Abstract 13559: Engineered Three-dimensional Cardiac Tissues Maturated by Dynamic Culture in a Rotating Wall Vessel Bioreactor Remodel the Diseased Heart in the Rat Infarction Model

Introduction: To completely repair the distressed heart with few myocytes it may be crucial how to construct massive engineered three-dimensional cardiac tissues (E3DCTs) and integrate them to myocardium after transplant. We hypothesized that E3DCTs maturated by dynamic culture could enhance to remodel diseased left ventricle (LV) in the distressed rat heart. Methods: We made E3DCT by seeding 2.0 х 10 6 human induced pluripotent stem cell derived cardiomyocytes on the 6mm х 6mm PLGA fiber sheet. It was cultured in a rotating wall vessel bioreactor for seven days (RWV group) or culture dishes for the control. After in vitro assessment, these tissues were transplanted to myocardial infarction model nude rats, and 4 weeks after transplantation, we evaluated functional recovery and histology. Results: In the RWV group, thickness of E3DCTs were around 400μm, and cardiomyocytes showed mature phenotype evidenced by significantly higher expression of Troponin T (TnT), sarcomeric α actinin (SAA), connexin 43 (Cx43) and myosin heavy chain 7 (MYH7) with upregulation of mammalian target of rapamycin by Western blots (TnT; 2.1±0.2 vs. 1.0±0.2, SAA; 2.0±0.2 vs. 1.0±0.2, Cx 43; 2.0±0.3 vs. 1.0±0.1, MYH7; 2.0±0.2 vs. 1.0±0.2, p<0.05, respectively) (Figure 1A). Moreover, the concentration of angiogenic cytokines was significantly higher in the RWV group. Four weeks after transplantation, the LV ejection fraction was significantly improved in the RWV group than in the control (47±4.9 vs. 38±6.9%, p<0.01). On histological analysis, transplanted E3DCT reached a thickness of 1mm and vascular-like structures were partially observed inside (Figure 1B). In the RWV group, LV remodeling was significantly attenuated and neovascularization was significantly noted (Figure 1C, 1D). Conclusions: E3DCTs maturated by dynamic culture in a RWV bioreactor could remodel the diseased LV in rat infarction model, proposing potent armamentarium for cardiomyogenesis therapy in failed heart.

Functional engineered heart tissue cultured in a rotating wall vessel bioreactor improve cardiac function in the distressed rat heart

Introduction How to construct massive cardiac tissue and culture it with functional improvement may be crucial as cardiomyogenesis in failed heart. We previously presented that dynamic culture in a rotating wall vessel (RWV) bioreactor could provide a better culture environment for maintenance of the engineered 3D cardiac tissue. However, it is unknown about the effect of the tissue cultured in a RWV bioreactor on engraftment and improvement of function in the distressed rat heart. Hypothesis We hypothesized that the engineered 3D cardiac tissue cultured in a RWV bioreactor could improve its engraftment and lead recovery of cardiac function in rat infarction model. Methods We made engineered cardiac tissue by seeding 2.0 × 106 human induced pluripotent stem cell derived cardiomyocytes on the PLGA fiber sheet. It was cultured in the RWV bioreactor for seven days (RWV group). For the control, static culture has been done. After in vitro assessment, these tissues were transplanted to myocardial infarction model nude rats (sham, control, and RWV group, n=10, respectively) and cardiac performance was evaluated by ultrasonography. Four weeks after transplantation, we evaluated their hearts by histological analysis. Results The RWV group demonstrated maturation of cardiomyocytes evidenced by significantly higher expression of Troponin T (TnT), sarcomeric α actinin (SAA), connexin 43 (Cx43) and myosin heavy chain 7 (MYH7) than the control by Western blots (TnT; 2.7±1.0 vs. 1.0±0.4, p&lt;0.01, SAA; 2.1±0.7 vs. 1.0±0.2, p&lt;0.01, Cx 43; 2.0±0.6 vs. 1.0±0.1, p&lt;0.05, MYH7; 10.9±2.7 vs. 1.0±0.1, p&lt;0.01). In the culture supernatant, the concentration of cytokines related to angiogenesis was significantly higher in the RWV group than in the control (VEGF; 29.6±7.4 vs. 12.2±4.3pg/ml, p&lt;0.01, HGF; 72.7±9.9 vs. 42.6±5.9pg/ml, p&lt;0.01). Four weeks after transplantation, the left ventricular ejection fraction was significantly improved in the RWV group than in the control (RWV vs. control; 47±4.9 vs. 38±6.9%, p&lt;0.01). On histological analysis, more engineered cardiac tissue survived in the RWV group than in the control (RWV vs. control; 7/10 vs. 3/10, p=0.18). A vascular-like structure double-stained with isolectin B4 and smooth muscle actin was partially observed in the transplanted tissue. LV remodeling exhibiting extracellular collagen deposition (fibrotic area, RWV vs. control; 17±4.3 vs. 24±5.2%, p&lt;0.05) and cardiomyocyte hypertrophy (RWV vs. control; 16±1.7 vs. 18±2.1μm, p&lt;0.05) was significantly attenuated in RWV group than in the control. Neovascularization was significantly noted in the RWV group compared with the control (capillary density, RWV vs. control; 545±113 vs. 356±92, p&lt;0.01). Conclusion Functional engineered 3D cardiac tissue cultured in a RWV bioreactor could induce angiogenesis and improved its engraftment, leading significant improvement of cardiac function in rat infarction model. Dynamic culture in a RWV bioreactor Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science