This October, publicly traded bioprinting firm Organovo Holdings announced that it has begun work on 3D printing human liver tissue for direct human transplant. If that doesn’t make you squeal with joy and ontological despair, then what will?
Once research and development has been completed, which should not be understated, the results of this work will take the form of a bioprinted liver “patch” that will be transplanted onto an existing liver in order to prolong the vitality of the diseased organ. These patches will represent a strategy to prolong the health of patients currently stuck on transplant waiting lists. The holy grail of this research, however, is the 3D printing of a fully functional liver, with a patient’s own cells, that can be transplanted directly into the body of someone suffering from liver failure.
So far, Organovo has demonstrated the ability to 3D print liver and kidney tissues, but what does it take to 3D print a liver patch, let alone an entire organ? Let’s dive into the perplexing world of playing god with bioprinting.
3D Printing Human Tissue
Among the first commercially available bioprinters was the industrial-grade 3D-Bioplotter from EnvisionTEC, but new start-ups, like BioBots and CELLINK, are introducing systems for as little as $10,000. These platforms often rely on an X-Y-Z gantry system that deposits biomaterials, such as water-based gels known as hydrogels, from syringe-style printheads.
While some materials printed with bioprinters can be used more immediately after printing, others require placement in an incubator before they are functional. For instance, hydroxyapatite, the natural occurring mineral that makes up the majority of the material in our bones, can be 3D printed as a bone implant and directly integrated into a body. Hydrogels seeded with stem cells, in contrast, act as scaffolds on which stem cells grow and proliferate into specific organic tissues.
There are now a number of companies and research labs evolving the tissue engineering technology known as bioprinting. Among the pioneers in the field is the Wake Forest Institute for Regenerative Medicine, which was the first lab to actually grow and successfully implant a working bladder into a human in 2006. Wake Forest is intent on creating tissues and therapies for over 30 different areas of the body, including a process for 3D printing skin directly onto the wounds of soldiers, as seen in the video below.
A number of others have made amazing progress in the field, such as the Shah Tissue Engineering And Manufacturing (TEAM) Lab, which bioprinted ovaries and successfully transplanted them in mice, but Organovo stands out as the first publicly traded bioprinting company.
Organovo first approached the market with the NovoGen MMX Bioprinter, a system developed in house for bioprinting human tissues. The company has since shifted its business model toward the tissues themselves. Maintaining the bioprinter in house, Organovo began producing liver cell arrays to be sold to pharmaceutical companies and researchers for drug testing and discovery.
3D Printing Liver Cells
Though Organovo was founded in 2009, the company first published results of its bioprinted human tissues in 2013, when the company announced the successful printing of viable liver cells.
To print the cells, the firm used its NovoGen technology, filling one syringe with an ink made up of spheroids, which contained tens of thousands of parenchymal hepatocytes (liver cells that make up 70 to 85 percent of the liver volume). Another syringe was filled with non-parenchymal liver cells (which make up 6.5 percent of the liver volume) to encourage cell growth and a hydrogel.
The second syringe first printed a honeycomb-shaped mold before the first syringe filled each hexagonal compartment of the mold with parenchymal cells. The resulting print was then placed into an incubator, where the hepatocytes proliferated to become 20 cells thick, measuring about 500 microns. With this result, Organovo demonstrated the ability to create thick, multicellular liver cells that actually possessed critical liver functions, such as the production of the proteins albumin, fibrinogen and transferrin, as well as enzyme activities in the biosynthesis of cholesterol.
From there, Organovo expanded its research, further proving the viability and usefulness of its liver tissues. The bioprinting firm was able to keep the cells alive for 40 days and, through partnerships with pharmaceutical companies like Roche, introduced acetaminophen, known for its toxic effects on the liver, to the cells and demonstrated that its 3D-printed liver arrays could be used to indicate drug toxicities in the liver.
By 2014, Organovo was able to commercialize the product fully, releasing the ExVive Human Liver Tissue to the market. The company is now able to 3D print batches of 24 liver cell arrays measuring 3 mm in diameter and 0.5 mm in thickness in about 45 minutes.
While these simple cellular arrays may not seem as immediately essential as a complete liver, the product was meant to speed up drug discovery for researchers in the pharmaceutical industry. The three-dimensional nature of the liver cells makes the array much more closely resemble the actual physical environment of the liver so that, not only do the cells survive longer, but they actually exhibit more accurate liver function than the 2D cell cultures typically used. For instance, when initially announced, the 3D-printed liver cells produced five to nine times the albumin production of 2D cellular arrays.
By providing researchers with more accurate representations of the liver with which to test new drugs, it may be possible to bring life-enhancing medications to market more quickly, as these medicines can move from animal models to human clinical trials with greater understanding of the effects they will have on humans. At the same time, it’s possible that drugs with negative effects will be rooted out more easily so that toxic medications don’t have to be recalled after they’ve already been released on the market.
Organovo then went on to announce the ability to 3D print kidney cells, commercializing its kidney proximal tubule model in September 2016. These cells exhibit functions associated with the proximal tubule area of the kidney for over four weeks and could be used to test kidney toxicity. Though the names have not been disclosed, Organovo claims that two of the top 25 pharmaceutical companies have ordered the ExVive Human Kidney Tissue as part of an early access program.
From Cells to Organs
The jump from viable cells to complete organs is no easy task. While the aforementioned proximal tubule may be a useful tool in testing drug toxicity, those cells alone are not enough to replicate a functioning organ.
Dr. Susan Hou, a leading nephrologist at Loyola University Medical Center who also happens to be this author’s mother, explained that the kidney itself is “made up of filtering units called glomerulae,” which filter the blood as the main function of the kidney. According to Hou, without glomerulae, it’s not possible to recreate the function of the kidney.
In other words, Organovo may still have a lot of work to do before it can create a complete functioning organ. In addition to the ability to culture other cells vital to the functioning of an organ, there lies the issue of getting these cells to coexist. To 3D print a liver, it’s not as easy as 3D printing the hepatocytes and then 3D printing the non-parenchymal cells (made up of the Kupffer, stellate, and liver sinusoidal endothelial cells) next to each other and waiting for them to produce bile.
The biggest obstacle to producing more complex tissues that are made up of multiple types of cells is the ability to bring nutrients and oxygen to the cells in order to keep them alive. To achieve this, it’s necessary to vascularize the tissue in order to pump blood to the cells. Typically, 3D-printed vasculature has not been robust enough to function within thick layers of tissue.
Numerous researchers are attempting to address the problem of pumping blood to bioprinted tissues, but among the farthest along may be Jennifer Lewis and her lab at Harvard’s Wyss Institute for Biologically Inspired Engineering. This March, Lewis’s team was able to fabricate thick tissue with an embedded vascular network. Within a 3D-printed silicone mold, her team printed a grid made up of a copolymer that turns liquid at 4 °C and an ink made up of gelatin, human blood cells and fibrinogen protein.
Once the network was printed, the copolymer is cooled down to a liquid state and flushed out, leaving behind a series of channels. Then, a liquid made up of fibroblasts and extracellular matrix was introduced to fill the areas between the 3D-printed tissue. Finally, bone growth factors were pumped through the channels, causing the stem cells to differentiate into bone marrow. Due to the vasculature, the researchers were able to feed nutrients to the 1-cm thick tissue, a remarkable size for bioprinting, enabling it to survive for over 100 days.
Lewis believes that this platform can be modified for a wide variety of shapes and tissues, and is in the process of exploring the viability of such a 3D-printed structure in animal models.
Wake Forest, too, has demonstrated great success with the fabrication of vascular channels within human tissue. Rather than printing a wash-away material to form the vasculature, the Wake Forest team 3D prints its hydrogel along with a stiffer material for structural support, while leaving empty the tunnels that will become the ultimate vessels.
With this technique, the team was able to create two human-sized ears made from cartilage, which were then implanted under the skin of mice. After two months, the blood vessels had formed within the ears. The team then went on to perform similar studies with muscle tissue and jaw bone fragments, which also demonstrated vascularization.
3D-Printed Liver Patches
Once vascularization has been achieved, there are still a number of milestones to achieve on the road towards 3D printing a complete liver. Between liver arrays and an entire organ, there is still the opportunity for presenting viable treatments for medical conditions.
A liver transplant is required in cases where the liver has failed and needs to be replaced, but Organovo believes that 3D-printed liver patches could both be used to treat certain liver diseases and to provide the approximately 17,000 patients currently on U.S. organ transplant waiting lists with a few more viable years before they receive a transplant.
Eric Michael David, chief strategy officer and executive vice president of Preclinical Development for Organovo, said of the company’s first therapeutic product, “Organovo’s approach is designed to overcome many of the challenges that cell therapies and conventional tissue engineering have struggled to address, including limited engraftment and significant migration of cells away from the liver. In our preclinical studies, we deliver a patch of functional tissue directly to the liver, which integrates well, remains on the liver and maintains functionality. We believe our tissues have the potential to extend the lives of patients on liver transplant lists, or those who do not qualify for transplants due to other factors.”
Dr. John Geibel, vice chairman of Surgery at Yale University, said of the potential of organ patches, “There are many conditions in areas such as liver, kidney, gastrointestinal, vascular, and lung disease where supplying a tissue patch may be curative, or bridge a patient a few more years before they need a transplant. The promise of 3D bioprinting human tissues to address these unmet needs is significant.”
According to the company, Organovo has already seen strong results in animal models, with implants exhibiting engraftment, vascularization and functionality. As Organovo continues research, the firm plans to submit an Investigational New Drug application to the U.S. Food and Drug Administration in three to five years, meaning that the company anticipates having the product ready for clinical trials by that time.
Estimates of when we can expect complete organs vary widely. For instance, 3D Bioprinting Solutions in Russia has claimed to 3D print and implant functional organoids, like the thyroid gland, and expects to be able to 3D print a functional kidney by 2018. Ibrahim Ozbolat, a professor of Engineering Science and Mechanics at Pennsylvania State University, suggested that complex organs could be bioprinted in the next 10 to 15 years, though he’s not sure if actual transplants in humans will occur quite so soon.
Sharon Presnell, Organovo’s chief scientific officer, was a bit less specific, telling ACS Central Science in a recent interview, “When will a surgeon open a box and find a fully formed printed kidney? I think I will see that in my lifetime.”