ROBOTIC BIOPRINTER

Biswajeet Champaty Department of Biotechnology & Medical Engineering, National Institute of Technology, Rourkela-769008, Odisha, India.

ABSTRACT Tissue/organ printing aims to recapitulate the intrinsic complexity of native tissues. For a number of tissues, in particular those of musculoskeletal origin, adequate mechanical characteristics are an important prerequisite for their initial handling and stability, as well as long-lasting functioning. Hence, organized implants, possessing mechanical characteristics similar to the native tissue, may result in improved clinical outcomes of regenerative approaches. Using a bioprinter, grafts were constructed by alternate deposition of thermoplastic fibers and (cell-laden) hydrogels. 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.

1. INTRODUCTION Bioprinting is a new emerging technology,that aims at achieving to develop new tissues and eventually organs. It is in the research phase as with any technology that takes time to completely develop. Currently, there is a lot of research being done on bioprinters. In bioprinting, devices are printed by feeding biological materials as deposits. With an ink jet printer instead of ink, cells are printed or deposited on a surface layer by layer. And in this way a full organ can be produced. Bioprinting is a huge shift from the traditional approaches adopted so far in tissue engineering. It does not aim at seeding cells on a biodegradable scaffold. It aims at organizing the elements of the tissue during the fabrication step individually through the process of depositing layers of biologically important components. Bioprinting is a new area of research and engineering that involves printing devices that deposit biological material. The long-term goal is that the technology could be used to create

replacement organs or even entire organisms from raw biological materials. Today, bioprinters are in the development stage and primarily are used as scientific tools. They lack the speed and fine-tuning necessary for commercial deployment, though that day might not be far off. The first bioprinters deposited drops as small as 100 picoliters (by comparison, the volume of a cell is around 3 picoliters and the best inkjet printers can deposit drops 1-5 picoliters in volume) at a rate of tens of thousands per second. More recent bioprinters can extrude individual cells from a micropipette at a lower speed. A bioprinter developed by Gabor Forgacs, a biophysicist at the University of Missouri in Columbia, used combinations of "bioink" and "biopaper" to print complex 3D structures, albeit not at cellular resolution. Operating at 10,000 dots per second (10 kHz), a 100 picoliter printer can produce 60 microliters of tissue each minute, or 86 milliliters per day, a quantity of tissue that could almost fill a typical test tube. The downside of the 100 picoliter printer is its low resolution - most organic tissue we're familiar with requires precise cell-level organization in order to operate properly.

2. BACKGROUND Previous tissue engineering scaffold-based approaches have aimed to induce tissue self-assembly in applications such as cell-sheet engineering, centrifugal casting and magnetically driven approaches. Manipulating the cellular environment (i.e. surface tension, restricted geometry etc) may stimulate cells to self-aggregate to desirable geometrical shapes or migrate such as is done in rotating-culture systems and hanging-drop method. These concepts have been extended to other areas of research; utilizing micro fluidics, for example, for the high throughput generation and screening of compact spheroids and cell encapsulation. The emphasis of understanding the process by which cells self-assemble to 3D constructs has continued to grow to be central to comprehending morphogenesis and the development of living tissues. Evolving from tissue engineering, self-assembly processes have reached to other areas of research with increasing speed. Fundamentally, bioprinting emphasizes the use of computer-aided technology and principles such as tissue self-assembly, synthesis and remodeling. Marketing an idea of "directed tissue self-assembly", organ printing utilizes these rapid prototyping technologies and biological principles to influence tissue development. Computer-aided design enables the precise and

controlled placement and layer-by-layer deposition (also known as solid free-form fabrication) of cells, cell aggregates, cell-encapsulated hydrogels or any biologically related complex 3D structure along with other important chemical components such as nutrients and growth factors. With the simultaneous delivery of these cells, the natural autonomous organization of cells may potentially lead to the controlled patterning and assembly of complex 3D constructs.

Fig.1 Biopaper, Bioink, Bioprinter

Fig .2 Bioink deposited from bioprinter onto hydrogel biopaper. Alternating layers of printed ring structure and gel (acts like a matrix) eventually form a smooth cylinder after gel relaxes and layers merge.

Theoretically, bioprinting technology consists of six essential components: A CAD drawing of the desired engineered organ (blueprint); cells, cell aggregates or cell-encapsulated hydrogels capable of natural self-assembly (bioink); robotic-aided device for delivering the bioink (bioprinter); a container enclosed of the material to be deposited (biocartridge); a

bioprocessible biomimetic hydrogel to transfer material on (biopaper); and a perfused container containing the resulting printed 3D tissue construct capable of post-conditioning (bioreactor). While only in the early stages of development, bioprinting has many potential applications and can be divided into three steps: pre-processing, processing and post-processing.

3. BIOPRINTING PIONEERS Several experimental bioprinters have already been built. For example, in 2002 Professor Makoto Nakamura realized that the droplets of ink in a standard inkjet printer are about the same size as human cells. He therefore decided to adapt the technology, and by 2008 had created a working bioprinter that can print out biotubing similar to a blood vessel. In time, Professor Nakamura hopes to be able to print entire replacement human organs ready for transplant. You can learn more about this groundbreaking work here orread this message from Professor Nakamura. The movie below shows in real-time the biofabrication of a section of biotubing using his modified inkjet technology. Another bioprinting pioneer is Organovo. This company was set up by a research group lead by Professor Gabor Forgacs from the University of Missouri, and in March 2008 managed to bioprint functional blood vessels and cardiac tissue using cells obtained from a chicken. Their work relied on a prototype bioprinter with three print heads. The first two of these output cardiac and endothelial cells, while the third dispensed a collagen scaffold -- now termed 'bio-paper' -- to support the cells during printing. Since 2008, Organovo has worked with a company called Invetech to create a commercial bioprinter called the NovoGen MMX. This is loaded with bioink spheroids that each contain an aggregate of tens of thousands of cells. To create its output, the NovoGen first lays down a single layer of a water-based bio-paper made from collagen, gelatin or other hydrogels. Bioink spheroids are then injected into this water-based material. As illustrated below, more layers are subsequently added to build up the final object. Amazingly, Nature then takes over and the bioink spheroids slowly fuse together. As this occurs, the biopaper dissolves away or is otherwise removed, thereby leaving a final bioprinted body part or tissue.

As Organovo have demonstrated, using their bioink printing process it is not necessary to print all of the details of an organ with a bioprinter, as once the relevant cells are placed in roughly the right place Nature completes the job. This point is powerfully illustrated by the fact that the cells contained in a bioink spheroid are capable of rearranging themselves after printing. For example, experimental blood vessels have been bioprinted using bioink spheroids comprised of an aggregate mix of endothelial, smooth muscle and fibroblast cells. Once placed in position by the bioprint head, and with no technological intervention, the endothelial cells migrate to the inside of the bioprinted blood vessel, the smooth muscle cells move to the middle, and the fibroblasts migrate to the outside. In more complex bioprinted materials, intricate capillaries and other internal structures also naturally form after printing has taken place. The process may sound almost magical. However, as Professor Forgacs explains, it is no different to the cells in an embryo knowing how to configure into complicated organs. Nature has been evolving this amazing capability for millions of years. Once in the right places, appropriate cell types somehow just know what to do. In December 2010, Organovo create the first blood vessels to be bioprinted using cells cultured from a single person. The company has also successfully implanted bioprinted nerve grafts into rats, and anticipates human trials of bioprinted tissues by 2015. However, it also expects that the first commercial application of its bioprinters will be to produce simple human tissue structures for toxicology tests. These will enable medical researchers to test drugs on bioprinted models of the liver and other organs, thereby reducing the need for animal tests. In time, and once human trials are complete, Organovo hopes that its bioprinters will be used to produce blood vessel grafts for use in heart bypass surgery. The intention is then to develop a wider range of tissue-on-demand and organs-on-demand technologies. To this end, researchers are now working on tiny mechanical devices that can artificially exercise and hence strengthen bioprinted muscle tissue before it is implanted into a patient.

Organovo anticipates that its first artificial human organ will be a kidney. This is because, in functional terms, kidneys are one of the more straight-forward parts of the body. The first bioprinted kidney may in fact not even need to look just like its natural counterpart or duplicate all of its features. Rather, it will simply have to be capable of cleaning waste products from the blood. You can read more about the work of Organovo and Professor Forgac's in this article from Nature.

4. BIOPRINTING Manipulation of picoliter to nanoliter droplets has been a challenge for several applications including biochemical surface patterning, tissue engineering, and direct placement of cells and biomaterials for wound dressing applications [14]. In this regard, ejecting droplets via an actuator has emerged as a valuable technological advance addressing the issue of precise manipulation and deposition. Bioprinting is dened as the use of printing technology to deposit living cells, extracellular matrix (ECM) components, biochemical factors, proteins, drugs, and biomaterials on a receiving solid or gel substrate or liquid reservoir [58]. In recent years, there has been a growing interest in bioprinting due to its various advantages over existing patterning methods such as photolithography, soft-lithography, and stamping. Among these advantages are: (i) simple to use; (ii) enabling researchers to generate geometrically well-dened scaffolds in a rapid and inexpensive manner using polymers or ceramics and other cell stimulating factors providing support and induction for seeded cells [9]; (iii) allowing high-throughput generation of replicas of spatially and temporally well-controlled complex constructs [10]; and (iv) providing 3D complexity by multilayer printing [6,7,10]. By using conventional single-step lithography and stamp printing methods, building 3D constructs at high-throughput is challenging because fabricated 2D layers have to be merged. Existing co-culture approaches either lack highthroughput (e.g., conventional multiwell plate co-culture) or require complex fabrication steps and peripheral systems (e.g., microuidic co-culture). Emerging methods to pattern and assemble microscale hydrogels fabricated by photolithography are promising in terms of mimicking the complexity of the native microenvironment [1115]. Although bioprinting is a young eld, it has experienced a rapid growth despite the initial challenges that an emerging eld experiences. First, biological challenges for bioprinting have been cell viability and long-term functionality post-printing.

Concerns regarding potential apoptotic effects after and during bioprinting have been raised by the potential future end-users of this technology such as biologists, who consider bioprinting as an enabling technology for various applications. These biological requirements have set standards on the technology, leading to the development of several bioprinting technologies focusing on cytocompatibility issues maintaining a control over cell positioning in 2D and 3D microenvironments. Among these bioprinting technologies are inkjet-based printing [16,17], laser printing [1822], acoustic cell encapsulation [5,23], and valve-based printing [7,2426]. In recent years, there has been an increasing interest in the use of bioprinting for applications in biology and medicine. An emerging area is integration of bioprinting technologies with stem cell research. Microenvironments with spatially controlled gradients of immobilized macromolecules have been engineered to direct stem cell fate [2733]. Stem cells such as embryonic stem cells (ESCs), human bone marrow stem cells, and adipose-derived stem cells (ASCs) have been also directly bioprinted onto substrates [3437].

Fig.3 Principles of bioprinting technology: a) bioprinter (general view); b) multiple bioprinter nozzles; c) tissue spheroids before dispensing; d) tissue spheroids during dispensing; e) continuous dispensing in air; f) continuous dispensing in fluid; g) digital dispensing in air; h) digital dispensing in fluid; i) scheme of bioassembly of tubular tissue construct using bioprinting of self-assembled tissue spheroids illustrating sequential steps of layer-by-layer tissue spheroid deposition and tissue fusion process.

5. BIOPRINTING TECHNOLOGIES: ROBOTIC APPROACH Bioprinting helps to replicate human organs. The long term goal is to create a whole organ. However, this technology is in its rudimentary stage. As the system still lacks fine-tuning, it is not yet well-accepted in the mainstream medical field. With a regular ink-jet printer a cartridge is moved back and forth on a petri dish. A liquid is kept in the petri dish and the cartridges have cells inside them instead of ink. Inside, it has a crosslinker for binding. The crosslinker turns the liquid into a gel like substance over which the cells are deposited. This process is repeated with addition of liquid and more layers of cells. In this way, cells are structured for the formation of an organ. Newly developed bioprinters work at lower speed and can extrude cells individually from a micropipette.

Fig.4 Sketch of bioprinting technologies. (a) Thermal and piezoelectric ink-jet printing. Two major methods to jet the bio-ink are demonstrated. The thermal technique heats a resistor and expands an air bubble. The piezoelectric technique charges crystals that expand. (b) Setup for acoustic pico-liter droplet generation. Droplets can be deposited drop-on-demand with predetermined separation and locations. Periodically spaced interdigitated gold rings of an acoustic picoliter droplet ejector are demonstrated. The wavelength of the acoustic wave (low f) is much larger than the cell size resulting in harmless ejection of cells [5]. (c) Sketch of the valvebased printing setup [7,2426]. (d) Sketch of the laser printing setup. (Left) Laser-guided direct cell printing [18,39]. The laser is focused into a cell suspension and the force due to the difference in refractive indexes moves the cells onto an acceptor substrate. (Right) The cellhydrogel compound is propelled forward as a jet by the pressure of a laser-induced vapor bubble.

Several bioprinting methods have been developed to deposit cells including acoustic [5,23], inkjet [16,17], valve-based [7,2426], and laser printing technologies [1822]. Initially,commercially available desktop inkjet printers have been modied and used as cell printers [38]. In these systems, cell suspensions are placed in a printer cartridge, and a computer controls the printing pattern. Another technique to generate cell-encapsulating hydrogel droplets is the valve-based droplet ejection method [24,25]. In this method, cell-encapsulating hydrogel droplets are ejected onto a surface drop-on-demand. Size and number of cells in a single droplet and amount of droplets are controlled by the valve opening duration and actuation frequency [26]. In the laser-guided direct writing method, photons from a laser beam trap and guide cells by exploiting the differences in refractive indexes of cells and cell media [18,39]. Exposure of excessive thermal energy via laser light onto the cell biolayer and overheating of the cell can be one of the challenges. To overcome these challenges, near-infrared wavelengths (7001000 nm) have been used [39]. Photons in the near-infrared lack the energy to generate free radicals and are not absorbed by DNA. Hence, laserguided direct printing is described to be unlikely to cause mutations or trigger apoptosis, although wavelength optimization remains an open issue [39]. As an alternative to laser-guided direct printing, a cell-encapsulating donor lm is previously deposited onto a substrate, and placed in parallel facing the receiving substrate [19,21,40]. The heat transfer from the laser pulse to the cellencapsulating donor lm leads to the transfer of material from the donor lm to the receiving substrate. Patterning is performed on the receiving substrate that is usually xed on a computerized stage and coated with a cell culture medium or a biopolymer layer for cellular adhesion. Among these methods are laser-induced forward transfer (LIFT), absorbing lm-assisted laser-induced forward transfer (AFA-LIFT), biological laser processing (BioLP), and matrix-assisted pulsed laser evaporation direct writing (MAPLE DW) [42].
Table I. Comparison of commonly used bioprinting technologies based on performance

Performance metric Valve-based bioprinting [7,2426]

Throughput Droplet size Spatial resolution Single Cell Control Medium 100m1 mm Medium Medium

Laser induced Laser-guided Inkjet bioprinting (LIFT, bioprinting bioprinting BioLP, MAPLE) [18,39] [16,17] [19,21,4044]
Medium >20 m Medium Medium Low >10 m High High High 50300 m Medium Low

Acoustic bioprinting [5,23]

High 10500 m Medium High

A picoliter droplet-based cell encapsulation technology via acoustics has been developed [5,23,42]. Acoustic droplet formation rate can reach up to 100 000 droplets per second [5] with high cell viability. Among the advantages of this technology over other printing approaches are: (i) no nozzle is required for droplet generation because droplets are created from an open liquid reservoir, which circumvents complications that may be related to shear or clogging; (ii) acoustic waves do not harm cells due to low power droplet generation with only a few microseconds of pulse duration; and (iii) acoustic ejectors can be combined in an adjustable array format as multiple ejectors [41]. This would allow to enhance the rate of printing and to deposit multiple cell and ECM types. These ejectors could potentially print several biomaterials such as living cells, ECM proteins, nutrients, therapeutic drugs, and growth factors (GFs) simultaneously from the same platform by integrating microuidic systems into these ejectors [42]. To obtain reproducible results for the deposition of cell-encapsulating droplets, bioprinting spatial precision should be comparable to the cell size, for example, 1020 m in suspension [5].

6. APPLICATIONS AND RECENT ADVANCES

6.1 Regenerative Scaffolds and Bones A further research team with the long-term goal of producing human organs-on-demand has created the Envisiontec Bioplotter. Like Organovo's NovoGen MMX, this outputs bio-ink 'tissue spheroids' and supportive scaffold materials including fibrin and collagen hydrogels. But in addition, the Envisontech can also print a wider range of biomaterials. These include biodegradable polymers and ceramics that may be used to support and help form artificial organs, and which may even be used as bioprinting substitutes for bone. Talking of bone, a team lead by Jeremy Mao at the Tissue Engineering and Regenerative Medicine Lab at Columbia University is working on the application of bioprinting in dental and bone repairs. Already, a bioprinted, mesh-like 3D scaffold in the shape of an incisor has been implanted into the jaw bone of a rat. This featured tiny, interconnecting microchannels that contained 'stem cell-recruiting substances'. In just nine weeks after implantation, these triggered the growth of fresh periodontal ligaments and newly formed alveolar bone. In time, this research may enable people to be fitted with living, bioprinted teeth, or else scaffolds that will cause the body to grow new teeth all by itself. You can read more about this development in this article from The Engineer.

n another experient, Mao's team implanted bioprinted scaffolds in the place of the hip

bones of several rabbits. Again these were infused with growth factors. As reported inThe Lancet, over a four month period the rabbits all grew new and fully-functional joints around the mesh. Some even began to walk and otherwise place weight on their new joints only a few weeks after surgery. Sometime next decade, human patients may therefore be fitted with bioprinted scaffolds that will trigger the grown of replacement hip and other bones. In a similar development, a team from Washington State Universityhave also recently reported on four years of work using 3D printers to create a bone-like material that may in the future be used to repair injuries to human bones. 6.2 In Situ Bioprinting The aforementioned research progress will in time permit organs to be bioprinted in a lab from a culture of a patient's own cells. Such developments could therefore spark a medical revolution. Nevertheless, others are already trying to go further by developing techniques that will enable cells to be printed directly onto or into the human body in situ. Sometime next decade, doctors may therefore be able to scan wounds and spray on layers of cells to very rapidly heal them.

Fig.5. Robotic bioprinter for in situ bioprinting

Already a team of bioprinting researchers lead by Anthony Alata at the Wake Forrest School of
Medicine have developed a skin printer. In initial experiments they have taken 3D scans of test

injuries inflicted on some mice and have used the data to control a bioprint head that has sprayed skin cells, a coagulant and collagen onto the wounds. The results are also very promising, with

the wounds healing in just two or three weeks compared to about five or six weeks in a control group. Funding for the skin-printing project is coming in part from the US military who are keen to develop in situ bioprinting to help heal wounds on the battlefield. At present the work is still in a pre-clinical phase with Alata progressing his research usig pigs. However, trials of with human burn victims could be a little as five years away. The potential to use bioprinters to repair our bodies in situ is pretty mind blowing. In perhaps no more than a few decades it may be possible for robotic surgical arms tipped with bioprint heads to enter the body, repair damage at the cellular level, and then also repair their point of entry on their way out. Patients would still need to rest and recuperate for a few days as bioprinted materials fully fused into mature living tissue. However, most patients could potentially recover from very major surgery in less than a week.

6.3 Cosmetic Applications As well as allowing keyhole bioprinters to repair organs inside a patient during an operation, in situ bioprinting could also have cosmetic applications. For example, face printers may be created. These would evaporate existing flesh and simultaneously replace it with new cells to exact patient specification. People could therefore download a face scan from the Internet and have it applied to themselves. Alternatively, some teenagers may have their own face scanned, and then reapplied every few years to achieve apparent perpetual youth.

Fig.6. face bioprinter

The idea of having the cells of your face slowly burnt away by a laser and reprinted to order may sound like a nightmare that nobody would ever choose to endure. However, as we all know, many people today go under the knife to achieve far less cosmetically. When the technology is available to create them, face printers capable of printing new muscles without the hassle of exercise.

6.4 3D Bio-printer to create arteries and organs An engineering firm has developed a 3D bio-printer that could one day be used to create organs on demand for organ replacement surgery. The device is already capable of growing arteries and its creators say that arteries "printed" by the device could be used in heart bypass surgery in as little as five years. Meanwhile, more complex organs such as hearts, and teeth and bone should be possible within ten years. The 3D bio-printer allows scientists to place cells of almost any type into a desired 3D pattern. It includes two print heads, one for placing human cells, and the other for placing a hydrogel, scaffold, or support matrix. The cells used by the device need to be the cells of what is being regenerated building an artery requires arterial cells for example. Because the patients own cells are used the new organ will not be rejected by the body. The printer fits inside a standard biosafety cabinet for sterile use.

Fig.7. 3D robotic bio-printers

Its creators say that one of the most complex challenges in the development of the printer was being able to repeatedly position the capillary tip, attached to the print head, to within microns. This was essential to ensure that the cells are placed in exactly the right position. A computer controlled, laser-based calibration system was developed to achieve the required repeatability. The 3D bio-printers include a software interface that allows engineers to build a model of the tissue construct before the printer commences the physical constructions of the organs cell-by-cell using the automated, laser-calibrated print heads. Researchers can place liver cells on a preformed scaffold, support kidney cells with a coprinted scaffold, or form adjacent layers of epithelial and stromal soft tissue that grow into a mature tooth. Ultimately the idea would be for surgeons to have tissue on demand for various uses, and the best way to do that is get a number of bio-printers into the hands of researchers and give them the ability to make three dimensional tissues on demand.

7. FUTURE CHALLENGES
Although the idea of building tissues and organs has long been envisioned, bioprinting is still a very new process. While the concept seems possible, its full potential has yet to be fully demonstrated. Despite much progress and a number of breakthroughs in this area, researchers have met many challenges. There are many design considerations, only a few are discussed below, to factor in the design and development of the process and bioprinting devices. Utilizing emerging technologies and advances (in all fields) and optimizing the current state-of-the-art process and tools will progress bioprinting further. An organ blueprint should not only provide an accurate representation of the desired tissue, but also factor in dynamic events such as tissue fusion, compaction and retraction and constant remodeling. However, designing a bioprint to consider post-processing fusion, remodeling, retraction and compaction after printing and solidification is a challenging task. To resolve this, one modification to the blueprint would require it to be larger and slightly shaped differently from the final desired tissue structure. The CAD drawing may also need to include coefficients that estimate these remodeling factors. Furthermore, to accurately capture and simulate dynamic tissue self-assembly and post-processing remodeling, computer simulations must be improved in order to be utilized in the organ printing process. However, novel software and blueprints may lead to more effective solutions.

There are also challenges to improving and optimizing the biofabrication process. Currently, biopaper is characterized as a bioprocessible biomimetic hydrogel that have been generally used as matrices or scaffolds for bioprinting. Ideally, biopaper must be capable of rapid solidification, be dispensible, functional with growth factors, non-toxic to enable high cell viability (biocompatible), stimuli-sensitive and cross-linkable, capable of tissue fusion, hydrophilic and able to maintain shape, biodegradable and low cost. Thus far, naturally derived hydrogels such as collagen have been used. Improvements in the biomaterials used as bioinks and biopaper are needed before real clinical application potential can be realized. It is necessary to optimize bioinks such as their deformation and fluidic (rheological) and surface properties. While studies have demonstrated the potential of dispensing and depositing single cells, more difficulty lies in developing a standardized scalable fabrication method for the robotic delivery of cell aggregates or tissue spheroids. Following that, biocartridge designs must also be modified. There are important challenges in designing and fabricating a bioprinter that is biologically "friendly" with rapid prototyping capabilities. Possible solutions that may address and answer these challenges include the use of temporary, removable porous needles. These type of needles may provide temporary mechanical support and perfusion of the printed scaffold essential in the post-processing step. In addition, enhancing the ability to develop vascular systems, these needles may enable the automatic printing of vascular-like tubular structures from self-assembling cell aggregates or fused vascular spheroids. It is not enough to be able to print an organ. It is necessary for the printed tissue construct to undergo post-conditioning to acquire properties similar to native tissue. Bioreactors used in classical tissue engineering research differ from the ideal bioreactor for bioprinting. The bioreactor must meet a number of challenges including continuous perfusion, enabling accelerated tissue maturation and functionality for intravascular perfusion initiation, maintaining cell viability and vascularization, and dynamic biomechanical conditioning. Ideally, all components in the biofabrication process should be smoothly integrated to allow for easy transitioning of the tissue construct from the printed state (in a wet environment) to the postconditioning state (transfer to bioreactor). The use of modern software and manufacutring device and process improvements with the aid of robotic biofabrication equipment may lead to revolutionary changes in this area.

8. DISCUSSION
As bioprinters enter medical application, so replacement organs will be output to individual patient specification. As every item printed will be created from a culture of a patient's own cells, the risk of transplant organ rejection should be very low indeed. Together with developments in nanotechnology and genetic engineering, bioprinting may also prove a powerful tool for those in pursuit of life extension. Mainstream bioprinting will also inevitably drive further the New Industrial Convergence, with doctors, engineers and computer scientists all increasingly learning to manipulate living tissue at its most basic cellular level.

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