Bioprinting Towards Organ Fabrication: Challenges and Future Trends Ibrahim Ozbolat and Yin Yu

This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. TBME-01840-2012 1

Bioprinting towards Organ Fabrication: Challenges and Future Trends Ibrahim Ozbolat and Yin Yu

 proliferation, and direct cell differentiation toward certain Abstract— Tissue engineering has been a promising field of lineages. Although significant success has been achieved in the research, offering hope for bridging the gap between organ past decades both in research and clinical applications [7], it is shortage and transplantation needs. However, building three- obvious that complex 3D organs require more precise multi- dimensional (3D) vascularized organs remains the main technological barrier to be overcome. Organ printing, which is cellular structures with vascular network integration, which defined as computer-aided additive biofabrication of 3D cellular cannot be fulfilled by traditional methods. tissue constructs, has shed light on advancing this field into a new era. Organ printing takes advantage of rapid prototyping (RP) technology to print cells, biomaterials, and cell-laden biomaterials individually or in tandem, layer by layer, directly creating 3D tissue-like structures. Here, we overview RP-based bioprinting approaches and discuss the current challenges and trends towards fabricating living organs for transplant in the near future.

Index Terms— Bioadditive manufacturing, bioprinting, organ fabrication, tissue engineering

I. INTRODUCTION RGAN shortage has become more problematic in spite of O an increase in willing donors. From July 2000 to July 2001, for example, approximately 80,000 people in the United States awaited an organ transplant, with less than a third receiving it [1]. The solution to this problem, as with the solutions to other grand engineering challenges, requires long- Fig. 1: Organ printing concept: 3D functional organs are printed from term solutions by building or manufacturing living organs from the bottom up using living cells in a supportive medium (image a person’s own cells. For the past three decades, tissue courtesy of Christopher Barnatt [8]). engineering has emerged as a multidisciplinary field involving A computer-aided bioadditive manufacturing process has scientists, engineers, and physicians, for the purpose of emerged to deposit living cells together with hydrogel-base creating biological substitutes mimicking native tissue to scaffolds for 3D tissue and organ fabrication. Bioprinting or replace damaged tissues or restore malfunctioning organs [2]. direct cell printing is an extension of tissue engineering, as it The traditional tissue engineering strategy is to seed cells onto intends to create de novo organs. It uses bioadditive scaffolds, which can then direct cell proliferation and manufacturing technologies, including laser-based writing [9], differentiation into three-dimensional (3D) functioning tissues. inkjet-based printing [10] and extrusion-based deposition [11]. Both synthetic and natural polymers have been used to Bioprinting offers great precision on spatial placement of the engineer various tissue grafts like skin, cartilage, bone, and cells themselves, rather than providing scaffold support alone bladder [3-6]. To be successfully used for tissue engineering, [12]. Although still in its infancy, this technology appears to be these materials must be biocompatible and biodegradable, with more promising for advancing tissue engineering towards the mechanical strength to support cell attachment, organ fabrication, ultimately mitigating organ shortage and saving lives. Figure 1 demonstrates the concept of futuristic Manuscript received December 12, 2012. This research was supported by 3D direct organ printing technology, where multiple living National Institutes of Health (NIH) and the Institute for Clinical and Translational Science (ICTS) grant number ULIRR024979. cells with the supportive media stored in cartridges are printed I.T. Ozbolat is with The University of Iowa Mechanical and Industrial layer by layer using inkjet printing technology. It offers a Engineering and Biomanufacturing Laboratory, Iowa City, IA, 52242 USA controllable fabrication process, which allows precise (corresponding author to provide phone: 319-384-0810; fax: 319-335-5669; e-mail: [email protected]). placement of various biomaterial and/or cell types Y. Yin is with The University of Iowa Biomedical Engineering simultaneously according to the natural compartments of the Department and Biomanufacturing Laboratory, Iowa City, IA 52242 USA (e- target tissue or organs. Multiple cell types, including organ- mail: yin-yu@ uiowa.edu). specific cells and blood vessel cells, i.e., smooth muscle and

Copyright (c) 2013 IEEE. Personal use is permitted. For any other purposes, permission must be obtained from the IEEE by emailing [email protected]. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. TBME-01840-2012 2 endothelial cells (ECs), constitute the entire organ. Although a certain pattern onto a substrate by a laser beam. This is the concept seems to be trivial considering the complexity and followed by deposition of hydrogel on top of each layer of functionality of the parts that can be manufactured using cells, and the process is repeated for multiple cycles to get a contemporary rapid prototyping (RP) technology [13], several 3D structure. Nahmias et al. successfully performed challenges impede the evolution of organ printing. This paper hepatocytes patterning in collagen and MatrigelTM using laser- discusses the current state of the art in bioprinting technology, guided 3D cell writing [16]. In their study, three layers of cells its advantages and limitations, and grand challenges and future and hydrogels were alternately deposited on top of each other, trends towards fabrication of living organs in both research forming a 3D cellular structure. Cell viability and proliferation and clinical scenarios. was well-maintained post-deposition. Inkjet-based bioprinting was introduced in the early 2000s II. BIOPRINTING: CURRENT STATE OF THE ART and built a great foundation for future organ printing Bioprinting, where living cells are precisely printed in a technologies. In this technique, living cells are printed in the certain pattern, has great potential and promise for fabricating form of droplets through cartridges instead of seeding them on engineered living organs. Based on their working principles, scaffolds (see Fig. 2(B)). It uses a non-contact reprographic bioprinting systems can be primarily classified as: (1) laser- technique that takes digital data from a computer representing based, (2) inkjet-based, or (3) extrusion-based. tissue or organs, and reproduces it onto a substrate using “bioink” made of cells and biomaterials [10]. Boland et al. used a thermal inkjet printer to successfully fabricate 3D cellular assemblies of bovine aortal ECs with thermosensitive gels [10]. Post-incubation, printed structures showed high cell viability and maintained cell phenotype. Cui and his coworkers [17] applied inkjet printing technology to repair human articular cartilage, showing its promising potential for high- efficiency direct tissue regeneration. Huang and his coworkers [18] developed a bipolar wave-based drop-on-demand jetting. In their studies, cell-encapsulated alginate microspheres were jetted and assembled to create vertically oriented, short, tubular structures [19]. Inkjet-based system allows printing single cells or cell aggregates [20, 21] by controlling process parameters such as cell concentration, drop volume, resolution, nozzle diameter and average diameter of printed cells [22]. Weiss and his coworkers [23] developed a multi-head inkjet- based bioprinting platform for fabricating heterogeneous structures with a concentration gradient changing from the Fig. 2: Bioprinting techniques including (A) laser-based writing of bottom up. Multiple growth factors such as fibrinogen and cells, (B) ink-jet based systems and (C) extrusion-based deposition. thrombin and cells were printed with spatial precision in a functionally graded manner into rat calvarial defect in-situ Laser technology has recently been applied in the cell [24]. They demonstrated the feasibility of in-situ printing; printing process, in which laser energy is used to excite the however, this technique did not seem to be a practical cells and give patterns to control spatially the cellular approach for clinicians due to complex nature of the process in environment. A laser-based system was first introduced in their study [24]. 1999 by Odde et al. to process 2D cell patterning [14]. Laser Another bioprinting technique has been introduced for direct-write (LDW) is a biofabrication method capable of printing living cells and is based on the extrusion of rapidly creating precise patterns of viable cells on petri dishes. continuous filaments made of biomaterials. It is a combination In LDW, cells suspended in a solution in donor slides are of a fluid-dispensing system and an automated three-axis transferred to a collector slide using laser energy. A laser pulse robotic system for extrusion and printing, respectively [11]. creates a bubble, and shock waves are generated by the bubble During printing, biomaterial is dispensed by a pressure- formation, which eventually propel cells towards the collector assisted system, under the control of “robots,” resulting in substrate (see Fig. 2(A)). Micro-scale cell patterning can be precise deposition of cells encapsulated in the cylindrical achieved through optimizing viscosity of biological material filaments of desired 3D structures (see Fig. 2(C)). Wang et al. (bioink), laser printing speed, laser energy, and pulse used a 3D syringe-based bioprinting system to deposit frequency [15]. Writing of multiple cell types is also feasible different cells with various biocompatible hydrogels [25, 26]. by selectively propelling different cells to the collector They used hepatocytes and adipose-derived stromal cells substrate. Laser printing technology is also integrated with (ADSCs) together with gelatin/chitosan hydrogels to engineer scaffold printing, where LDW is performed in tandem with an artificial liver. Sun and his coworkers [27-29] built a multi- photopolymerization of hydrogels. It basically deposits cells in

Copyright (c) 2013 IEEE. Personal use is permitted. For any other purposes, permission must be obtained from the IEEE by emailing [email protected]. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. TBME-01840-2012 3 nozzle bioprinting system with the capacity to simultaneously other techniques require gentle handling of a flat deposition deposit cells and multiple biomaterials. Their rheology study surface that limits their direct applicability in surgery rooms. and cell viability assay were performed to investigate Despite their great advantages, inkjet printers suffer from mechanical-stress-induced cell damage during the printing drawbacks including significant cell damage and death as well process [30]. The results showed that cell viability was as cell sedimentation and aggregation due to small orifice influenced by material flow rate, material concentration, diameter that restricts printing cells in high densities (<5x106 dispensing pressure, and nozzle geometry. Their findings can cell/ml) [22, 39, 40]. In addition, structural integrity of the serve as a guideline for future studies and optimization of the printed structures is another obstacle, where the droplets do deposition system. Kachouie et al. proposed a method using not fuse into each other easily and the shape of droplets cannot hydrogel-encapsulated cells as tissue units to make a construct be controlled precisely. Adequate structural integrity is crucial with geometric patterns specific to target tissue types [31]. to retain the designed and the fabricated shape. Although bioprinting is a promising way as a methodical Extrusion-based systems provide relatively better structural interface between tissue and engineering, each technique has integrity due to continuous deposition of cylindrical struts. It is its own limitations. Laser-based systems have high resolution the most convenient technique in rapidly fabricating 3D and enable precise patterning of living cells, where cells can be porous cellular structures [12]. Although this technology lays maintained registry within 5.6±2.5µm to the initial pattern foundation for cell patterning for scale-up tissue and organ [32]. This resolution, certainly, cannot be achieved by other fabrication technologies, it constitutes several limitations such bioprinting techniques that makes laser-based cell writing as a as shear stress induced cell deformation and limited material great potential for micro-cellular features in organs and tissues selection due to need for rapid encapsulation of cells via i.e. micro-vascularate. In the authors opinion, prolonged gelation. Shear stress on nozzle tip wall induces significant fabrication time, laser shock related thermally and drop in the number of living cells when the cell density is high; mechanically induced cell deformation, interactions of cell however, this can be partially alleviated using optimum components with light, gravitational and random setting of process parameters such as biomaterial concentration, nozzle cells in the precursor solution, limitations in printing in third pressure (ideally minimum), nozzle diameter and loaded cell dimension and the need for photo-crosslinkable biomaterials density [41]. Restricted biomaterial selection and low should be overcome for future developments in laser-based resolution and accuracy brings limited applicability of systems [15, 33, 34]. In order to improve resolution further extrusion-based systems [12, 42]. Besides, sufficiently high and increase throughput, parameters related to laser pulse viscosity is essential for the biomaterial suspension to characteristics (i.e. pulse duration, wavelength, repetition rate, overcome surface tension-driven droplet formation and be energy and beam focus diameter), precursor solution drawn in form of straight filaments. High viscosity, on the properties such as viscosity, thickness and surface tension and other hand, triggers clogging inside the nozzle tip and should substrate properties should be optimized [34]. One-directional be optimized considering the diameter of the nozzle tip. propulsion of cells toward the collector substrate (top to Considering prolonged fabrication time for printing scale-up bottom) certainly limits development of 3D structures with tissues and organs, one of the important disadvantages of complex heterocellular architectures. In order to expand this encapsulating living cells in biomaterials is that cell- technology in the third dimension, a rotating donor-side biomaterial suspension needs to be stored considerable time in carousel leveling system with rotational and linear stages can the material reservoir that compromise cell viability and limits be developed that allows printing multiple cells in different their bioactivity. Thus, a more automated way of loading and layers for the development of heterocellular structures. Similar ejecting cell-biomaterial suspension is required for scale-up technology has been recently demonstrated with multi-material tissue and organ fabrication. Recently, Novogen MMX stereolithography process [35]. In addition, the fabrication BioprinterTM [43] developed the technology that the printer speed can be improved by increasing the laser pulse rate or head has both suction and ejection capability enabling integrating multiple laser beams [36]. automatic loading of suspension through the nozzle tip Inkjet-based systems are versatile and affordable that favors occasionally instead of loading it manually prior to fabrication. cells encapsulation. For instance, Xu et al. [37] developed a Mechanical strength and structural integrity of the fabricated fabrication platform with a different working principle as structures is a common drawback among bioprinting opposed to traditional inkjet printers, where droplets of techniques, which mainly use hydrogels due to high water crosslinker chemical were deposited onto a suspension of content and biocompatibility that allows permeation of cardiomyocytes and alginate for 3D cardiac pseudo tissue nutrients into and cellular products out of the gel [44]. Water fabrication. In addition, the traditional setup allows integrating content increases their biocompatibility; however, it multiple print heads easily to deposit multiple cell types, which deteriorates mechanical properties and processability is one of the pivotal steps in fabricating heterocellular tissues significantly. Although hydrogels possess unique properties, and organs. The other advantage of inkjet printing is that they are intrinsically weak due to high water content and do surfaces where the cells are printed and patterned do not have not withstand mechanical loading during and after gelation to be flat that favors cell printing in situ [38]. In general, all process. Gelation of cell encapsulated hydrogels is a

By using tissue spheroids, Forgacs and his coworkers at the University of Missouri, Columbia, used RP-based bioprinting

Fig. 3: (A) Hanging drop cultures with 20,000 chondrocytes together with multicellular spheroids made from smooth showing (B) spheroid fusion 7 days after harvesting, creating a muscle cells and fibroblasts, producing scaffold-free vascular larger scale tissue. constructs [55]. Figure 4 illustrates vascular construct printing, where tissue spheroids are printed sequentially in cylindrical

Cell aggregates or cell-laden hydrogels as building blocks at filaments from the bottom up. Upon fusion of tissue spheroids the microscale are selectively deposited to create larger tissues followed by a tissue maturation process of three days post- [48, 50, 51]. In this approach, these “minitissues” are printing, the support material is pulled away manually to considered as a biomaterial with certain measurable and generate the lumen. Multiple cell types, including human controllable properties and are assembled into tissues with umbilical vein smooth muscle cells and human skin fibroblast specific features through layer-by-layer stacking [52] or direct cells, were printed together to fabricate multicellular

Copyright (c) 2013 IEEE. Personal use is permitted. For any other purposes, permission must be obtained from the IEEE by emailing [email protected]. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. TBME-01840-2012 5 constructs. The bioprinting platform used in this study, the paper discusses major challenges in the context of bioprinting Novogen MMX BioprinterTM [43], has been recently towards organ fabrication. commercialized and specializes in developing bioprinting Despite the progress in tissue engineering, several across a broad array of cell types to create functional 3D challenges must be addressed for organ printing to become a tissues. When building large-scale organ structures, the reality. The most critical challenge in organ printing is the mechanical integrity of the printed structures as well as the integration of a vascular network, which is also a problem that integration of the vascular network with the rest of the organ the majority of tissue engineering technologies are facing. seems to be the major challenges to expanding the technology Without vascularization, engineered 3D thick tissue or organs for further applications. cannot get enough nutrients, gas exchange, and waste removal, all of which are needed for maturation during perfusion. This results in low cell viability and malfunction of artificial organs. III. ORGAN PRINTING AND ITS CHALLENGES Systems must be developed to transport nutrients, growth Organ printing is a computer-aided process in which cells factors, and oxygen to cells while extracting metabolic waste and/or cell-laden biomaterials are placed in the form of products such as lactic acid, carbon dioxide, and hydrogen aggregates, which then serve as building blocks and are further ions so the cells can grow and fuse together, forming the assembled into a 3D functional organ. It’s an automated organ. Cells in a large 3D organ structure cannot maintain their approach that offers a pathway for scalable, reproducible mass metabolic functions without vascularization, which is production of engineered living organs where multiple cell traditionally provided by blood vessels. Bioprinting types can be positioned precisely to mimic their natural technology, on the other hand, currently does not allow multi- counterparts. Developing a functional organ requires advances scale tissue fabrication where bifurcated vessels are required to in and integration of three types of technology [56]: (1) cell be manufactured with capillaries to mimic natural vascular technology, which addresses the procurement of functional anatomy. Although several researchers have investigated cells at the level needed for clinical applications, (2) developing vascular trees using computer models [64], only a biomanufacturing technology, which involves combining the few attempts have been made toward fabricating bifurcated or cells with biomaterials in a functional 3D configuration, and branched channels [55]. Successful maturation towards (3) technologies for in vivo integration, which addresses the functional mechanically integrated bifurcated vessels is still a issue of biomanufactured construct immune acceptance, in challenge. vivo safety and efficacy, and monitoring of construct integrity In order to closely mimic natural organs, 3D vascularized and function post-implantation. Success in fabrication of organs need to be fabricated using heterocellular aggregates, functional organs highly depends on advancements in stem cell which was discussed extensively in the literature [48]. In that technology. Stem cells, which are found in several tissues in study, intraorgan vascular branched networks are envisioned to the human body [57], can self-renew to produce more stem be printed and maturated with the rest of the organ. Instead of cells and differentiate into diverse specialized cell types to biomimetically designing and fabricating a vascular tree, form various organs [58]. A variety of cell types can be used which seems to be one of the impediments down the road for for this application, such as embryonic stem cells (ESCs) [57], organ printing, we alternatively propose printable adult stem cells (ASCs) [59], most recently, induced semipermeable microfluidic channels to mimic a vascular pluripotent stem (iPS) cells [60], and tissue-specific cell lines network in perfusing media and facilitating oxygenation for [61, 62]. Although a patient’s stem cells can be differentiated cell viability, coaxing tissue maturation and formation. into organ-specific cells for organ printing, there is still risk of Figure 5 illustrates our conceptual model of organ printing tissue rejection by the receiver [56]. Stem cell behaviors can through integration of vessel-like micro-fluidic channels with even change during the bioprinting process. In addition, organ cellular assembly, which has been featured in the literature [1, fabrication necessitates various types of organ-specific cells, 65]. The concept allows constructing structures with micro- which is not currently feasible considering the current isolation fluidic channels acting like blood vessels, allowing media and differentiation technologies. Although stem cells offer perfusion through an extracellular matrix that can support cell great promise as an unlimited source of cells, a greater viability in 3D. In Fig. 5, vessel-like micro-fluidic channels are understanding of and control over the differentiation process is printed in a 0°-90° lay-down pattern to develop 3D structures. required in order to generate expandable organ-specific cells Oxygenized perfusion media can be pumped into channels for in consistent quality with the desired phenotype. In this way, circulation purposes. Round ends are considered for the zigzag rejection by the recipient side will be minimized post- deposition pattern to prevent blockage of media flow. After transplantation. Moreover, imaging modalities such as parallel printing of each micro-fluidic channel layer, cellular computed tomography (CT), positron emission tomography spheroids, i.e., tissue spheroids or cell encapsulated (PET), and nuclear magnetic resonance (NMR) imaging microspheres, can be deposited between fluidic channels in the should be used to monitor the transplanted organ form of droplets using another robotic arm; semipermeable noninvasively; NMR offers a unique advantage in monitoring fluidic channels allow transport and diffusion of media to the organ integrity and function without the need to modify the cellular environment. The proposed strategy enables printing cells genetically [63]. Although cell and transplantation semi-permeable micro-fluidic channels in tandem with printing technology plays a crucial role in organ fabrication, this review cellular assembly. Concurrent printing has the potential to focuses on biomanufacturing technology, and the rest of the

Advantages of tissue spheroids have already been discussed aggregates and hydrogels as support material), and the Multi- in Section 2; spheroids significantly enhance cell viability of Arm BioPrinter (MABP) (which can print tissue spheroids in stem-cell-derived organ-specific cells, allowing the tandem with vessel-like microfluidic channels). These development of further organ fabrication techniques in the technologies can be applied for cellular aggregate near future. Printing encapsulated cells in spheroids also biofabrication in such a way as to facilitate self-assembly of greatly reduces shear-stress-induced cell damage compared to the tissue spheroids to mimic the natural process of tissue and printing cells directly loaded within biomaterial media. In organ morphogenesis. general, cells are subjected to shear stress during the printing A B process, and cell injury and DNA damage should be minimized. The proposed concept can also allow inclusion of multiple cell types in a spatially organized way by integrating another printer unit mounted on the robotic arm to print a secondary type of spheroids precisely.

Fig. 6: Printed cellular microfluidic channels: (A) perfusion of oxygenized cell type media, (B) media flow with intentionally generated air bubbles and (C) the laser confocal image of the mid- plane showing single lumen channel with live/dead staining where CPCs are labeled with calcein AM and ethidium homodimer.

Figure 6 illustrates oxygenized media perfusion through a Fig. 7: Bioprinters: (A) 3D Bioplotter® (designed by envisionTEC GmbH, Gladbeck, Germany [66]), (B) Novogen MMX BioprinterTM printed cellular micro-fluidic channel 44cm in length and (designed by Organovo, San Diego, California, USA [43]) and (C) 1mm wide with a 500µm lumen diameter. The cell media is Multi-Arm Bioprinter-“MABP” (designed by the University of Iowa, circulated through the channel without any blockage or Iowa City, Iowa, USA). swirling, which shows a great potential for developing embedded channels and serving as a vascular network for In order to fabricate scalable organs such as a mouse liver thick tissue fabrication. Direct printing of these channels which has 1.3x108 cells per gram [67], the bioprinter needs to allows them to be integrated within a hybrid bioprinting run for several hours considering the resolution of the system platform and also facilitates patterning them into very as in microscale. During prolonged fabrication time, it should complex shapes. Currently, mechanobiological properties of deliver biological substances through micro-nozzle or other printed channels are under investigation, and electrospun means without clogging problems or collision issues between nanofiber reinforcement is performed to match the mechanical the nozzle tip and the printed structure. In addition, properties with that of blood vessels. Elasticity and tensile sterilization is also crucial during bioprinting and necessitates strength are essential to be biomimetically mediated. Viability construction of bioprinters on a small scale to fit in standard of cartilage progenitor cells (CPCs) encapsulated within biosafety cabinets. In general, commercial bioprinters can cost

Copyright (c) 2013 IEEE. Personal use is permitted. For any other purposes, permission must be obtained from the IEEE by emailing [email protected]. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. TBME-01840-2012 7 substantially high around $100-200k depending on their intravascular device to secrete insulin to the bloodstream. unique capabilities, where home-made bioprinters on the other Although the pancreas serves two functions, which are carried hand roughly cost less than quarter of their commercial out by two different cell groups within the organ, a patient counterparts. with diabetes will be interested in correcting hyperglycemia Another challenging site for organ printing technology is the through fabrication and transplantation of a pancreatic organ rapid or accelerated tissue maturation process, where printed that can restore the function of the endocrine portions of the organ constructs should be rapidly fused, remodeled, and pancreas, which makes only about 2% of pancreas cells. The maturated toward a solid construct, ensuring mechanical endocrine portion is made of approximately a million cell rigidity for transplantation. Collagen and elastin are some of clusters called islets of Langerhans [70]. Four main cell types the proteins in the extracellular matrix of human organs, and exist in the islets. α, β, delta and gamma cells secrete glucagon, they are abundant in the connective tissue stromal of insulin, somatostatin (regulates/stops α and β cells) and parenchymal organs [68, 69]. Thus, production and deposition pancreatic polypeptide, respectively. Miniature organs can also of these proteins during tissue maturation are essential to enhancing the mechanical properties of the printed organs. be designed and fabricated to bring new functionalities and After the organ-printing process, the fabricated structure needs superiorities in the human body rather than restoring the to be transferred to the bioreactor. Bioreactors such as an functionality of their natural counterparts, such as living irrigation dripping perfusion bioreactor can be used to organs that can continuously generate electricity to eliminate expedite the tissue maturation and organ formation process, the use of batteries for internal devices such as peace makers. providing an optimal environment. The transfer of a printed Another future trend in organ printing is in-situ printing, organ to the bioreactor should be performed gently without where living organs can be printed in the human body during inducing any vibration that can degenerate the integrity of operations. Currently, in-situ bioprinting has already been fragile gel-based structures. Thus, future bioprinters can be tested for repairing external organs such as skin [38], where enclosed in a bioreactor system that will allow direct and rapid the wounded section is filled with multiple cells, including connection of printed structures to perfusion channels. human keratinocytes and fibroblast, with stratified zones throughout the wound bed. A transitional approach seems to IV. PROPOSED ENVISIONED FUTURE be logical to advance the state of the art through printing and Considering the pathway from isolation of stem cells to repairing partially damaged, diseased, or malfunctioning transplantation into a human, seamlessly automated protocols internal organs that do not have self-repair characteristics, such and systems are essential for customized functional organ as the liver. With the recent advancements in robot-assisted fabrication. This pathway includes (i) blueprint modeling of an surgery, computer-controlled robotic bioprinters will lead the organ with its vascular architecture, (ii) generation of a process evolution of this technology in the very near future. plan for bioprinting, (iii) isolation of stem cells, (iv) differentiation of stem cells into organ-specific cells, (v) V. CONCLUSIONS preparation and loading of organ-specific cells and blood This paper discusses the current state of the art in vessel cells as well as support medium, and (vi) bioprinting bioprinting with recent trends in organ printing technology process followed by organogenesis in a bioreactor for towards fabrication of living organs for transplant, where transplantation. In order to accelerate the entire process, one prototype organs can be developed using layer-by-layer can envision a bioreactor enclosing a bioprinter that will technology in the very near future. Although the technology immediately facilitate fusion of tissues and keep the printed shows a great deal of promise, there is still a long way to go to organ until desired maturation is achieved. In this way, a practically realize this ambitious vision. Overcoming current customized and automated system will facilitate delivering impediments in cell technology, biomanufacturing technology, artificial organs to transplant patients in a reasonable amount and technologies for in-vivo integration is essential for of time, preferably at earlier stages of their diseases when developing seamlessly automated technology from stem cell patients are healthier so that they are better able to withstand isolation to transplantation. surgical interventions. Miniature organs can be considered a future trend in organ ACKNOWLEDGMENT printing and might be a transition towards fully functioning The authors would like to thank A. Lehman from the organs. 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