Bioprinting organoids for functional cardiac constructs: Progress and unmet challenges

Abstract

Developing physiologically relevant cardiac-engineered in vitro models has been a longstanding challenge in cardiac tissue engineering. Bioprinting technologies have been utilized to recreate the complex architecture of the human heart via the precise placement of cells and biomaterials. Concurrently, self-organizing cardiac organoids have emerged as powerful tools for developing cardiac tissues accurately mimicking the heart’s biological composition. This review explores the merging of these two rapidly evolving fields to produce functionally mature engineered cardiac tissues. Together, bioprinting can provide spatial control and mechanical support to guide cardiac self-organization, including strategies to directly print cardioids or incorporate them as modular units, while cardioid differentiation protocols promote multi-cellular complexity and developmental relevance to improve the functionality of engineered cardiac constructs. In this review, we discuss the key processing challenges and goals across the bioprinting workflow—spanning pre-processing, processing, and post-processing—and evaluate how they intersect with cell viability, structural integrity, and electromechanical function. We then explore the formation and functional features of self-organized cardioids, outlining major differentiation protocols, signaling cues, and functional outcomes. Finally, we propose a convergence between bioprinting and cardioid technologies to produce the next generation of in vitro cardiac models.

Keywords

Bioprinting / Cardiac organoid / Regenerative medicine / Cardiac tissue engineering

Funding

The authors acknowledge funding for this project from the Betkowski Family Fund and the Carlos Family Fund through Children’s Healthcare of Atlanta.

Conflict of interest

The authors declare no competing interests.

References

[1] Lindstrom M, DeCleene N, Dorsey H, et al. Global burden of cardiovascular diseases and risks collaboration, 1990-2021. J Am Coll Cardiol. 2022; 80(25):2372-2425.doi: 10.1016/j.jacc.2022.11.001

[2] Cesare MD, Perel P, Taylor S, et al. The Heart of the World. Glob Heart. 2024; 19(1):11.doi: 10.5334/gh.1288

[3] Tsao CW, Aday AW, Almarzooq ZI, et al. Heart disease and stroke statistics—2023 update: a report from the American Heart Association. Circulation. 2023; 147(8):e93-e621.doi: 10.1161/CIR.0000000000001123

[4] Bergmann O, Bhardwaj RD, Bernard S, et al. Evidence for cardiomyocyte renewal in humans. Science. 2009; 324(5923):98-102.doi: 10.1126/science.1164680

[5] Heidenreich PA, Albert NM, Allen LA, et al. Forecasting the impact of heart failure in the United States. Circ Heart Fail. 2013; 6(3):606-619.doi: 10.1161/HHF.0b013e318291329a

[6] Kazi DS, Elkind MSV, Deutsch A, et al. Forecasting the economic burden of cardiovascular disease and stroke in the United States through 2050: a presidential advisory from the American Heart Association. Circulation. 2024; 150(4):e89-e101.doi: 10.1161/CIR.0000000000001258

[7] Thomas D, Choi S, Alamana C, Parker KK, Wu JC. Cellular and engineered organoids for cardiovascular models. Circ Res. 2022; 130(12):1780-1802.doi: 10.1161/CIRCRESAHA.122.320305

[8] Cho S, Discher DE, Leong KW, Vunjak-Novakovic G, Wu JC. Challenges and opportunities for the next generation of cardiovascular tissue engineering. Nat Methods. 2022; 19(9):1064-1071.doi: 10.1038/s41592-022-01591-3

[9] Wang Z, Wang L, Li T, et al. 3D bioprinting in cardiac tissue engineering. Theranostics. 2021; 11(16):7948-7969.doi: 10.7150/thno.61621

[10] Sung K, Patel NR, Ashammakhi N, Nguyen KL. 3-Dimensional bioprinting of cardiovascular tissues: emerging technology. JACC: Basic Transl Sci. 2021; 6(5):467-482.doi: 10.1016/j.jacbts.2020.12.006

[11] Zimmermann WH, Schneiderbanger K, Schubert P, et al. Tissue engineering of a differentiated cardiac muscle construct. Circ Res. 2002; 90(2):223-230.doi: 10.1161/hh0202.103644

[12] Wu F, He Q, Li F, Yang X. A review of protocols for engineering human cardiac organoids. Heliyon. 2023; 9(9):e19938.doi: 10.1016/j.heliyon.2023.e19938

[13] Zhao D, Lei W, Hu S. Cardiac organoid—a promising perspective of preclinical model. Stem Cell Res Ther. 2021; 12(1):272.doi: 10.1186/s13287-021-02340-7

[14] Zhu L, Liu K, Feng Q, Liao Y. Cardiac Organoids: A 3D technology for modeling heart development and disease. Stem Cell Rev Rep. 2022; 18(8):2593-2605.doi: 10.1007/s12015-022-10385-1

[15] El Sabbagh A, Eleid MF, Al-Hijji M, et al. The various applications of 3D printing in cardiovascular diseases. Curr Cardiol Rep. 2018; 20(6):47.doi: 10.1007/s11886-018-0992-9

[16] Hsieh PCH, Davis ME, Lisowski LK, Lee RT. Endothelial- cardiomyocyte interactions in cardiac development and repair. Annu Rev Physiol. 2006; 68:51-66.doi: 10.1146/annurev.physiol.68.040104.124629

[17] Tian Y, Morrisey EE. Importance of myocyte-nonmyocyte interactions in cardiac development and disease. Circ Res. 2012; 110(7):1023-1034.doi: 10.1161/CIRCRESAHA.111.243899

[18] Michel NA, Ljubojevic-Holzer S, Bugger H, Zirlik A. Cellular heterogeneity of the heart. Front Cardiovasc Med. 2022;9:868466.doi: 10.3389/fcvm.2022.868466

[19] Liu X, Huang J, Chen T, et al. Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Cell Res. 2008; 18(12):1177-1189.doi: 10.1038/cr.2008.309

[20] Lyra-Leite DM, Gutiérrez-Gutiérrez Ó, Wang M, Zhou Y, Cyganek L, Burridge PW. A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming. STAR Protoc. 2022; 3(3):101560.doi: 10.1016/j.xpro.2022.101560

[21] Lian X, Hsiao C, Wilson G, et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A. 2012; 109(27):E1848-E1857.doi: 10.1073/pnas.1200250109

[22] Burridge PW, Matsa E, Shukla P, et al. Chemically defined and small molecule-based generation of human cardiomyocytes. Nat Methods. 2014; 11(8):855-860.doi: 10.1038/nmeth.2999

[23] Kupfer ME, Lin WH, Ravikumar V, et al. In situ expansion, differentiation, and electromechanical coupling of human cardiac muscle in a 3D bioprinted, chambered organoid. Circ Res. 2020; 127(2):207-224.doi: 10.1161/CIRCRESAHA.119.316155

[24] Esser TU, Anspach A, Muenzebrock KA, et al. Direct 3D-bioprinting of hiPSC-derived cardiomyocytes to generate functional cardiac tissues. Adv Mater. 2023; 35(52):2305911.doi: 10.1002/adma.202305911

[25] Wolfe JT, He W, Kim MS, et al. 3D-bioprinting of patient- derived cardiac tissue models for studying congenital heart disease. Front Cardiovasc Med. 2023;10:1162731.doi: 10.3389/fcvm.2023.1162731

[26] Köhne M, Behrens CS, Stüdemann T, et al. A potential future Fontan modification: preliminary in vitro data of a pressure-generating tube from engineered heart tissue. Eur J Cardiothorac Surg. 2022; 62(2):ezac111.doi: 10.1093/ejcts/ezac111

[27] MacQueen LA, Sheehy SP, Chantre CO, et al. A tissue- engineered scale model of the heart ventricle. Nat Biomed Eng. 2018; 2(12):930-941.doi: 10.1038/s41551-018-0271-5

[28] Goldfracht I, Protze S, Shiti A, et al. Generating ring-shaped engineered heart tissues from ventricular and atrial human pluripotent stem cell-derived cardiomyocytes. Nat Commun. 2020; 11(1):1-15.doi: 10.1038/s41467-019-13868-x

[29] Querdel E, Reinsch M, Castro L, et al. Human engineered heart tissue patches remuscularize the injured heart in a dose-dependent manner. Circulation. 2021; 143(20): 1991-2006.doi: 10.1161/CIRCULATIONAHA.120.047904

[30] Mannhardt I, Breckwoldt K, Letuffe-Brenière D, et al. Human engineered heart tissue: analysis of contractile force. Stem Cell Rep. 2016; 7(1):29-42.doi: 10.1016/j.stemcr.2016.04.011

[31] Jabbour RJ, Owen TJ, Pandey P, et al. In vivo grafting of large engineered heart tissue patches for cardiac repair. JCI Insight. 2021; 6(15):e144068.doi: 10.1172/jci.insight.144068

[32] Lappi H, Kauppila M, Aalto-Setälä K, Mörö A. The 3D bioprinted human induced pluripotent stem cell-derived cardiac model: toward functional and patient-derived in vitro models for disease modeling and drug screening. Bioprinting. 2023;36:e00313.doi: 10.1016/j.bprint.2023.e00313

[33] Rehman S, Khan A, Rehman A. Physiology, coronary circulation. In: StatPearls. Treasure Island, FL: StatPearls Publishing; 2025. http://www.ncbi.nlm.nih.gov/books/NBK482413/. Accessed January 10, 2025.

[34] Maiullari F, Costantini M, Milan M, et al. A multi-cellular 3D bioprinting approach for vascularized heart tissue engineering based on HUVECs and iPSC-derived cardiomyocytes. Sci Rep. 2018; 8(1):13532.doi: 10.1038/s41598-018-31848-x

[35] Zhang YS, Arneri A, Bersini S, et al. Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip. Biomaterials. 2016; 110:45-59.doi: 10.1016/j.biomaterials.2016.09.003

[36] Song HHG, Rumma RT, Ozaki CK, Edelman ER, Chen CS. Vascular tissue engineering: progress, challenges, and clinical promise. Cell Stem Cell. 2018; 22(3): 40-354.doi: 10.1016/j.stem.2018.02.009

[37] Kennedy CC, Brown EE, Abutaleb NO, Truskey GA. Development and application of endothelial cells derived from pluripotent stem cells in microphysiological systems models. Front Cardiovasc Med. 2021;8:625016.doi: 10.3389/fcvm.2021.625016

[38] Gu M. Efficient differentiation of human pluripotent stem cells to endothelial cells. Curr Protoc Hum Genet. 2018; 98(1):e64.doi: 10.1002/cphg.64

[39] Hamad S, Derichsweiler D, Gaspar JA, et al. High-efficient serum-free differentiation of endothelial cells from human iPS cells. Stem Cell Res Ther. 2022; 13(1):251.doi: 10.1186/s13287-022-02924-x

[40] Williams IM, Wu JC. Generation of endothelial cells from human pluripotent stem cells. Arterioscler Thromb Vasc Biol. 2019; 39(7):1317-1329.doi: 10.1161/ATVBAHA.119.312265

[41] Ayoubi S, Sheikh SP, Eskildsen TV. Human induced pluripotent stem cell-derived vascular smooth muscle cells: differentiation and therapeutic potential. Cardiovasc Res. 2017; 113(11):1282-1293.doi: 10.1093/cvr/cvx125

[42] Souders CA, Bowers SLK, Baudino TA. Cardiac fibroblast: the renaissance cell. Circ Res. 2009; 105(12):1164-1176.doi: 10.1161/CIRCRESAHA.109.209809

[43] Li RA, Keung W, Cashman TJ, et al. Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells. Biomaterials. 2018; 163:116-127.doi: 10.1016/j.biomaterials.2018.02.024

[44] Tsuruyama S, Matsuura K, Sakaguchi K, Shimizu T. Pulsatile tubular cardiac tissues fabricated by wrapping human iPS cells-derived cardiomyocyte sheets. Regen Ther. 2019; 11:297-305.doi: 10.1016/j.reth.2019.09.001

[45] Kawai Y, Tohyama S, Arai K, et al. Scaffold-free tubular engineered heart tissue from human induced pluripotent stem cells using bio-3D printing technology in vivo. Front Cardiovasc Med. 2022;8:806215.doi: 10.3389/fcvm.2021.806215

[46] Whitehead AJ, Hocker JD, Ren B, Engler AJ. Improved epicardial cardiac fibroblast generation from iPSCs. J Mol Cell Cardiol. 2022; 164:58-68.doi: 10.1016/j.yjmcc.2021.11.011

[47] Zhang J, Tao R, Campbell KF, et al. Functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors. Nat Commun. 2019; 10(1):2238.doi: 10.1038/s41467-019-09831-5

[48] Bao X, Lian X, Qian T, Bhute VJ, Han T, Palecek SP. Directed differentiation and long-term maintenance of epicardial cells from human pluripotent stem cells under fully defined conditions. Nat Protoc. 2017; 12(9):1890-1900.doi: 10.1038/nprot.2017.080

[49] Lee A, Hudson AR, Shiwarski DJ, et al. 3D bioprinting of collagen to rebuild components of the human heart. Science. 2019; 365(6452):482-487.doi: 10.1126/science.aav9051

[50] Bliley JM, Vermeer MCSC, Duffy RM, et al. Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype. Sci Transl Med. 2021; 13(603):eabd1817.doi: 10.1126/scitranslmed.abd1817

[51] Miller KL, Xiang Y, Yu C, et al. Rapid 3D BioPrinting of a human iPSC-derived cardiac micro-tissue for high-throughput drug testing. Organs Chip. 2021;3:100007.doi: 10.1016/j.ooc.2021.100007

[52] Fullenkamp DE, Maeng WY, Oh S, et al. Simultaneous electromechanical monitoring in engineered heart tissues using a mesoscale framework. Sci Adv. 2024; 10(37):eado7089.doi: 10.1126/sciadv.ado7089

[53] Finkel S, Sweet S, Locke T, et al. FRESH^TM 3D bioprinted cardiac tissue, a bioengineered platform for in vitro pharmacology. APL Bioeng. 2023; 7(4):046113.doi: 10.1063/5.0163363

[54] Noor N, Shapira A, Edri R, Gal I, Wertheim L, Dvir T. 3D printing of personalized thick and perfusable cardiac patches and hearts. Adv Sci (Weinh). 2019; 6(11):1900344.doi: 10.1002/advs.201900344

[55] Lock RI, Graney PL, Tavakol DN, et al. Macrophages enhance contractile force in iPSC-derived human engineered cardiac tissue. Cell Rep. 2024; 43(6):114302.doi: 10.1016/j.celrep.2024.114302

[56] Szepes M, Melchert A, Dahlmann J, et al. Dual function of IPSC-derived pericyte-like cells in vascularization and fibrosis-related cardiac tissue remodeling in vitro. Int J Mol Sci. 2020; 21(23):8947.doi: 10.3390/ijms21238947

[57] Karvinen J, Kellomäki M. Design aspects and characterization of hydrogel-based bioinks for extrusion-based bioprinting. Bioprinting. 2023;32:e00274.doi: 10.1016/j.bprint.2023.e00274

[58] Kabirian F, Mozafari M. Decellularized ECM-derived bioinks: prospects for the future. Methods. 2020; 171:108-118.doi: 10.1016/j.ymeth.2019.04.019

[59] Gillispie G, Prim P, Copus J, et al. Assessment methodologies for extrusion-based bioink printability. Biofabrication. 2020; 12(2):022003.doi: 10.1088/1758-5090/ab6f0d

[60] Blaeser A, Duarte Campos DF, Puster U, Richtering W, Stevens MM, Fischer H. Controlling shear stress in 3D bioprinting is a key factor to balance printing resolution and stem cell integrity. Adv Healthc Mater. 2016; 5(3):326-333.doi: 10.1002/adhm.201500677

[61] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006; 126(4):677-689.doi: 10.1016/j.cell.2006.06.044

[62] Caliari SR, Burdick JA. A practical guide to hydrogels for cell culture. Nat Methods. 2016; 13(5):405-414.doi: 10.1038/nmeth.3839

[63] Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012; 37(1):106-126.doi: 10.1016/j.progpolymsci.2011.06.003

[64] Li Z, Ruan C, Niu X. Collagen-based bioinks for regenerative medicine: Fabrication, application and prospective. Med Nov Technol Devices. 2023;17:100211.doi: 10.1016/j.medntd.2023.100211

[65] Osidak EO, Kozhukhov VI, Osidak MS, Domogatsky SP. Collagen as bioink for bioprinting: a comprehensive review. Int J Bioprint. 2020; 6(3):270.doi: 10.18063/ijb.v6i3.270

[66] Shpichka A, Osipova D, Efremov Y, et al. Fibrin-based bioinks: new tricks from an old dog. IJB. 2024; 6(3):269. doi: 10.18063/ijb.v6i3.269

[67] Kumar SA, Alonzo M, Allen SC, et al. A visible light-cross-linkable, fibrin-gelatin-based bioprinted construct with human cardiomyocytes and fibroblasts. ACS Biomater Sci Eng. 2019; 5(9):4551-4563.doi: 10.1021/acsbiomaterials.9b00505

[68] Zhang D, Shadrin IY, Lam J, Xian HQ, Snodgrass HR, Bursac N. Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. Biomaterials. 2013; 34(23):5813-5820.doi: 10.1016/j.biomaterials.2013.04.026

[69] de Melo BAG, Jodat YA, Cruz EM, Benincasa JC, Shin SR, Porcionatto MA. Strategies to use fibrinogen as bioink for 3D bioprinting fibrin-based soft and hard tissues. Acta Biomater. 2020; 117:60-76.doi: 10.1016/j.actbio.2020.09.024

[70] Bejleri D, Davis ME. Decellularized extracellular matrix materials for cardiac repair and regeneration. Adv Healthc Mater. 2019; 8(5):1801217.doi: 10.1002/adhm.201801217

[71] Jain P, Rauer SB, Möller M, Singh S. Mimicking the natural basement membrane for advanced tissue engineering. Biomacromolecules. 2022; 23(8):3081-3103.doi: 10.1021/acs.biomac.2c00402

[72] Zhao KY, Du YX, Cao HM, Su LY, Su XL, Li X. The biological macromolecules constructed Matrigel for cultured organoids in biomedical and tissue engineering. Colloids Surf B Biointerfaces. 2025;247:114435.doi: 10.1016/j.colsurfb.2024.114435

[73] Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, Martin GR. Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry. 1982; 21(24):6188-6193.doi: 10.1021/bi00267a025

[74] Yue K, Santiago GT de, Alvarez MM, Tamayol A, Annabi N, Khademhosseini A. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials. 2015; 73:254-271.doi: 10.1016/j.biomaterials.2015.08.045

[75] Khoeini R, Nosrati H, Akbarzadeh A, et al. Natural and synthetic bioinks for 3D bioprinting. Adv NanoBiomed Res. 2021; 1(8):2000097.doi: 10.1002/anbr.202000097

[76] Wang X. Advanced polymers for three-dimensional (3D) organ bioprinting. Micromachines. 2019; 10(12):814.doi: 10.3390/mi10120814

[77] Hockaday LA, Kang KH, Colangelo NW, et al. Rapid 3D printing of anatomically accurate and mechanically heterogeneous aortic valve hydrogel scaffolds. Biofabrication. 2012; 4(3):035005.doi: 10.1088/1758-5082/4/3/035005

[78] Shin YJ, Shafranek RT, Tsui JH, Walcott J, Nelson A, Kim DH. 3D bioprinting of mechanically tuned bioinks derived from cardiac decellularized extracellular matrix. Acta Biomater. 2021; 119:75-88.doi: 10.1016/j.actbio.2020.11.006

[79] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials. 2003; 24(24):4385-4415.doi: 10.1016/s0142-9612(03)00343-0

[80] Zhu J. Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering. Biomaterials. 2010; 31(17):4639-4656.doi: 10.1016/j.biomaterials.2010.02.044

[81] Nourse MB, Halpin DE, Scatena M, et al. VEGF induces differentiation of functional endothelium from human embryonic stem cells. Arterioscler Thromb Vasc Biol. 2010; 30(1):80-89.doi: 10.1161/ATVBAHA.109.194233

[82] Seliktar D, Zisch AH, Lutolf MP, Wrana JL, Hubbell JA. MMP-2 sensitive, VEGF-bearing bioactive hydrogels for promotion of vascular healing. J Biomed Mater Res A. 2004; 68(4):704-716.doi: 10.1002/jbm.a.20091

[83] Balatskyi VV, Sowka A, Dobrzyn P, Piven OO. WNT/β- catenin pathway is a key regulator of cardiac function and energetic metabolism. Acta Physiol. 2023; 237(3):e13912.doi: 10.1111/apha.13912

[84] Lee YB, Polio S, Lee W, et al. Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture. Exp Neurol. 2010; 223(2):645-652.doi: 10.1016/j.expneurol.2010.02.014

[85] Poldervaart MT, Gremmels H, van Deventer K, et al. Prolonged presence of VEGF promotes vascularization in 3D bioprinted scaffolds with defined architecture. J Control Release. 2014; 184:58-66.doi: 10.1016/j.jconrel.2014.04.007

[86] Mann BK, Schmedlen RH, West JL. Tethered- TGF-β increases extracellular matrix production of vascular smooth muscle cells. Biomaterials. 2001; 22(5):439-444.doi: 10.1016/S0142-9612(00)00196-4

[87] DeLong SA, Moon JJ, West JL. Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration. Biomaterials. 2005; 26(16):3227-3234.doi: 10.1016/j.biomaterials.2004.09.021

[88] Bliley J, Tashman J, Stang M, et al. FRESH 3D bioprinting a contractile heart tube using human stem cell-derived cardiomyocytes. Biofabrication. 2022; 14(2):10.1088/1758- 5090/ac58be.doi: 10.1088/1758-5090/ac58be

[89] Ren J, Han P, Ma X, et al. Canonical Wnt5b signaling directs outlying Nkx2.5+ mesoderm into pacemaker cardiomyocytes. Dev Cell. 2019; 50(6):729-743.e5.doi: 10.1016/j.devcel.2019.07.014

[90] Liu J, He J, Liu J, et al. Rapid 3D bioprinting of in vitro cardiac tissue models using human embryonic stem cell-derived cardiomyocytes. Bioprinting. 2019;13:e00040.doi: 10.1016/j.bprint.2019.e00040

[91] Jia W, Gungor-Ozkerim PS, Zhang YS, et al. Direct 3D bioprinting of perfusable vascular constructs using a blend bioink. Biomaterials. 2016; 106:58-68.doi: 10.1016/j.biomaterials.2016.07.038

[92] Pati F, Jang J, Lee JW, Cho DW. Chapter 7—extrusion bioprinting. In: Atala A, Yoo JJ, eds. Essentials of 3D Biofabrication and Translation. Academic Press; 2015:123-152.doi: 10.1016/B978-0-12-800972-7.00007-4

[93] Li H, Dai J, Wang Z, et al. Digital light processing (DLP)- based (bio)printing strategies for tissue modeling and regeneration. Aggregate. 2023; 4(2):e270.doi: 10.1002/agt2.270

[94] Hosseinabadi HG, Dogan E, Miri AK, Ionov L. Digital light processing bioprinting advances for micro-tissue models. ACS Biomater Sci Eng. 2022; 8(4):1381-1395.doi: 10.1021/acsbiomaterials.1c01509

[95] Gudapati H, Dey M, Ozbolat I. A comprehensive review on droplet-based bioprinting: past, present and future. Biomaterials. 2016; 102:20-42.doi: 10.1016/j.biomaterials.2016.06.012

[96] Shin S, Kwon Y, Hwang C, et al. Visible-light-driven rapid 3d printing of photoresponsive resins for optically clear multifunctional 3D objects. Adv Mater. 2024; 36(19):2311917.doi: 10.1002/adma.202311917

[97] Salem T, Frankman Z, Churko JM. Tissue engineering techniques for induced pluripotent stem cell derived three-dimensional cardiac constructs. Tissue Eng Part B Rev. 2022; 28(4):891-911.doi: 10.1089/ten.teb.2021.0088

[98] You S, Xiang Y, Hwang HH, et al. High cell density and high-resolution 3D bioprinting for fabricating vascularized tissues. Sci Adv. 2023; 9(8):eade7923.doi: 10.1126/sciadv.ade7923

[99] Hinton TJ, Jallerat Q, Palchesko RN, et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci Adv. 2015; 1(9):e1500758.doi: 10.1126/sciadv.1500758

[100] Mirdamadi E, Tashman JW, Shiwarski DJ, Palchesko RN, Feinberg AW. FRESH 3D bioprinting a full-size model of the human heart. ACS Biomater Sci Eng. 2020; 6(11): 6453-6459.doi: 10.1021/acsbiomaterials.0c01133

[101] Skylar-Scott MA, Uzel SGM, Nam LL, et al. Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels. Sci Adv. 2019; 5(9):eaaw2459.doi: 10.1126/sciadv.aaw2459

[102] Bhusal A, Dogan E, Nguyen HA, et al. Multi-material digital light processing bioprinting of hydrogel-based microfluidic chips. Biofabrication. 2021; 14(1):014103.doi: 10.1088/1758-5090/ac2d78

[103] Liu J, Miller K, Ma X, et al. Direct 3D bioprinting of cardiac micro-tissues mimicking native myocardium. Biomaterials. 2020;256:120204.doi: 10.1016/j.biomaterials.2020.120204

[104] Yu C, Ma X, Zhu W, et al. Scanningless and continuous 3D bioprinting of human tissues with decellularized extracellular matrix. Biomaterials. 2019; 194:1-13.doi: 10.1016/j.biomaterials.2018.12.009

[105] Zhu W, Qu X, Zhu J, et al. Direct 3D bioprinting of prevascularized tissue constructs with complex microarchitecture. Biomaterials. 2017; 124:106-115.doi: 10.1016/j.biomaterials.2017.01.042

[106] Izadifar M, Babyn P, Kelly ME, Chapman D, Chen X. Bioprinting pattern-dependent electrical/mechanical behavior of cardiac alginate implants: characterization and ex vivo phase-contrast microtomography assessment. Tissue Eng Part C Methods. 2017; 23(9):548-564.doi: 10.1089/ten.tec.2017.0222

[107] Kotadia I, Whitaker J, Roney C, et al. Anisotropic cardiac conduction. Arrhythm Electrophysiol Rev. 2020; 9(4):202-210.doi: 10.15420/aer.2020.04

[108] Bera AK, Rizvi MS, KN V, Pati F. Engineering anisotropic tissue analogues: harnessing synergistic potential of extrusion-based bioprinting and extracellular matrix-based bioink. Biofabrication. 2024; 17(1):015003.doi: 10.1088/1758-5090/ad86ec

[109] Dwyer KD, Coulombe KLK. Cardiac mechanostructure: using mechanics and anisotropy as inspiration for developing epicardial therapies in treating myocardial infarction. Bioact Maters. 2021; 6(7):2198-2220.doi: 10.1016/j.bioactmat.2020.12.015

[110] Chikae S, Kubota A, Nakamura H, et al. Three-dimensional bioprinting human cardiac tissue chips of using a painting needle method. Biotechnol Bioeng. 2019; 116(11):3136-3142.doi: 10.1002/bit.27126

[111] Xu T, Baicu C, Aho M, Zile M, Boland T. Fabrication and characterization of bio-engineered cardiac pseudo tissues. Biofabrication. 2009; 1(3):035001.doi: 10.1088/1758-5082/1/3/035001

[112] El Khoury R, Nagiah N, Mudloff JA, Thakur V, Chattopadhyay M, Joddar B. 3D bioprinted spheroidal droplets for engineering the heterocellular coupling between cardiomyocytes and cardiac fibroblasts. Cyborg Bionic Syst. 2021;2021:9864212.doi: 10.34133/2021/9864212

[113] Datta P, Ayan B, Ozbolat IT. Bioprinting for vascular and vascularized tissue biofabrication. Acta Biomater. 2017; 51:1-20.doi: 10.1016/j.actbio.2017.01.035

[114] Elkhoury K, Zuazola J, Vijayavenkataraman S. Bioprinting the future using light: a review on photocrosslinking reactions, photoreactive groups, and photoinitiators. SLAS Technol. 2023; 28(3):142-151.doi: 10.1016/j.slast.2023.02.003

[115] GhavamiNejad A, Ashammakhi N, Wu XY, Khademhosseini A. Crosslinking strategies for three-dimensional bioprinting of polymeric hydrogels. Small. 2020; 16(35):e2002931.doi: 10.1002/smll.202002931

[116] Lee JE, Heo SW, Kim CH, Park SJ, Park SH, Kim TH. In-situ ionic crosslinking of 3D bioprinted cell-hydrogel constructs for mechanical reinforcement and improved cell growth. Biomater Adv. 2023;147:213322.doi: 10.1016/j.bioadv.2023.213322

[117] Chatterjee S, Hui PCL, Kan CW, Wang W. Dual-responsive (pH/temperature) Pluronic F-127 hydrogel drug delivery system for textile-based transdermal therapy. Sci Rep. 2019; 9(1):11658.doi: 10.1038/s41598-019-48254-6

[118] Sarker M, Izadifar M, Schreyer D, Chen X. Influence of ionic crosslinkers (Ca2+/Ba2+/Zn2+) on the mechanical and biological properties of 3D bioplotted hydrogel scaffolds. J Biomater Sci Polym Ed. 2018; 29(10):1126-1154.doi: 10.1080/09205063.2018.1433420

[119] Kim MH, Lee YW, Jung WK, Oh J, Nam SY. Enhanced rheological behaviors of alginate hydrogels with carrageenan for extrusion-based bioprinting. J Mech Behav Biomed Mater. 2019; 98:187-194.doi: 10.1016/j.jmbbm.2019.06.014

[120] Gu Y, Zhang L, Du X, et al. Reversible physical crosslinking strategy with optimal temperature for 3D bioprinting of human chondrocyte-laden gelatin methacryloyl bioink. J Biomater Appl. 2018; 33(5):609-618.doi: 10.1177/0885328218805864

[121] Bahney CS, Lujan TJ, Hsu CW, Bottlang M, West JL, Johnstone B. Visible light photoinitiation of mesenchymal stem cell-laden bioresponsive hydrogels. Eur Cell Mater. 2011; 22:43-55; discussion 55.doi: 10.22203/ecm.v022a04

[122] Basara G, Ozcebe SG, Ellis BW, Zorlutuna P. Tunable Human myocardium derived decellularized extracellular matrix for 3D bioprinting and cardiac tissue engineering. Gels. 2021; 7(2):70.doi: 10.3390/gels7020070

[123] Budharaju H, Sundaramurthi D, Sethuraman S. Efficient dual crosslinking of protein-in-polysaccharide bioink for biofabrication of cardiac tissue constructs. Biomater Adv. 2023;152:213486.doi: 10.1016/j.bioadv.2023.213486

[124] Karbassi E, Fenix A, Marchiano S, et al. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol. 2020; 17(6):341-359.doi: 10.1038/s41569-019-0331-x

[125] Wu P, Deng G, Sai X, Guo H, Huang H, Zhu P. Maturation strategies and limitations of induced pluripotent stem cell-derived cardiomyocytes. Biosci Rep. 2021; 41(6):BSR20200833.doi: 10.1042/BSR20200833

[126] Guo Y, Pu W. Cardiomyocyte maturation: new phase in development. Circ Res. 2020; 126(8):1086-1106.doi: 10.1161/CIRCRESAHA.119.315862

[127] Tulloch NL, Muskheli V, Razumova MV, et al. Growth of engineered human myocardium with mechanical loading and vascular coculture. Circ Res. 2011; 109(1):47-59.doi: 10.1161/CIRCRESAHA.110.237206

[128] Zhao Y, Rafatian N, Feric NT, et al. A platform for generation of chamber specific cardiac tissues and disease modelling. Cell. 2019; 176(4):913-927.e18.doi: 10.1016/j.cell.2018.11.042

[129] Ronaldson-Bouchard K, Ma SP, Yeager K, et al. Advanced maturation of human cardiac tissue grown from pluripotent stem cells. Nature. 2018; 556(7700):239-243.doi: 10.1038/s41586-018-0016-3

[130] Godier-Furnémont AFG, Tiburcy M, Wagner E, et al. Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation. Biomaterials. 2015; 60:82-91.doi: 10.1016/j.biomaterials.2015.03.055

[131] Scuderi GJ, Butcher J. Naturally engineered maturation of cardiomyocytes. Front Cell Dev Biol. 2017;5:50.doi: 10.3389/fcell.2017.00050

[132] Bliley JM, Stang MA, Behre A, Feinberg AW. Advances in 3D bioprinted cardiac tissue using stem cell-derived cardiomyocytes. Stem Cells Transl Med. 2024; 13(5):425-435.doi: 10.1093/stcltm/szae014

[133] Paez-Mayorga J, Hernández-Vargas G, Ruiz-Esparza GU, et al. Bioreactors for cardiac tissue engineering. Adv Healthc Mater. 2019; 8(7):1701504.doi: 10.1002/adhm.201701504

[134] Kim H, Kamm RD, Vunjak-Novakovic G, Wu JC. Progress in multicellular human cardiac organoids for clinical applications. Cell Stem Cell. 2022; 29(4):503-514.doi: 10.1016/j.stem.2022.03.012

[135] Drakhlis L, Biswanath S, Farr CM, et al. Human heart-forming organoids recapitulate early heart and foregut development. Nat Biotechnol. 2021; 39(6):737-746.doi: 10.1038/s41587-021-00815-9

[136] Lewis-Israeli YR, Wasserman AH, Gabalski MA, et al. Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease. Nat Commun. 2021; 12(1):5142.doi: 10.1038/s41467-021-25329-5

[137] Rossi G, Broguiere N, Miyamoto M, et al. Capturing cardiogenesis in gastruloids. Cell Stem Cell. 2021; 28(2):230-240.e6.doi: 10.1016/j.stem.2020.10.013

[138] Hofbauer P, Jahnel SM, Papai N, et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell. 2021; 184(12):3299-3317.e22.doi: 10.1016/j.cell.2021.04.034

[139] Silva AC, Matthys OB, Joy DA, et al. Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids. Cell Stem Cell. 2021; 28(12):2137-2152.e6.doi: 10.1016/j.stem.2021.11.007

[140] Branco MA, Dias TP, Cabral JMS, Pinto-do-Ó P, Diogo MM. Human multilineage pro-epicardium/foregut organoids support the development of an epicardium/myocardium organoid. Nat Commun. 2022; 13(1):6981.doi: 10.1038/s41467-022-34730-7

[141] Olmsted ZT, Paluh JL. A combined human gastruloid model of cardiogenesis and neurogenesis. iScience. 2022; 25(6):104486.doi: 10.1016/j.isci.2022.104486

[142] Meier AB, Zawada D, De Angelis MT, et al. Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease. Nat Biotechnol. 2023; 41(12):1787-1800.doi: 10.1038/s41587-023-01718-7

[143] Volmert B, Kiselev A, Juhong A, et al. A patterned human primitive heart organoid model generated by pluripotent stem cell self-organization. Nat Commun. 2023; 14(1):8245.doi: 10.1038/s41467-023-43999-1

[144] Mohr E, Thum T, Bär C. Accelerating cardiovascular research: recent advances in translational 2D and 3D heart models. Eur J Heart Fail. 2022; 24(10):1778-1791.doi: 10.1002/ejhf.2631

[145] Kehat I, Kenyagin-Karsenti D, Snir M, et al. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest. 2001; 108(3):407-414.doi: 10.1172/JCI12131

[146] Burridge PW, Anderson D, Priddle H, et al. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel v-96 plate aggregation system highlights interline variability. Stem Cells. 2007; 25(4):929-938.doi: 10.1634/stemcells.2006-0598

[147] Voges HK, Mills RJ, Elliott DA, Parton RG, Porrello ER, Hudson JE. Development of a human cardiac organoid injury model reveals innate regenerative potential. Development. 2017; 144(6):1118-1127.doi: 10.1242/dev.143966

[148] Lemon LS, Bodnar LM, Garrard W, et al. Ondansetron use in the first trimester of pregnancy and the risk of neonatal ventricular septal defect. Int J Epidemiol. 2020; 49(2): 648-656.doi: 10.1093/ije/dyz255

[149] Richards DJ, Li Y, Kerr CM, et al. Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity. Nat Biomed Eng. 2020; 4(4):446-462.doi: 10.1038/s41551-020-0539-4

[150] Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns HJ, Moens AL. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol. 2012; 52(6):1213-1225.doi: 10.1016/j.yjmcc.2012.03.006

[151] Brassard JA, Nikolaev M, Hübscher T, Hofer M, Lutolf MP. Recapitulating macro-scale tissue self-organization through organoid bioprinting. Nat Mater. 2021; 20(1):22-29.doi: 10.1038/s41563-020-00803-5

[152] Skylar-Scott MA, Huang JY, Lu A, et al. Orthogonally induced differentiation of stem cells for the programmatic patterning of vascularized organoids and bioprinted tissues. Nat Biomed Eng. 2022; 6(4):449-462.doi: 10.1038/s41551-022-00856-8

[153] Lawlor KT, Vanslambrouck JM, Higgins JW, et al. Cellular extrusion bioprinting improves kidney organoid reproducibility and conformation. Nat Mater. 2021; 20(2):260-271.doi: 10.1038/s41563-020-00853-9

[154] Yang H, Sun L, Pang Y, et al. Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure. Gut. 2021; 70(3):567-574.doi: 10.1136/gutjnl-2019-319960

[155] Choi Y mi, Lee H, Ann M, Song M, Rheey J, Jang J. 3D bioprinted vascularized lung cancer organoid models with underlying disease capable of more precise drug evaluation. Biofabrication. 2023; 15(3):034104.doi: 10.1088/1758-5090/acd95f

[156] Wang D, Guo Y, Zhu J, et al. Hyaluronic acid methacrylate/ pancreatic extracellular matrix as a potential 3D printing bioink for constructing islet organoids. Acta Biomater. 2023; 165:86-101.doi: 10.1016/j.actbio.2022.06.036

[157] He C, Yan J, Fu Y, Guo J, Shi Y, Guo J. Organoid bioprinting strategy and application in biomedicine: a review. IJB. 2023; 9(6):0112.doi: 10.36922/ijb.0112

[158] Liu F, Liu C, Chen Q, et al. Progress in organ 3D bioprinting. Int J Bioprint. 2018; 4(1):128.doi: 10.18063/IJB.v4i1.128

[159] Romanazzo S, Nemec S, Roohani I. iPSC bioprinting: where are we at? Materials (Basel). 2019; 12(15):2453.doi: 10.3390/ma12152453

[160] Ahrens JH, Uzel SGM, Skylar‐Scott M, et al. Programming cellular alignment in engineered cardiac tissue via bioprinting anisotropic organ building blocks. Adv Mater. 2022; 34(26):2200217.doi: 10.1002/adma.202200217

[161] Ren Y, Yang X, Ma Z, et al. Developments and opportunities for 3D bioprinted organoids. Int J Bioprint. 2021; 7(3):364.doi: 10.18063/ijb.v7i3.364

[162] Bernal PN, Delrot P, Loterie D, et al. Volumetric bioprinting of complex living-tissue constructs within seconds. Adv Mater. 2019; 31(42):1904209.doi: 10.1002/adma.201904209

[163] Bernal PN, Bouwmeester M, Madrid-Wolff J, et al. Volumetric bioprinting of organoids and optically tuned hydrogels to build liver-like metabolic biofactories. Adv Mater. 2022; 34(15):2110054.doi: 10.1002/adma.202110054