Transplantation of Human Brain Organoids: Revisiting the Science and Ethics of Brain Chimeras
2019; Elsevier BV; Volume: 25; Issue: 4 Linguagem: Inglês
10.1016/j.stem.2019.09.002
ISSN1934-5909
AutoresH. Isaac Chen, John A. Wolf, Rachel Blue, Mingyan Maggie Song, Jonathan D. Moreno, Guo‐li Ming, Hongjun Song,
Tópico(s)Neuroethics, Human Enhancement, Biomedical Innovations
ResumoRecent demonstrations of human brain organoid transplantation in rodents have accentuated ethical concerns associated with these entities, especially as they relate to potential “humanization” of host animals. Consideration of established scientific principles can help define the realistic range of expected outcomes in such transplantation studies. This practical approach suggests that augmentation of discrete brain functions in transplant hosts is a more relevant ethical question in the near term than the possibility of “conscious” chimeric animals. We hope that this framework contributes to a balanced approach for proceeding with studies involving brain organoid transplantation and other forms of human-animal brain chimeras. Recent demonstrations of human brain organoid transplantation in rodents have accentuated ethical concerns associated with these entities, especially as they relate to potential “humanization” of host animals. Consideration of established scientific principles can help define the realistic range of expected outcomes in such transplantation studies. This practical approach suggests that augmentation of discrete brain functions in transplant hosts is a more relevant ethical question in the near term than the possibility of “conscious” chimeric animals. We hope that this framework contributes to a balanced approach for proceeding with studies involving brain organoid transplantation and other forms of human-animal brain chimeras. The advent of brain organoids derived from human pluripotent stem cells has generated an avalanche of enthusiasm and interest in the neurobiological and biomedical communities. These entities emulate normal neurodevelopment, producing brain-specific architecture such as neural progenitor zones and rudimentary cortical layers through the principles of self-organization (Lancaster et al., 2013Lancaster M.A. Renner M. Martin C.A. Wenzel D. Bicknell L.S. Hurles M.E. Homfray T. Penninger J.M. Jackson A.P. Knoblich J.A. Cerebral organoids model human brain development and microcephaly.Nature. 2013; 501: 373-379Crossref PubMed Scopus (2805) Google Scholar, Paşca et al., 2015Paşca A.M. Sloan S.A. Clarke L.E. Tian Y. Makinson C.D. Huber N. Kim C.H. Park J.Y. O’Rourke N.A. Nguyen K.D. et al.Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.Nat. Methods. 2015; 12: 671-678Crossref PubMed Scopus (869) Google Scholar, Qian et al., 2016Qian X. Nguyen H.N. Song M.M. Hadiono C. Ogden S.C. Hammack C. Yao B. Hamersky G.R. Jacob F. Zhong C. et al.Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.Cell. 2016; 165: 1238-1254Abstract Full Text Full Text PDF PubMed Scopus (1264) Google Scholar). Because of their recapitulation of certain brain structures, brain organoids could enable the study of human neurodevelopment and cerebral disorders in novel, previously unimaginable ways (Di Lullo and Kriegstein, 2017Di Lullo E. Kriegstein A.R. The use of brain organoids to investigate neural development and disease.Nat. Rev. Neurosci. 2017; 18: 573-584Crossref PubMed Scopus (364) Google Scholar, Kelava and Lancaster, 2016Kelava I. Lancaster M.A. Dishing out mini-brains: Current progress and future prospects in brain organoid research.Dev. Biol. 2016; 420: 199-209Crossref PubMed Scopus (192) Google Scholar, Kretzschmar and Clevers, 2016Kretzschmar K. Clevers H. Organoids: Modeling Development and the Stem Cell Niche in a Dish.Dev. Cell. 2016; 38: 590-600Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, Lancaster and Knoblich, 2014Lancaster M.A. Knoblich J.A. Organogenesis in a dish: modeling development and disease using organoid technologies.Science. 2014; 345: 1247125Crossref PubMed Scopus (1505) Google Scholar). One example is the role played by brain organoids in defining the pathogenic mechanisms of Zika virus during the recent global health emergency (Garcez et al., 2016Garcez P.P. Loiola E.C. Madeiro da Costa R. Higa L.M. Trindade P. Delvecchio R. Nascimento J.M. Brindeiro R. Tanuri A. Rehen S.K. Zika virus impairs growth in human neurospheres and brain organoids.Science. 2016; 352: 816-818Crossref PubMed Scopus (825) Google Scholar, Ming et al., 2016Ming G.L. Tang H. Song H. Advances in Zika Virus Research: Stem Cell Models, Challenges, and Opportunities.Cell Stem Cell. 2016; 19: 690-702Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, Qian et al., 2016Qian X. Nguyen H.N. Song M.M. Hadiono C. Ogden S.C. Hammack C. Yao B. Hamersky G.R. Jacob F. Zhong C. et al.Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.Cell. 2016; 165: 1238-1254Abstract Full Text Full Text PDF PubMed Scopus (1264) Google Scholar) Moreover, there are several potential clinical applications of brain organoids, including personalized models of pathogenesis, therapeutic screening, and repair of damaged cerebral circuitry (Chen et al., 2019Chen H.I. Song H. Ming G.L. Applications of human brain organoids to clinical problems.Dev. Dyn. 2019; 248: 53-64Crossref PubMed Scopus (66) Google Scholar). Although their scientific and translational promise is great, brain organoids also have sparked intense debate among academics (Farahany et al., 2018Farahany N.A. Greely H.T. Hyman S. Koch C. Grady C. Pașca S.P. Sestan N. Arlotta P. Bernat J.L. Ting J. et al.The ethics of experimenting with human brain tissue.Nature. 2018; 556: 429-432Crossref PubMed Scopus (90) Google Scholar) and in the press (Begley, 2017Begley S. Tiny Human Brain Organoids Implanted into Rodents, Triggering Ethical Concerns.STAT. 2017; (November 6, 2017)https://www.statnews.com/2017/11/06/human-brain-organoids-ethics/Google Scholar, Moody, 2017Moody O. Human brain cells thrive inside the skull of a rat.. The Times, 2017https://www.thetimes.co.uk/article/human-brain-cells-thrive-inside-the-skull-of-a-rat-6dk8lsmbxGoogle Scholar) regarding the potential ethical challenges they pose. These concerns stem from the ethical and moral implications of generating and using neural tissues that are increasingly similar to the human brain, the source of the higher-order cognitive capacities that are most often equated with being human. From a purely scientific perspective, it may be tempting to dismiss the ethical considerations of brain organoids as not currently relevant. After all, as we will discuss below, the likelihood that current iterations of organoids or animals transplanted with these organoids can develop more complex cognitive abilities is minute. However, engagement of neurobiologists and neuroscientists in brain organoid ethics is important for several reasons. Scientists should help develop the appropriate frameworks for these ethical discussions to prevent faulty conclusions from being drawn, especially in the realm of public policy. There is also the need for scientists to clearly articulate the scientific and translational benefits of brain organoids to society so that any ethical or moral risks can be properly weighed. Finally, there is wisdom in understanding the relevant ethical considerations to avoid potential pitfalls that may arise as organoid technology advances. The emerging ethical debate regarding brain organoids centers on issues pertaining to organoids themselves, animals subjected to transplantation procedures, and socio-legal governance of organoid generation and storage. These three areas were broadly discussed in a recent summary of two workshops supported by the Duke Initiative for Science and Society and the NIH Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative (Farahany et al., 2018Farahany N.A. Greely H.T. Hyman S. Koch C. Grady C. Pașca S.P. Sestan N. Arlotta P. Bernat J.L. Ting J. et al.The ethics of experimenting with human brain tissue.Nature. 2018; 556: 429-432Crossref PubMed Scopus (90) Google Scholar). Other commentaries on brain organoid ethics have also begun to appear in the literature (Lavazza and Massimini, 2018aLavazza A. Massimini M. Cerebral organoids and consciousness: how far are we willing to go?.J. Med. Ethics. 2018; 44: 613-614Crossref PubMed Scopus (15) Google Scholar, Lavazza and Massimini, 2018bLavazza A. Massimini M. Cerebral organoids: ethical issues and consciousness assessment.J. Med. Ethics. 2018; 44: 606-610Crossref PubMed Scopus (66) Google Scholar, Munsie et al., 2017Munsie M. Hyun I. Sugarman J. Ethical issues in human organoid and gastruloid research.Development. 2017; 144: 942-945Crossref PubMed Scopus (48) Google Scholar, Shepherd, 2018Shepherd J. Ethical (and epistemological) issues regarding consciousness in cerebral organoids.J. Med. Ethics. 2018; 44: 611-612Crossref PubMed Scopus (21) Google Scholar). In this article, we focus on transplantation of brain organoids into animal hosts, which has been the subject of a number of recent publications (Daviaud et al., 2018Daviaud N. Friedel R.H. Zou H. Vascularization and Engraftment of Transplanted Human Cerebral Organoids in Mouse Cortex.eNeuro. 2018; 5 (ENEURO.0219-18.2018)Crossref PubMed Scopus (109) Google Scholar, Mansour et al., 2018Mansour A.A. Gonçalves J.T. Bloyd C.W. Li H. Fernandes S. Quang D. Johnston S. Parylak S.L. Jin X. Gage F.H. An in vivo model of functional and vascularized human brain organoids.Nat. Biotechnol. 2018; 36: 432-441Crossref PubMed Scopus (576) Google Scholar) and scientific abstracts (D. Jgamadze, 2017, Soc. Neurosci., abstract; O. Revah, 2018, Soc. Neurosci., abstract). We first summarize recent progress in the field of brain organoids and provide our perspective on areas of advancement on the horizon. Within this context as well as that of prior literature on the ethics of human-animal brain chimeras, we then evaluate the scientific possibility of enhancing host animal brain function using brain organoid transplantation. Separately, we discuss some of the ethical ramifications of enhanced animals if their creation should become feasible. We hope that this discussion of pertinent ethical issues and associated scientific frameworks facilitates participation of the scientific community in the public discourse on brain organoid development and transplantation. The modern era of human brain organoids began with the development of whole-brain organoids (Lancaster et al., 2013Lancaster M.A. Renner M. Martin C.A. Wenzel D. Bicknell L.S. Hurles M.E. Homfray T. Penninger J.M. Jackson A.P. Knoblich J.A. Cerebral organoids model human brain development and microcephaly.Nature. 2013; 501: 373-379Crossref PubMed Scopus (2805) Google Scholar) and the demonstration that stratified cortical epithelium could arise through self-organization of human pluripotent stem cells (Kadoshima et al., 2013Kadoshima T. Sakaguchi H. Nakano T. Soen M. Ando S. Eiraku M. Sasai Y. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.Proc. Natl. Acad. Sci. USA. 2013; 110: 20284-20289Crossref PubMed Scopus (583) Google Scholar). In the latter case, some of the temporal and spatial features of neocorticogenesis were recapitulated. Subsequent studies refined these aspects of normal neurodevelopment in region-specific organoids, resulting in rudimentary segregation of superficial and deep cortical layers (Paşca et al., 2015Paşca A.M. Sloan S.A. Clarke L.E. Tian Y. Makinson C.D. Huber N. Kim C.H. Park J.Y. O’Rourke N.A. Nguyen K.D. et al.Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.Nat. Methods. 2015; 12: 671-678Crossref PubMed Scopus (869) Google Scholar, Qian et al., 2016Qian X. Nguyen H.N. Song M.M. Hadiono C. Ogden S.C. Hammack C. Yao B. Hamersky G.R. Jacob F. 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Takata N. Oiso Y. Tsuji T. Sasai Y. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells.Nat. Commun. 2016; 7: 10351Crossref PubMed Scopus (122) Google Scholar), hypothalamus (Qian et al., 2016Qian X. Nguyen H.N. Song M.M. Hadiono C. Ogden S.C. Hammack C. Yao B. Hamersky G.R. Jacob F. Zhong C. et al.Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.Cell. 2016; 165: 1238-1254Abstract Full Text Full Text PDF PubMed Scopus (1264) Google Scholar), and cerebellum (Muguruma et al., 2015Muguruma K. Nishiyama A. Kawakami H. Hashimoto K. Sasai Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells.Cell Rep. 2015; 10: 537-550Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) have also been reported. Several approaches have been used to assess the similarity of brain organoids to the human brain. Genetic (Camp et al., 2015Camp J.G. Badsha F. Florio M. Kanton S. Gerber T. Wilsch-Bräuninger M. Lewitus E. Sykes A. Hevers W. Lancaster M. et al.Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.Proc. Natl. Acad. Sci. USA. 2015; 112: 15672-15677Crossref PubMed Scopus (92) Google Scholar, Paşca et al., 2015Paşca A.M. Sloan S.A. Clarke L.E. Tian Y. Makinson C.D. Huber N. Kim C.H. Park J.Y. O’Rourke N.A. Nguyen K.D. et al.Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.Nat. Methods. 2015; 12: 671-678Crossref PubMed Scopus (869) Google Scholar, Qian et al., 2016Qian X. Nguyen H.N. Song M.M. Hadiono C. Ogden S.C. Hammack C. Yao B. Hamersky G.R. Jacob F. Zhong C. et al.Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.Cell. 2016; 165: 1238-1254Abstract Full Text Full Text PDF PubMed Scopus (1264) Google Scholar), epigenetic (Luo et al., 2016Luo C. Lancaster M.A. Castanon R. Nery J.R. Knoblich J.A. Ecker J.R. Cerebral Organoids Recapitulate Epigenomic Signatures of the Human Fetal Brain.Cell Rep. 2016; 17: 3369-3384Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), and epitranscriptomic (Yoon et al., 2017Yoon K.J. Ringeling F.R. Vissers C. Jacob F. Pokrass M. Jimenez-Cyrus D. Su Y. Kim N.S. Zhu Y. Zheng L. et al.Temporal Control of Mammalian Cortical Neurogenesis by m(6)A Methylation.Cell. 2017; 171: 877-889.e17Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar) analyses indicate a high degree of concordance between brain organoids and the human fetal cortex through the second trimester. However, brain organoids are distinctly different from the human brain in several ways. Their maximal size is on the order of millimeters because of the limits of nutrient, gas, and waste exchange via diffusion, and organoids lack endothelial cells, microglia, and other cell types that contribute to the microenvironment of the brain. Furthermore, even within whole-brain organoids, organized structural nodes and the white matter connections among them are absent. Data are emerging regarding the electrical activity of brain organoids, but our understanding of their neurophysiology is still underdeveloped. Slow neuronal calcium waves, post-synaptic potentials, and induced action potentials have been reported in organoids (Lancaster et al., 2013Lancaster M.A. Renner M. Martin C.A. Wenzel D. Bicknell L.S. Hurles M.E. Homfray T. Penninger J.M. Jackson A.P. Knoblich J.A. Cerebral organoids model human brain development and microcephaly.Nature. 2013; 501: 373-379Crossref PubMed Scopus (2805) Google Scholar, Paşca et al., 2015Paşca A.M. Sloan S.A. Clarke L.E. Tian Y. Makinson C.D. Huber N. Kim C.H. Park J.Y. O’Rourke N.A. Nguyen K.D. et al.Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.Nat. Methods. 2015; 12: 671-678Crossref PubMed Scopus (869) Google Scholar, Qian et al., 2016Qian X. Nguyen H.N. Song M.M. Hadiono C. Ogden S.C. Hammack C. Yao B. Hamersky G.R. Jacob F. Zhong C. et al.Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.Cell. 2016; 165: 1238-1254Abstract Full Text Full Text PDF PubMed Scopus (1264) Google Scholar). Spontaneous action potentials require extended periods of time to appear (Quadrato et al., 2017Quadrato G. Nguyen T. Macosko E.Z. Sherwood J.L. Min Yang S. Berger D.R. Maria N. Scholvin J. Goldman M. Kinney J.P. et al.Cell diversity and network dynamics in photosensitive human brain organoids.Nature. 2017; 545: 48-53Crossref PubMed Scopus (652) Google Scholar), and addition of GABAergic neurons to glutamatergic populations promotes synaptic inputs and more robust induced action potentials (Birey et al., 2017Birey F. Andersen J. Makinson C.D. Islam S. Wei W. Huber N. Fan H.C. Metzler K.R.C. Panagiotakos G. Thom N. et al.Assembly of functionally integrated human forebrain spheroids.Nature. 2017; 545: 54-59Crossref PubMed Scopus (659) Google Scholar). There is some evidence to suggest that brain organoids form local neural networks. Light stimulation of photosensitive cells in brain organoids attenuates the activity of a subpopulation of neurons, and statistical analyses of late-organoid activity indicate the interdependency of neural activity (Quadrato et al., 2017Quadrato G. Nguyen T. Macosko E.Z. Sherwood J.L. Min Yang S. Berger D.R. Maria N. Scholvin J. Goldman M. Kinney J.P. et al.Cell diversity and network dynamics in photosensitive human brain organoids.Nature. 2017; 545: 48-53Crossref PubMed Scopus (652) Google Scholar). So far, there has been no direct evidence of communication across multiple network nodes in these entities or computational processing required to generate more complex information. Brain organoids have facilitated the study of human neurodevelopment, modeling of congenital brain conditions and neuropsychiatric disorders, and exploration of differences in brain formation among species (Chen et al., 2019Chen H.I. Song H. Ming G.L. Applications of human brain organoids to clinical problems.Dev. Dyn. 2019; 248: 53-64Crossref PubMed Scopus (66) Google Scholar, Qian et al., 2019Qian X. Song H. Ming G.L. Brain organoids: advances, applications and challenges.Development. 2019; 146 (dev166074)Crossref Scopus (244) Google Scholar). However, current iterations of these organoids are still imperfect facsimiles of the human brain. Although they faithfully recapitulate certain aspects of cerebral architecture, others, such as the layers of the cerebral cortex, remain rudimentary. Related to this incomplete structure is the relative transcriptomic immaturity of the organoids, which approximate, at best, the late second trimester of human fetal development (Camp et al., 2015Camp J.G. Badsha F. Florio M. Kanton S. Gerber T. Wilsch-Bräuninger M. Lewitus E. Sykes A. Hevers W. Lancaster M. et al.Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.Proc. Natl. Acad. Sci. USA. 2015; 112: 15672-15677Crossref PubMed Scopus (92) Google Scholar, Paşca et al., 2015Paşca A.M. Sloan S.A. Clarke L.E. Tian Y. Makinson C.D. Huber N. Kim C.H. Park J.Y. O’Rourke N.A. Nguyen K.D. et al.Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture.Nat. Methods. 2015; 12: 671-678Crossref PubMed Scopus (869) Google Scholar, Qian et al., 2016Qian X. Nguyen H.N. Song M.M. Hadiono C. Ogden S.C. Hammack C. Yao B. Hamersky G.R. Jacob F. Zhong C. et al.Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure.Cell. 2016; 165: 1238-1254Abstract Full Text Full Text PDF PubMed Scopus (1264) Google Scholar, Quadrato et al., 2017Quadrato G. Nguyen T. Macosko E.Z. Sherwood J.L. Min Yang S. Berger D.R. Maria N. Scholvin J. Goldman M. Kinney J.P. et al.Cell diversity and network dynamics in photosensitive human brain organoids.Nature. 2017; 545: 48-53Crossref PubMed Scopus (652) Google Scholar, Sloan et al., 2017Sloan S.A. Darmanis S. Huber N. Khan T.A. Birey F. Caneda C. Reimer R. Quake S.R. Barres B.A. Pasca S.P. Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells.Neuron. 2017; 95: 779-790.e6Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, Velasco et al., 2019Velasco S. Kedaigle A.J. Simmons S.K. Nash A. Rocha M. Quadrato G. Paulsen B. Nguyen L. Adiconis X. Regev A. et al.Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.Nature. 2019; 570: 523-527Crossref PubMed Scopus (402) Google Scholar). It is also the case that current brain organoids are comprised of multiple redundant units rather than being a unified organ and that higher-order features, including gyrification and white matter tracts, are missing. These deficiencies have motivated a major push to engineer next-generation organoids with a greater degree of maturity and complexity. Modeling later stages of brain development is especially relevant for cerebral disorders that manifest later in life, such as schizophrenia (young adulthood) and neuro-degenerative diseases (late adulthood). The following sections will discuss areas in need of progress to achieve the objective of “better” brain organoids that expand our capacity to study human cerebral development and disease. Importantly, organoids that more accurately recapitulate the brain provide scientific context for the ethical discussion below. One of the fundamental challenges in generating more mature brain organoids that reflect later stages of development is the constraint imposed by diffusion. Organoids can grow up to 4 mm in size using orbital shakers and high-oxygen incubators (Kadoshima et al., 2013Kadoshima T. Sakaguchi H. Nakano T. Soen M. Ando S. Eiraku M. Sasai Y. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.Proc. Natl. Acad. Sci. USA. 2013; 110: 20284-20289Crossref PubMed Scopus (583) Google Scholar, Lancaster et al., 2017Lancaster M.A. Corsini N.S. Wolfinger S. Gustafson E.H. Phillips A.W. Burkard T.R. Otani T. Livesey F.J. Knoblich J.A. Guided self-organization and cortical plate formation in human brain organoids.Nat. 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Mierau S.B. Gibbons G.M. Wenger L.M.D. Masullo L. Sit T. Sutcliffe M. Boulanger J. Tripodi M. Derivery E. et al.Cerebral organoids at the air-liquid interface generate diverse nerve tracts with functional output.Nat. Neurosci. 2019; 22: 669-679Crossref PubMed Scopus (249) Google Scholar) or physically constraining growth in the z dimension using an on-chip method (Karzbrun et al., 2018Karzbrun E. Kshirsagar A. Cohen S.R. Hanna J.H. Reiner O. Human Brain Organoids on a Chip Reveal the Physics of Folding.Nat. Phys. 2018; 14: 515-522Crossref PubMed Scopus (229) Google Scholar). These approaches have resulted in improved neuronal survival, axon growth and alignment, and surface wrinkling reminiscent of cortical folds. However, it should be noted that data showing that these organoids are more mature than previous versions are so far lacking. Introducing a perfusion system into brain organoids is an alternative strategy that bypasses the diffusion problem altogether. Incorporation of endothelial cells into brain organoids leads to primitive vascular networks that are likely not functional (Pham et al., 2018Pham M.T. Pollock K.M. Rose M.D. Cary W.A. Stewart H.R. Zhou P. Nolta J.A. Waldau B. Generation of human vascularized brain organoids.Neuroreport. 2018; 29: 588-593Crossref PubMed Scopus (257) Google Scholar). More advanced vascular networks can be derived from biomaterial casting techniques (Miller et al., 2012Miller J.S. Stevens K.R. Yang M.T. Baker B.M. Nguyen D.H. Cohen D.M. Toro E. Chen A.A. Galie P.A. Yu X. et al.Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.Nat. Mater. 2012; 11: 768-774Crossref PubMed Scopus (1432) Google Scholar), 3D printing (Mirabella et al., 2017Mirabella T. MacArthur J.W. Cheng D. Ozaki C.K. Woo Y.J. Yang M. Chen C.S. 3D-printed vascular networks direct therapeutic angiogenesis in ischaemia.Nat. Biomed. 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