Cell prints

US-based medical R&D company Organovo produces Novotissues, which are living human tissues, by using 3D bioprinting. The company expects to launch its first product – 3D human liver tissue – in 2014.

Recently, Organovo demonstrated that its 3D liver tissues retain key liver functions, including production of liver-specific proteins, such as albumin and transferrin, and biosynthesis of cholesterol, comparable with human liver functionality for up to 40 days and exhibit dose-dependent responses to paracetamol [acetaminophen], a known liver toxicant; the toxic effects can be assessed using both standard screening assays and histopathological assessment.

The fact that these tissues demonstrate similar activity to native liver when presented with a known drug is an encouraging indication of their use in drug development, according to Michael Renard, executive vp of commercial operations with Organovo.

‘Our 3D tissues offer several advantages over standard cell-culture platforms because they are constructed purely of human cells found in native tissues and contain the different cells types, and can be precisely placed to have cell density and microvascular networks similar to native tissues. In addition, automated 3D printing enables the highly reproducible production of the tissues,’ Renard notes.

James Yoo, professor of regenerative medicine at Wake Forest Institute for Regenerative Medicine, in  North Carolina, US, points to the precision of 3D bioprinting as its main advantage over conventional tissue engineering methods. He explains: ‘The way tissues are often currently engineered is to fabricate a scaffold using biocompatible materials and place cells manually. In contrast, with 3D printing, the printer precisely places cells and biomaterials to create tissue constructs in a controlled manner. It is likely that 3D printing will one day play an important role in tissue engineering because it enables the construction of a tissue that is similar in configuration to native tissue.’

‘Access to consistent, human tissues for drug assays will have a huge impact not only on reducing drug development times, but also on increasing the reliability of the screening process, and thus reducing the failure rate for drug candidates,’ adds Andrew Hessel, a researcher with the software company Autodesk, based in Hampshire, UK, which developed computer-aided design (AutoCAD) and other 3D design software.

Researchers at Wake Forest are currently working on printing tissues that include nose and ear structures (cartilage), muscle, bone and trachea tissue. One of their long-term projects is to print a human kidney. They have already shown it is possible to form a mini-kidney that, when implanted in a castrated bullock or steer, produced a urine-like fluid.

Organovo, meanwhile, is working with several different partners to develop both healthy- and disease-tissue models, including breast and prostate cancer tissues. So far the company has bioprinted tissues, such as heart muscle patches, nerve grafts, blood vessels for bypass, and tissues with shapes: tubes, sheets, clusters of globules etc, using the current technology.

The 3D bioprinting process comprises three stages: (i) selection, culture, expression and growth of the appropriate human stem cells that can differentiate into the cell types required for a given tissue type; (ii) formulation and printing of the bioinks containing these stem cells, layer-by-layer, onto a matrix, typically a hydrogel, up to ~500 um-thick, which is controlled by a computer; and (iii) feeding the formed pre-tissue with nutirents to encourage growth. ‘We didn’t coin the term, but we now refer to such bioprinting as a 4D printing process, with the fourth dimension being time, because the created tissue is not static and continues to change even after the cells are deposited,’ Hessel comments.

One of the key features of this process, according to Renard, is the role of developmental biology. ‘Once a small aggregate of cells is created with the correct placement and arrangement of the cells, then they will naturally do what they are supposed to do and begin communicating with one another, organising into the targeted tissue type,’ he says.

‘Overall, the goal of our 3D printing projects is to engineer tissues that can be used in reconstructive procedures or to replace diseased organs in human patients,’ says Yoo. ‘We are in the early phases of research, though, so it is premature to predict when products may be commercialised. It is safe to say, however, that solid and complex organs such as the kidney present many challenges and will take some time.’

There are a number of significant hurdles that must be overcome before larger tissues and whole organs can be produced via 3D bioprinting. One of the key issues is the need to create a vascular network for blood and nutrient supply and waste removal, which has yet to be accomplished, according to Renard. ‘The right cells must be found and grown to resist the forces of the printing process and then deposited in the right place in order to enable the cell-cell signaling that will lead to network construction,’ he observes. While many new types of adult human stem cells have been discovered recently, there is a need for additional types, as well as the development of methods for obtaining high quality cells suitable for the printing environment.

The team at Wake Forest, for example, is taking on the challenge of providing oxygen to engineered tissues and organs. ‘We are pursuing several strategies to address this issue, including refining delivery systems, developing biocompatible biomaterials that might be used to print blood vessels, and using oxygen-generating materials to provide a temporary oxygen supply,’ says Yoo.

Furthering our understanding of biological development will be critical to success in this area, according to Anthony Vicari, an analyst with US market research firm Lux Research. Hessel agrees, adding: ‘Complex cell and tissue development does not naturally occur outside of the body. There is much we still need to learn about keeping the cells alive, enabling them to grow, and the process of cell differentiation.’

Hessel also notes that the 3D printers used for bioprinting are first-generation systems, and there is much opportunity for improvement. ‘The advances in technology related to 3D bioprinting are occurring at an exponential rate, and there are many projects focused on developing better printers. Existing software tools can take data from magnetic resonance imaging (MRI) and computed tomography (CT) scans for use as model designs for 3D bioprinting. We just need 3D bioprinters that can process and utilise the information. Fortunately, there is tremendous investment in the field, and the prospects are quite exciting,’ he adds.

Software is another area open to development. Autodesk, for example, is partnering with Organovo to develop 3D design software for the bioprinting of more complex tissues, and ultimately organs. The hope is that the improved software will increase the usability and functionality of bioprinters and make it possible for biologists without software backgrounds to pursue 3D bioprinting projects, thus opening up the technology to a broader group of users, says Renard.

To encourage more widespread development of 3D printing techniques, Autodesk has launched Project Cyborg, a cloud-based platform of design tools for programming matter, including tissue engineering. ‘The idea is provide access to software tools for 3D printing of all types to a wider array of researchers, developers, and students and potentially open up the opportunity for hybrid 3D printing of mechanical structures and cellular material integrated with electronics,’ Hessel explains.

The software tools enable design at the nanoscale/molecular level, and make it possible to create new systems through the combination of 3D bioprinting of cells (synthetic biology) and nanomaterials. Such systems might, in the future, include fully functioning replacement limbs comprised of 3D-printed cells, micromachines and microelectronics.

The printing of hybrid structures is also an area of interest for Yoo, whose team has designed a hybrid printing system as a way to print a durable cartilage structure. Constructs made of gels that promote cell growth and synthetic materials that provide strength were fabricated and then inserted into mice for two, four and eight weeks to see how they performed in a real life system. ‘After eight weeks,’ according to Yoo, ‘the constructs appeared to have developed the structures and properties that are typical of elastic cartilage, demonstrating their potential for insertion into a patient.’ Based on this concept, the team later developed a robotic 3D system that allows for the printing of multiple cell types and biomaterials.

The original founder of Organovo, Andras Forgacs, has since founded another company that will use 3D bioprinting in non-medical applications, specifically the production of synthetic meat and skin.

The cultured leather and meat products manufactured using such advanced tissue engineering techniques require no animal slaughter and have much lower inputs of land, water, energy and chemicals, according to the company. Importantly, these products are simpler to construct and are not intended to be living materials. Therefore, commercialisation of such technology should occur more rapidly and there should be fewer regulatory hurdles to overcome.

Many of the applications of 3D bioprinting are still at the R&D stage and as Yoo says: ‘We are always working on new ideas, and though it is hard to predict how things will progress , my guess is that 10 years from now, we’ll be doing things we wouldn’t have imagined today.’ 

Lee Cronin, regius chair of chemistry at the University of Glasgow, UK, has a vision for 3D printing that could significantly impact the drug discovery and development process. ‘We have shown that it is possible to use 3D printing to create specifically designed microreactors and use them for what we call “configurational chemistry”, in which we control the reaction by controlling the shape and size of the reactor and the timing of the interaction of the reagents,’ he notes.

A sealant is used to print microreactors with specific geometries. Once the sealant sets, the chemical inks containing the reagents are injected in a defined order and at defined intervals. The printer can be programmed to create various reactor shapes and inject a set of standard inks in different ratios, orders, and at different rates. Thus, using 3D printing, it is possible to create a wide range of very specific reaction conditions that influence the outcome of the reaction and lead to the production of a large number of different products. As a result, the reactor becomes a dynamic parameter, according to Cronin.

He envisages the combination of such a 3D printed microreactor system being used to synthesise combinatorial libraries of active pharmaceutical ingredients, with the 3D printing of formulated drug matrices and tissues for testing of the formulated therapies. ‘We are very excited about the triangular development of tissue engineering with synthetic biology and configurational chemistry. Such a 3D printed system would enable the synthesis, formulation and screening of new drug candidates in one system, which could dramatically speed up drug discovery.’