3D Bioprinting Human Tissues, One Layer of Cells at a Time

In 2011, the Congressional Budget Office reported that over 1,000 American soldiers required an amputation, due in large part to improvised explosive devices. Some lost legs; gone are the feelings of an ocean washing over their feet. Others, an ear, binding them to auditory imperfection and forever altering their mirrored reflection. But must these losses last forever? The integration of stem cell science with new tissue fabrication techniques is tantalizingly close to achieving a feat seemingly pulled from the pages of science fiction. Can we regrow those soldiers’ limbs and ears?

Tissue engineers combine expertise in biomedical engineering, materials science, stem cell biology, and chemistry to construct functional biological tissues. These manufactured tissue structures are already used to test potential drug candidates, as 3D tissues mimic the real thing better than cells grown in a petri dish. Lab-grown tissue constructs can also infiltrate existing organs to patch-up injured body parts. Researchers have had success restoring tissue function after implanting lab-grown tissue for a variety of organs such as the skin [1] and the heart [2].

But the Holy Grail for tissue engineers is the manufacture of whole organs. According to the U.S. Department of Health and Human Services, there are currently over 120,000 people in need of an organ transplant. Organs are incredibly complex structures and notoriously difficult to reproduce, but 3D printing has proven to be a powerful new tool for the tissue engineer’s toolbox. Major breakthroughs continue to emerge from two of the leading research centers in 3D bioprinting - the Harvard Wyss Institute and the Wake Forest Institute for Regenerative Medicine.

Building Biology
3D printing, or “additive manufacturing,” has come a long way from the plastic trinkets it often brings to mind. Additive manufacturing can refer to several different techniques, all designed to produce three-dimensional objects from the ground up. Desktop 3D printers, like MakerBot, commonly melt small volumes of plastic filament as it is extruded through a heating element. The plastic re-solidifies as each subsequent pass of the printer-head deposits more material on top of the underlying layer. The position at which the printer chooses to deposit material is predetermined from computer drawings.

While additive manufacturing reduces material waste, the ability to design and create custom parts is what excites biomedical engineers. Patients have unique anatomical dimensions that make it difficult to use a one-size-fits-all model. The ease of creating one-off solutions allows far greater customization for patients. For example, a 3D printed, reconstructive jaw was made of titanium with dimensions obtained from a patient’s personal medical imaging data. And unique pharmaceutical needs have led to the development of the first FDA-approved, 3D printed pharmaceutical tablet.

But bioengineers have been ditching the plastic and titanium for a gelatinous mixture of cells and nutrients dubbed ‘bio-ink.’ Bio-inks must be tailored for each specific type of cell and each tissue structure. The gel material must be soft enough to match the host tissue but stiff enough to provide structural support during printing. Shooting bio-ink through a print-head nozzle improperly can shear fragile cells, and an imbalanced selection of nutrients and cell densities can impair growth. The trick is getting the right ratio of ingredients to enable structural integrity without killing the cells during printing.

Hyun-Wook Kang and his colleagues at Wake Forest University recently used their own bio-ink concoction laden with cartilage cells to print, layer-by-layer, a full-sized human ear [3]. Kang used biodegradable materials that gave the cells enough time to proliferate and form their own structural support while it slowly degraded away, leaving behind only the healthy tissue. Initial experimentation on mice showed that their 3D printed tissue structures developed a network of blood vessels and nerves - a monumental task that has proven to be a persistent struggle in the field. Tissue thicker than around 1mm typically won’t survive because oxygen and nutrients can’t reach all of the cells. This network of blood vessels was necessary to provide oxygen and nutrients throughout the entire ear to connect it to the rest of the body and keep the entire ear healthy. By intentionally leaving microchannels networked throughout the tissue structure using 3D printing’s intricate precision, these life-supporting tributaries flourished.

(Photo: Wake Forest Institute for Regenerative Medicine)

This accomplishment demonstrates the ability to create custom structures for each patient that can restore both cosmetic and functional applications. The near future may offer hope to the soldier returning home, marred from combat. After scanning the dimensions of a remaining ear, a 3D-printed replacement could be grown with perfect symmetry. After implementation, it would once again feel and hear as it once did.

Using a slightly different approach, researchers at the Harvard Wyss Institute used the precision and additive nature of 3D printing to print blood vessels directly into the tissue structure during fabrication. As each deposition layer passed, Kolesky and his team of researchers used an extra print-head filled with endothelial cells to periodically deposit rows of cells that would later form the walls of blood vessels [4]. This technique, like Kang’s method, also resulted in thick, viable tissue growth.

New approaches to printing biological tissue have not gone unnoticed in other industries. Modern Meadow, a company headquartered in New York, is developing 3D printed leather for consumer products like purses and belts. And although it is still not cheap or tasty enough for the market, Mosa Meats is one of a few companies creating lab-grown hamburgers. If 3D printed meat could be produced economically, the environmental and ethical issues plaguing the cattle industry may become a thing of the past.

Stop the Presses!
Although researchers have been making incredible strides in bioprinting technologies, organ manufacturing is not yet ready to print a liver and undo a lifetime’s worth of drinking. Most organs are still too complex to reproduce, and full functionality is yet to be seen. Beyond the technical hurdles, other issues may emerge. Lengthy and costly clinical trials for FDA approval will likely persist for decades, and legal debates on patent eligibility may also come to the forefront. Furthermore, Gartner research has predicted that 3D bioprinting “will ignite a major debate on ethics and regulation” [5]. Replacing a soldier’s lost limb seems benign – even morally just. But should we use this technology for human enhancement? Should we design and print muscles, bones, and lungs that are stronger, less brittle, and that oxygenate blood in ways that are superior to anything Homo sapiens have experienced before?

While it’s wise to encourage debate and ethically responsible science, the potential of 3D bioprinting is too great to let controversy stall progress. Tissue engineering has the capacity to replace lost limbs, repair diseased tissue, and even help the environment by improving the meat industry. The proper conditions for enabling cells to grow organs have proven difficult to recreate, but it won’t be long before organs become manufacturable. 3D printing has come a long way from creating plastic pencil holders - it may soon be saving your life.

Brian Bender (@BFBender, LinkedIn)
Guest Contributor, Signal to Noise Magazine
PhD, Bioengineering

Brian earned his doctorate in bioengineering at UCLA researching lab-on-a-chip systems for tissue engineering. He earned an MS in biomedical engineering at UCLA and a BS in materials science and engineering at North Carolina State University. Previously he has worked as a materials engineer for the DoD and in manufacturing engineering and product development roles at Corning. He currently works in academic technology transfer at UCLA and is building a RepRap 3D printer at home in his spare time...plastics only.

[1] Groeber, F. et al. Skin tissue engineering — In vivo and in vitro applications. Adv Drug Deliver Rev 63(4-5), 352-366 (2011).
[2] Radisic, M. and K.L. Christman. Materials science and tissue engineering: repairing the heart. Mayo Clin Proc 88(8), 884-898 (2013).
[3] Kang, H.-W. et al. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat Biotechnol 34(3), 312-319 (2016).
[4] Kolesky, D. et al. Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci 113(12) 3179-3184 (2016).
[5] Basiliere, P. et al. Predicts 2014: 3D Printing at the Inflection Point. Gartner (2013).