In Vitro Perfusion of Engineered Heart Tissue Through Endothelialized Channels Ingra Vollert, PhD, 1–3, * Moritz Seiffert, MD, 2–4, * Johanna Bachmair, 5,6 Merle Sander, 1–3 Alexandra Eder, PhD, 1–3 Lenard Conradi, MD, 2–4 Alexander Vogelsang, 1–3 Thomas Schulze, 1–3 June Uebeler, 1–3 Wolfgang Holnthoner, PhD, 5 Heinz Redl, MD, 5 Hermann Reichenspurner, MD, PhD, 2–4 Arne Hansen, MD, 1–3 and Thomas Eschenhagen, MD 1–3 In engineered heart tissues (EHT), oxygen and nutrient supply via mere diffusion is a likely factor limiting the thickness of cardiac muscle strands. Here, we report on a novel method to in vitro perfuse EHT through tubular channels. Adapting our previously published protocols, we expanded a miniaturized fibrin-based EHT-format to a larger six-well format with six flexible silicone posts holding each EHT (15 · 25 · 3 mm 3 ). Thin dry alginate fibers (17 · 0.04 · 0.04 mm) were embedded into the cell–fibrin–thrombin mix and, after fibrin polymerization, dissolved by incubation in alginate lyase or sodium citrate. Oxygen concentrations were measured with a microsensor in 14-day-old EHTs (37°C, 21% oxygen) and ranged between 9% at the edges and 2% in the center of the tissue. Perfusion rapidly increased it to 10%–12% in the immediate vicinity of the microchannel. Continuous perfusion (20 mL/h, for 3 weeks) of the tubular lumina (100–500 mm) via hollow posts of the silicone rack increased mean dystrophin-positive cardiomyocyte density (36% – 6% vs. 10% – 3% of total cell number) and cross sectional area (73 – 2 vs. 48 – 1 mm 2 ) in the central part of the tissue compared to nonperfused EHTs. The channels were populated by endothelial cells present in the reconstitution cell mix. In conclusion, we developed a novel approach to generate small tubular structures suitable for perfusion of spontaneously contracting and force-generating EHTs and showed that prolonged perfusion improved cardiac tissue structure. Introduction A lthough the methods and techniques for generating engineered heart tissue (EHT) have been improved over the past decade, 1,2 their limited size remains a serious re- striction. Mere oxygen and nutrient supply via diffusion limits the size of compact muscle strands in engineered tissues to a maximum thickness of 100–200 mm, 3,4 whereas cardiac tissue replacement therapy likely requires a transplant thick- ness of several millimeters to functionally support the injured heart. In human myocardium, a dense capillary network surrounding cardiomyocytes supplies a microcirculatory unit providing blood. 5 Inadequate perfusion immediately stops cardiomyocytes to work. Thus, methods are needed that permit tissue perfusion and/or increase tissue permeability. Current approaches rest upon the use of porous scaffolds, 6,7 decellularized tissues, 8 in situ tissue engineering onto isolated A/V loops, 9,10 sandwich culture of different cell layers, 11,12 polysurgery of cell sheet grafts, 13 addition of vascularization promoting factors, 14 co-culture with endothelial cells and fi- broblasts, 15,16 and interstitial flow stress. 17 Small capillary-like vascular networks were recently achieved in an elegant ap- proach using vascular explants and a thymosin b4-loaded hydrogel seeded with cardiomyocytes. 18 Another approach is based on gene-modified myoblasts added to cardiomyo- cytes. 19 Here, we report on a new method, which uses fila- mentous alginate templates to create a tubular lumen in EHTs and allows, for the first time, prolonged in vitro perfusion of large contractile EHTs. Materials and Methods We adapted our previously published protocol 20 and ex- panded the fibrin-based mini-EHT from a 24-well format An abstract of this work was published before: Vollert et al., J Tissue Eng Regen Med 6, Supp. 1, 2012. 1 Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 2 Cardiovascular Research Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 3 DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lu ¨ beck, Hamburg, Germany. 4 Department of Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany. 5 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria. 6 Institute for Production Engineering and Laser Technology, Vienna University of Technology, Vienna, Austria. *These authors contributed equally to this study. TISSUE ENGINEERING: Part A Volume 20, Numbers 3 and 4, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2013.0214 854