Construction of cardiomyoblast sheets for cardiac tissue repair: comparison of three different approaches

Abstract

Recently, cell sheet engineering has emerged as one of the most accentuated approaches of tissue engineering and cardiac tissue is the pioneering application area of cell sheets with clinical use. In this study, we cultured rat cardiomyoblasts (H9C2 cell line) to obtain cell sheets by using three different approaches; using (1) thermo-responsive tissue culture plates, (2) high cell seeding density/high serum content and (3) ascorbic acid treatment. To compare the outcomes of three methods, morphologic examination, immunofluorescent stainings and live/dead cell assay were performed and the effects of serum concentration and ascorbic acid treatment on cardiac gene expressions were examined. The results showed that cardiomyoblast sheets were successfully obtained in all approaches without losing their integrity and viability. Also, the results of RT-PCR analysis showed that the types of tissue culture surface, cell seeding density, serum concentration and ascorbic acid treatment affect cardiac gene expressions of cells in cell sheets. Although three methods were succeeded, ascorbic acid treatment was found as the most rapid and effective method to obtain cell sheets with cardiac characteristics.

Keywords: Ascorbic acid; Cardiac tissue; Cell sheet engineering; H9C2 cell line; Thermo-responsive surface.

Fig. 1

H9C2 cells characterization studies. Crystal violet staining (a, b, c; 32X), immunofluorescence staining (d, e, f; 32X), MTT results (g), cell growth curve (h) of H9C2 cells (scale bars: 50 µm). (Color figure online)

Fig. 2

The morphology of cell sheets obtained by thermo-responsive surface and high cell density/high serum content methods. a, e Before detachment (20X); b, f after detachment (20X); c g 3 days and 1 day after re-adhesion (20X); d, h 3 days and 1 day after re-adhesion (4X) (scale bars: 20X 100 µm; 4X 500 µm). Black arrows show sprouting of cells from cell sheets

Fig. 3

Morphologies of AA-treated cell sheets with high serum content (20X, scale bars: 100 µm). Black arrows show sprouting of cells from cell sheets

Fig. 4

Morphologies of AA-treated cell sheets with normal serum content (20X, scale bars: 100 µm). Black arrows show sprouting of cells from cell sheets

Fig. 5

Fluorescence staining of cell sheets obtained by thermo-responsive surface (a, b), high cell density/high serum content (c) and AA treatment (high serum content: d, e, f, g, h, i; normal serum content: j, k, l, m, n, o). Actin filaments and nuclei are green and bright blue coloured, respectively (20X, scale bars: 100 µm). (Color figure online)

Fig. 6

Live/dead staining of cell sheets obtained by thermo-responsive surface (a, b), high cell density/high serum content (c) and AA treatment (high serum content: d, e, f, g, h, i; normal serum content: j, k, l, m, n, o). Live and dead cells are green and red coloured, respectively (10X, scale bars: 200 µm). (Color figure online)

Fig. 7

RT-PCR analyses for Col1a1, Tnnt2, Cacna1c, Cacna1s and Slc29a1 genes. Comparison of thermo-responsive and TCPS surface (a), high and low cell seeding density at TCPS (b) and AA treatment groups in two different FBS content (c). TCPS surface and low cell seeding density groups are the same. Statistically significant differences are denoted by symbols; a, b n = 4; *p < 0.05, **p < 0.01 and ***p < 0.001; c n = 5; N-FBS and H-FBS groups were compared according to control among themselves *p < 0.05, **p < 0.01 and ***p < 0.001, 20 µg/mL AA served as control ^#p < 0.05; N-FBS groups served as control versus H-FBS groups ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001