Study On The Promotion And Characterization Of Cardiomyocyte Maturation In Vitro

The traditional in vitro culture technique of cardiomyocytes is difficult to mature the stem cell-derived cardiomyocytes,and the obtained cardiomyocytes are similar in gene expression,electrophysiology and morphology to human fetal heart cells.Compared with adult cardiomyocytes,cardiomyocytes cultured in vitro have smaller volume,decreased electrical excitability,impaired excitation-contraction coupling,and incomplete expression of adrenergic receptors.If immature cardiomyocytes are used to construct a series of pharmacological models in myocardial tissue engineering,the results do not reflect the true state of the mature cardiomyocytes for the drug.Therefore,the promotion of in vitro cardiomyocyte maturation and its characterization methods are important links in myocardial tissue engineering.The current determination of cardiomyocyte maturity is based primarily on the qualitative characterization of cell morphology.Combining digital image processing technology,this thesis designs and develops a set of methods and software for evaluating the maturity of cardiomyocytes,which realizes the size of cell morphology,the expression density of myocardial marker proteins,the molecular structure of proteins,and the transport rate of calcium ions.In view of the excellent conductivity and the non-toxicity of Pt,a set of Pt-PDMS column electrode platform was constructed using platinum nanoparticles as a conductive carrier.The platform can be used not only to apply electrical stimulation,but also to monitor electrical signals and pharmacological effects of myocardial tissue.The short-term electrical stimulation was applied to the cardiomyocytes cultured in vitro by a constant current pulse wave.After six days of electrical stimulation for 30 minutes,the cardiomyocytes of the electro-stimulation group were better than the control group in terms of cell morphology,protein expression and electrophysiological function.Experiments have shown that electrical stimulation can promote cardiomyocyte maturation in vitro.On the other hand,this thesis combines the microfluidic chip technology to explore a reasonable modification method for the growth of cardiomyocytes on PDMS.The self-designed loop stretching device can provide stable and controllable force stimulation for the cardiomyocytes cultured in the microfluidic chip.A long-term force stimulation wasapplied to the cardiomyocytes cultured for 2 days with a deformation pull of 3%,and the cell morphology was significantly improved after 3 days of tensile stimulation.A preliminary discussion on the effects of mechanical forces on cardiomyocytes has opened the way for the design and fabrication of electrical-force chips.