Successful organ printing relies heavily upon advancements in stem cell technology as native tissues and organs are heterocellular. Acquisition of functional primary cells from patients with visceral dysfunction such as liver or heart failure or other devastating diseases such as type I diabetes as autologous beta (β) cells is not possible. Stem cells, which are found in several tissues in human body, can self-renew to produce more stem cells and have the remarkable potential to differentiate into diverse specialized cell types to form various organs. A variety of cell types can be used for this application, such as embryonic stem (ES) cells (Thomson et al., 1998), adult stem cells (ASCs) (Baglioni et al., 2009), iPSCs (Takahashi et al., 2007), human adipocyte stem cells (ADSCs) (Gimble et al., 2007), and tissue-specific cell lines (Pan et al., 2009). Although ES cells are pluripotent, their availability has been hampered by controversial ethical concerns due to their derivation from early embryos (Wert, 2003). Induced pluripotent stem cells have eliminated those issues, as somatic cells from patients can be easily isolated with minimally invasive procedures such as skin biopsy. The obtained skin fibroblasts can be reprogrammed into adult cells from all three germ layers in vitro, which have a great potential in regenerative medicine for cell therapy applications or tissue or organ fabrication. Reprogramming of somatic cells to obtain iPSCs, depending on the method used, may pose substantial risk and thus limit their clinical translation. Human adipocyte stem cells also eliminate the ethical concerns as abundant amount of adipose tissue can be easily isolated from subcutaneous adipose deposits with safe, minimal invasive procedures in an outpatient setting. Additionally, adipocyte stem cells retain pluripotency in vitro (Bunnell et al., 2008). Thus these stem cell types provide a wide range of cell sources for organ printing; however, there are still some impediments with their use. Although autologous stem cells can be differentiated into organ-specific cells for organ printing, there is still a risk of tissue rejection by the recipient (Lanza et al., 2007). Phenotypic behavior of stem cells can even change during the bioprinting process. In addition, organ fabrication requires various types of organ-specific cells, which is not currently feasible with current stem cell isolation and differentiation technologies. Although stem cells offer great promise as an unlimited source of cells for organ printing, a greater understanding of and control over the differentiation process is required to generate expandable organ-specific cells of consistent quality with the desired phenotype and genotype thereby minimizing organ rejection by the recipient posttransplantation. In addition, immunobarrier devices or molecular level interventions in the form of DNA modifications are essential to overcome immunologic rejection of organs. Moreover, imaging modalities such as positron emission tomography and nuclear magnetic resonance (NMR) imaging should be used to monitor the functionality of seeded stem cells noninvasively; NMR offers a unique advantage in monitoring organ integrity and cell function without the need to modify the cells genetically (Stabler et al., 2005).

Although current stem cell technologies can expand stem cells into adequate numbers for laboratory experiments, future organ printing technologies will require scale-up manufacturing of stem cells for larger-scale human tissues for transplantation and generation of tissue samples for pharmaceutical use. To attain sufficient numbers of cells for human organ printing, advanced bioprocessing technologies should be implemented to enable practical expansion of stem cells in compliance with good manufacturing practices with appropriate quality assurance measures, bioprocess monitoring control, and automation (Placzek et al., 2009). Each stem cell type has a different expansion potential. Embryonic stem cells have unlimited expansion capacity; however, other stem cells do not. For example, mesenchymal stem cells (MSCs) from young donors have 40 population doubling; whereas MSCs from older donors are limited to 25 population doubling (Stenderup et al., 2003). As previously discussed, the use of ES cells have other issues such as ethical considerations while MSCs have other limitations such as the need for an invasive procedure to obtain a limited sample volume as they are primarily isolated from bone marrow. Therefore other stem cells such as iPSCs and ADSCs stand as promising cell sources that can be obtained in large quantities from abundant tissue volumes isolated using minimally invasive procedures. After isolation, stem cells should be plated and expanded in appropriate culture conditions. In general, expansion culture environments are maintained at 5% carbon dioxide (CO₂), 20% oxygen, 37°C, and pH 7.4; however, growth and differentiation of different stem cells may vary under different conditions. For example, it has been demonstrated that a lower pH level of 7.1 increased the expansion of megakaryocyte progenitor cells (Yang et al., 2002). Large volumes of cells need to be expanded in a bioreactor culture with appropriate nutrient and waste exchange, accommodations for high cell density, and a continuous monitoring and feedback mechanism. For organ printing, two different strategies can be used in loading stem cells including (1) bioprinting differentiated stem cells during the organ printing process or (2) bioprinting predifferentiated stem cells followed by differentiation into organ-specific cell lineages within appropriate sectors of the printed organ constructs. The former approach is preferred over the latter one, in general, as differentiation of multiple cell types into different lineages is extremely challenging requiring spatiotemporal delivery of growth factors or plasmid DNA (Ozbolat and Hospodiuk, 2016).

Upon expansion to sufficient numbers, cells are processed for bioink preparation. Depending on the bioink type utilized, such as hydrogels, cell aggregates, microcarriers, or decellularized matrix components as discussed in Chapter 3, different cell quantities are required. Quality assessment of the cells is essential to ensure that their purity, phenotype, genotype, and functionality are acceptable.

To bioprint an organ construct, a blueprint model is essential to guide the bioprinting process. Organ bioprinting processes are complex compared to the bioprinting of cells within bulk- or lattice-shaped hydrogel scaffolds. These endeavors include scaffold-free bioprinting (Yu et al., 2016; Norotte et al., 2009), integration of scaffold-free approach with vascular network (Yu et al., 2014), a scaffold-based approach with integrated vascular network (Zhang et al., 2013a), and the use of a hard polymeric frame to support cell-laden hydrogel sections (Kang et al., 2016). All these approaches are highly complicated in their construction, where multiple materials need to be bioprinted, hence requiring a complex blueprint model. Such complex blueprint models can be developed using one or more of available techniques such as CAD-based systems, image-based design, freeform design, implicit surfaces, and space filling curves as discussed in Section 2.3. For scaffold-free approaches, particularly, where the bioprinting process guide the tissue self-assembly, tissue remodeling, fusion, and contraction are commonly observed, resulting in significant deviations from the originally bioprinted construct. Therefore the blueprint model and the associated toolpath plan should compensate for the postbioprinting changes.

Upon generation of the blueprint model for the target organ, a process plan should be performed to determine how to bioprint the organ construct including its compartments and components, such as stromal, parenchyma, blood vessels, and support material. An appropriate toolpath plan is thus essential to provide the bioprinter with information about the robot motion and the deposition patterns so that exact deposition of different bioink constituents can be determined. This information is then transferred from toolpath planning software to the machine control software via digital signals that control the motion and dispensing mechanisms, such as actuation of motors and air pressure, respectively (Ozbolat et al., 2014). For toolpath planning, both Cartesian- and parametric-based approaches, as discussed in Section 2.5, can be considered. As organ printing may necessitate bioprinting multiple bioink materials or even the support material, Cartesian-based toolpath planning is preferred due to its simplicity.

At this step of organ fabrication, organ constructs can be 3D bioprinted using any single or combinations of existing bioprinting modalities including EBB, DBB, and LBB as presented in Chapters 4–6, respectively. As EBB modality facilitates fabrication of 3D constructs at the clinically relevant volumes, it is currently preferred over other methods, but can also be integrated with others if required. To print organs at clinically relevant volumes, robust technologies and protocols should be developed to enable bioprinting of vascular constructs in multiple-scale ranges from arteries and veins down to capillaries. Since it is difficult to print capillaries at submicron scale using the current bioprinting technology, one alternative strategy can be bioprinting the macrovasculature with the expectation that the interconnecting capillaries will be formed on their own (Yu et al., 2014). In this regard, two alternative approaches have been considered in the literature including indirect bioprinting utilizing a fugitive ink that is removed by thermally induced decrosslinking leaving a vascular network behind (Kolesky et al., 2014; Lee et al., 2014a,b) and direct bioprinting of vasculature network in tandem with the rest of the tissue constructs (Yu et al., 2014).

After the organ bioprinting process, as discussed previously, the fabricated construct needs to be transferred to a bioreactor for long-term culture to facilitate cell growth and proliferation, vascularization, and organ maturation. A bioreactor can be defined as a system in which the conditions are closely controlled with respect to physiological conditions to induce certain behavior in living cells, tissues, and organs (Korossis et al., 2005). Three major bioreactor types have been used in culturing engineered tissue constructs including static and mixed flasks, rotating wall, and perfusion bioreactors (Gaspar et al., 2012). Mixed flask culture or rotating wall utilizes a convection mass transfer mechanism; however, perfusion bioreactors enable both convection and diffusion-mediated mass transfer. Complex-organ systems such as decellularization-based approaches or bioprinted vascularized organ constructs require perfusion culture that forces culture medium through the vascular inlets and collects it from outlets using a suction mechanism. The ideal bioreactor should provide the appropriate physical stimulation to cells, a continuous supply of nutrients (e.g., glucose, amino acids) and removal of by-products of cellular metabolism. The bioreactor should also facilitate sufficient convection and diffusion of biochemical factors and oxygen, as well as provide mechanical stimuli to induce mechanotransduction and the appropriate deposition of ECM proteins with controlled orientation and structure. In addition, such a bioreactor should maintain aseptic conditions, allow for a high media volume to tissue construct volume ratio, fail to generate any toxic products, and be easy to clean after the culture period. In general, different organ and tissue types, possessing different anatomical, biological, mechanical, and structural characteristics, may need specialized bioreactor designs; however, the three major requirements for bioreactor design include (1) mass transport, (2) mechanical, and (3) electrical stimulation

During the bioreactor culture, bioprinted organ constructs exhibit different biological and morphological changes over time depending on the bioink and bioprinting strategy utilized in the organ printing process. For example, organ constructs made using a scaffold-free approach undergo a different process than cells suspended and bioprinted in hydrogels.

Bioprinted organs, depending on their type, may pose difficulties associated with transplantation such as cellular ischemia requiring the printed organ to be transported on ice, the need for various flush solutions to prevent cellular edema, delay cell destruction, and maximize functions of organs after perfusion is reestablished. Once a bioprinted organ arrives in the operating room, it needs to be implanted in the appropriate site and attached to an uninjured intact vascular pedicle so that the organ can be perfused. Larger organs require larger vascular pedicles for immediate parenchymal perfusion; however, as the bioprinted organs need to be fully engrafted with the host, angiogenic integration with the recipient microcirculation is also essential. Since it is challenging to bioprint capillaries within bioprinted organ using current bioprinting technologies, one alternative approach is to bioprint endothelial cells within the construct and have the microcirculatory system developed naturally within the host postimplantation (Ozbolat, 2015). Ischemic resilient organs, such as kidney, may be more amenable to this strategy than other visceral organs such as liver and pancreas. In addition, the vascular network within bioprinted organs should be lined with an endothelium layer to prevent blood coagulation. Tight junctions of endothelial cells can be achieved in vitro or such organization can be performed in vivo post-implantation if the size of the printed organs is small (Itoh et al., 2015). The other important requirement for bioprinted organs is their innervation capability depending on their type and function. During transplantation, it has been shown that transplants denervate for a period and recover nerve function over time posttransplantation. For example, histological staining of transplanted kidneys demonstrated evidence of nerve function restoration after weeks posttransplant; recovery continues for years (Mulder et al., 2013). Similar recovery of nerve function can be seen in other organs such as heart (Murphy et al., 2000). Thus nerve tissue should be incorporated into bioprinted organs as one of the future goals to augment currently existing allogeneic transplants.