| Objectives1.To master the techniques of cell culture about muscle-derived stem cells(MDSC)in suspension culture,and establish a model of MDSC suspension culture in order to lay the foundation for the different local density suspension culture.2.On the basis of suspension culture model of muscle-derived stem cells(MDSC),the effects of clustering and dispersion culture(partial density difference)on the proliferation activity of MDSCs were initially investigated to screen the culture methods that are more conductive to MDSCs' proliferation.3.Based on the suspension clustering culture method,the in vivo MDSC self-renewal and proliferation environment was simulated.Explore the effects of fibroblasts and satellite cells as feeder cells on the peoliferation of the MDSCs.Initially explore the mechanism of suspension co-culture on the proliferation of the MDSCs.4.Compare the established suspension co-culture with satellite cells model with the pure suspension culture method and the most commonly used traditional pre-plating culture method through observing the differences in the amount of MDSCs and the effect on MDSCs in different culture methods.To verify the advantages of suspension co-culture model.Methods1.The muscle-derived stem cells were isolated through mechanical method and multi-enzyme digestion from skeletal muscles of 2wk old C57BL/6 mice.The cells were expanded by a non-adherent proliferation culture system.Thoughout MDSC expansion,cell number and CD34,Sca-1 expression were assessed by a hemocytometer imaged with bright field and flow cytometry.2.The muscle-derived stem cells were isolated through mechanical method and multi-step enzymes digestion from skeletal muscles of 2wk old C57BL/6 mice.Cell suspension was prepared for non-adherent culture system and were classed as suspension clusters and dispersed cells.Throughout MDSC expansion,cell number and CD34,Sca-1 expression were assessed by a hemocytometer imaged with bright field and flow cytometry.3.The muscle-derived stem cells were isolated through mechanical method and multi-step enzymes digestion from skeletal muscles of 2wk old C57BL/6 mice.They were divided into ABCD groups.AB groups co-cultured with fibroblasts.CD groups co-cultured with satellite cells.Groups A and C were in the condition medium dishes.Groups C and D were in the direct-contact dishes.Each group has its control.Throughout MDSC expansion in 42 days,cell number,differentiation potential and CD34,Sca-1 expression were assessed by a hemocytometer imaged with bright field,differentiation experiments and flow cytometry.4.The muscle-derived stem cells were isolated through mechanical method and multi-step enzymes digestion from skeletal muscles of 2wk old C57BL/6 mice.The cells were divided into 3 groups.Group E co-cultured with satellite cells.Group F applied pure suspension culture.Group G adopted traditional pre-plating technique.Throughout MDSC expansion in 21 days,cell number,differentiation potential and CD34,Sca-1 expression were assessed by a hemocytometer imaged with bright field,differentiation experiments and flow cytometry.Results1.Single cell suspension were cultivated by a non-adherent proliferation culture,namely suspension culture.Cultured cells had potential of self-proliferation and could form small aggregates.CD34 and Sca-1 expression increased along with the proliferation time.Cell-spheres could differentiate into bone cells and Schwann cells which tested through p75 NGF receptor immunofluorescence and Alizarin red S.2.In the cluster culture group,the MDSCs were cluster-like,with smooth cell surface,small sphere,and high refraction.The cells in the dispersion culture group showed single or small clusters,smooth cell surface,small sphere,high refraction,and occasional cell wrinking.The proliferation of MDSCs in the cluter group was more than the other.CD34 expression in the cluster group was 45.77%,Sca-1 was 27.98%,both positive was 18.90%and Pax7 was 23.4%.CD34 expression in the dispersion group was 10.17%,Sca-1 was 19.15%,both positive was 5.52%and Pax7 was 8.88%.The markers were higher in the cluster group.Cell-spheres in both groups could differentiate into bone cells and Schwann cells which tested through p75 NGF receptor immunofluorescence and Alizarin red S.3.The amount of cells in groups A,C and D increased with the prolongation of culture time.After initial increase in group B,the amount of cells decreased rapidly,and the amount of cells in group D increased the most.The experimental group had more proliferation than the control group(P<0.05).Cells were cluster-like5 spherical,and had high refractive index.In group A and B,MDSC was easily adherent and differentiated into muscle fibers.In group C and D,a small number of cells adhered to the dish and no obvious cell fusion or differentiation was observed.Flow cytometry showed that the expression levels of CD34,Sca-1,and Pax7 in cultured cells in each group were slightly different,yet the difference was not statistically significant.Cell-spheres in all groups could differentiate into bone cells and Schwann cells which tested through p75 NGF receptorimmunofluorescence and Alizarin red S.4.The amount of cells in the three groups increased with the prolongation of the culture time.The proliferation in the E group was the largest and the difference was statistically significant.The F group was higher than the G group in the 1st week,and then was lower than the other two groups.Cells are cluster-like,spherical,and have a high refractive index.No obvious cell fusion and differentiation were observed.Flow cytometry showed that the expression of CD34,Sca-1 and Pax7 on the MDSCs in each group was slightly different,but the difference was not statistically significant.Cell-spheres in all groups could differentiate into bone cells and Schwann cells which tested through p75 NGF receptor immunofluorescence and Alizarin red S.Conclusions1.The MDSCs cultured by suspension culture system have potential of self-renewal,express stem factor markers(CD34,Sca-1)and have differentiation potential to the mesoderm and ectoderm.2.The MDSC cultured with clusters have stronger potential of self-renewal and multi-differentiation ability than dispersed cells.The clusters can express stem factor markers(CD34,Sca-1)and facilitate the differentiation of MDSC to hierarchically downstream satellite cells.MDSCs in both group can differentiate into the mesoderm and ectoderm.3.The amount of MDSCs obtained by co-culture with satellite cells is much more than the other.MDSCs co-cultured with fibroblasts tend to cause terminal differentiation or lysis.MDSCs in all groups can express stem factor markers(CD34,Sca-1)and differentiate into the mesoderm.Prolonged culture time can facilitate the differentiation of MDSC to hierarchically downstream satellite cells.4.Compared with traditional pre-plating technique and pure suspension culture,more MDSCs were obtained through suspended co-culturing with satellite cells.MDSCs in all groups can express stem factor markers(CD34,Sca-1)and differentiate into the mesoderm and ectoderm.The differentiation of MDSC to hierarchically downstream satellite cells is related to the prolongation of culture time and has little to do with culture methods. |
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