| Objective:To investigate the biocompatibility of porcine omental derived extracellular matrix hydrogel(PO-ECM)on human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)and its feasibility as an injectable hydrogel for cardiac tissue engineering,so as to provide a cellular research basis for subsequent in vivo experiments.Methods:The PO-ECM was prepared by a series of acellular processes,and the temperature response of the hydrogel was observed in vitro.The residual cellular components and the retention of extracellular matrix components of the hydrogel was detected by hematoxylin-eosin(HE),sirius scarlet,alcian blue and other histological staining methods.The microstructure of the hydrogel was observed by scanning electron microscopy.The hydrogel was injected into the myocardium of mice under the guidance of ultrasound,and the ability of hydrogel in situ gelation in vivo was detected by subsequent HE staining of myocardium tissue.hiPSCs were directed to cardiac differentiation using a small molecule induction method,followed by purification.Then,the cells were identified by immunofluorescence staining of cardiomyocyte markers.The differentiated hiPSC-CMs were then co-cultured with hydrogel.The cell viability and survival status of cardiomyocytes were detected by CCK-8 assay and living/dead staining.The cytoskeletal and growth morphology of cardiomyocytes were observed by phalloidin staining.The beats of cardiomyocytes were observed under light microscope.Finally,the survival and phenotype maintenance of cardiomyocytes were analyzed by immunofluorescence staining and Western blotting based detection of their respective biomarker.Results:The prepared PO-ECM was temperature responsive,being liquid at 4℃ and gel at37℃.The results of histological staining indicated that there was no significant cellular residue in the hydrogel,while collagen and glycosaminoglycan,the main components of extracellular matrix,were retained.The results of scanning electron microscopy showed that the micromorphology of hydrogel was composed of interlaced fibers and irregular pores.The results of injection of hydrogel and subsequent HE staining of myocardial tissue in mice suggested that hydrogel could form in situ gelled in vivo.In this study,hiPSCs were successfully differentiated into beating cardiomyocytes,and this result was confirmed by immunofluorescence staining of cardiomyocyte markers including c Tn T and α-actinin.CCK-8 assay and live/dead staining indicated that hiPSC-CMs had good viability when co-cultured with hydrogel,and most cells survived.The results of phalloidin staining showed that the morphology of cardiomyocytes in the hydrogel group was similar to that in the control group,and the expression area of F-actin in cardiomyocytes of the hydrogel group was not significantly influenced by hydrogel.The results of immunofluorescence staining and Western blotting of cardiomyocyte markers showed that the hydrogel supported the survival of cardiomyocytes and phenotype maintenance,which was beneficial to synchronize electromechanical activities of cardiomyocyte clusters.Moreover,there was no significant difference in the beating frequency of cardiomyocyte clusters between the two groups under light microscope.Conclusion:In this study,we successfully prepared the temperature-responsive PO-ECM,which undergone decellularization,retained the main biochemical components of the natural extracellular matrix,and had a complex fibrous structure.In addition,the hydrogel has the characteristics of minimally invasive injection feasibility and in vivo gelation,and has the advantages of good biocompatibility and negligible cytotoxicity.Besides,it can be used as an effective carrier and supporting material to support the survival,contractile function and phenotype maintenance of hiPSC-CMs.It is expected that the hydrogel is a promising injectable cell scaffold for the future cardiac repair. |
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