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Organ tissue printing is the layer-by-layer computer-aided biofabrication of functional 3-D tissues and organs. It is distinct from conventional tissue engineering as most existing techniques require the usage of a solid scaffold. This technology has high level of potential for manufacturing of viable, transferable organs.

Overview
This technique surfaced as modern computer-guided 3D printing developed. Layer-to-layer printing of biomaterial would not have been possible without the already developed computer guiding technology. From early modified inkjet printers, printing simple monolayer mammalian cells pattern, to custom made 3-D robotic dispenser that doubles as a bioreactor, organ printing has come a long way since the last decades.

Organ printing is a bottom-up approach which employs self-assembling tissue spheroids or cell aggregates. Unlike traditional tissue engineering, it is a solid-scaffold-free technology that provides with maximum cell density essential for efficient tissue assembly and maturation. The 2-D sheets of cells and ECM polymers are built layer by layer to form tissues, similar to additive photopolymerization process. This approach minimizes inflammatory responses upon implantation of the organ due to the cultured cells’ ability to grow own ECM and mediate cell-cell adhesion.

Traditional Tissue Engineering
The traditional tissue engineering is a top-down. approach which utilizes the biodegradable polymeric scaffolds that are populated with stem or differentiated cells for the development of targeted extracellular matrix and microarchitecture. The provided numerous regulatory factors and adequate molecular and structural environment increase sophistication and complexity of the scaffolds to mimic the targeted type of tissue. Yet, the biodegradable nature of the scaffolds leads to disruption of the natural organization of the neighbouring walls. Controlling the degradation rate still remains as a big challenge. Moreover, due to the non-automated assembly of the tissue constructs, the process is very slow and costly as it requires continuous regulation and control. The placement of cells remains imprecise as well especially during construction and vascularization of multicellular structures.

Organ Printing Over Scaffold Tissue Engineering
Organ printing on the other hand is fast and automated. It mimics embryonic development of tissue fusion during the post processing of printed tissue. It does not require a scaffold. The automated printing allowed for reproducible results. Its control over cell placements is significantly better than the slow cell distribution within scaffold base tissue engineering. Organ printing showed promising results towards vascularization of thick tissues while scaffold-based tissue engineering struggled. This is because with organ printing, structures are pre-vascularized in vitro while scaffold-based tissue engineering depends heavily on angiogenesis. Most importantly, organ printing has the potential to allow for mass production of robotic biofabrication of complex vascularized living human organs suitable for clinical implantation. This is something scaffold based technology will never achieve as it is slow and results are often non reproducible.

Procedure
Organ printing is a bottom-up approach. which employs self-assembling tissue spheroids or cell aggregates. Unlike traditional tissue engineering, it is a solid-scaffold-free technology that provides with maximum cell density essential for efficient tissue assembly and maturation. The 2-D sheets of cells and ECM polymers are built layer by layer to form tissues, similar to additive photopolymerization process. is approach minimizes inflammatory responses upon implantation of the organ due to the cultured cells’ ability to grow own ECM and mediate cell-cell adhesion.

General processes regarding most organ printing techniques


 * 1) Pre-processing: Creation of organ ‘blueprint’- conversion of computer-aided design  (CAD, put wiki link) created based on clinical bioimaging into a bioprinter friendly file for instructive software to print a patient-specific human organ.
 * 2) Processing: Actual printing and biofabrication- fusion of spheroid cells into tissue constructs
 * 3) Post-processing: Transformation of printed tissue construct into a functional organ for implantation; achieving adequate functionality and tissue maturation speed is aimed at this step

Tissue Spheroids in Organ Printing


The usage of tissue spheroids is one of the techniques for organ printing. Tissue spheroids are living materials that are analogous to aggregates of cells with evolving and controllable material properties. Organ printing uses tissue spheroids as building blocks to construct vascularized functional human organs. During human development, cells interact with surrounding cells via extracellular matrix and other signalling factors; their initial fusion driven by surface tension forces forms appropriate tissues, which eventually build into organs accordingly. Fluid-like tissue spheroids follow a similar mechanism and undergo tissue fusion when placed close to each other, mimicking the spontaneous self-assembly nature of the cells.

Production of Tissue Spheroids
Tissue spheroid production involves gravitational and hydrodynamic forces applied on single cells loaded on micromolded-wells in a non-adhesive hydrogel. To retain the self-assembly nature, large-scale production of uniformly sized tissue spheroids with appropriate incubation length is essential.

Organogenesis using organ printing requires millions of different types of tissue spheroids as building blocks. Thus, effective and flexible biofabrication of tissue spheroids is required. The current common methods are potentially automatable hanging drop culture and large-scale microfluidics-based biofabrication. The success of tissue spheroid fusion relies on the acceleration of tissue maturation, during when the initial fluid-like state of the spheroids are transited to solid-like state immediately following the fusion. Due to the absence of a solid scaffold, accelerated tissue maturation is critical to maintain shape, composition, and integrity. Studies have shown that incubation length of tissue spheroids is directly proportional to the degree of tissue cohesion, maturation, and extracellular matrix accumulation.

Application of Tissue Spheroids: Vascularization
Over the past few decades, effective vascularization of thick organ constructs has been a challenge in tissue engineering. However, the recent application of tissue spheroids on organ printing has given hopes to resolve the problem. Vascularization involves the formation of a complex network of perfusable endothelial walls and hierarchical branches with different diameters that are capable of transporting oxygen, nutrients, and waste product. Due to the complexity of the system, a combination of 3 types of vascular tissue spheroids are required for the construction of intra-organ vascular tree to derive branches with different sizes: solid (non-lumenized), mono-lumenized, and hystotypical micro-vascularized tissue spheroids. Solid and lumenized vascular tissue spheroids are mainly used for engineering small fragments of the branched vascular tree.

Challenges

 * Organ blueprint: Bioprinted soft tissues are subjected to postprocess remodeling due to fusion, thus organ blueprint design can not be directly extracted from 3D clinical images.  The blueprints of engineered structures have to consider processes such as compaction, remodeling, retraction and postprocessing fusion of the soft tissue in the printed organ.   The blueprint must account for the post processing changes of the printed structure.
 * In silico tissue self-assembly: Tissue self-assembly is essential for the production of functional organs. The dynamic aspect of the process makes it difficult to be accounted for in the computer aided designs (CAD).
 * Biopaper: Biopaper is functionally important to provides mechanical support for the development of the printed tissue. It has to be bioprocessible, biomimetic, biocompatible etc.  Different cell aggregates/spheroids require different hydrogel in order to survive and achieve appropriate fusion.   The challenge is to develop hydrogels for every tissue types in human biology.
 * Bioink: Bioinks have to survive the printing and post processing process. They also have to be different for different types of tissues. Some bioinks like the vascular tissue spheroids can be extremely complicated.
 * Bioprinters and bioreactors: bioprinters and bioreactors are both essential equipments for 3D bioprinting. Commercial, thermal-based printers are widely used but the modifications are challenging. Thermal printing has the potential to damage/lyse the dispensed cells since mammalian cells are highly heat and stress sensitive. Bioprinters also need to be programmable for various tissues/organs as well as self-sufficient. In addition, ink leakage and mist formation can be problematic during the printing process. In order to biofabricate complex mammalian organs, bioreactors are required to allow the maturation of vascular system. The special modifications of bioreactors for the 3D bioprinting from the traditional models are notably complicated.
 * Viability & vascularization: Cell viability is an ultimate issue to solve for 3D bioprinting. In order to obtain a functional and mature organ, cells need to stay alive to become a working organ. Preprocessing during cartridges loading, processing while printing, and postprocessing during fusion and maturation are the three particularly important periods. Cell viability is also related to the biopaper it is dispensed onto, the mixture of bioink, the condition of bioprinter etc. Due to the extreme complexity of vascularization, the success of vascular system printing comes with tremendous challenges. The development of the lumen, epithelial and endothelial of the vascular trees requires precise dispensing of the bioink and careful maintenance in the postprocsssing stage.

Other Possibilities
Although organ printing still has a long way to go before being medically viable, its possible contributions to existing studies are numerous. One of them would be the integration of organ printing to the existing scaffold based tissue engineering. Scaffold based tissue engineering research has been stalled by the inability to vascularize thick tissues. With organ printing, this issue might be resolved. Since scaffold based tissue engineering has been used medically for over a decade, the resolution of the vascularization of thick tissues can quickly introduce more engineered organs such as the liver and kidney.