Biomaterial processes: DIY tissue engineering for dummies

Checking on the growth of my new cell cultures in the Pelling Lab.

I always vowed to myself throughout my academic career that I would never, ever, ever do a PhD. It seemed ludicrous. It seemed like way too much academia, and I don't exactly love the structure of academia. I didn't think I would ever have a single idea that could sustain 5-7 years worth of research. Plus, an MFA is officially a terminal degree for artists. However, the tissue engineering workshop at the Pelling Lab changed my thinking completely. I gained a very clear sense that my entire art practice thus far has led me to this work, and that everything I've ever done now makes perfect sense. Then I discovered that SymbioticA has a PhD program in creative tissue engineering. Oh, dear.

Oron Catts is one of the world leaders and innovators of the intersection between tissue engineering and art. I really enjoyed working with him at the Pelling Lab. It wasn't just the technical processes he so openly and generously shared, but also his philosophy towards science and scientific research: that the ongoing mystification and media hype needs to be debunked, and that scientific research is accessible, feasible and important for artists. Also, artists are important for science. He did, however, inform us that we were quite privileged in being selected to participate in the workshop and in actually being given this kind of access so openly.

One friend, with an academic background in sciences, asked me outright how it was that I, someone with no training whatsoever in science, had access to these kinds of labs (type II containment labs necessary for working with biohazardous materials), and this kind of highly specialized work. I explained that through this workshop, I was paired up with biologists to learn the 'craft' (as Oron calls it) of tissue engineering. For me, as an artist with a background in fine craft practices, this is speaking my language. Interdisciplinary practice is hot in the art world and in the research funding world so more and more we are seeing different fields of research being paired up with art practices. This is ultimately very enriching. Oron is interested in disseminating information that will enable artists to use semi-living matter (definition: that which comes from a living entity but can only exist in a laboratory environment) as art material. This art material is essentially disembodied life. And, as an artist, I can muck about with wet biology processes to dream up and create a new body for that disembodied life! I can hear that [here unnamed] church congregation praying once again for the salvation of my soul. I joke about that but in all seriousness, there are ethical implications with this type of work, and those problematics are extremely interesting to me, and part of the work that Oron explored with us. More on that later.

In the first day of the workshop, we learned to construct DIY incubators to grow biological cultures in, and air purifier hoods for creating a sterile work environment. Oron brought everyone to the labs, showed us a pile of materials that he'd selected for us, and instructed us to collaboratively figure out how to use the materials to build the lab equipment we'd need for growing cell cultures. 

The inside of our DIY incubator.

I was in the incubator working group. We riffled through the pile and chose a styrofoam cooler, a heating pad, a heating wire with a temperature sensor, a thermostat, a digital thermometer/ barometer, and foil tape. We also had scissors and a hand saw. None of us had any experience with these things previous to this. With those materials and basic pieces of equipment, we turned the cooler into a successfully functioning incubator that kept a fairly evenly-regulated temperature of 38˚ (body temperature). Animal cells need to be kept at body temperature to stay alive and grow. Our incubator had a plastic window in the door, built from the packaging that the thermostat came in. Oron was tough and gave us a hypothetical grade of 51%. Our incubator was not cell culture quality, he said, but we could grow mushrooms or culture yogurt/bacteria in it if we really wanted to. In truth, the materials he gave us would likely never produce an incubator suitable for cell cultures, but the exercise of collaborating to put one together with no previous knowledge whatsoever was empowering.

In the second day, we learned the processes of decellularization and creating new cell cultures. Decellularization is absolutely fascinating. Animal tissues, when all cells are removed, exist as nothing but a scaffolding of collagen strands. Plant matter (as opposed to animal tissue), when decellularized, becomes cellulose scaffolding. Animal = collagen. Plant = cellulose. Those are the matrices that all living matter grow on, the underlying structures that hold everything together. I thought of Marvin the Martian in Bugs Bunny cartoons, with his disintegrator ray gun, and imagined that it must simply eliminate collagen. Without the collagen scaffolding, we might collapse into a pile of loose cells with nowhere to go. 

A decellularized apple with only the cellulose scaffolding remaining.

A decellularized piece of tissue (pork) showing just the remaining collagen scaffolding (top) - it appears as a translucent, random mesh of fibres.

Decellularization is important because it allows you to create a raw material to work with, which can then be repopulated with any kind of living cell culture you want (FYI, many different types of living cells can be ordered online). One of the projects that was being worked on in the Pelling Lab was to repopulate an apple cellulose scaffolding with mouse muscle tissue. The apple was stripped of all its apple cells, then enculturated with the C2C12s (mouse muscle tissue cells), to produce an apple-shaped mouse muscle. They were interested in producing an apple that would twitch. Cells behave the way they're supposed to behave, meaning that heart cells will beat with the pulse of the heart they're taken from and muscle cells will know to expand and contract when stimulated. One thing that they discovered at the Pelling Lab was that a decellularized mouse heart repopulated with muscle tissue cells actually shrunk to half its size because the new muscle tissues contracted and pulled the heart form in. 

This is work in progress by Andrew Pelling's research crew. The container on the left is a decellularized apple and the container on the right is a decellularized apple that has been repopulated with C2C12s (mouse muscle tissue cells). It's in a red liquid because this is the nutrient solution necessary to feed and keep cell cultures alive and growing. The nutrient solution is made of fetal calf serum (blood) and antibiotics (since in vitro cell cultures do not have immune systems to protect them from contamination by diseases).

Once my piece of tissue was in the appropriate solution* in the magnetic stirrer to decellularize, I learned next how to create and grow my own cell cultures. We worked in the lab to create new HeLa (cervical cancer cells) and C2C12 cultures. HeLa cells are often used for testing in labs because they are so robust and grow very quickly. There is a history of complicated ethics around the use of HeLa cells (which were harvested from a dying woman without her knowledge or consent, or that of her family).

With a partner, I learned how to trypsonize an existing cell culture to separate the individual cells from each other (they grow in a tight, single layer on the bottom of the pitre dish), extract the free-floating cells from the old liquid, select a small sample of cells and transplant them to a new pitre dish, replenish the nutrient solution to feed them and establish a new culture (which has to go into the incubator to grow and thrive). This all has to be done in extremely sterile conditions in order to not contaminate the cells and cause them to sicken and die. Sick cell cultures will discolour the nutrient solution, so it is easy to tell which cultures are successful and which aren't. We came back to the lab the next day to check on our cultures - mine (a C2C12 culture) was growing remarkably well and looking very healthy, and I felt wondrously proud as I viewed them under the microscope. I'd grown my first in vitro life.

A successful C2C12 tissue culture grown collaboratively with my lab partner, Ottawa-based artist Daphne Enns, along with the cooperation of the cells themselves, of course. You cannot see the cells here because they are microscopic - all you can see is the hydrogel, but it is in fact swarming with hungry cells ready to proliferate under the right conditions.

You will notice that the above image of the cell culture indicates that it is suspended in hydrogel. Creating a hydrogel suspension was one of three techniques we learned for creating three-dimensional cell culture forms (decellularization/repopulating collagen scaffolding was the first technique). Hydrogel is extremely accessible, and in fact, Oron brought in a package of disposable baby diapers and showed us how to cut them open to collect the hydrogel beads that are embedded inside (used to cause baby urine to coagulate to keep the diapers 'dry'). So, the above cell culture is suspended in a hydrogel solution that I made from no-name diapers. Apropos for a culture of new (baby) cells, no? The laboratory (false) motherhood metaphor was not lost on me. As I mentioned, cell cultures grow in a single layer, typically, on the bottom of a pitre dish and stop reproducing when they run out of room. But, creating a hydrogel suspension and enculturating it with cells will provide the cells a 'shape' to grow into and through, and they will eventually take over the whole form in multiple layers. Were the cells pictured above allowed to continue to grow for an extended period of time, they would create a large, cylindrical muscle tissue the same shape as the entire pitre dish up to the edge of the hydrogel. This technique has fantastic possibilities. One could essentially create a mold of any shape and size, fill it with hydrogel suspension, enculturate it, incubate it and grow a semi-living form of one's own design.

The third technique for growing tissue in three dimensions is via a 3-D printer. In the Pelling Lab, there is a 3-D printer that extrudes a polymer cellulose in successive layers, building up whatever form you program it to print. We printed human vertebrae. Remember that cellulose is the plant-based matrix that cells can grow on, so this cellulose polymer is a friendly place for a new cell culture to grow, into the form you've printed. Eventually the polymer breaks down and the cells replace the underlying scaffold just as with the hydrogel.

These are the (two) vertebra we printed using the cellulose polymer extrusion process. If you look closely (or click the image to enlarge), you can see the layers of polymer that were built up. Polymer means 'plastic', but this is a vegetable- (cellulose-) based plastic that is biodegradable. Perfect for cells.

Some specialized labs are 3-D printing human organs. This is done with a different type of 3-D printer, one that prints cell-enculturated droplets of hydrogel into the form of a liver, etc. These experiments have been successful for reproducing organs in the lab.

A microscopic image of C2C12s on a computer screen at the Pelling Lab. These muscle tissue cells are being manipulated through a process of microscopic magnetic suction to attempt to align them all into the same direction or to change their shape. This is important for growing a muscle that will actually work properly to expand and contract in one direction.

Here an old iMac casing is being upcycled into an incubator (Pelling Lab).

This shows my (mostly) decellularized tissue after 24 hours in the magnetic stirrer, along with all of the others.

What happened with our living, thriving cell cultures? They were eventually disposed of (killed). We were asked first to think about what this really means and to collectively agree to allow those cultures to die. Personally, I did not have a problem with this decision, but ultimately, I did wistfully imagine taking it home and continuing to nurture this semi-living material the same as one would a plant or a pet or a child or any other living thing we care for, I suppose. This is impossible, of course, but I did still entertain the thought. Ideas of attachment to life and responsibility for that life, even in the process of engineering life in a false environment, is an important consideration, among others.

*I do have the recipe for the chemical bath that decellularizes tissue, along with all of the other recipes for feeding and sustaining cell cultures, etc.