Prevascularization and in vitro perfusion of engineered heart tissue

Aims: Although methods for developing engineered heart tissue (EHT) have been improved intensively over the last decade, one major constraint for their potential clinical use constitutes the limited size of these constructs. Oxygen and nutrient supply via diffusion limits the thickness of compact muscle strands to 100 – 200 µm. To advance EHT towards cardiac tissue replacement, requiring a tissue thickness of several millimeters, we report on our recent efforts to integrate functional vessels into EHT to overcome this limitation.

Methods: In adaption of the previously published protocol, we expanded the fibrin-based EHT-format from a 24- to a 6-well-format. Posts of the silicon rack featured hollow tubes allowing for perfusion of the EHT generated adjacent to these racks. Thin calcium-alginate fibers were generated and placed into the two hollow middle-posts of the silicon racks, connecting both sides of the silicon rack at the bottom of the casting mold. The reconstitution mix (neonatal rat heart cells, fibrinogen, thrombin) was pipetted into the casting mold, enclosing the lower part of the silicon posts including the alginate fiber. After polymerization of the EHT the alginate fibers were dissolved leaving a tube (diameter ˜100 µm) inside the EHT and perfusion with cell culture medium was initiated.

Results: Alginate fibers inside the EHT could be fully disintegrated by incubation in sodium citrate or alginate lyase without significantly affecting cardiomyocyte viability. Perfusion was achieved using a micro-pump with continuous flow starting at the first day of cell culture. EHT started to beat coherently around day 6 of culture. Histological evaluation revealed a patent lumen within the EHT and endothelial cell lineage. Cardiomyocyte density in the central region of the EHT was evaluated by anti-dystrophin staining and confirmed higher counts in the circumference of the perfused lumen if compared to controls without perfusion.

Conclusion: We report on our first efforts to generate thin tube-like vessels within EHT suitable for the perfusion of living, actively contracting and force-generating 3D engineered tissues that is, in principle, applicable to all types of tissue engineering. The new technique represents an important first step towards generating EHT at sizes suitable for cardiac tissue replacement therapy.