International Journal of Stem Cells

Korean Society for Stem Cell Research (한국줄기세포학회)
권호 표지
DOI QR코드

DOI QR Code

In Vitro Generation of Luminal Vasculature in Liver Organoids: From Basic Vascular Biology to Vascularized Hepatic Organoids

  • Hyo Jin Kim (Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Gyeongmin Kim (Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Kyun Yoo Chi (Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University) ;
  • Jong-Hoon Kim (Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University)
  • Received : 2022.09.13
  • Accepted : 2022.09.30
  • Published : 2023.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

Liver organoids have gained much attention in recent years for their potential applications to liver disease modeling and pharmacologic drug screening. Liver organoids produced in vitro reflect some aspects of the in vivo physiological and pathological conditions of the liver. However, the generation of liver organoids with perfusable luminal vasculature remains a major challenge, hindering precise and effective modeling of liver diseases. Furthermore, vascularization is required for large organoids or assembloids to closely mimic the complexity of tissue architecture without cell death in the core region. A few studies have successfully generated liver organoids with endothelial cell networks, but most of these vascular networks produced luminal structures after being transplanted into tissues of host animals. Therefore, formation of luminal vasculature is an unmet need to overcome the limitation of liver organoids as an in vitro model investigating different acute and chronic liver diseases. Here, we provide an overview of the unique features of hepatic vasculature under pathophysiological conditions and summarize the biochemical and biophysical cues that drive vasculogenesis and angiogenesis in vitro. We also highlight recent progress in generating vascularized liver organoids in vitro and discuss potential strategies that may enable the generation of perfusable luminal vasculature in liver organoids.

Keywords

Acknowledgement

This research was supported by a National Research Foundation of Korea (NRF) Grant (No. 2018M3A9H1019504), the Technology Innovation Program (No. 20009350, Development of the standard metrology for organoid based drug metabolism and toxicity testing) of the Korea Evaluation Institute of Industrial Technology funded by the Ministry of Science & ICT (MSIT), Republic of Korea, and a Korea University Grant (No. K2106861).

References

  1. Ho H, Zhang E. Virtual lobule models are the key for multiscale biomechanical and pharmacological modeling for the liver. Front Physiol 2020;11:1061 
  2. Matsumoto K, Yoshitomi H, Rossant J, Zaret KS. Liver organogenesis promoted by endothelial cells prior to vascular function. Science 2001;294:559-563 
  3. Si-Tayeb K, Lemaigre FP, Duncan SA. Organogenesis and development of the liver. Dev Cell 2010;18:175-189 
  4. Iwakiri Y, Shah V, Rockey DC. Vascular pathobiology in chronic liver disease and cirrhosis - current status and future directions. J Hepatol 2014;61:912-924 
  5. Elaut G, Henkens T, Papeleu P, Snykers S, Vinken M, Vanhaecke T, Rogiers V. Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Curr Drug Metab 2006;7:629-660 
  6. Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, Sato T, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, Clevers H. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 2013;494:247-250 
  7. Huch M, Gehart H, van Boxtel R, Hamer K, Blokzijl F, Verstegen MM, Ellis E, van Wenum M, Fuchs SA, de Ligt J, van de Wetering M, Sasaki N, Boers SJ, Kemperman H, de Jonge J, Ijzermans JN, Nieuwenhuis EE, Hoekstra R, Strom S, Vries RR, van der Laan LJ, Cuppen E, Clevers H. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 2015;160:299-312 
  8. Hu H, Gehart H, Artegiani B, LOpez-Iglesias C, Dekkers F, Basak O, van Es J, Chuva de Sousa Lopes SM, Begthel H, Korving J, van den Born M, Zou C, Quirk C, Chiriboga L, Rice CM, Ma S, Rios A, Peters PJ, de Jong YP, Clevers H. Long-term expansion of functional mouse and human hepatocytes as 3D organoids. Cell 2018;175:1591-1606.e19 
  9. Shinozawa T, Kimura M, Cai Y, Saiki N, Yoneyama Y, Ouchi R, Koike H, Maezawa M, Zhang RR, Dunn A, Ferguson A, Togo S, Lewis K, Thompson WL, Asai A, Takebe T. High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids. Gastroenterology 2021;160:831-846.e10 
  10. Kim H, Im I, Jeon JS, Kang EH, Lee HA, Jo S, Kim JW, Woo DH, Choi YJ, Kim HJ, Han JS, Lee BS, Kim JH, Kim SK, Park HJ. Development of human pluripotent stem cell-derived hepatic organoids as an alternative model for drug safety assessment. Biomaterials 2022;286:121575 
  11. Mun SJ, Ryu JS, Lee MO, Son YS, Oh SJ, Cho HS, Son MY, Kim DS, Kim SJ, Yoo HJ, Lee HJ, Kim J, Jung CR, Chung KS, Son MJ. Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids. J Hepatol 2019;71:970-985 
  12. Collardeau-Frachon S, Scoazec JY. Vascular development and differentiation during human liver organogenesis. Anat Rec (Hoboken) 2008;291:614-627 
  13. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, Zhang RR, Ueno Y, Zheng YW, Koike N, Aoyama S, Adachi Y, Taniguchi H. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013;499:481-484 
  14. Camp JG, Sekine K, Gerber T, Loeffler-Wirth H, Binder H, Gac M, Kanton S, Kageyama J, Damm G, Seehofer D, Belicova L, Bickle M, Barsacchi R, Okuda R, Yoshizawa E, Kimura M, Ayabe H, Taniguchi H, Takebe T, Treutlein B. Multilineage communication regulates human liver bud development from pluripotency. Nature 2017;546:533-538 
  15. Jain RK, Au P, Tam J, Duda DG, Fukumura D. Engineering vascularized tissue. Nat Biotechnol 2005;23:821-823 
  16. Takebe T, Sekine K, Kimura M, Yoshizawa E, Ayano S, Koido M, Funayama S, Nakanishi N, Hisai T, Kobayashi T, Kasai T, Kitada R, Mori A, Ayabe H, Ejiri Y, Amimoto N, Yamazaki Y, Ogawa S, Ishikawa M, Kiyota Y, Sato Y, Nozawa K, Okamoto S, Ueno Y, Taniguchi H. Massive and reproducible production of liver buds entirely from human pluripotent stem cells. Cell Rep 2017;21:2661-2670 
  17. Velazquez JJ, LeGraw R, Moghadam F, Tan Y, Kilbourne J, Maggiore JC, Hislop J, Liu S, Cats D, Chuva de Sousa Lopes SM, Plaisier C, Cahan P, Kiani S, Ebrahimkhani MR. Gene regulatory network analysis and engineering directs development and vascularization of multilineage human liver organoids. Cell Syst 2021;12:41-55.e11 
  18. Rafii S, Butler JM, Ding BS. Angiocrine functions of organ-specific endothelial cells. Nature 2016;529:316-325 
  19. Trefts E, Gannon M, Wasserman DH. The liver. Curr Biol 2017;27:R1147-R1151 
  20. DeSesso JM. Vascular ontogeny within selected thoracoabdominal organs and the limbs. Reprod Toxicol 2017;70:3-20 
  21. Zhang X, Tang L, Yi Q. Engineering the vasculature of stem-cell-derived liver organoids. Biomolecules 2021;11:966 
  22. Poisson J, Lemoinne S, Boulanger C, Durand F, Moreau R, Valla D, Rautou PE. Liver sinusoidal endothelial cells: physiology and role in liver diseases. J Hepatol 2017;66:212-227 
  23. Ober EA, Lemaigre FP. Development of the liver: insights into organ and tissue morphogenesis. J Hepatol 2018;68:1049-1062 
  24. DeLeve LD, Wang X, Hu L, McCuskey MK, McCuskey RS. Rat liver sinusoidal endothelial cell phenotype is maintained by paracrine and autocrine regulation. Am J Physiol Gastrointest Liver Physiol 2004;287:G757-G763 
  25. Yoshida M, Nishikawa Y, Omori Y, Yoshioka T, Tokairin T, McCourt P, Enomoto K. Involvement of signaling of VEGF and TGF-beta in differentiation of sinusoidal endothelial cells during culture of fetal rat liver cells. Cell Tissue Res 2007;329:273-282 
  26. Klein D, Demory A, Peyre F, Kroll J, Augustin HG, Helfrich W, Kzhyshkowska J, Schledzewski K, Arnold B, Goerdt S. Wnt2 acts as a cell type-specific, autocrine growth factor in rat hepatic sinusoidal endothelial cells cross-stimulating the VEGF pathway. Hepatology 2008;47:1018-1031 
  27. Geraud C, Koch PS, Zierow J, Klapproth K, Busch K, Olsavszky V, Leibing T, Demory A, Ulbrich F, Diett M, Singh S, Sticht C, Breitkopf-Heinlein K, Richter K, Karppinen SM, Pihlajaniemi T, Arnold B, Rodewald HR, Augustin HG, Schledzewski K, Goerdt S. GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis. J Clin Invest 2017;127:1099-1114 
  28. DeLeve LD, Maretti-Mira AC. Liver sinusoidal endothelial cell: an update. Semin Liver Dis 2017;37:377-387 
  29. Hammoutene A, Rautou PE. Role of liver sinusoidal endothelial cells in non-alcoholic fatty liver disease. J Hepatol 2019;70:1278-1291 
  30. Gracia-Sancho J, Caparros E, Fernandez-Iglesias A, Frances R. Role of liver sinusoidal endothelial cells in liver diseases. Nat Rev Gastroenterol Hepatol 2021;18:411-431 
  31. Gracia-Sancho J, Lavina B, Rodriguez-Vilarrupla A, Garcia-Caldero H, Bosch J, Garcia-Pagan JC. Enhanced vasoconstrictor prostanoid production by sinusoidal endothelial cells increases portal perfusion pressure in cirrhotic rat livers. J Hepatol 2007;47:220-227 
  32. Deleve LD, Wang X, Guo Y. Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence. Hepatology 2008;48:920-930 
  33. Xie G, Choi SS, Syn WK, Michelotti GA, Swiderska M, Karaca G, Chan IS, Chen Y, Diehl AM. Hedgehog signalling regulates liver sinusoidal endothelial cell capillarisation. Gut 2013;62:299-309 
  34. Ding BS, Cao Z, Lis R, Nolan DJ, Guo P, Simons M, Penfold ME, Shido K, Rabbany SY, Rafii S. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis. Nature 2014;505:97-102 
  35. Coulon S, Heindryckx F, Geerts A, Van Steenkiste C, Colle I, Van Vlierberghe H. Angiogenesis in chronic liver disease and its complications. Liver Int 2011;31:146-162 
  36. Patan S. Vasculogenesis and angiogenesis. Cancer Treat Res 2004;117:3-32 
  37. Potente M, Makinen T. Vascular heterogeneity and specialization in development and disease. Nat Rev Mol Cell Biol 2017;18:477-494 
  38. Arroyo AG, Iruela-Arispe ML. Extracellular matrix, inflammation, and the angiogenic response. Cardiovasc Res 2010;86:226-235 
  39. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell 2011;146:873-887 
  40. Naito H, Iba T, Takakura N. Mechanisms of new blood-vessel formation and proliferative heterogeneity of endothelial cells. Int Immunol 2020;32:295-305 
  41. Choi HJ, Zhang H, Park H, Choi KS, Lee HW, Agrawal V, Kim YM, Kwon YG. Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2. Nat Commun 2015;6:6943 
  42. Ong YT, Andrade J, Armbruster M, Shi C, Castro M, Costa ASH, Sugino T, Eelen G, Zimmermann B, Wilhelm K, Lim J, Watanabe S, Guenther S, Schneider A, Zanconato F, Kaulich M, Pan D, Braun T, Gerhardt H, Efeyan A, Carmeliet P, Piccolo S, Grosso AR, Potente M. A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth. Nat Metab 2022;4:672-682 
  43. Wright GL, Maroulakou IG, Eldridge J, Liby TL, Sridharan V, Tsichlis PN, Muise-Helmericks RC. VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase. FASEB J 2008;22:3264-3275 
  44. Guo D, Wang Q, Li C, Wang Y, Chen X. VEGF stimulated the angiogenesis by promoting the mitochondrial functions. Oncotarget 2017;8:77020-77027 
  45. Lamalice L, Le Boeuf F, Huot J. Endothelial cell migration during angiogenesis. Circ Res 2007;100:782-794 
  46. Serbo JV, Gerecht S. Vascular tissue engineering: biodegradable scaffold platforms to promote angiogenesis. Stem Cell Res Ther 2013;4:8 
  47. Saharinen P, Eklund L, Miettinen J, Wirkkala R, Anisimov A, Winderlich M, Nottebaum A, Vestweber D, Deutsch U, Koh GY, Olsen BR, Alitalo K. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts. Nat Cell Biol 2008;10:527-537 
  48. Sulpice E, Ding S, Muscatelli-Groux B, Berge M, Han ZC, Plouet J, Tobelem G, Merkulova-Rainon T. Cross-talk between the VEGF-A and HGF signalling pathways in endothelial cells. Biol Cell 2009;101:525-539 
  49. Kaga T, Kawano H, Sakaguchi M, Nakazawa T, Taniyama Y, Morishita R. Hepatocyte growth factor stimulated angiogenesis without inflammation: differential actions between hepatocyte growth factor, vascular endothelial growth factor and basic fibroblast growth factor. Vascul Pharmacol 2012;57:3-9 
  50. Oladipupo S, Hu S, Kovalski J, Yao J, Santeford A, Sohn RE, Shohet R, Maslov K, Wang LV, Arbeit JM. VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting. Proc Natl Acad Sci U S A 2011;108:13264-13269 
  51. Brindle NP, Saharinen P, Alitalo K. Signaling and functions of angiopoietin-1 in vascular protection. Circ Res 2006;98:1014-1023 
  52. Williams PA, Stilhano RS, To VP, Tran L, Wong K, Silva EA. Hypoxia augments outgrowth endothelial cell (OEC) sprouting and directed migration in response to sphingosine-1-phosphate (S1P). PLoS One 2015;10:e0123437 
  53. Qin D, Trenkwalder T, Lee S, Chillo O, Deindl E, Kupatt C, Hinkel R. Early vessel destabilization mediated by Angiopoietin-2 and subsequent vessel maturation via Angiopoietin-1 induce functional neovasculature after ischemia. PLoS One 2013;8:e61831 
  54. Takuwa Y, Du W, Qi X, Okamoto Y, Takuwa N, Yoshioka K. Roles of sphingosine-1-phosphate signaling in angiogenesis. World J Biol Chem 2010;1:298-306 
  55. Crosby CO, Zoldan J. Mimicking the physical cues of the ECM in angiogenic biomaterials. Regen Biomater 2019;6:61-73 
  56. Edgar LT, Underwood CJ, Guilkey JE, Hoying JB, Weiss JA. Extracellular matrix density regulates the rate of neovessel growth and branching in sprouting angiogenesis. PLoS One 2014;9:e85178 
  57. Mason BN, Starchenko A, Williams RM, Bonassar LJ, Reinhart-King CA. Tuning three-dimensional collagen matrix stiffness independently of collagen concentration modulates endothelial cell behavior. Acta Biomater 2013;9:4635-4644 
  58. Bao M, Chen Y, Liu JT, Bao H, Wang WB, Qi YX, Lv F. Extracellular matrix stiffness controls VEGF165 secretion and neuroblastoma angiogenesis via the YAP/RUNX2/SRSF1 axis. Angiogenesis 2022;25:71-86 
  59. Guo Y, Mei F, Huang Y, Ma S, Wei Y, Zhang X, Xu M, He Y, Heng BC, Chen L, Deng X. Matrix stiffness modulates tip cell formation through the p-PXN-Rac1-YAP signaling axis. Bioact Mater 2021;7:364-376 
  60. Paszkowiak JJ, Dardik A. Arterial wall shear stress: observations from the bench to the bedside. Vasc Endovascular Surg 2003;37:47-57 
  61. Dekker RJ, van Soest S, Fontijn RD, Salamanca S, de Groot PG, VanBavel E, Pannekoek H, Horrevoets AJ. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2). Blood 2002;100:1689-1698 
  62. Yuan P, Hu Q, He X, Long Y, Song X, Wu F, He Y, Zhou X. Laminar flow inhibits the Hippo/YAP pathway via autophagy and SIRT1-mediated deacetylation against atherosclerosis. Cell Death Dis 2020;11:141 
  63. Takebe T, Koike N, Sekine K, Fujiwara R, Amiya T, Zheng YW, Taniguchi H. Engineering of human hepatic tissue with functional vascular networks. Organogenesis 2014;10:260-267 
  64. Ramachandran SD, Schirmer K, Munst B, Heinz S, Ghafoory S, Wolfl S, Simon-Keller K, Marx A, Oie CI, Ebert MP, Walles H, Braspenning J, Breitkopf-Heinlein K. In vitro generation of functional liver organoid-like structures using adult human cells. PLoS One 2015;10:e0139345 
  65. Li J, Xing F, Chen F, He L, So KF, Liu Y, Xiao J. Functional 3D human liver bud assembled from MSC-derived multiple liver cell lineages. Cell Transplant 2019;28:510-521 Erratum in: Cell Transplant 2022;31:9636897221126859 
  66. Takebe T, Enomura M, Yoshizawa E, Kimura M, Koike H, Ueno Y, Matsuzaki T, Yamazaki T, Toyohara T, Osafune K, Nakauchi H, Yoshikawa HY, Taniguchi H. Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell 2015;16:556-565 
  67. Asai A, Aihara E, Watson C, Mourya R, Mizuochi T, Shivakumar P, Phelan K, Mayhew C, Helmrath M, Takebe T, Wells J, Bezerra JA. Paracrine signals regulate human liver organoid maturation from induced pluripotent stem cells. Development 2017;144:1056-1064 
  68. Ayabe H, Anada T, Kamoya T, Sato T, Kimura M, Yoshizawa E, Kikuchi S, Ueno Y, Sekine K, Camp JG, Treutlein B, Ferguson A, Suzuki O, Takebe T, Taniguchi H. Optimal hypoxia regulates human iPSC-derived liver bud differentiation through intercellular TGFB signaling. Stem Cell Reports 2018;11:306-316 
  69. Goulart E, de Caires-Junior LC, Telles-Silva KA, Araujo BHS, Kobayashi GS, Musso CM, Assoni AF, Oliveira D, Caldini E, Gerstenhaber JA, Raia S, Lelkes PI, Zatz M. Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-β. Stem Cell Res Ther 2019;10:258 
  70. Jin Y, Kim J, Lee JS, Min S, Kim S, Ahn DH, Kim YG, Cho SW. Vascularized liver organoids generated using induced hepatic tissue and dynamic liver-specific microenvironment as a drug testing platform. Adv Funct Mater 2018;28:1801954 
  71. Peng WC, Logan CY, Fish M, Anbarchian T, Aguisanda F, Alvarez-Varela A, Wu P, Jin Y, Zhu J, Li B, Grompe M, Wang B, Nusse R. Inflammatory cytokine TNFα promotes the long-term expansion of primary hepatocytes in 3D culture. Cell 2018;175:1607-1619.e15 
  72. Ardalani H, Sengupta S, Harms V, Vickerman V, Thomson JA, Murphy WL. 3-D culture and endothelial cells improve maturity of human pluripotent stem cell-derived hepatocytes. Acta Biomater 2019;95:371-381 
  73. Yap KK, Gerrand YW, Dingle AM, Yeoh GC, Morrison WA, Mitchell GM. Liver sinusoidal endothelial cells promote the differentiation and survival of mouse vascularised hepatobiliary organoids. Biomaterials 2020;251:120091 
  74. Son JS, Park CY, Lee G, Park JY, Kim HJ, Kim G, Chi KY, Woo DH, Han C, Kim SK, Park HJ, Kim DW, Kim JH. Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs. Biomaterials 2022;283:121429 
  75. Sasaki K, Akagi T, Asaoka T, Eguchi H, Fukuda Y, Iwagami Y, Yamada D, Noda T, Wada H, Gotoh K, Kawamoto K, Doki Y, Mori M, Akashi M. Construction of three-dimensional vascularized functional human liver tissue using a layer-by-layer cell coating technique. Biomaterials 2017;133:263-274 
  76. Guye P, Ebrahimkhani MR, Kipniss N, Velazquez JJ, Schoenfeld E, Kiani S, Griffith LG, Weiss R. Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6. Nat Commun 2016;7:10243 
  77. Lee JW, Choi YJ, Yong WJ, Pati F, Shim JH, Kang KS, Kang IH, Park J, Cho DW. Development of a 3D cell printed construct considering angiogenesis for liver tissue engineering. Biofabrication 2016;8:015007 
  78. Kang D, Hong G, An S, Jang I, Yun WS, Shim JH, Jin S. Bioprinting of multiscaled hepatic lobules within a highly vascularized construct. Small 2020;16:e1905505 
  79. Yanagi Y, Nakayama K, Taguchi T, Enosawa S, Tamura T, Yoshimaru K, Matsuura T, Hayashida M, Kohashi K, Oda Y, Yamaza T, Kobayashi E. In vivo and ex vivo methods of growing a liver bud through tissue connection. Sci Rep 2017;7:14085 
  80. Rennert K, Steinborn S, Groger M, Ungerbock B, Jank AM, Ehgartner J, Nietzsche S, Dinger J, Kiehntopf M, Funke H, Peters FT, Lupp A, Gartner C, Mayr T, Bauer M, Huber O, Mosig AS. A microfluidically perfused three dimensional human liver model. Biomaterials 2015;71:119-131 
  81. Li X, George SM, Vernetti L, Gough AH, Taylor DL. A glass-based, continuously zonated and vascularized human liver acinus microphysiological system (vLAMPS) designed for experimental modeling of diseases and ADME/TOX. Lab Chip 2018;18:2614-2631 
  82. Zhang B, Montgomery M, Chamberlain MD, Ogawa S, Korolj A, Pahnke A, Wells LA, Masse S, Kim J, Reis L, Momen A, Nunes SS, Wheeler AR, Nanthakumar K, Keller G, Sefton MV, Radisic M. Biodegradable scaffold with built-in vasculature for organ-on-a-chip engineering and direct surgical anastomosis. Nat Mater 2016;15:669-678 
  83. Baptista PM, Siddiqui MM, Lozier G, Rodriguez SR, Atala A, Soker S. The use of whole organ decellularization for the generation of a vascularized liver organoid. Hepatology 2011;53:604-617 
  84. Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nat Methods 2019;16:255-262 
  85. Cakir B, Xiang Y, Tanaka Y, Kural MH, Parent M, Kang YJ, Chapeton K, Patterson B, Yuan Y, He CS, Raredon MSB, Dengelegi J, Kim KY, Sun P, Zhong M, Lee S, Patra P, Hyder F, Niklason LE, Lee SH, Yoon YS, Park IH. Engineering of human brain organoids with a functional vascular-like system. Nat Methods 2019;16:1169-1175 
  86. Sun XY, Ju XC, Li Y, Zeng PM, Wu J, Zhou YY, Shen LB, Dong J, Chen YJ, Luo ZG. Generation of vascularized brain organoids to study neurovascular interactions. Elife 2022;11:e76707 
  87. Kook MG, Lee SE, Shin N, Kong D, Kim DH, Kim MS, Kang HK, Choi SW, Kang KS. Generation of cortical brain organoid with vascularization by assembling with vascular spheroid. Int J Stem Cells 2022;15:85-94 
  88. Heo JH, Kang D, Seo SJ, Jin Y. Engineering the extracellular matrix for organoid culture. Int J Stem Cells 2022;15:60-69