Functional engineered heart tissue cultured in a rotating wall vessel bioreactor improve cardiac function in the distressed rat heart
Authors: Nakazato T , Miyagawa S , Uemura T , Liu L , Li J , Sasai M , Harada A , Toda K , Sawa Y 
Published: November 2020

Abstract

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