Organovo's NovoGen MMX BioprinterThe human cell represents the smallest functional unit of life; it is able to absorb oxygen and nutrients, produce life-sustaining proteins, undergo cell division and replicate, and adapt in response to its microenvironment. All tissues in the body are composed of multiple cell types, typically arranged in a three-dimensional architecture that is relevant to the functions they carry out. For example, our bodies contain many tubular structures composed of a multi-layered wall surrounding a lumen through which some biological substance (blood, urine, air, etc.) passes, and solid tissues have patterns as well, such as the repeating hexagonal functional units (lobules) of the liver, or the distinct layers present in the skin or retina. Since cells were first isolated and grown in the laboratory environment, biologists and engineers have pursued the utilization of these tiny building blocks of life in the reconstruction and regeneration of functional tissue. Whether used in a controlled laboratory setting to model specific diseases and test the effects of drugs, or delivered into the body as therapeutics for the treatment of disease, the common goal is to establish (or re-establish) in vivo-like function.

The field of Tissue Engineering, an interdisciplinary field focused on building or regenerating functional three-dimensional tissues, has deployed several fabrication strategies aimed at bringing cells and structure together to generate tissue. Biomaterial scaffolding, which provides structural support and can be formed into biologically relevant shapes, has been combined with cells to generate hybrid 3D structures that can be used as tissue surrogates in vitro and in vivo.1 Protocols have been developed that enable removal of living cells from native tissues, leaving only a natural scaffolding of extracellular matrix, which can then be re-seeded with cells to reconstruct or partially reconstruct 3D tissues.2 Another approach to soft tissue reconstruction has been the development of cell-laden hydrogels, which are often cast into a specific shape and placed into a permissive environment in vitro or in vivo that allows maturation and establishment of tissue-specific characteristics.3 In recent years, with the advancement of 3D printing technologies for the on-demand fabrication of complex polymer-based objects, efforts have been underway to adapt 3D printing technologies and engineer bioprinting instruments that can leverage similar 3D replication concepts and accommodate the incorporation of living cells.

First-generation 3D prototyping techniques relied on subtractive processes—the removal of material from a solid block using filing, milling, drilling, cutting and grinding methods. Advanced 3D prototyping technologies utilize additive processes in which the desired part is built up—or “printed” layer by layer. Objects of virtually any shape can now be fabricated from a fairly wide range of non-biological materials using additive technologies such as stereolithography (SLA), fused deposition modeling (FDM), selective laser sintering (SLS), laminated object manufacturing (LOM), and digital light processing (DLP). Additive manufacturing generally involves using computer assisted design (CAD) software to design the desired part. The 3D drawing of the newly designed part is then sliced apart layer by layer and the appropriate printer drive commands are created using computer methods and used to control the print job. Objects can be created very rapidly using additive methods and intricate, detailed features that would have been difficult or impossible to machine are often easily incorporated.

The power and utility of 3D printing in the non-biological materials area has sparked the imaginations of biologists and engineers alike and fueled research and development activities aimed at producing intricate biological 3D structures. Consequently, precise, automated, layer-by-layer fabrication of tissue (i.e. Bioprinting) is now possible using only living cells as building blocks. This is resulting in simultaneous achievement of unique features such as true three-dimensionality, tissue-like cellular densities, and reproduction of native tissue architecture through the spatially directed placement of distinct cell types.

Bioprinting hardware requires unique features that ensure success at the interface of engineering and biology; low-shear deposition mechanisms are essential to maintaining viability and function of living cells; fabrication speed must be rapid enough to prevent destruction of the bioprinted tissue due to nutrient or oxygen deprivation; all components that contact living cells must be non-toxic and either disposable or sterilizable to prevent cross-contamination between runs, and tissues must be generated in a format that enables them to be manipulated and used in application(s) after fabrication. Bioprinting combines the synergistic potential of engineering and biology to create living human tissues that mimic the form of native tissue and, therefore, achieve unique tissue-specific metabolic functions.

Organovo’s NovoGen MMX Bioprinter precisely dispenses “bio-ink”—tiny building blocks composed of living cells—generating tissues layer-by-layer according to user-defined designs. Built for flexibility, the NovoGen Bioprinter enables fabrication of tissues with a wide array of cellular compositions and geometries; side-by-side comparison of multiple tissue prototypes facilitates optimization and selection of specific designs geared toward a particular application. Working within the confines of an object library, bio-ink building blocks of various shapes, sizes, and compositions are assembled into architectures that recapitulate the form of native tissue. Tubes, layered sheets, and patterned structures have been bioprinted, yielding three-dimensional tissues that are free of biomaterial scaffolding and characterized by tissue-like microarchitecture, including the development of intercellular junctions and endothelial networks.

In the short-term, 3D human tissues are being deployed in the laboratory setting as models of human physiology and pathology; cell-based assays are a mainstay of the drug discovery and development process, and multi-cellular / multi-tissue systems may serve as more predictive indicators of clinical outcomes. Longer-term applications of 3D tissue technologies will extend our knowledge of how to build the smallest functional units of a tissue to the fabrication of larger-scale tissues useful for surgical grafts to repair or replace damaged tissues and organs in the body. What are the next steps in the evolution of bioprinting? Scaling up and down—increasing the resolution of specific features while advancing fabrication hardware and techniques to produce larger-scale tissues. Enhancing the complexity of designs—building the tool set that enables conceptual or visual inputs to be translated rapidly to executable bioprinting programs that select from a library of bio-ink building blocks to translate the vision into reality. Driven by a myriad of unmet needs and opportunities, 3D bioprinting promises to be a rapidly evolving field for years to come.

1 Chan, B.P., et al. Eur Spine J 2008 17:S467
2 Badylak, S.F., et al. Ann Rev Biomed Eng 2011 13:27
3 Li, Y., et al. Chem Soc Rev 2012 41:2193