The Construction Of Vascularized Tissue Engineering Cardic In Vitro

Obiectives The separation and cultivation of SD rat bone marrow mesenchymal stemcells and bone marrow-derived endothelial progenitor cells, induced into cardiomyocytecells and endothelial cells. Two kinds of cells in proportions of2:1with liquid Matrigelmatrix glue mixed in a5%CO2incubator at37℃for in vitro co-culture, to explore thefeasibility of constructing of vascularized tissue engineering cardic in vitro.Methods1Separated and cultured bone marrow mesenchymal stem cells by the wholebone marrow culture, and5-azacytidine induce into cardiomyocyte cells. Before and afterof induction, identified the cells by the expression of cells surface marker CD29, CD44,CD34; and cTnT by immunocytochemistry.2Separation3-weeks SD rats bilateral femur,tibia bone marrow, the equivalent lymphocytes layered liquid separation, collection ofmononuclear cells, bone marrow-derived endothelial progenitor cells were cultured in theEGM-2MV conditions medium. Observe cellular morphology which are after inducedcultured, and identified the cells by the expression of cell surface marker CD31, CD34,and CD133by immunocytochemistry.3Bone marrow-derived CMs and bone marrow-derived endothelial progenitor cells in proportions of2:1,1:0with liquid Matrigel matrixglue mixed in a5%CO2incubator at37℃for in vitro co-culture. Respectively indifferent time inverted phase contrast microscope to observe the morphology,immunofluorescence microscopy observation and hematoxylin-eosin staining method toobserve the distribution of two kinds of cells, and then take cardiomyocyte apoptosisdetection. All data was analyzed by the SPSS19.0. The measurement data weredemonstrated by (mean±SD). T tests was used in two measurement data. A value ofP<0.005was considered as statistical significant.Results1The cell morphology of primary cultured cells were showed in fusiform, orirregular shape, eventually were fusiform, arranged in a direction. After passage, BMSCsgrowth evenly distributed, more single morphology, and growth speeder. Theimmunohistochemical showed, the positive expression of CD44and CD29, the negativeexpression of CD34.2The growth of BMSCs: the subcultured after2days, due to adaptto the new culture environment and trypsin digestion, cell volume decreased slightly,3days later a proliferation of cells,6days for cells in logarithmic growth phase,9days intothe platform. Cell growth is relatively stable.3Some cells droped from the culture flaskafter24h induced. One week latee, a few cells became larger and fusiform. Two weekslater, some cells appeared to be short columnar and multi—angular while the cellscontacted closely. Four weeks later, cells were fusiform, arranged in a direction. After4weeks induce, cTnT is showed positive expression, and the masculine cell rate was above 90%.4Most of EPCs were showed in fusiform, spindle-shaped or irregular shape. About5th days, the colony of EPCs appeared. At14th days, the cells showed a typical"cobblestone" structure, which was the typical morphological characteristics of EPCs.The immunohistochemical showed that the surface markers factor CD31, CD34andCD133were all in positive expression, and the masculine cell rate was above90%.5Thegrowth of EC: the subcultured after1days, due to adapt to the new culture environmentand trypsin digestion, cell volume decreased slightly,3days later a proliferation of cells,5days for cells in logarithmic growth phase,8days into the platform. Cell growth isrelatively stable.6The immunofluorescence and hematoxylin-eosin staining resultsshowed that the cell density in the2:1mixed group was relatively uniform. Theimmunofluorescence display the nucleus of myocardial cell is rounded blue fluorescence,endothelial cell is red.7Inverted phase contrast microscope observation shows that co-culture different proportions of cells mixed with Matrigel, the2:1mixed group appearedtypical “vascular phenomenon” of endothelial cells24hafter cultured, while the “vascularphenomenon”.8Cell apoptosis detection were showed that the quantity of died CMs inco-cultured group more than myocardial cell culture group.9It is demonstrated that thevascular-like structure are formed increasingly in the engineered myocardium blendedwith vascular endothelial cell.Conclusions1Through the induce of5-azacytidine, pure cardiomyocyte cells, can besuccessfully obtained.2Through the induction culture of conditioned medium, goodactivity and pure bone marrow-derived endothelial progenitor cells can be successfullyobtained.3Co-culture of the bone marrow-derived cardiomyocyte cells, and bonemarrow-derived Endothelial cells with2:1can successfully build vascularized tissueengineering cardic in vitro.