Portal de Periódicos da CAPES

Using Remote Fields for Complex Tissue Engineering

2019; Elsevier BV; Volume: 38; Issue: 3 Linguagem: Inglês

10.1016/j.tibtech.2019.07.005

0167-9430

James P. K. Armstrong, Molly M. Stevens,

Tissue Engineering and Regenerative Medicine

Remote fields have recently emerged as an alternative to contact force application and microfabrication in the engineering of complex tissue structures.New methods have been developed that use magnetic fields to manipulate nanoparticles for biochemical patterning, material assembly, and biological actuation.A series of recent reports in optical manipulation have shown high-level control over gene expression, epigenetics, material degradation, and reversible protein immobilization.Recent advances have been made in preserving acoustically patterned cell arrays, which have enabled the engineering skeletal muscle, cardiac, and neural tissue. Great strides have been taken towards the in vitro engineering of clinically relevant tissue constructs using the classic triad of cells, materials, and biochemical factors. In this perspective, we highlight ways in which these elements can be manipulated or stimulated using a fourth component: the application of remote fields. This arena has gained great momentum over the last few years, with a recent surge of interest in using magnetic, optical, and acoustic fields to guide the organization of cells, materials, and biochemical factors. We summarize recent developments and trends in this arena and then lay out a series of challenges that we believe, if met, could enable the widespread adoption of remote fields in mainstream tissue engineering. Great strides have been taken towards the in vitro engineering of clinically relevant tissue constructs using the classic triad of cells, materials, and biochemical factors. In this perspective, we highlight ways in which these elements can be manipulated or stimulated using a fourth component: the application of remote fields. This arena has gained great momentum over the last few years, with a recent surge of interest in using magnetic, optical, and acoustic fields to guide the organization of cells, materials, and biochemical factors. We summarize recent developments and trends in this arena and then lay out a series of challenges that we believe, if met, could enable the widespread adoption of remote fields in mainstream tissue engineering. Tissue engineers today can draw on a host of new scientific and technological tools, such as cell reprogramming, gene editing, and advanced cell screening [1Armstrong J.P.K. Stevens M.M. Emerging technologies for tissue engineering: from gene editing to personalized medicine.Tissue Eng. Part A. 2019; 25: 688-692Crossref PubMed Scopus (13) Google Scholar]. These advances have enabled unprecedented levels of biological control and characterization, however, it is striking to note how the core principles of this discipline have remained largely unchanged from the early tissue engineering experiments carried out by Bell and coworkers in 1979 [2Bell E. et al.Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1274-1278Crossref PubMed Scopus (1678) Google Scholar]. In particular, cells, biomaterials, and biochemical factors are still widely held up as the three pillars of tissue engineering. If we set aside certain practical flaws, such as nutrient transport gradients [3Armstrong J.P.K. et al.Artificial membrane binding proteins stimulate oxygenation of stem cells during tissue engineering of large cartilage constructs.Nat. Commun. 2015; 6: 7405Crossref PubMed Scopus (49) Google Scholar], then we can consider most tissue engineering protocols to be equilibrium systems: cells dispersed homogenously throughout isotropic materials, with nutrients and biochemical factors supplied in a global fashion by bulk diffusion from the surrounding culture medium. The absence of any imposed constraints or directionality means that these strategies generally yield homogenous tissue constructs that exhibit very little structural organization. Evidently, this situation is far removed from the vivid complexity of natural systems. Tissues often exhibit multiscale and hierarchical structure, with anisotropic, inhomogeneous, and directional arrangement of both cells and extracellular matrix components. Structural organization is almost always inextricably linked to physiological function, for instance, the anisotropic packing of myofibers ensures directional actuation of skeletal muscle [4Frontera W.R. Ochala J. Skeletal muscle: a brief review of structure and function.Calcif. Tissue Int. 2015; 96: 183-195Crossref PubMed Scopus (346) Google Scholar], the orientation of cardiomyocytes guides the propagation of contractile waves in myocardial tissue [5Nitsan I. et al.Mechanical communication in cardiac cell synchronized beating.Nat. Phys. 2016; 12: 472-478Crossref Scopus (58) Google Scholar], and the zonal organization of cartilage and bone allows effective load transmission across the osteochondral interface [6Bergholt M.S. et al.Raman spectroscopy reveals new insights into the zonal organization of native and tissue-engineered articular cartilage.ACS Cent. Sci. 2016; 2: 885-895Crossref PubMed Scopus (51) Google Scholar]. Moreover, it is widely appreciated that tissue grafts engineered with mature vasculature and structural profiles that match the defect site have an improved chance of successful integration and survival [7Ben-Shaul S. et al.Mature vessel networks in engineered tissue promote graft – host anastomosis and prevent graft thrombosis.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 2955-2960Crossref PubMed Scopus (30) Google Scholar, 8Patel S. et al.Integrating soft and hard tissues via interface tissue engineering.J. Orthop. Res. 2018; 36: 1069-1077Crossref PubMed Scopus (38) Google Scholar]. It is clear that the functional performance of an engineered tissue graft will be negatively impacted by a failure to address structural complexity. Indeed, it is our view that in vitro tissue engineering is unlikely to become established as a mainstream clinical practice without addressing the issue of structural organization. This point can be illustrated by considering the nature of some of the major tissue engineering products and procedures that have reached the clinic: skin grafts (e.g., Epicel®, Apligraf®, Dermagraft®), cartilage implants (e.g., MACI®), and corneal sheets (e.g., Holoclar®) [1Armstrong J.P.K. Stevens M.M. Emerging technologies for tissue engineering: from gene editing to personalized medicine.Tissue Eng. Part A. 2019; 25: 688-692Crossref PubMed Scopus (13) Google Scholar]. The clinical success of these products is owed to the fact that they can still perform certain functions without possessing optimal tissue structure. Skin grafts can provide a basic physical barrier for patients with burns or ulcers, despite lacking natural features, such as sweat glands. Cellularized implants that fill small articular cartilage defects can improve joint function and relieve pain, despite these constructs lacking the intricate organization of cells and extracellular matrix fibers. Meanwhile, corneal sheets can be used to improve visual acuity in cases of limbal stem cell deficiency, despite the absence of zonal organization. These examples serve as a marker of the current status of tissue engineering: products that offer clinical benefits 'despite' a lack of structural organization. Whether we are looking to improve the clinical utility of existing products or seeking translational solutions for more structurally demanding targets (e.g., liver, neural, cardiac tissue), it is critical that the next generation of tissue engineers focus on methods that can fully replicate physiological functions by faithfully recreating native structural complexity. There are a host of fabrication tools that can be used to spatially arrange cells, materials, and biochemical factors. For instance, 3D bioprinting is commonly used to create precision architecture and patterned multicellular structures [9Graham A.D. et al.High-resolution patterned cellular constructs by droplet-based 3D printing.Sci. Rep. 2017; 7: 7004Crossref PubMed Scopus (78) Google Scholar]. Techniques such as aligned electrospinning [10Xia H. et al.Oriented growth of rat Schwann cells on aligned electrospun poly(methyl methacrylate) nanofibers.J. Neurol. Sci. 2016; 369: 88-95Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar], melt electrospinning writing [11Castilho M. et al.Melt electrospinning writing of poly-hydroxymethylglycolide-co-ε-caprolactone-based scaffolds for cardiac tissue engineering.Adv. Healthc. Mater. 2017; 61700311Crossref Scopus (63) Google Scholar], and unidirectional freeze drying [12Wang D. et al.Highly flexible silica/chitosan hybrid scaffolds with oriented pores for tissue regeneration.J. Mater. Chem. B. 2015; 3: 7560-7576Crossref Google Scholar] can be used to create anisotropic substrates, while materials can also be molded [13Wang X-Y. et al.Engineering interconnected 3D vascular networks in hydrogels using molded sodium alginate lattice as the sacrificial template.Lab Chip. 2014; 14: 2709-2716Crossref PubMed Google Scholar], layered into zonal structures [14Steele J.A.M. et al.Combinatorial scaffold morphologies for zonal articular cartilage engineering.Acta Biomater. 2014; 10: 2065-2075Crossref PubMed Scopus (86) Google Scholar], or cast with mechanical, compositional, or morphogen gradients [15Li C. et al.Buoyancy-driven gradients for biomaterial fabrication and tissue engineering.Adv. Mater. 2019; 31: 1900291Crossref Scopus (26) Google Scholar]. These fabrication-driven approaches are either used to spatially organize components prior to tissue culture or as a means of creating material or biochemical cues that can drive directional or local cell responses during tissue culture. An alternative strategy that has been investigated for complex tissue engineering is the use of externally applied forces. For example, electrical or mechanical stimulation can be used to guide cell alignment or differentiation during tissue engineering [16Rangarajan S. et al.Use of flow, electrical, and mechanical stimulation to promote engineering of striated muscles.Ann. Biomed. Eng. 2014; 42: 1391-1405Crossref PubMed Scopus (63) Google Scholar]. These examples use apparatus that directly interface with either the cells, media, or tissue constructs. In this article, we wish to highlight a related strategy that has recently come to prominence: the use of remotely applied force fields (see Glossary) (e.g., optical, acoustic, magnetic) that can be used to guide structural complexity without any tangible contact. While appreciating that these fields can be used to promote bulk effects (e.g., global cell differentiation), we have focused on strategies that seek to disrupt the equilibrium balance of cells, materials, or biochemical factors (Figure 1, Key Figure). In particular, we highlight recent approaches that use remote fields to: (i) spatially assemble different tissue engineering components; (ii) initiate local responses, such as cell differentiation or material degradation; or (iii) exert directional responses, such as cell or matrix fiber alignment. Strong magnetic fields have been widely used to orient matrix fibers during gelation, for example, Eguchi and coworkers recently used an 8 T field to fabricate aligned collagen hydrogels that could guide Schwann cell orientation [17Eguchi Y. et al.Effectiveness of magnetically aligned collagen for neural regeneration in vitro and in vivo.Bioelectromagnetics. 2015; 36: 233-243Crossref PubMed Scopus (10) Google Scholar] (Figure 2A ). However, the recent trend has seen the introduction of magnetically susceptible components that allow alignment using much weaker magnetic fields. For instance, Antman-Passig and coworkers used superparamagnetic nanoparticles to guide the alignment of collagen fibers in a 26 mT field [18Antman-Passig M. Shefi O. Remote magnetic orientation of 3D collagen hydrogels for directed neuronal regeneration.Nano Lett. 2016; 16: 2567-2573Crossref PubMed Scopus (127) Google Scholar] (Figure 2B). This aligned system was used for neural cell orientation, while a similar approach was employed by Betsch and coworkers for the 4D bioprinting of cartilage tissue [19Betsch M. et al.Incorporating 4D into bioprinting: real-time magnetically directed collagen fiber alignment for generating complex multilayered tissues.Adv. Healthc. Mater. 2018; 71800894Crossref Scopus (44) Google Scholar]. Meanwhile, magnetic surfactant conjugation has been used to magnetize proteins [20Brown P. et al.Magnetizing DNA and proteins using responsive surfactants.Adv. Mater. 2012; 24: 6244-6247Crossref PubMed Scopus (57) Google Scholar], and superparamagnetic nanoparticles have been used as field-responsive carriers for RNA and proteins [21Cruz-Acuna M. et al.Magnetic nanoparticles loaded with functional RNA nanoparticles.Nanoscale. 2018; 10: 17761-17770Crossref PubMed Google Scholar, 22Li C. et al.Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering.Biomaterials. 2018; 176: 24-33Crossref PubMed Scopus (37) Google Scholar, 23Zhang W. et al.Magnetically controlled growth-factor-immobilized multilayer cell sheets for complex tissue regeneration.Adv. Mater. 2017; 291703795Crossref Scopus (47) Google Scholar]. The latter approach was used by Li and coworkers to create gradients of bone morphogenetic protein 2 (BMP-2) within cellularized hydrogels, in order to promote localized osteogenesis and mineralization during osteochondral tissue engineering [22Li C. et al.Glycosylated superparamagnetic nanoparticle gradients for osteochondral tissue engineering.Biomaterials. 2018; 176: 24-33Crossref PubMed Scopus (37) Google Scholar] (Figure 2C). An interesting route to cellular manipulation was recently reported by Tasoglu and coworkers, who used external magnetic fields for the levitational self-assembly of cell-seeded materials in a paramagnetic suspension [24Tasoglu S. et al.Magnetic levitational assembly for living material fabrication.Adv. Healthc. Mater. 2015; 4: 1469-1476Crossref PubMed Scopus (57) Google Scholar]. A more common approach is direct cell magnetization, however, this strategy necessitates the use of cytocompatible protocols that can label cells with a large quantity of paramagnetic material [25Correia Carreira S. et al.Ultra-fast stem cell labelling using cationised magnetoferritin.Nanoscale. 2016; 8: 7474-7483Crossref PubMed Google Scholar]. Bulk cell magnetization has been used to engineer vocal folds [26Pöttler M. et al.Magnetic tissue engineering of the vocal fold using superparamagnetic iron oxide nanoparticles.Tissue Eng. Part A. 2019; (Published online May 2, 2019. https://doi.org/10.1089/ten.TEA.2019.0009)Crossref PubMed Scopus (8) Google Scholar], secretory epithelial organoids [27Adine C. et al.Engineering innervated secretory epithelial organoids by magnetic three-dimensional bioprinting for stimulating epithelial growth in salivary glands.Biomaterials. 2018; 180: 52-66Crossref PubMed Scopus (27) Google Scholar], and embryoid bodies [28Du V. et al.A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation.Nat. Commun. 2017; 8: 400Crossref PubMed Scopus (72) Google Scholar], however, superparamagnetic nanoparticles can also interact with cells in a more refined manner. Historically, superparamagnetic nanoparticles have been targeted to specific integrin receptors or ion channels on the cell membrane surface allowing remote field actuation [29Dobson J. Remote control of cellular behaviour with magnetic nanoparticles.Nat. Nanotechnol. 2008; 3: 139-143Crossref PubMed Scopus (372) Google Scholar]. More recently, magnetic fields have been used to manipulate the position of functionalized, intracellular superparamagnetic nanoparticles in order to modulate processes such as cytoskeletal assembly, mitochondrial dynamics and gene expression [30Etoc F. et al.Subcellular control of Rac-GTPase signalling by magnetogenetic manipulation inside living cells.Nat. Nanotechnol. 2013; 8: 193-198Crossref PubMed Scopus (95) Google Scholar, 31Stanley S.A. et al.Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles.Nat. Med. 2015; 21: 92-98Crossref PubMed Scopus (129) Google Scholar, 32Liße D. et al.Engineered ferritin for magnetogenetic manipulation of proteins and organelles inside living cells.Adv. Mater. 2017; 291700189Crossref Scopus (21) Google Scholar]. With further development, this method could potentially offer a remotely activated magnetogenetic switch for complex tissue engineering. Tissue engineering components can be manipulated with much higher precision using optical technologies. Light irradiation can be used to form, cleave, or rearrange chemical bonds, a characteristic ideally suited to patterning materials [33Ruskowitz E.R. DeForest C.A. Photoresponsive biomaterials for targeted drug delivery and 4D cell culture.Nat. Rev. Mater. 2018; 317087Crossref Scopus (157) Google Scholar]. Historically, simple photomask-based systems have been used to present an uneven distribution of light, however, the recent trend has been towards more dynamic 3D patterning techniques, such as multiphoton lithography. This method was used by Arakawa and coworkers to precisely sculpt vascular networks in hydrogels crosslinked with photodegradable peptides [34Arakawa C.K. et al.Multicellular vascularized engineered tissues through user-programmable biomaterial photodegradation.Adv. Mater. 2017; 291703156Crossref Scopus (71) Google Scholar] (Figure 3A ). A similar method was used by DeForest and Tirrell, who used two bio-orthogonal photochemistries to immobilize and release proteins within a synthetic hydrogel (Figure 3B). Applying this approach to vitronectin enabled the reversible and spatiotemporal differentiation of human mesenchymal stem cells [35DeForest C.A. Tirrell D.A. A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels.Nat. Mater. 2015; 14: 523-531Crossref PubMed Scopus (289) Google Scholar]. An alternative method is the use of localized heating caused by infrared irradiation to trigger temperature-mediated processes. This strategy was employed by Stowers and coworkers to release liposomal cargo that could either stiffen or soften alginate hydrogels with spatiotemporal control [36Stowers R.S. et al.Dynamic phototuning of 3D hydrogel stiffness.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 1953-1958Crossref PubMed Scopus (156) Google Scholar]. Local heating by infrared light was also used by Martin-Saavedra and coworkers to spatiotemporally trigger the transgene expression of vascular endothelial growth factor (VEGF) [37Martin-Saavedra F.M. et al.Temporal and spatial patterning of transgene expression by near-infrared irradiation.Biomaterials. 2014; 35: 8134-8143Crossref PubMed Scopus (17) Google Scholar]. This study used a heat-activated gene switch, however, a more direct method for optically controlling cells is the use of optogenetic technology. Indeed, optogenetics has enabled the direct photo-activation of processes such as neurite outgrowth, myogenic differentiation, and angiogenesis [38Park S. et al.Optogenetic control of nerve growth.Sci. Rep. 2015; 5: 9669Crossref PubMed Scopus (45) Google Scholar, 39Polstein L.R. et al.An engineered optogenetic switch for spatiotemporal control of gene expression, cell differentiation, and tissue morphogenesis.ACS Synth. Biol. 2017; 6: 2003-2013Crossref PubMed Scopus (21) Google Scholar], while Reis and coworkers recently reported that optoepigenetic probes could be used for light-controlled gene expression [40Reis S.A. et al.Light-controlled modulation of gene expression by chemical optoepigenetic probes.Nat. Chem. Biol. 2016; 12: 317-323Crossref PubMed Scopus (45) Google Scholar]. While optogenetic/optoepigenetic tissue engineering requires further development, these reports offer the enticing prospect of using optical switches to guide complex tissue formation. Meanwhile, other light-based technologies have been used to precisely assembly different tissue engineering components. For instance, optical tweezers can trap and maneuver individual cells or microtubules into customized arrays [41Kirkham G.R. et al.Precision assembly of complex cellular microenvironments using holographic optical tweezers.Sci. Rep. 2015; 5: 8577Crossref PubMed Scopus (57) Google Scholar, 42Bergman J. et al.Constructing 3D microtubule networks using holographic optical trapping.Sci. Rep. 2015; 518085Crossref PubMed Scopus (15) Google Scholar]. Although optical tweezers offer extremely high spatial precision, it is clear that significant improvements in throughput are required if this method is to be applied to the engineering of full-size tissue constructs. A more high-throughput method for cell manipulation is acoustic patterning. This method typically uses one or more ultrasound standing waves to create uneven pressure fields that can pattern cells en masse into well-defined geometric assemblies [43Marx V. Biophysics: using sound to move cells.Nat. Methods. 2015; 12: 41-44Crossref PubMed Scopus (42) Google Scholar]. Early work in this field focused on patterning cells on 2D culture substrates, however, a major development in this area was the use of hydrogelation to encapsulate the patterned cell arrays [44Garvin K.A. et al.Vascularization of three-dimensional collagen hydrogels using ultrasound standing wave fields.Ultrasound Med. Biol. 2011; 37: 1253-1264Abstract Full Text Full Text PDF Scopus (30) Google Scholar]. This method allowed cell arrays to be preserved for long-term in vitro tissue engineering after removal of the acoustic field. Since 2016, devices have been developed to generate lines of myoblasts for skeletal muscle tissue engineering [45Armstrong J.P.K. et al.Engineering anisotropic muscle tissue using acoustic cell patterning.Adv. Mater. 2018; 301802649Crossref Scopus (53) Google Scholar] (Figure 4A ), assemblies of beating cardiomyocytes for cardiac tissue engineering [46Naseer S.M. et al.Surface acoustic waves induced micropatterning of cells in gelatin methacryloyl (GelMA) hydrogels.Biofabrication. 2017; 9015020Crossref PubMed Scopus (72) Google Scholar, 47Serpooshan V. et al.Bioacoustic-enabled patterning of human iPSC-derived cardiomyocytes into 3D cardiac tissue.Biomaterials. 2017; 131: 47-57Crossref PubMed Scopus (41) Google Scholar], levitated sheets of neuroprogenitors for neural tissue engineering [48Bouyer C. et al.A bio-acoustic levitational (BAL) assembly method for engineering of multilayered, 3D brain-like constructs, using human embryonic stem cell derived neuro-progenitors.Adv. Mater. 2016; 28: 161-167Crossref PubMed Scopus (80) Google Scholar] (Figure 4B) and arrays of endothelial cells for neovascularization [49Comeau E.S. et al.Ultrasound patterning technologies for studying vascular morphogenesis in 3D.J. Cell Sci. 2017; 130: 232-242Crossref PubMed Scopus (12) Google Scholar, 50Kang B. et al.High-resolution acoustophoretic 3D cell patterning to construct functional collateral cylindroids for ischemia therapy.Nat. Commun. 2019; 9: 5402Crossref Scopus (48) Google Scholar] (Figure 4C). These examples use simple geometric arrays to create lines, columns, or sheets, however, in the future it may be possible to create customized tissue architecture by employing more flexible holographic assembly routes [51Marzo A. et al.Holographic acoustic elements for manipulation of levitated objects.Nat. Commun. 2015; 6: 8661Crossref PubMed Scopus (352) Google Scholar, 52Melde K. et al.Holograms for acoustics.Nature. 2016; 537: 518-522Crossref PubMed Scopus (259) Google Scholar]. A limitation to acoustic cell patterning is that the forces generated are relatively weak [53Bassindale P.G. et al.Measurements of the force fields within an acoustic standing wave using holographic optical tweezers.Appl. Phys. Lett. 2014; 104163504Crossref Scopus (19) Google Scholar]. As a result, acoustic patterning can be readily disrupted by factors such as mechanical agitation, thermal currents, acoustic streaming, viscosity, and gravity [54Armstrong J.P.K. et al.Spatiotemporal quantification of acoustic cell patterning using Voronoï tessellation.Lab Chip. 2019; 19: 562-573Crossref PubMed Google Scholar]. Moreover, the forces used for patterning are proportional to the object volume and thus it can be extremely challenging to pattern nanoscale entities, such as biochemical factors or matrix fibers. This limitation has led to the development of strategies that use microscale carriers to host biomolecular cargo or template material fabrication [55Nichols M.K. et al.Fabrication of micropatterned dipeptide hydrogels by acoustic trapping of stimulus-responsive coacervate droplets.Small. 2018; 141800739Crossref Scopus (22) Google Scholar, 56Melde K. et al.Acoustic fabrication via the assembly and fusion of particles.Adv. Mater. 2018; 301704507Crossref Scopus (49) Google Scholar]. An alternative approach is the use of focused ultrasound that can provide highly localized stimuli capable of modulating different tissue engineering components. The local heating caused by focused ultrasound has been used to modulate fibril size during collagen gelation [57Garvin K.A. et al.Controlling collagen fiber microstructure in three-dimensional hydrogels using ultrasound.J. Acoust. Soc. Am. 2013; 134: 1491-1502Crossref PubMed Scopus (24) Google Scholar] and trigger the transgene expression of growth factors (BMP-2 and VEGF) in a spatially controlled manner [58Wilson C.G. et al.Patterning expression of regenerative growth factors using high intensity focused ultrasound.Tissue Eng. Part C. 2014; 20: 769-779Crossref PubMed Scopus (14) Google Scholar]. Focused ultrasound can also be used to initiate nonthermal effects, such as the liberation of growth factors from acoustically responsive droplets or scaffolds [59Fabiilli M.L. et al.Acoustic droplet-hydrogel composites for spatial and temporal control of growth factor delivery and scaffold stiffness.Acta Biomater. 2013; 9: 7399-7409Crossref PubMed Scopus (49) Google Scholar, 60Moncion A. et al.Controlled release of basic fibroblast growth factor for angiogenesis using acoustically-responsive scaffolds.Biomaterials. 2017; 140: 26-36Crossref PubMed Scopus (44) Google Scholar]. It is evident that remote field systems have the potential to greatly advance in vitro tissue engineering. However, the convergence of magnetic, optical, and acoustic technologies with in vitro tissue engineering is a relatively new development and, as a result, many studies exist only in the academic sphere and are far from disrupting the clinical status quo. Here, we propose three key challenges that must be addressed in order to realize the benefit of remote fields in translational tissue engineering (see Outstanding Questions). First, it is imperative that 'proof-of-concept' studies using arbitrary feature dimensions and model cell types are replaced with methods that enable the engineering of mature, multicellular tissues with native structural dimensions. For instance, while ultrasound standing waves have been used to create multilayer sheets of endothelial cells in collagen hydrogels [44Garvin K.A. et al.Vascularization of three-dimensional collagen hydrogels using ultrasound standing wave fields.Ultrasound Med. Biol. 2011; 37: 1253-1264Abstract Full Text Full Text PDF Scopus (30) Google Scholar], the next step for this technology is the creation of functional networks of blood vessels, replete with support cells (e.g., smooth muscle cells), formed not in a bare hydrogel but in a real tissue structure (e.g., muscle, bone, liver). The second challenge is to use materials and protocols that support remote manipulation without affecting other aspects of the engineered tissue. This statement is made in light of the fact that many remote field technologies necessitate the use materials with defined properties: certain levels of optical transparency, viscosity, magnetic susceptibility, photoresponsivity, or acoustic attenuation. As a result, many strategies rely on either: (i) a customized material, designed and synthesized with the appropriate set of characteristics; or (ii) the selection of an existing material system that has compatible properties within an operational parameter space. While a carefully designed or selected material can evidently be used to enable remote organization of structural features, this benefit must not be at the expense of the biological, physical, and mechanical properties required to support cell survival, differentiation, and extracellular matrix production. Ideally, a tissue engineering protocol that is recognized as the academic or clinical gold standard would be used with remote field application as the sole change to the established procedure. The final challenge is to improve the accessibility of remote field instrumentation. Apparatus is often assembled in house [45Armstrong J.P.K. et al.Engineering anisotropic muscle tissue using acoustic cell patterning.Adv. Mater. 2018; 301802649Crossref Scopus (53) Google Scholar, 55Nichols M.K. et al.Fabrication of micropatterned dipeptide hydrogels by acoustic trapping of stimulus-responsive coacervate droplets.Small. 2018; 141800739Crossref Scopus (22) Google Scholar], however, the need for users to assemble and operate their own devices restricts usage to a small number of groups with specialist expertise. This situation could be alleviated by more active dissemination of academic knowledge through protocols and methods papers, or by making devices available through user collaboration or product commercialization. Alternatively, many remote field technologies use high-end equipment that is already commercially available, such as multiphoton lithography [35DeForest C.A. Tirrell D.A. A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels.Nat. Mater. 2015; 14: 523-531Crossref PubMed Scopus (289) Google Scholar], optical tweezers [41Kirkham G.R. et al.Precision assembly of complex cellular microenvironments using holographic optical tweezers.Sci. Rep. 2015; 5: 8577Crossref PubMed Scopus (57) Google Scholar], or focused ultrasound systems [60Moncion A. et al.Controlled release of basic fibroblast growth factor for angiogenesis using acoustically-responsive scaffolds.Biomaterials. 2017; 140: 26-36Crossref PubMed Scopus (44) Google Scholar]. These systems, which can require considerable expense and expertise to operate and maintain, tend to be sold as multifunctional apparatus rather than tailored to particular end-user applications. Therefore, a major challenge can be tuning and integrating commercial apparatus to meet biological requirements (cytocompatibility, sterility, etc.). Overall, the creation of more integrated, accessible technologies would enable research groups around the world to embrace remote fields as a mainstream tool for complex tissue engineering.Outstanding QuestionsCan we progress from 'proof-of-concept' studies towards the engineering of clinically relevant tissues with native multicellularity and natural feature dimensions?Can we avoid a reliance on customized systems and instead develop methods that employ materials capable of supporting both structural organization and tissue development?Can we move away from specialized techniques towards more accessible technologies, in order to allow adoption of remote fields in mainstream tissue engineering? Can we progress from 'proof-of-concept' studies towards the engineering of clinically relevant tissues with native multicellularity and natural feature dimensions? Can we avoid a reliance on customized systems and instead develop methods that employ materials capable of supporting both structural organization and tissue development? Can we move away from specialized techniques towards more accessible technologies, in order to allow adoption of remote fields in mainstream tissue engineering? J.P.K.A. was funded by the Medical Research Council ( MR/S00551X/1 ). M.M.S. acknowledges support from the grant from the UK Regenerative Medicine Platform 'Acellular / Smart Materials – 3D Architecture' ( MR/R015651/1 ), the European Research Council (ERC) Seventh Framework Programme Consolidator grant 'Naturale CG' ( 616417 ), the Rosetrees Trust , and the Wellcome Trust Senior Investigator Award ( 098411/Z/12/Z ). The authors would also like to acknowledge Chunching Li and Valeria Nele for their insight and edits. No competing financial interests exist. the loss of energy of a sound wave as it propagates through a medium. the use of sound waves to create pressure gradients that can move particles (or cells) into nodes or antinodes. a technique that uses lenses or curved transducers to create local regions of high intensity sound energy with frequencies greater than 20 kHz. vector maps describing the noncontact forces experienced by particles at different spatial coordinates. a technique that uses a focused laser to drive local changes (e.g., photocrosslinking, photopolymerization, photodegradation) with electronic excitation requiring the near-simultaneous absorption of two or more photons. a technique that uses highly focused laser beams to spatially trap and manipulate small particles with a refractive index that is different to the surrounding medium. the use of light to control epigenetic mechanisms, for instance, by using photochromic inhibitors of chromatin-modifying enzymes. the use of light to modulate molecular events in living cells using genes encoding photoresponsive proteins. a class of magnetism in which a material has unpaired electrons that can align with an external magnetic field. a magnetic behavior exhibited by small ferrimagnetic/ferromagnetic materials, which have single magnetic domains that can align with an external magnetic field. the biosynthesis of functional products (usually proteins) from genes artificially introduced into a cell. a sound wave that oscillates with a frequency of more than 20 kHz but has stationary pressure nodes.