Morphological Observation Of Bone Marrow Mesenchymal Stem Cells Under The Condition Of Three-dimensional Culture

Objective:Matrigel contains collagen,laminin,nestin and heparin sulfate proteoglycan,which can promote similar three-dimensional growth condition in vivo by establishing a three-dimensional cell culture environment.For the purpose of exploratory research and discovery,bone marrow mesenchymal stem cells were coated in matrigel.Phase contrast microscope was used to select the time points of obvious cell morphological changes and proliferation.In order to research the survival,morphology and proliferation of bone marrow mesenchymal stem cells under three-dimensional culture condition,HE staining was used to observe cell morphological changes,while live / dead cell staining was used to analyze cell viability,Methods:After resuscitation,the purchased bone marrow mesenchymal stem cells were cultured and passaged normally.The 3rd and 4th generation cells with good morphology were mixed with the melted matrigel suspension and placed in a 24-well plate.When it is solidified into matrigel in a incubator,add a small amount of complete medium to culture.The growth and proliferation of cells on the 1st,3rd,7th and 14 th day were observed under phase contrast microscope,and the frozen sections of the cells in matrigel on the1 st,3rd and 7th day were selected to observe the cell morphology by HE staining experiment.Then the cells at the same time node were stained with live / dead cell kit,and the cell activity in the matrigel culture environment was determined by fluorescence microscope.Using Adobe Photoshop CS4 software to cut the phase contrast microscope observation picture,NIS-Elements Viewer 4.50 trimming,cutting live / dead cell staining pictures,using Graph Pad Prism5 to draw the column chart of cell activity changing with time,collating the experimental results,and finally making statistical analysis,using inter-group Fisher test to compare and statistics the cell survival state at different time points.When the p value is less than 0.05,it shows that the difference of cell activity has statistical difference with the extension of time.When the p value was less than 0.01,it showed that the difference of cell activity had significant statistical difference with time.Results:On the first day of culture under phase contrast microscope,most of the cells were short fusiform in matrigel;on the third day of culture,the number of cells became more,and the elongation shape of cells was long fusiform;on the 7th day of culture,the cells continued to elongate and interweave into a network;on the 14 th day of culture,the matrigel softened and collapsed,and the number of cells was less than that on the 7th day.The results of HE staining showed that the cells and cytoplasm were gradually lengthened and intertwined into a network with the extension of culture time on the 1st,3rd and 7th day.The results of live / dead cell staining experiments showed that the longer the culture time was,the higher the proportion of green fluorescence(living cells)was,which was 90.33%,93.33% and 96.61%,respectively,indicated that the matrigel had good biocompatibility.Conclusion:Bone marrow mesenchymal stem cells wrapped in matrigel can continue to grow and proliferate.They can elongate,cross and wind into a network within a certain period of time,and the matrigel combines well with the cells.The cell survival rate is high,but with the extension of culture time,the matrigel will gradually collapse,the cells will no longer increase.