Halloween project. So last year, half a dozen strong-stomached biohackers got an assortment of chicken hearts and gizzards from the grocery store and made an impromptu run at decellularization.Every participant got a chicken heart to play with and tried a unique combination of detergent choice, number of distilled water and saline rinses, etc. In the end, we wound up with several jars of chicken heart in detergent soup on the shake platform. Shaking, and shaking, and shaking, for days on end with very little effect. Sure, we had a few hearts that were getting noticeably paler, but the whole mess was definitely getting noticeably stinkier as well! Lessons learned. We decided to table the experiment for the year and try again for Halloween 2013. Next time, we plotted, we would use a (more human-like) pig heart, and perfuse detergents through the organ, to make sure all the cells in the tissue are broken up and flushed out equally.

Fast forward one year: Cameron at BioCurious managed to get us some pig hearts from his sister’s farm, so we planned to do a test run at Science Hack Day San Francisco. Unfortunately, hearts that have been butchered for food typically have the arteries at the top of the heart cut off and a slash through the chambers of the heart to let the blood drain out, which makes them entirely unsuited for our perfusion experiment. Instead, we cut some slices from the heart and tested a range of digestive enzymes on them.

What we needed was a pig heart butchered specifically for biological experiments (called a "bioheart" in the trade). So we got ourselves some bloody pig bio-hearts from a local meat wholesaler, ordered some simple reagents, located a pump (thanks, Eric!), got some plumbing supplies from Home Depot, assembled a motley crew of coconspirators from Counter Culture Labs and BioCurious, and decided to try again. With mere days before Halloween, we descended on BioCurious for a full-day experiment.

Some assembly required: pig heart with hose clamp and barbed coupler attached to the aorta, ready for retrograde coronary perfusion
Figure 10-2. Some assembly required: pig heart with hose clamp and barbed coupler attached to the aorta, ready for retrograde coronary perfusion

The published protocols take 10–13 hours to complete, but since it took us hours to get everything setup correctly, we ended up adjusting the durations on the fly to fit into the time remaining. And since we were just making a showpiece rather than a ghost heart that could be successfully reseeded with stem cells, we made some judicious substitutions in the reagents: using plain tap water instead of gallons and gallons of reagent-grade water, kitchen salt in tap water instead of phosphate buffered saline, OxyClean (sodium percarbonate) instead of peracetic acid, and 151-proof Everclear instead of reagent-grade ethanol. If you wanted to do a real hardcore DIY version, you should even be able to replace the initial trypsin perfusion with digestive enzymes from the health food store (bromelain seemed to work quite well) and replace the lab-grade detergent with a concentrated shampoo! We didn’t have access to a high-volume peristaltic pump, but a buffer recirculation pump seemed to work OK. Again, you could completely DIY this part by using a fountain pump from the hardware store, or even just an elevated bucket (six-foot elevation should give you about the right fluid pressure).

Residual blood leaking out of the heart during the initial perfusion steps, as we push fluids through the heart muscle
Figure 10-3. Residual blood leaking out of the heart during the initial perfusion steps, as we push fluids through the heart muscle

One nice aspect about this experiment is that it uses fairly mild reagents, since the end result should be able to support cellular growth. But you’re also working with lots of fluids under pressure, and you really, really don’t want assorted pig heart juices squirting into your eyes. So make sure to wear some goggles at least. Also, if you’re using a fountain pump, keep in mind that they’re typically immersion pumps and not designed to handle conductive saline solutions. So try at your own risk…

This was probably one of the most ambitious single-day experiments we have been involved in—especially since none of us had any experience in doing this kind of thing. We frankly surprised ourselves with how well it worked (including some successful emergency cardiac surgery using super glue!), and we ended up with a stunning showpiece. Although initially daunting, the techniques are well within reach of the DIY community and can be used to educate and begin discussions on the latest in organ engineering research.

Check out our Ghost Heart in a Jar Instructable for all the details. After all, Valentine’s Day is just around the corner.