ABSTRACT
BACKGROUND: Increasing studies have shown that cell-sheet engineering is a more promising method for the treatment of myocardial infarction. OBJECTIVE: To construct sandwich-like myocardial cell sheets with electrospun chitosan-collagen membrane as support membrane and with adipose-derived stem cells as seed cells. METHODS: In this study, we prepared electrospun chitosan-collagen membranes at different mass ratio (7:3, 5:5) as a support membrane. Adipose-derived stem cells at passage 4 that were cultured in a thermo-sensitive petri dish for 3 days were seeded onto the two kinds of electrospun membranes at 20 ℃ for 15 minutes until the cell sheets rolled up. Then, the electrospun membranes and cell sheets with the cell layers facing up were placed together onto another thermo-sensitive petri dish in which adipose-derived stem cells were confluent completely. After 30 minutes of culture, the cell sheets were cultured in myocardial cell medium. Two weeks later, cell morphology was observed using multi-photon microscopy. Immunocytochemistry, western blot and flow cytometry were used to detect the expression of TnI and Cx43 in the myocardium. Real-time quantitative PCR was used to detect cardiomyocyte-specific gene expression. RESULTS AND CONCLUSION: From the results of scanning electronic microscopy (SEM) and propidium iodide (PI) staining, we could found that ADSCs grew easily on the chitosan-collagen (5:5) membrane, and there were more dead cells on the chitosan-collagen (7:3) membrane than on the chitosan-collagen (5:5) membrane. After 2 weeks of differentiation with the myocardial cell medium, higher troponin I and Cx43 protein expressions were observed on the chitosan-collagen (5:5) membrane (P＜0.05). Moreover, the mRNA levels of α-skA, β-MHC, TnI, Cx43, ANP, GATA-4 and Nkx2.5 were higher in the chitosan-collagen (5:5) membrane group than the chitosan-collagen (7:3) membrane group. All these results indicate that the chitosan-collagen (5:5) membrane is better for the growth and differentiation of adipose-derived stem cells, as well as for sandwich-like cell-sheet construction.