Commercial 3D Bioprinters
The bioprinters are robotic devices, which enable 3D bioprinting. In this chapter, we provide classification of already existing commercially available 3D bioprinters and outline basic principles of their construction and functionalities. The emerging trends in the design and development of 3D bioprinters, perspectives of creation of new types of commercial 3D bioprinters based on new physical principles, including in situ bioprinters, as well as completely integrated organ biofabrication lines or “human organ factories” will be also discussed.
Keywords3D Bioprinters Ink-jet bioprinting Extrusion-based bioprinting Laser-based bioprinting In situ bioprinting Organ biofabrication line
List of leading bioprinting companies producing commercial bioprinters (https://3dprintingindustry.com/news/top-10-bioprinters-55699/)
EnvsionTech (Germany) https://envisiontec.com
RegenHu (Switzerland) https://www.regenhu.com
Poetis (France) http://poietis.com
Organovo (USA) http://organovo.com
Sciperio/nScript (USA) http://www.sciperio.com
Cellink (Sweden/USA) https://cellink.com
Allevi (formerly BioBots) (USA) https://biobots.io
TeVido BioDevices (USA) http://tevidobiodevices.com
3Dynamics systems (USA) http://www.bioprintingsystems.com
Aspect BioSystems (Canada) https://www.aspectbiosystems.com
Rokit (South Korea) http://en.3disonprinter.com
3D Bioprinting solutions (Russia) http://www.bioprinting.ru
Cyfuse Biomedics (Japan) https://www.cyfusebio.com/en/
Rikoh (Japan) (https://www.ricoh.com
Regenovo (China) http://regenovo.com/english/
SunP biotech international (China/USA) http://sunpbiotech.com/about-us-2/
2 Definition of 3D Bioprinters
One possible definition of a 3D bioprinter is an automated device for robotic additive biofabrication of 3D functional tissue and organs based on digital models. This definition implies following criteria for such devices. First, it must be automated and robotic device. In this context, manually controlled biomaterials, cells, or minitissues dispensing and/or cell spraying do not fit to definition. Second, it must be able to produce not just 2D cell patterning but rather 3D tissue and organ constructs. Third, it must enable bioprinting both biomaterials (printable bioinks) and living cells or minitissues. Thus, certain robotic devices, for example, 3D printer based on so-called fused deposition modeling or melt electrospinning, which can print cell-free scaffolds and are not able to print or dispense living cells should not be considered as 3D bioprinters. Finally, bioprinting process is based on using digital models. Manually controlled dispensing, pipetting, or spraying even using hydrogel containing living cells without employment digital model (usually in STL file) does not exactly fit to our definition. Moreover, as it will be shown later, there are new 3D biofabrication methods based on using novel physical principles which do not fit to the above definition. It does not mean that these methods have disadvantages, but merely that the main focus of this chapter is on commercial 3D bioprinters as we have defined them. There are many different types of bioprinting devices, and popular hybridization of technological approaches constantly creates new types of devices, which could be eventually commercialized. For the sake of focus, we limit our scope only to consideration devices, which are already implemented and commercially available or at least under development. There are several textbooks on bioprinting (Lee et al. 2015; Ringeisen et al. 2010; Zhang et al. 2015; Atala and Yoo 2015; Chua and Yeong 2015; Ozbolat 2016) as well as excellent reviews (Mironov et al. 2009a; Derby 2012; Murphy and Atala 2014; Mandrycky et al. 2016; Mironov et al. 2008; Mironov et al. 2003; Mironov et al. 2009b; Melchels et al. 2012) where readers will be able to find all necessary information about the most recent developments in this sector. Finally, we also do not review in this chapter related patents which readers could find elsewhere .
3 Anatomy of 3D Bioprinters
4 Classification of 3D Bioprinters
The classification is a logical part of systematization of any types of knowledge. It is already generally accepted classification of 3D bioprinters on three main groups: (i) ink-jet bioprinters, (ii) extrusion-based bioprinters, and (iii) laser-based bioprinters (Melchels et al. 2012). Some authors and especially industry reports often include an additional group of so-called magnetic bioprinting technology developed and commercialized by USA company n3D Biosciences, USA (http://www.n3dbio.com/products/magnetic-levitation/). However, devices which are working without digital model do not fit our definition of bioprinters.
Although magnetic (Durmus et al. 2015; Tasoglu et al. 2015; Tocchio et al. 2018) and acoustic levitations (Bouyer et al. 2016) as well as dielectrophoresis (Albrecht et al. 2004; Lin et al. 2006) and using electric field have enormous potential for development of new types of 3D bioprinters or more correctly say 3D biofabricators or 3D assemblers, according our best knowledge there is no published evidence for any attempts of commercialization of these very perspective biofabrication technologies yet. The potential advantage of these technologies is using magnetic, acoustic, and electric fields as some sort of temporal and removable support which we (using obvious analogy with scaffold which is usually defined as a temporal and removable or biodegradable support) suggest to call scaffields-based bioassembly.
3D Bioprinting Solutions (Russia) is now developing a first commercial variant of magnetic and acoustic bioprinting devices based on the principles of diamagnetic and acoustic levitational bioassembly. However, for using nontoxic concentration of paramagnetic salts (such as gadolinium slats) which are enabling diamagnetic levitation, it is necessary either to use very expensive supermagnets (30 Tesla) on the Earth or to perform magnetic levitation in the condition of microgravity in Space at The International Space Station, which is also very expensive.
Although they often employ digital models, it would be more logical to call such new evolving types of devices 3D bioassemblers.
5 Ink-Jet 3D Bioprinters
The first ink-jet bioprinter has been developed by Thomas Boland at Clemson University (USA) using modified standard Hewlett-Packard 2D ink-jet printer (Wilson Jr and Boland 2003) and related patent belongs to company Organovo (USA). However, according our best knowledge until now there were no indications of commercialization and developing production of ink-jet 3D bioprinters by this company. An early attempt to develop a multinozzle ink-jet cell printer has also been reported in California, USA, at the famous Xerox Institute, but again they chose not to commercialize technology due to the absence of obvious market at that time. Similarly, Japanese company Cannon tried to develop an ink-jet bioprinters, but decided to focus their attention of using ink-jet technology for 2D protein and cell patterning. The main technological impediment for development of commercial ink-jet technology was transition from 2D to 3D printing. Although world leaders in the development of ink-jet bioprinting technology such as Thomas Boland (USA), Brain Derby (UK), and Makoto Nakamura (Japan) (Boland et al. 2006; Arai et al. 2011; Saunders et al. 2008) made great efforts in the advancing this technology, they were not able to move it to the market with commercial product. However, there are some good news. Thomas Boland recently co-founded a new startup 3D bioprinting company TeVido Biodevices (http://tevidobiodevices.com) in Texas (USA), and it is logical to expect the development of commercial ink-jet bioprinter by this company in the nearest future. Even more interesting and exciting developments are ongoing now in Japan. At recent biofabrication meeting in Austria, representative of printing company Rikoh (Japan) (https://www.ricoh.com) for first time reported publically about the development of a new modified ink-jet 3D bioprinting technology and its potential commercialization. The great advantage of presented technology is that they elegantly solved the problem of transition from 2D to 3D ink-jet bioprinting using as printable biomaterials gelatin microbeads. Cheap commercially available ink-jet 3D bioprinters are highly desirable.
6 Extrusion-Based 3D Bioprinters
The extrusion-based bioprinters are most popular versions of 3D bioprinters. A publication by Robert Klebe from the University of Texas in San Antonio (Texas, USA) about so-called cytoscribing published in 1988 is often mistakenly considered as a first pioneering publication about 3D bioprinting technology (Klebe 1988). Robert Klebe is also a holder of first patent about extrusion-type 3D bioprinter named apparatus for the precise positioning of cells (Klebe 1987). However, the careful reading of his publication indicates, beside the title of his paper, that he used printing of proteins for 2D cell patterning and never actually bioprint living cells or 3D tissues. Moreover, what is most important in the context of this chapter that his cytoscribing technology has never been commercialized.
The adaptation of fused deposition modeling for 3D printing of at first biodegradable tissue engineered scaffold and later hydrogel was the next important step and simultaneously developed by Dietmar Hutmacher’ s group in National University of Singapore (Singapore) (Zein et al. 2002), Anthony Mikos’s group in Rice University in Texas (USA) (Cooke et al. 2003), and Ralf Mulhaupt’s group in University of Freiburg (Germany) (Landers et al. 2002). However, only the German group was able to successfully commercialize their bioplotter technology through German company Envisiontec (https://envisiontec.com). Envisiontec 3D bioplotter became a first commercial 3D bioprinter. Modified fused deposition modeling technology for fabrication of solid biodegradable scaffold was not truly bioprinting technology because cell was seeded on printed scaffold later not by robotic bioprinter but simply manually. Hydrogel-based extrusion type bioprinter could not provide desirable initial high cell density, and material properties of construct were also inferior.
The next main advance developed initially by Jos Malda’s group in Utrecht University (The Netherlands) (Schuurman et al. 2011) and later by Antony Atala’s group in Wake Forest University in NC, USA (Kang et al. 2016), was based on using hybrid approach or combination of fused deposition modeling of solid biodegradable scaffold with simultaneous bioprinting of hydrogel loaded with living cells. This technology has been successfully commercialized and became a standard for the most advanced extrusion-type commercial 3D bioprinters.
The low initial cell density forced some researchers to think about possible alternatives to extrusion bioprinting based on using original concept of organ printing introduced by Vladimir Mironov et al. in 2003, using tissue spheroids as building blocks (Mironov et al. 2009c). According our best knowledge at least several groups and companies tried to develop bioprinting technology based on using tissue spheroid as building blocks. Organovo (USA) which holds the original patent on this technology developed jointly by Gabor Forgacs’s group at University of Missouri (Columbia, USA) and Vladimir Mironov’s group at The Medical University of South Carolina (Charleston, USA) failed to develop a reproducible tissue spheroid-based biotechnology because tissue spheroids usually fused before they have a chance to be dispensed. Thus, Gabor Forgacs’s group and Organovo later switched to development of rod-like continuous dispensing (Owens et al. 2013).
7 Laser-Based Bioprinters
Among the most commercially ripe methods is laser-induced forward transfer (LIFT), also referred to as biological laser printing. We refer the readers to the chapter “Laser-Based Cell Printing” of this book for detailed introduction and overview of the current state of the art of this method. One of the main advantages of LIFT is its relatively high bioprinting resolution (down to several picoliters and one cell per drop) and the fact that it is a nozzle-free technology, avoiding shear-forces potentially damaging to cells. The printing resolution of LIFT depends on several factors, which include laser pulse energy, repetition rate, thickness of the bioprintable material layer and its viscosity, the distance between donor and collector substrates, and the substrate wettability. This technology was in fact successfully commercialized by a French company Poetis founded by Fabien Guillemot (http://www.poietis.com). However, Poetis decided do not sell their bioprinters because their business model is based on providing bioprinting service such as bioprinting of human skin to the large cosmetic, chemical, and pharmacological companies (see Chapter “Emerging Business Models Toward Commercialization of Bioprinting Technology” of this book).
In 1999 David Odde from University of Minnesota (USA) introduced a laser-assisted bioprinting method using optical cell strapping (Odde and Renn 1999). This laser-guided direct writing approach was demonstrated to be capable of producing 2D cell patterns. However, the subsequent attempts to adapt it for 3D bioprinting practically failed, and to the best our best knowledge this technology has been never commercialized.
Stereolithography (SLA) was also shown to produce complex 3D constructs form photopolymerizable materials containing living cells (Chan et al. 2010). This photopatterning technique can be adapted to process multiple material types and potentially offers spatial resolution superior to extrusion-based methods. Digital light processing (DLP) is another lithography-based technique, which can be conveniently adapted to live cell patterning (Ma et al. 2016). Despite the fact that SLA and DLP are among of the most advanced technologies very widespread throughout industrial 3D printing market, their use for 3D bioprinting has not been commercialized so far. A combination of optical trapping and stereolithography for cell patterning has also been reported in the literature (Linnenberger et al. 2013).
Multiphoton processing, often referred to as two-photon polymerization (2PP) or multiphoton-excited microfabrication, is a lithography-based technique providing even higher spatial resolution down to subcellular level (Ovsianikov et al. 2012). The group of Paul Campagnola demonstrated already in 2005 that this method can be used for cross-linking of cytoplasmic proteins in live cells (Basu et al. 2005). Interestingly, among the main bottlenecks for developing 2PP towards biofabrication is again its high spatial resolution, resulting in long processing times when it comes to the 3D constructs of tissue-relevant size. The need to substantially increase the throughput of this method for bioprinting necessitates the development of novel biomaterials and highly efficient photoinitiators (Qin et al. 2014). A recent report has demonstrated that macromolecular photoinitiators, designed to exhibit large two-photon absorption cross-sections, can be used for efficient 2PP in the presence of cells (Tromayer et al. 2017). Despite the fact that several companies sell 2PP-based devices, none of them is currently suitable for bioprinting .
8 Organ Biofabrication Line
In 2011 this concept indeed looked a little bit futuristic and even close to domain of science fiction rather than to profit-oriented pragmatic and realistic commercialization. Now it is possible to state that practically all components of this proposed integrated organ biofabrication line are commercially available. Moreover, according to Rokit (South Korea) a similar concept of integrated tissue and organ factory is currently under commercial development. It is safe to predict now that similar approach will be explored by other European and USA companies. It is interesting that in this context there is a room for potentially very interesting and potentially very profitable emerging business model when one intermediate service company will collect and integrate all necessary for 3D tissue and organ biofabrication devices developed by other companies into one integrated and automated organ biofabrication line and will sell it including necessary service as one package at a very good price to all hospitals and clinics interested in well-equipped intrahospital GMP facilities for regenerative medicine, biofabrication, and 3D bioprinting.
9 In Situ Bioprinters and Biofabricators
10 Emerging Trends in the Design and Functionalities of Commercial 3D Bioprinters
The fact that there are already several dozens of 3D bioprinting companies around the world already producing commercially available 3D bioprinters is a direct manifestation and objective evidence that we are facing the emerging and rapid development of new very perspective bioengineering industry. Thus, all these conversations about bioprinting as some sort of science fiction and statement that bioprinting technology is still in infancy are in essence nothing more than a subjective opinion which is probably based either on unsufficient knowledge or on limited access to objective information. We hope that this review provides at least some objective documented evidence against this still persisting view. Instead of dealing with such types of baseless and often unprofessional futuristic predictions and similar statements about enormous complexity of human tissues and organs is much more productive actually to work hard on the development of 3D bioprinting technology and, thus, advance this exciting and perspective field.
Moreover, the analysis of the development of commercial 3D bioprinters can also show some emerging important trends in the designing of commercial 3D bioprinters. The first trend strongly indicates on obvious fact that number of commercial 3D bioprinters as well as number companies and countries producing 3D bioprinters continue to grow and that 3D bioprinting technology is already in the process of global commercialization. Thus, we are observing the emergence of new bioprinting and biofabrication industry. The second trend clearly demonstrates that there are different types of 3D bioprinters based on different physical principles of their work. The third recent trend demonstrates that companies started to develop not only expensive but also affordable 3D bioprinters which will enable further spreading of this very popular technology in the academic circles. The hybridization of bioprinting technologies as well as increasing functionalities of commercially available 3D bioprinters are the next evolving trends .
11 Challenges and Future Perspectives
The started commercialization of 3D bioprinting technology does not automatically mean that there are no problems and challenges left. It just indicates that bioprinting technology is already mature enough to initiate potentially profitable commercialization. However, relatively young 3D bioprinting technology still has a lot of problems and limitations. First of all, there are no bioprinted human organs and certified 3D bioprinters approved for clinical use. The bioprinting resolution and speed need further improvement. There are at least several basic questions related to commercial 3D bioprinters which we will face in nearest future.
First such question – Can we use one type of commercial bioprinter for bioprinting of any types of human tissue and organs? The answer is probably no. The second question – Is it enough just 3D bioprinter to print tissue and organ constructs? The answer is also probably no. As we argued in previous sections just development of only 3D bioprinters is not enough and we will need development of integrated ideally automated and robotic organ biofabrication line. The third question – Did we already discover and systematically explore all possible physical principles for 3D bioprinting and biofabrication of human tissue and organs? The answer is again no, at least not yet. Another interesting but still unanswered question – Do we need one bioprinter to print whole entire organ in one step or we need several distributed biofabricators, bioassemblers, and bioprinters which will print at first small living building blocks such a minitissues, tissue modules, organ lobules, and even lobes and then we will need to design some types of bioassemblers which will put these building blocks together? Final question – Do we need to create all human tissue and organs only ex vivo and only then transplant them into human body or we must try to print at least some of human tissue directly in vivo in operation room combining advanced surgical robotics with 3D bioprinting and 3D biofabrication? The answer is we must definitely try to develop in vivo bioprinting.
The sufficient and long term sustainable funding, properly orchestrated multidisciplinary efforts, creation of national centers of excellence in bioprinting and biofabrication as well as transnational and may be even global initiatives are essential for success. The well-funded multidisciplinary efforts as well as competition between emerging 3D bioprinting companies will guarantee that transplantation of bioprinted functional and vascularized 3D human tissues and organs will eventually became a highly desirable clinical reality and will ensure that commercialization of 3D bioprinting technology will be successful. It is safe to predict that second generation of commercial 3D bioprinters will be more advanced and sophisticated and we will face the emerging of profitable bioprinting industry.
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