Abstract 15337: The Development of Cryo-Vetrification Method in Human Myoblast Cell Sheet for Propagation of Regeneration Therapy
Introduction: Although tissue engineered cardiac construct have some impacts on the functional recovery in heart failure, how to preserve tissue engineered cardiac constructs may be crucial for industrialization and widely-use of this treatment.
Hypothesis: In this study we hypothesized whether tissue engineered cardiac constructs may be histologically and functionally preserved by new cryopreservation method.
Methods: Scaffold-free cardiac cell-sheet constructs were prepared by skeletal myoblast (SMB) using temperature response culture dish, and were immersed carboxyl poly-L-lysine-based vitrification solution, wrapped by a thin film and frozen above a liquid nitrogen pool to achieve cryo-vitrification of the cell construct (CV, n=6) and frozen in the slow freezing method using 10% DMSO (SF, n=6) and data were compared with fresh SMB sheet (F, n=6). The cryopreserved cell-sheet constructs were thawed at 2, 7 and 28 days after vitrification for assessment.
Results: Cell viability in the cardiac constructs were slightly reduced in the CV and SF group compared with the F group (F:91%±3%, CV*:83%±3%, SF*:84%±2%,; *P <0.05). Real time PCR revealed that expression of cytokines such as VEGF, HIF-1 and SDF-1 were maintained between these groups for 28 days. The structures of cell-sheet construct were maintained in the CV group, but were not in the SF group resulting in the difficulty for implantation. Electron microscopy revealed that Cell-cell junctions such as desmosomes, extracellular matrix(ECM) and structure of the cell membrane were well preserved in the CV group and F group, but not in the SF group. Western blotting analysis revealed that expression of fibronectin, integrin α5, and collagen1 were well preserved in the CV and SF group compared to the F group (Fibronectin / GAPDH ; F*:1, CV*:1.08±0.18, SF*:0.97±0.21; *n.s.). Extracellular matrix, such as fibronectin, integrin α5, collagen1 and collagen3, were maintained in the CV group, but they were collapsed in the SF group by immunohistological analysis.
Conclusions: Vitrification method preserved functionality and structure of scaffold-free cardiac cell-sheet constructs using human SMB after thaw, suggesting the possibility in propagation of cell sheet therapy in clinical scenario.
Author Disclosures: H. Ohkawara: None. S. Miyagawa: None. S. Fukushima: None. A. Saito: None. Y. Sawa: None.
- © 2016 by American Heart Association, Inc.