Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and, most lately, bioprinting. To be successful in this endeavor, it is crucial to provide in vitro micro-environmental clues for the cells resembling those in the organism. Therefore, scaffolds, populated with differentiated cells or stem cells, of increasing complexity and sophistication are being fabricated. However, no matter how sophisticated scaffolds are, they can cause problems stemming from their degradation, eliciting immunogenic reactions and other a priori unforeseen complications. It is also being realized that ultimately the best approach might be to rely on the self-assembly and self-organizing properties of cells and tissues and the innate regenerative capability of the organism itself, not just simply prepare tissue and organ structures in vitro followed by their implantation. Here we briefly review the different strategies for the fabrication of three-dimensional biological structures, in particular bioprinting. We detail a fully biological, scaffoldless, print-based engineering approach that uses self-assembling multicellular units as bio-ink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion.

Tissue engineering tools and technologies are critical for regenerative medicine and the translational research supporting development of cell-based therapies. 3D cell bioprinting is a relatively new engineering tool being used to design 3D cell constructs (rather than cell suspensions) for transplantation therapies. In this review, we describe a broad range of printing technologies now being used to deliver cells and biomaterials in preclinical studies. We focus on 3D cell bioprinting, in which the building blocks (or ‘bioink’) used in printing process are three-dimensional cell structures, that are placed by the bioprinter into precise architectures to generate small tissues or organs. 3D cell bioprinting is a flexible research tool for basic and translational stem cell biology.

Tissue engineering is emerging as a possible alternative to methods aimed at alleviating the growing demand for replacement tissues and organs. A major pillar of most tissue engineering approaches is the scaffold, a biocompatible network of synthetic or natural polymers, which serves as an extracellular matrix mimic for cells. When the scaffold is seeded with cells it is supposed to provide the appropriate biomechanical and biochemical conditions for cell proliferation and eventual tissue formation. Numerous approaches have been used to fabricate scaffolds with ever-growing complexity. Recently, novel approaches have been pursued that do not rely on artificial scaffolds. The most promising ones utilize matrices of decellularized organs or methods based on multicellular self-assembly, such as sheet-based and bioprinting-based technologies. We briefly overview some of the scaffold-free approaches and detail one that employs biological self-assembly and bioprinting. We describe the technology and its specific applications to engineer vascular and nerve grafts.

Biofabrication of living structures with desired topology and functionality requires the interdisciplinary effort of practitioners of the physical, life and engineering sciences. Such efforts are being undertaken in many laboratories around the world. Numerous approaches are pursued, such as those based on the use of natural or artificial scaffolds, decellularized cadaveric extracellular matrices and, most lately, bioprinting. To be successful in this endeavor, it is crucial to provide in vitro micro-environmental clues for the cells resembling those in the organism. Therefore, scaffolds, populated with differentiated cells or stem cells, of increasing complexity and sophistication are being fabricated. However, no matter how sophisticated scaffolds are, they can cause problems stemming from their degradation, eliciting immunogenic reactions and other a priori unforeseen complications. It is also being realized that ultimately the best approach might be to rely on the self-assembly and self-organizing properties of cells and tissues and the innate regenerative capability of the organism itself, not just simply prepare tissue and organ structures in vitro followed by their implantation. Here we briefly review the different strategies for the fabrication of three-dimensional biological structures, in particular bioprinting. We detail a fully biological, scaffoldless, print-based engineering approach that uses self-assembling multicellular units as bio-ink particles and employs early developmental morphogenetic principles, such as cell sorting and tissue fusion.

Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cell–cell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication techniques for production of scaffold-free engineered tissue constructs have recently emerged. Here we report on a fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. Various vascular cell types, including smooth muscle cells and fibroblasts, were aggregated into discrete units, either multicellular spheroids or cylinders of controllable diameter (300–500 μm). These were printed layer-by-layer concomitantly with agarose rods, used here as a molding template. The post-printing fusion of the discrete units resulted in single- and double-layered small diameter vascular tubes (OD ranging from 0.9 to 2.5 mm). A unique aspect of the method is the ability to engineer vessels of distinct shapes and hierarchical trees that combine tubes of distinct diameters. The technique is quick and easily scalable.

Understanding the principles of biological self-assembly is indispensable for developing efficient strategies to build living tissues and organs. We exploit the self-organizing capacity of cells and tissues to construct functional living structures of prescribed shape. In our technology, multicellular spheroids (bio-ink particles) are placed into biocompatible environment (bio-paper) by the use of a three-dimensional delivery device (bio-printer). Our approach mimics early morphogenesis and is based on the realization that the genetic control of developmental patterning through self-assembly involves physical mechanisms. Three-dimensional tissue structures are formed through the postprinting fusion of the bio-ink particles, in analogy with early structure-forming processes in the embryo that utilize the apparent liquid-like behavior of tissues composed of motile and adhesive cells. We modeled the process of self-assembly by fusion of bio-ink particles, and employed this novel technology to print extended cellular structures of various shapes. Functionality was tested on cardiac constructs built from embryonic cardiac and endothelial cells. The postprinting self-assembly of bio-ink particles resulted in synchronously beating solid tissue blocks, showing signs of early vascularization, with the endothelial cells organized into vessel-like conduits.

Present efforts in tissue engineering are aimed at building living structures by employing the self-organizing properties of cells and tissues and automated technologies. One such technology is bioprinting that utilizes three-dimensional delivery devices for the rapid and accurate placement of biological materials into biocompatible environments, where post-printing self-assembly takes place. This Application article summarizes the scientific basis of this approach and some of the recent developments.

By examining the formative role of physical processes in modern-day developmental systems, we infer that although such determinants are subject to constraints and rarely act in a “pure” fashion, they are identical to processes generic to all viscoelastic, chemically excitable media, non-living as well as living. The processes considered are free diffusion, immiscible liquid behavior, oscillation and multistability of chemical state, reaction-diffusion coupling and mechanochemical responsivity. We suggest that such processes had freer reign at early stages in the history of multicellular life, when less evolution had occurred of genetic mechanisms for stabilization and entrenchment of functionally successful morphologies. From this we devise a hypothetical scenario for pattern formation and morphogenesis in the earliest metazoa. We show that the expected morphologies that would arise during this relatively unconstrained “physical” stage of evolution correspond to the hollow, multilayered and segmented morphotypes seen in the gastrulation stage embryos of modern-day metazoa as well as in Ediacaran fossil deposits of ~600 Ma. We suggest several ways in which organisms that were originally formed by predominantly physical mechanisms could have evolved genetic mechanisms to perpetuate their morphologies.

Organs form during morphogenesis, the process that gives rise to specialized biological structures of specific shape and function in early embryonic development. Morphogenesis is under strict genetic control, but shape evolution itself is a physical process. Here we report the results of experimental and modeling biophysical studies on in vitro biological structure formation. Experimentally, by controlling the interaction between cells and their embedding matrices, we were able to build living structures of definite geometry. The experimentally observed shape evolution was reproduced by Monte Carlo simulations, which also shed light on the biophysical basis of the process. Our work suggests a novel way to engineer biological structures of controlled shape.

COMPUCELL is a multi-model software framework for simulation of the development of multicellular organisms known as morphogenesis. It models the interaction of the gene regulatory network with generic cellular mechanisms, such as cell adhesion, division, haptotaxis and chemotaxis. A combination of a state automaton with stochastic local rules and a set of differential equations, including subcellular ordinary differential equations and extracellular reaction–diffusion partial differential equations, model gene regulation. This automaton in turn controls the differentiation of the cells, and cell–cell and cell–extracellular matrix interactions that give rise to cell rearrangements and pattern formation, e.g. mesenchymal condensation. The cellular Potts model, a stochastic model that accurately reproduces cell movement and rearrangement, models cell dynamics. All these models couple in a controllable way, resulting in a powerful and flexible computational environment for morphogenesis, which allows for simultaneous incorporation of growth and spatial patterning.