Introduction
The field of cell and organ printing had humble beginnings near the turn of the millennium when a few groups of scientists had a common interest in merging the physical and biological worlds with design engineering.Reference Wilson and Boland1–Reference Odde and Renn4 From the physical world came well-defined energetic mechanisms such as laser ablation, piezoelectric stimulation, and thermal actuators. Light-based mechanisms have emerged as powerful tools to create and modify biomaterials, so much so that MRS Bulletin Reference Narayan and Goering5 dedicated an entire issue to this area in 2011. From the biological world came the emerging fields of stem cell culture, tissue engineering (TE), and three-dimensional tissue culturing. Through design engineering, information was added a priori to guide the development of rapid prototyping and computer-aided design/computer-aided manufacturing. It was difficult to see an immediate connection or synergy between the fields, but through creative insight and determination, the field of cell and organ printing was born from researchers melding the three together.
The goal is the same now as it was 15 years ago; only today, researchers are getting closer to actually replicating the three-dimensional structure and function of natural tissue and organs in vitro.Reference Nerem6 The mainstream TE perspective began with creating polymer scaffolds to recruit or differentiate cells that then adhere, grow, and ultimately transform distinct starting material into living tissue. Over time, the approach has become more sophisticated so as to incorporate stem cells and disparate biochemical factors to ultimately create a tissue construct that could use autologous cues and differentiation or recruitment to create synthetic tissue. This research has succeeded for thin tissues (bladder,Reference Atala, Bauer, Soker, Yoo and Retik7 skinReference Marston, Hanft, Norwood and Pollak8,Reference Bello and Falabella9 ) with appropriate functionality to become viable alternatives to transplantation. The larger problem of regenerating truly 3D organs remains problematic primarily due to the inability to replicate the intricate heterogeneous structure and maturity of natural organs, including vascular and microvascular subsystems as well as stroma.Reference Berthiaume, Maguire and Yarmush10
If one could arrange and assemble different types of cells, scaffold, and factors the way nature intended, not just in two dimensions but in three, then could a synthetic tissue’s ability to replicate both structure and function in vitro and ultimately in vivo also be replicated? Enter cell and organ printingReference Mironov, Boland, Trusk, Forgacs and Markwald11,wherein early studies focused on whether cell arrangement and assembly caused deleterious effects on the deposited cells—physical energetics have the potential to destroy fragile membranes containing even more fragile cytoskeletal and supramolecular assemblies holding the framework to life.Reference Ringeisen, Othon, Barron, Young and Spargo12 Many were skeptical of the cell printer’s chance of success, because the acceleration and deceleration caused with the printers would result in a “bugs on a windshield” scenario. If that did not happen, then the actuation energy used to manipulate the cells could result in cell lysis (the breaking down or destruction of the cell) or DNA damage, or both. Preliminary results from separate printing approaches, including modified thermal inkjet, modified laser-induced forward transfer (LIFT), and extrusion pens, proved that neither potential source of injury would kill nor damage the cells.Reference Ringeisen, Spargo and Wu13 Several approaches have now moved forward to arrange and assemble cells into 3D patterns, opening up the prospect of engineering made-to-order heterogeneous cell constructs.Reference Mironov, Boland, Trusk, Forgacs and Markwald11,Reference Mironov, Kasyanov, Drake and Markwald14,Reference Mironov, Reis and Derby15
In general, cell and organ printing is one approach in the much broader TE community.Reference Mironov, Visconti and Markwald16 The bioprinting approach has specific advantages that include direct cell and biomaterial deposition to create multicellular 3D structures and patterns that mimic natural tissue, unique scaffold material design, and the prospect for printing both vascular and microvascular networks throughout the engineered tissue. Figure 1 compares bioprinting technologies to traditional cell seeded scaffold-based TE approaches. Cell and organ printing technologies differ because they are used to engineer heterogeneous cell microenvironments. In general, printers can organize cells, scaffold, and signaling molecules in micron-scale configurations over mm to cm length structures. With the aid of layer-by-layer processing, printers have the ability to create heterogeneous three-dimensional cellular constructs. Cell-cell communication (e.g., paracrine and juxtacrine signaling) and/or biofactor co-printing can then be used to induce differentiation, including lumen/vascular formation and development of an entirely synthetic cellular microniche (the microenvironment around cells that make individual tissues unique—extracellular matrix, cell-cell junctions).
Overview of how organ printing fits into the larger discipline of tissue engineering.
Since those early days, cell and organ printing has blossomed into a research field that spans the globe. There are three primary approaches currently being explored: layer-by-layer biofabrication, scaffold-only printing with subsequent cell seeding, and printing cell aggregates followed by cellular self-assembly. These approaches are attacked by five primary printing tools (and numerous minute variations), each with their own advantages and disadvantages. After over a decade, animal studies have begun in some labs, and there are now several companies either pursuing organ printing products or selling organ printers alone. It is truly an exciting time, and even after 15 years, the field is still in its infancy with burgeoning potential far outweighing deficiencies. This article will introduce the rich past of cell and organ printing, and then delve into both the current day tools and approaches being used to print organs. We will conclude by offering future prospects and predictions for the field, including a brief description of emerging commercial ventures.
Approaches to print complex 3D cellular constructs
Generally speaking, the fabrication of complex 3D constructs/structures can be classified into two main categories: solvent casting/molding and direct writing. Using a casting/molding approach, 3D constructs can be fabricated in two ways: filling porous construct biomaterials into a sacrificial mold; or making a mold using construct biomaterials and then directly melting away sacrificial patterns/cores, resulting in a mold construct with cavities inside, such as a vascular construct.Reference Miller, Stevens, Yang, Baker, Nguyen, Cohen, Toro, Chen, Galie, Yu, Chaturvedi, Bhatia and Chen17 Both approaches usually follow the molding steps with uniform cell seeding, resulting in a living 3D construct. Unfortunately, the inability to make changes quickly or create heterogeneity and sufficient vascularization of thick tissue constructs limit broad applications of this technique. In addition, it is difficult to precisely seed different multiple cell types within 3D constructs with a desirable cell density and orientation.
Alternatively, using a direct writing approach, complex and heterogeneous structures can be created without the need for part-specific tooling or masks. Direct writing, commonly known as “3D printing” or “additive manufacturing,” is a suite of computer automated technologies to fabricate 3D structural and functional parts from different engineering as well as biological materials. Novel components and structures of any complex geometries and material compositions can be fabricated without difficulty. Most liquid-based 3D direct-write techniques, which may be potentially applicable for biofabrication applications,Reference Ringeisen, Othon, Barron, Young and Spargo12 can be classified into two main types: droplet-based (discrete) and filament-based (continuous). The former can be implemented by using modified inkjetReference Boland, Tao, Damon, Manley, Kesari, Jalota and Bhaduri18–Reference Xu, Chai, Huang and Markwald20 or LIFTReference Schiele, Corr, Huang, Raof, Xie and Chrisey21,Reference Koch, Deiwick, Schlie, Michael, Gruene, Coger, Zychlinski, Schambach, Reimers, Vogt and Chichkov22 to generate droplets as building blocks for deposition. The latter approach primarily uses extruded filaments instead of droplets to build parts, and this technology is represented by fused deposition modeling,Reference Zein, Hutmacher, Tan and Teoh23 extrusion,Reference Hamid, Snyder, Wang, Timmer, Hammer, Guceri and Sun24 and micropen printing.Reference Lewis25 With various recent advances in 3D printing, including the ability to deposit patterns of living cells (e.g., cell and organ printing), direct writing is a promising and emerging technological avenue for the fabrication of unique and complex 3D cellular constructs for regenerative medicine applications.
Layer-by-layer biofabrication
Layer-by layer biofabrication of cellular constructs can be approached in two ways: either by direct, serial bioink deposition onto the previous printed layer in an additive fashion or by printing bioink onto several “biopaper” scaffolds individually and then assembling them at a later time into a 3D construct. Both approaches have been demonstrated. Figure 2 shows a representative 3D alginate tube (6 mm tall, wall thickness of 0.8 mm, and outer diameter of 3.3 mm) printed with a modified LIFT technique using additive layer-by-layer printing of scaffold only.Reference Yan, Huang and Chrisey26 This approach can also simultaneously print scaffolds and living cells and will be described in more detail in the LIFT section. Inkjet and extrusion pens have also been used to create stand-alone layer-by-layer constructs from various hydrogel materials and cells.Reference Schiele, Corr, Huang, Raof, Xie and Chrisey21
Serial deposition layer-by-layer biofabrication using a laser-induced forward transfer printing approach. Images are of a freestanding acellular alginate scaffold with a diameter of 3 mm. (a) A tube imaged from the side; (b) a tube imaged from the top. Reproduced with permission from Reference 26. © 2013 Institute of Physics Publishing.
The biopaper approach to layer-by-layer biofabrication relies on prefabricated sheets of biocompatible polymer or hydrogel to be loaded into a cell printer to create cellular patterns on each sheet. This approach was recently demonstrated using the laser printing technique termed BioLP to print endothelial cells into cord-like structures growing on poly(lactic-co-glycolic acid) (PLGA) biopapers, thus engineering endothelial networks throughout an entire 3D construct.Reference Pirlo, Wu, Liu and Ringeisen27 The PLGA biopapers were mechanically stable enough to be loaded and unloaded into the cell printing system. Figure 3 shows single biopaper sheets and stacked layer-by-layer constructs (a–d, respectivelyReference Pirlo, Wu, Liu and Ringeisen27).
Biopaper layer-by-layer biofabrication to create a four-layer poly(lactic-co-glycolic acid) scaffold. (a) Single biopaper sheets. (b–d) Building layer-by-layer 3D biopaper constructs. (b), (c), and (d) show one, two, and four layers of biopaper, respectively. Squares are approximately 1.5 cm. Reproduced with permission from Reference 27. © 2011 Wiley Periodicals.
There are benefits and limitations for both approaches. The additive cell printing approach can either simultaneously deposit scaffold and cells or serially print scaffold followed by seeding or patterning cells on top of the previously printed scaffold layer. By printing all components of the system, complex 3D shapes, including pores, overhanging structures, and through vias, can be created in real time. The type of materials capable of being printed, however, limits the serial/additive approach to hydrogels and soft materials with limited sophistication. Some advantages of using the biopaper-based approach are that the scaffold can be fabricated using non-biocompatible methods and solvents as well as more time-intensive processing. This expands the potential options and complexity of the scaffold, allowing for processes such as nanofabrication, micromolding, electrospinning,Reference Catros, Guillemot, Nandakumar, Ziane, Moroni, Habibovic, van Blitterswijk, Rousseau, Chassande, Amedee and Fricain28 porogen leaching (dissolving materials or particles to enhance and/or create a porous material),Reference Catros, Guillemot, Nandakumar, Ziane, Moroni, Habibovic, van Blitterswijk, Rousseau, Chassande, Amedee and Fricain28 and photopolymerization. Additionally, time independence allows for post-processing of the printed biopapers, including natural or biofactor-induced maturation and development of cells and tissue structures, such as microvasculature prior to inclusion in a thicker tissue construct. By not printing the scaffold material, macroscopic structure and shape changes, however, are limited during the cell and construct engineering.
Scaffold printing with subsequent cell seeding
As discussed previously, simultaneous scaffold deposition and living cell printing can limit the type of materials used to only soft gel-based polymers. The low viscosity and room-temperature processing of the gel-based polymers, however, enables the end user to encapsulate cells within the scaffold as it is being fabricated. To model harder tissues such as bone, printing or direct write tools use polymers with a melting point of at least 60°C, which usually also has a higher Young’s modulus. Unfortunately, due to the solvents and/or temperatures required during hard material fabrication processes, living cells have not been embedded within the scaffold during fabrication.Reference Yeong, Chua, Leong and Chandrasekaran29,Reference Peltola, Melchels, Grijpma and Kellomäki30 In order to print scaffolds for hard tissues, simultaneous material and cell deposition are impossible unless layer-by-layer biopaper approaches are used instead. Hard fabrication processes, however, hold advantages, including the ability to perform post-processing surface enhancements to change the topology (including nanotopology) or add functional groups for enhanced or specific cell attachment. Cells are then traditionally seeded onto hard printed scaffolds by either directly pipetting them onto the scaffold or by using a cell printer to spatially control the deposition of cells within the scaffold.Reference Xu, Han, Dong, Shen, Chai, Yu, Lang and Ai31,Reference Klein and Glatzer32
Cell aggregate printing and cellular self-assembly
An approach often used in cell culture is to form large (>500 μm diameter) 3D cell aggregates, including spheres, blocks, cylinders, and rings. Spherical cell aggregates or spheroids are particularly common in the culture of many stem cells,Reference Cerwinka, Sharp, Boyan, Zhau, Chung and Yates33 where enhanced differentiation has been demonstrated over alternative culturing conditions. In a modular or self-assembling approach to organ printing, such aggregates are printed or arranged to form a larger ordered structure with volumes and mechanical and maturation properties not immediately possible by printing dissociated cell suspensions. In such a process, order at the cellular sub-200 μm level is left to spontaneous cell arrangement by intrinsic signaling (i.e., self-assembly).
Sheets and cellular tubes have been demonstrated using continuous printing of Chinese hamster ovary (CHO) cell spherical aggregates.Reference Jakab, Damon, Neagu, Kachurin and Forgacs34Figure 4a–c is an example of extrusion pen printed spherical CHO aggregates, demonstrating the initial printed structure and self-assembly into a contiguous ring structure. Additional bioprinting work has demonstrated scaffold-free vascular tissue fused from cylindrical aggregates of smooth muscle cells and fibroblasts.Reference Norotte, Marga, Niklason and Forgacs35Figure 4d–f shows the potential of using heterogeneous (2 cell type) cell aggregate approaches for TE. These non-printed structures (hanging drop aggregate formation) demonstrate the fusion of cellular spheroids comprising endothelial (inner cells, PECAM stain green) and smooth muscle cells (outer cells, SMαA red stain).Reference Fleming, Argraves, Gentile, Neagu, Forgacs and Drake36 The combined results shown in Figure 4 indicate the potential of luminal vessel fabrication via spheroid printing and self-assembly.
(a–c) Printed Chinese hamster ovary cell aggregates via an extrusion pen tool. (a–c) Show how the individually deposited aggregates fuse into a ring structure over time. The average diameter of the aggregates is 500 μm. Reproduced with permission from Reference 34. © 2006 IOS Press. (d–e) Non-printed (hanging drop method) bi-culture aggregates of endothelial and smooth muscle cells. (d–f) Show fusion and lumen development over one hour, three hours, and eight hours, respectively (scale bar = 150 μm). Reproduced with permission from Reference 36. © 2010 Wiley.
It has been notedReference Fleming, Argraves, Gentile, Neagu, Forgacs and Drake36 that a deficiency of spheroid printing is the time-consuming preparation of spherical aggregates. Recently, valve-based printers that use intermittent open/close cycles have enabled rapid fabrication of uniform spherical aggregates of human embryonic stem cells (hESC).Reference Faulkner-Jones, Greenhough, King, Gardner, Courtney and Shu37 A second limitation of cell aggregate printing is the resolution of the printing process (∼0.5 mm). This trait is advantageous in that realistic cell numbers can be printed into 3D structures at relevant time scales, but it is yet to be shown whether the microstructures found in natural tissue (microcapillaries, cell-cell junctions, membranes, sinuses) can be replicated by relying on self-assembly alone. It is possible that cell aggregate approaches could be hybridized with higher resolution printing approaches to create macroscopic constructs that include microscopic cellular interfaces and structures.
Printing tools
Extrusion pens
Extrusion pens are rapid prototyping systems that use liquid inks pushed (extruded) through micropens, usually with diameters exceeding 200 μm, to direct write 3D structures. These pens can be classed as pneumatic, thermal, or mechanical cell printing approaches. Thermal extrusion pens have been used to fabricate tissue constructs from polymers with high melting points.Reference Shor, Guceri, Wen, Gandhi and Sun38,Reference Shor, Guceri, Chang, Gordon, Kang, Hartsock, An and Sun39 This work demonstrated the bio-mimicry, precision, and architectural capabilities of the thermal extrusion pen by fabricating a human femur tissue scaffold with subsequent cell/bone growth. Due to the high temperature extrusion, thermal pens lack the ability to embed biomolecules within the tissue constructs during fabrication. Pneumatic extrusion pens have also been used to embed living cells within tissue constructs.Reference Chang, Emami, Wu and Sun40 Cells were mixed with hydrogels and then extruded via the pneumatic pen to develop 3D liver micro- and milli-organs for in vitro drug metabolism studies. One drawback of pneumatic pens for cell printing is that they are typically limited to low viscosity polymers, often causing a lack of structural stability and integrity for large-scale models. Mechanical pens are similar to pneumatic pens in that both have the capability to embed cells within their respective tissue constructs. Due to the characteristics of the mechanical pens, this tool has the capability to extrude polymers of a higher viscosity compared to the pneumatic pens, but these scaffolds still usually lack structural stability on large-scale scaffold models. As mentioned previously, extrusion pens can also deposit patterns of cell aggregates into larger 3D constructs.
Inkjet
A biomimetic approach proposed over a decade agoReference Mironov, Boland, Trusk, Forgacs and Markwald11 suggested building tissues layer-by layer using inkjet printing and related techniques. These tools were ideal for building cell constructs with the soft, low temperature materials needed for biological assembly. The inkjet printer could be used to deposit sheets of cells that in turn may generate both soft and hard matrices. This first required the development of methods to keep cells alive and functioning during the storage, printing, and attachment processes.
Most of the work to date has been done using thermal inkjet printers, which have some advantages for laboratory use. Initial work focused on cell survival,Reference Xu, Jin, Gregory, Hickman and Boland41 quantifying the process,Reference Cui and Boland42 appropriate printing fluid selections,Reference Boland, Xu, Damon and Cui43 and the effect of heat and shear on printed cells.Reference Cui, Dean, Ruggeri and Boland44 Inkjet cell printing approaches have a wide range of printing variables spanning 103 to 105 drops/sec (one to ten cells/drop) and droplet resolutions from tens to hundreds of microns. Figure 5 shows a zig-zag pattern of fibroblasts printed via a thermal inkjet using an alginate bioink.Reference Xu, Chai, Huang and Markwald20 The overhang structure was printed at 200 mm/min with a total of 210 sequential layers, each printed on top of a previously gelatinized layer. More recently, the technique has allowed for the construction of microvasculature, and this breakthrough has spawned several TE applications, including bilayered skin constructsReference Yanez, Rincon, Cortez, Boland and Maria45 as well as cartilage.Reference Cui, Breitenkamp, Finn, Lotz and D’Lima46
Overhanging zig-zag alginate/fibroblast construct formed via thermal inkjet. The structure was formed using a serial layer-by-layer approach. Reproduced with permission from Reference 20. © 2012 Wiley.
Electrospray
Over 100 years ago, Rayleigh and others discovered that a fluid droplet at the end of a capillary subjected to an electric field can form a cone jet.Reference Rayleigh47–Reference Zeleny49 A jet can be formed at the end of a conducting needle containing a fluid by applying a potential difference between the needle and a ground plane (i.e., the substrate). This process is called electrohydrodynamic jetting or electrospray. The physical attributes of the fluid jet (i.e., size, speed, shape) are easily controlled, and under the correct conditions, the jet can evolve into dropletsReference Zeleny49 that can be used for cell printing.
In 2006, Jayasinghe et al. demonstrated that electrospray is suitable as a non-contact jetting technique of printing biological materials, referred to as bioelectrospray (BES).Reference Jayasinghe50–Reference Eagles, Qureshi and Jayasinghe52 When combined with electrospinning techniques, scaffolding materials and cells can be simultaneously deposited.Reference Jayasinghe53,Reference Jayasinghe54 Typical conditions for BES cell deposition are a flow rate of 10–7 to 10–12 m3/s, bioink cell concentrations of 104–107 cells/ml, and a resolution size >100 μm.Reference Eagles, Qureshi and Jayasinghe52,Reference Jayasinghe53 Under these conditions, no cellular damage was detected, but it was also shown that BES has difficulties in stabilizing jetting conditions, controlling the number of cells per spot, and maintaining print location.Reference Eagles, Qureshi and Jayasinghe52,Reference Jayasinghe53
Modified laser-induced forward transfer
The modified LIFT process has been well published over the past 15 years and therefore will not be detailed here.Reference Kántor, Tóth and Szörényi55 Modified LIFT is based on a focused laser pulse that energetically induces the forward transfer of a droplet of material from a bioink-coated optically transparent glass plate, effectively using the focused laser energy to control the droplet size rather than an orifice. The first modified LIFT cell printing studies showed near 100% viability as well as retained genetic and phenotypic functionality of cells processed under a range of conditions, receiving substrates and cell types.Reference Schiele, Corr, Huang, Raof, Xie and Chrisey21,Reference Catros, Guillemot, Nandakumar, Ziane, Moroni, Habibovic, van Blitterswijk, Rousseau, Chassande, Amedee and Fricain28,Reference Ringeisen, Kim, Barron, Krizman, Chrisey, Jackman, Auyeung and Spargo56–Reference Barron, Wu, Ladouceur and Ringeisen61 Printed droplets can have a range of cell numbers (single cells to hundreds per drop) depending on the operating conditions of the laser pulse and cell concentration of the bioink. Demonstrated cell throughputs can range from 102 to 106 cells per second due to the wide range of possible pulsed laser repetition rates (102 to 105).Reference Guillotin, Souquet, Catros, Duocastella, Pippenger, Bellance, Bareille, Remy, Bordenave, Amedee and Guillemot62 A wide variety of bioinks with diverse viscosities have also been used, including modified cell culture media, hydrogels, and inorganic pastes.Reference Ringeisen, Chrisey, Piqué, Young, Modi, Bucaro, Jones-Meehan and Spargo2,Reference Schiele, Chrisey and Corr63
More recent studies have focused on building 3D tissue constructs via layer-by-layer approaches (Figures 226 and 327) and utilizing the small droplet size and single cell resolution of the modified LIFTReference Barron, Krizman and Ringeisen64 to print cellular microstructures unattainable by bulk cell seeding approaches.Reference Wu and Ringeisen65 One demonstrated example of this high resolution printing is shown in Figure 6a, where sub-50 micron diameter lines of human umbilical vein endothelial cells (HUVECs) were printed in a branched pattern to a hydrogel.Reference Ringeisen, Spargo and Wu13,Reference Wu and Ringeisen65 A day after printing, the HUVECs self-recognized and began to form tube-like structures, connecting the printed pattern into a branched network. Similar experiments have addressed multiple sheets of porous PLGA biopaper with zig-zag lines of HUVECs with multiple crossings (Figure 6b–c).Reference Pirlo, Wu, Liu and Ringeisen27 These experiments demonstrate how printing macroscale patterns (mm to cm lines) with micron-scale resolution can yield a template for cellular self-assembly to create heterogeneous cellular structures throughout scaffolds. Challenges for modified LIFT approaches to organ printing include achieving high enough cell and material throughput as well as printing stable 3D constructs. These challenges are being addressed by finding appropriate 3D bioinks (Figure 2) and by using high repetition rate lasers.Reference Guillotin, Souquet, Catros, Duocastella, Pippenger, Bellance, Bareille, Remy, Bordenave, Amedee and Guillemot62
(a) Printed human umbilical vein endothelial cells (HUVECs) in a vascular tree pattern showing lumen-like growth after 24 hours. Cells were printed onto Matrigel via BioLP, a laser-induced forward transfer-based process. Reproduced with permission from Reference 65. © 2010 Institute of Physics Publishing. (b–c) Crisscross HUVECs pattern printed onto a PLGA biopaper via BioLP. Lumen-like growth is observed with multiple crossings after 24 hours in (b) and (c). Spacing between printed lines is 150 μm. Reproduced with permission from Reference 27. © 2012 Wiley.
Selective photopolymerization
Selective photopolymerization in electronics or biofabrication is commonly achieved by a photomask and a UV lamp source. Alternatively, a computer-aided design or “virtual mask” can be used with a laser as the radical initiating energy source. Conventionally, these processes are limited to 2D and/or additive structures (i.e., layer-by-layer) and may commonly be referenced as stereolithography.Reference Zhang, Qu, Soman, Hribar, Lee, Chen and He66 However, polymerization of 3D structures within a gel is possible with tight control over the voxels of photoinitiation by using a strongly focused laser beam with a small, high intensity waist or with even more precision by multi-photon systems.Reference Doraiswamy, Jin, Narayan, Mageswaran, Mente, Modi, Auyeung, Chrisey, Ovsianikov and Chichkov67,Reference Farsari, Vamvakaki and Chichkov68 Two photon polymerization (TPP, 2PP) can be used to directly write 3D tissue scaffolds with complex, uniform pore architectures,Reference Käpylä, Aydogan, Virjula, Vanhatupa, Miettinen, Hyttinen and Kellomäki69 or TPP may also be used in conjunction with micro-molding to create 3D tissue scaffolds.Reference Koroleva, Gill, Ortega, Haycock, Schlie, Gittard, Chichkov and Claeyssens70,Reference Koroleva, Gittard, Schlie, Deiwick, Jockenhoevel and Chichkov71 TPP has also been used to print 3D polymer–protein hybrid microstructures combining the mechanical properties of a polymer with the signaling properties of proteins.Reference Engelhardt, Hoch, Borchers, Meyer, Krüger, Tovar and Gillner72
Comparison and contrast of cell printing tools
Table I shows a comparison of different cell and organ printing tools. Rather than attempt a quantitative comparison when resolutions and speeds can vary over several orders of magnitude, this chart shows a qualitative comparison using data defined in previous sections. It is immediately evident that certain tools are more suited for different approaches; modified LIFT tools, with their fine resolution, are well suited for layer-by-layer (scaffold plus cells and biopaper) approaches. LIFT approaches are also the only orifice-free technique, greatly broadening the viscosity range for bioinks. Inkjet is perhaps the most versatile technique, with good droplet resolution and the ability to utilize nearly every approach, including self-assembly of cell aggregates and layer-by-layer. Electrospray is a continuous approach with excellent cell throughput but limited resolution and control over placement/patterning of material. Electrospray aims to create sheets and layers of cell-laden biomaterials rather than discrete cell patterns. Extrusion pens have the fastest speed and higher cell throughput based on their large bore diameter orifices. These approaches are best suited for cell aggregate work and scaffold plus cell printing in a layer-by-layer fashion. Photopolymerization techniques do not print living cells, but have the ability to very rapidly create intricate and high-resolution scaffolds layer-by-layer.
Comparison between different cell printing tools.
Dashed or continuous line = discrete or continuous deposition. Arrow head size = relative resolution (qualitative). Length of line(s) = relative deposition speed or cell throughput.
Commercial efforts
Recent advances in the research world have enabled several commercial bioprinting ventures to expand. Sciperio was one of the first companies to develop a 3D bioprinter, termed the Biological Assembly Tool or BAT. nScrypt was founded in 2002 as a joint venture with Sciperio to manufacture, sell, and service Sciperio’s Micro Dispense Direct Write (MDDW) technology. nScrypt manufactures its TE series tabletop dispense system for biological applications, having demonstrated both live cell printing and layer-by-layer scaffold construction.Reference Xie, Parkhill, Warren and Smay73
Roslin Cellab is a stem cell technology company that applies new technologies to human stem cell systems and is developing a pneumatic valve based printerReference Faulkner-Jones, Greenhough, King, Gardner, Courtney and Shu37 (extrusion pen) for commercial fabrication of stem cell aggregates. Roslin Cellab has recently been funded to develop human mini-liver tissues for use in pharmaceutical drug testing. The hope is that early versions of mini-tissues could be substituted for portions of early-stage animal work in drug and toxicity testing, while later applications could be for organ replacement.
Organovo has developed the NovoGen MMX Bioprinter, designed specifically to direct write three-dimensional cellular structures while maintaining a high cellular viability throughout the printing process. The printing mechanism is based on continuous extrusion instead of drop-on-demand. In addition to bioprinting of cellular aggregates, its continuous deposition module enables integrated use of commercially available syringes for the extrusion of hydrogels, which can optionally contain living cells in a wide range of cell concentrations. The printer has been successfully demonstrated to construct perfusable blood vessels (tubes capable of transporting and diffusing nutrients) and implantable nerve conduits with mechanical integrity and acceptable tensile strength.
TeVido BioDevices is using 3D bio-printers to construct autologous adipose tissue implants for reconstructive options after cancer lumpectomy. A second product, utilizing skin cells, has applications in rebuilding areola that have been removed or radically changed due to mastectomy or lumpectomy procedures.
Future prospects
Looking back over the last 15 years, cell and organ research has grown out of its infancy and into adolescence. Nearly gone are the days of reporting cell viability and functionality alone. The bar should now be set higher—building 3D cell constructs and testing the functionality of those constructs to compare with natural human tissue. The first printed tissues and organs of use to the tissue engineering community will most likely be in vitro 3D human organ mimics that could be used for any number of applications, including biomarker discovery as well as pharmaceutical and cosmetic testing. True regenerative medicine applications (e.g., printing and growing organs as an alternative to relying on the organ donor programs currently in use) are still a decade or more in the future, as these products will require FDA approval. The first printed organs for regenerative medicine will likely be non-vascularized systems such as cartilage, skin grafts, or bladders.Reference Michael, Sorg, Peck, Koch, Deiwick, Chichkov, Vogt and Reimers74 The greatest promise, however, is for organ printing to solve the most difficult problem today in regenerative medicine—3D vascularization. Because organ printing approaches can generate heterogeneous (multi-cell type), CAD/CAM generated 3D constructs with cell placement at the 100 μm scale, it is feasible that fully vascularized organs could be generated by these technologies either through direct cell printing of vascular structures or printing of vascular spheroids capable of self-assembly and autonomous differentiation. Companies now sell 3D biological printers, expanding opportunities beyond the relatively small research groups that initially developed the different technologies described here, and are using these technologies with the hope that the promise of printed organs will not only save lives and limit animal testing but also bear financial reward. With time and systematic experimentation, researchers in this field will continue to develop technologies and earn a broader role throughout the TE community.
Bradley Ringeisen is head of the Bioenergy and Biofabrication Section at the US Naval Research Laboratory (NRL). He earned a PhD degree in physical chemistry from the University of Wisconsin–Madison in 2000 and was a postdoctoral fellow at NRL from 2000 to 2002. He has been a research chemist at NRL since 2002. His research interests include the study of microbial extracellular electron transfer, biofuel production, the bio/nano interface and cell and tissue printing for regenerative medicine applications. Ringeisen can be contacted by email at ringeisen@nrl.navy.mil.
Russell Kirk Pirlo is a research biologist at the US Naval Research Laboratory. He has a BS degree in physics from West Virginia University and a PhD degree in bioengineering from Clemson University. His research has focused on laser optics, instrument design, control system programming, cell culture, and microscopy/bioimaging on developing cell patterning technologies for developmental research and tissue engineering. Pirlo can be contacted by email at russell.pirlo@nrl.navy.mil.
Peter K. Wu is a professor of Physics and Materials Science at Southern Oregon University. He earned his BS degree in physics and mathematics from Macalester College in St. Paul, Minn. He received his MS degrees in physics and materials science and a PhD degree in materials science from the University of Wisconsin–Madison. His current research includes laser-assisted deposition of biological materials, microbial fuel cells, and remote sensing technology. Wu can be contacted by email at wu@sou.edu.
Thomas Boland is a professor in the Department of Metallurgy and Materials Engineering at the University of Texas at El Paso (UTEP). He also serves as the director of UTEP’s Biomedical Engineering Program. He received his BS in chemical engineering from the Ecole Nationale Supérieure d’Ingénieurs de Genie Chimique in Toulouse, France, in 1990, and his PhD degree in chemical engineering from the University of Washington, Seattle, Wash., in 1995. Boland’s research interests are applying engineering principles to automate, predict, and build three-dimensional structures that show biological function. He is the author of more than 45 publications, and is a member of the AVS, MRS, the Society for Imaging Science and Technology, and the Tissue Engineering and Regenerative Medicine International Society. Boland can be contacted by email at tboland@utep.edu.
Yong Huang is a professor of Mechanical and Aerospace Engineering at the University of Florida, Gainesville. His current research topics focus on direct writing of three-dimensional structures using laser and inkjet, precision engineering of medical implants, and fabrication of polymeric microspheres/microcapsules/hollow fiber membranes. He received numerous awards, including the NSF CAREER Award (2008) and the ASME Blackall Machine Tool and Gage Award (2005). He is a Fellow of ASME. Huang can be contacted by email at yongh@ufl.edu.
Wei Sun is the Albert Soffa Chair Professor of Mechanical Engineering at Drexel University and 1000plan Chair Professor and Director of Biomanufacturing Engineering at the Research Institute of Tsinghua University, Beijing, China. Sun’s research has focused on biofabrication, computer-aided tissue engineering, CAD/CAM, and additive manufacturing. He has published more than 300 journal and conference papers/abstracts. Sun is currently president of the International Society of Biofabrication and editor-in-chief of the journal Biofabrication. Sun can be contacted by email at sunwei@drexel.edu.
Qudus Hamid is pursuing his PhD degree in mechanical engineering at Drexel University. His areas of interest include precision polymer extrusion, rapid prototype, photolithography, and electrospinning. He is currently working on an integrated additive biofabrication system. Hamid can be contacted by email at qh25@drexel.edu.
Douglas B. Chrisey is a professor of Physics and Engineering Physics at Tulane University. After receiving his PhD degree in engineering physics from the University of Virginia in 1987, he spent the next 17 years at the US Naval Research Laboratory before joining RPI in 2006 and Tulane University as the Jung Chair in Materials Engineering in 2012. He has more than 400 cited publications and 18 granted patents. Chrisey can be contacted by email at douglasbchrisey@gmail.com.
