The original version of this article, Prototyping with Living Cellsran as an installment in a series on the state of biohacking by Biohacking Safari. The version below appears in Make: Vol. 56.

The greatest bridge between the world of makers and the world of biohackers is probably the mighty 3D printer. The main difference is instead of using plastics, they’re using biomaterials to build three-dimensional structures, and using special bioinks made of living cells to print messages and patterns.

Human cells cultured into a decellularized apple slice (left) and an apple carved into an ear shape (right) from Pelling Labs. Photo by Bonnie Findley

How BioCurious Started Bioprinting

BioCurious is a mandatory stop among biohacker communities in North America. This pioneering space, located in Sunnyvale, California, hosts a number of great people collaborating on the DIY BioPrinter project. Their bioprinting adventure started in 2012, when they had their first meetups. According to Patrik D’haeseleer, who is leading the project with Maria Chavez, they were looking for community projects that could bring new people into the space and let them quickly collaborate. None of the project leaders had a specific bioprinting application in mind, nor did they have previous knowledge on how to build this kind of printer. Still, it appeared to be a fairly approachable technology that people could play with.

“You can just take a commercial inkjet printer. Take the inkjet cartridges and cut off the top essentially. Empty out the ink and put something else in there. Now you can start printing with that,” D’haeseleer explains.

The BioCurious group started by printing on big coffee filters, substituting ink with arabinose, which is a natural plant sugar. Then they put the filter paper on top of a culture of E. coli bacteria genetically modified to produce a green fluorescent protein in the presence of arabinose. The cells started to glow exactly where arabinose was printed.

Modifying commercial printers for this, as they were doing, presented challenges. “You may need to reverse engineer the printer driver or disassemble the paper handling machinery in order to be able to do what you want,” says D’haeseleer.

First major success with BioCurious’ $150 DIY BioPrinter: fluorescent E. coli printed on agar with an inkjet printhead. Photo by Patrik Dʼhaeseleer

So the group decided to build their own bioprinter from scratch. Their second version uses stepper motors from CD drives, an inkjet cartridge as a print head, and an open source Arduino shield to drive it — a DIY bioprinter for just $150 that you can find on Instructables.

The next and still current challenge deals with the consistency of the ink. Commercial cartridges work with ink that is pretty watery. But bioink requires a more gel-like material with high viscosity. The DIY BioPrinter group has been experimenting with different syringe pump designs that could allow them to inject small amounts of viscous liquid through the “bio print head.”

BioCurious’ early printer: $11 syringe pumps mounted on a platform made from DVD drives. Photo by Patrik Dʼhaeseleer

Moving to 3D

Starting with an already existing 3D platform seemed like the best way to go beyond 2D patterns. The group first tried to modify their existing 3D printer by adding a bio print head directly on it. However, their commercial machine required some difficult reverse engineering and software modification to perfect the process. After a couple of months, this led to a dead end.

The RepRap family of 3D printers influenced the next step. After buying an affordable open source printer kit, the bioprinting team was able to switch out the plastic extruding print head for a print head with flexible tubes that connected to a set of stationary syringe pumps. It worked.

Converting a RepRap into BioCurious’ latest 3D BioPrinter platform, with an Open Syringe Pump. Photo by Maria Chavez

“The RepRap community is really what has made the whole 3D printing revolution possible,” says D’haeseleer.

Soon enough there was a community around 3D bioprinting, tinkering at home and in biohackerspaces such as BioCurious, BUGSS, and Hackteria, all sharing their experiments.

Working With Life

The holy grail of bioprinting is generating 3D organs for transplants. Working with human or mammalian cells is complex. You need to have someone in the lab every day taking care of the cells and to keep everything as sterile as possible. Because of these obstacles, the BioPrinter group’s current long-term project is to create a proof-of-concept functional plant organ and get it to photosynthesize. This will be an artificial leaf!

There hasn’t been much work with plant cells, raising a lot of open avenues for research. You need to figure out what kind of cell types you will use, how to connect them together, what a 3D structure of a leaf looks like, etc. According to D’haeseleer, 3D printing with plant cells fits much better for a DIY community lab than actual mammalian cells.

Whether it works or not, the interest here is to test things and see how they grow. A commercial application is not the only purpose for biohackers, even though some scientists are a bit overwhelmed by the potential of their research.

“We are not very goal oriented, like we want to make a startup out of bioprinting and sell a product, make millions of dollars … There are not too many plants in desperate need of leaf transplants! We participate in this project because it’s a fun thing to do. We make some progress week after week,” says D’haeseleer.

3D Bioprinting With Plant Cells

When printing with plant cells, the first step is to figure out the material in which the cells are going to be held in place until they grow and make connections. Some current experiments at BioCurious use a gel-like material called alginate, which has very interesting properties. Sodium alginate is soluble in water, but viscous whereas calcium alginate solidifies instantly. It is similar to the spherification techniques seen in food science, where a solid droplet is full of liquid on the inside (check out this cold oil spherification technique you can make as a bruschetta topping).

Testing alginate as a promising DIY-able and accessible bioink at BioCurious. Photo by Maria Chavez

Several syringe pump designs are in testing now, all using the same comparison: one syringe pump contains the cells within an alginate solution, and the second contains calcium chlorite. When the two materials come in contact, the structure solidifies. Then you actually print a solid with embedded cells. Optimization is in progress.

Another challenge is deciding what cell type is needed. “Should we differentiate all the cells first and print the cells where we think they should go? Should we print undifferentiated cells and growth factors at the same time to let them differentiate and rearrange in situ?” The question is still open for D’haeseleer. The DIY group experimented with diverse cell types and did not recommend using carrot cells as people usually do. These stem cells are undifferentiated, which means they can give rise to different cell types under good conditions, but they are often contaminated.

Blob of hand-extruded layered alginate gel made at BioCurious. Photo by Maria Chavez

Other Groups Working on Bioprinting

BUGSS — Baltimore

Closeup of a photopolymer print made with BUGGS’ biocompatible resin. BUGGS

Baltimore Underground Science Space is currently building a platform call 3DP.BIO that aims to connect scientists, engineers, and designers to accelerate research and development. They focus on resin printers, developing the control software along with a biocompatible resin that can be used to make 3D scaffolds for cell growth.

London Biohackspace

JuicyPrint uses G. hansenii and juice to make useful shapes from bacterial cellulose. Photo by Alasdair Allan

The London Biohackspace’s JuicyPrint machine prints using the Gluconacetobacter hansenii, a bacteria that is easy to grow using fruit juice as a food source. G. hansenii produces a layer of bacterial cellulose, a strong and exceptionally versatile biopolymer. However, the bacteria have been genetically modified to make them unable to produce cellulose under a light source. By shining different patterns of light onto successive layers of the culture, the structure of the final product can be manipulated, resulting in useful shapes made of cellulose.

Pelling Lab

Pelling Lab’s “Apple ears” during the decellularization process. Photo by Andrew Pelling

Another way to grow tissues or organs would be to use an already existing 3D structure as a scaffold for cells. Andrew Pelling describes the process: “You slice an apple, wash it in soap and water, then sterilize it. What’s left is a fine mesh of cellulose into which you can inject human cells — and they grow.” His lab is now doing that to grow human ear prototypes.

Counter Culture Labs

Counter Culture Labs’ ghost heart only has connective tissue — all cellular material is removed. Photo by Patrik D’haeseleer

Why 3D print when you can use already shaped forms? Case in point, the surprising example of a pig heart project from Oakland, California’s Counter Culture Labs.

They make it by stripping out all the cells from a donor organ ­— a pig heart ­— leaving only the connective tissue to make it a “ghost” organ. Then, the idea is to repopulate it with the cells they want to grow.