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BACKGROUND:Cold trypsin digestion is rarely reported to culture umbilical cord mesenchymal stem cells. OBJECTIVE:To compare the biological characteristics of umbilical cord mesenchymal stem cells cultured by cold trypsin digestion and tissue explant method. METHODS:Human umbilical cord mesenchymal stem cells were isolated, purified and passaged using cold trypsin digestion and tissue explant method, and then the first adhesion time and cellcycle were recorded. Morphology of umbilical cord mesenchymal stem cells was observed under inverted phase contrast microscope to draw growth curve of cells at passage 3. Flow cytometry was used to detect the surface markers of passage 3 umbilical cord mesenchymal stem cells, and Nestin expression was detected in passage 3 cells after 3 days culture in neural induction medium by fluorescence immunochemistry staining. RESULTS AND CONCLUSION:These two methods were both successful to harvest umbilical cord mesenchymal stem cells, but the first adhesion time was earlier in cells cultured by cold trypsin digestion than tissue explant method (P0.05). Under the inverted microscope, cells grew adherently and presented fibroblast-like shape. However, the minority of primary cells under induction of cold trypsin digestion was polygonal, irregular, and had larger cellvolume than those cultured by tissue explant method. Passage 3 cells cultured by tissue explant method showed faster proliferation rate than those cultured by cold trypsin digestion, and at logarithmic growth phase, cells cultured by these two methods were significant different in cellnumber (P<0.05). Two types of cells had a uniform cellphenotype, both of which expressed CD29, CD105, but did not express CD34, CD45. Under induction, passage 3 cells cultured by these two methods were both positive for nestin. These findings indicate that these two methods can both be used to culture umbilical cord mesenchymal stem cells, but the tissue explant method is more suitable for culture of umbilical cord mesenchymal stem cells and exhibits less damage to cells.
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BACKGROUND:After co-culture with osteoblasts, bone marrow stem cells can be induced to differentiate into osteoblasts. Whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts or not? OBJECTIVE:To observe whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts. METHODS:Adipose-derived stem cells and osteoblasts were isolated from New Zealand white rabbits. Then, passage 3 adipose-derived stem cells were co-cultured with passage 2 osteoblasts in 10%or 5%fetal bovine serum for 14 days. RESULTS AND CONCLUSION:After 7 days of co-culture, some adipose-derived stem cells became round in the two groups. After 14 days of co-culture, adipose-derived stem cells highly differentiated and differentiated cells were similar to mature osteoblasts that were positive for alkaline phosphatase staining and alizarin red staining. The mRNA expression of type I col agen and osteocalcin increased in both two group, especial y in the 10%fetal bovine serum group. These findings indicate that adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts induced by high-concentration serum culture.
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BACKGROUND:The umbilical cord mesenchymal stem cels can be obtained conveniently, noninvasively, without ethical restrictions, which are more original that stem cels from other sources and have little immunogenicity. Therefore, umbilical cord mesenchymal stem cels are a kind of ideal seed cels with promising application prospects. OBJECTIVE: To induce the differentiation of mesenchymal stem cels derived from human umbilical cord into adipocytes and osteoblasts. METHODS: The umbilical cord mesenchymal stem cels were isolated by tissue explants method. The morphology, proliferation and immunophenotype of the 3rd passage cels were analyzed, and then cels were induced to adipocytes and osteoblastsin vitro. The expressions of CD90, CD105, CD34 and CD15 were detected by flow cytometry. RESULTS AND CONCLUSION:The umbilical cord mesenchymal stem cels isolated by tissue explants method could be cultured and proliferatedin vitro. Flow cytometry analysis revealed that the cels were strongly positive for CD90 and CD105, but negative for CD34 and CD45. The umbilical cord mesenchymal stem cels could be induced to adipocytes and osteoblastsin vitro. These findings indicate that mesenchymal stem cels can be successfuly obtained from human umbilical cord by tissue explants method and have the potential of multi-directional differentiation.
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BACKGROUND:Human umbilical cord mesenchymal stem cells with capabilities for self-renewal and multi-differentiation have attracted widespread attention. OBJECTIVE:To develop an efficient method for isolation and culture of human umbilical cord mesenchymal stem cells, and to analyze the cellbiological features. METHODS:Mesenchymal stem cells were isolated and cultured from human umbilical cord by improved tissue cultivation. Immunophenotype and cellcycle were analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated by in vitro osteogenic and adipogenic induction as wel . RESULTS AND CONCLUSION:Some fusiform cells crawled out from human umbilical cord tissues after cultivation for 5 days and formed colonies about 10 days later. When the removed tissues were further cultured, more cells appeared again within 2 days and formed colonies after 5 days. The isolated cells exhibited similar morphology of fibroblast-like shape after passage. Furthermore, the cells expressed CD90, CD105, but were negative for the markers of CD34, CD45, HLA-DR. Population doubling time of the cells calculated from the result of MTT was about 50 hours and cellcycle analysis showed that 41.24%cells were in the G 2/S phrase. Therefore, the isolated cells had a high prolification ability. In addition, the isolated cells could be induced into osteoblasts and adipocytes in vitro. In a word, the results of this study demonstrated that the cells from the second tissues culture possessed the biological characteristics of mesenchymal stem cells and more primary umbilical cord mesenchymal stem cells were acquired through the improved method.
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BACKGROUND:Stem cells expansion technology in vitro is affected by the microenvironment of the culture system. So, to find an effective method is particularly important to promote celladherent and growth. OBJECTIVE:To compare the effects of different culture media on cellexpansion. METHODS:Human umbilical cord mesenchymal stem cells at a density of 5.0×104 cells/cm2 were inoculated into Corning? polystyrene culture dishes coated with or without poly-L-ornithine and Corning? cellBIND culture dishes. celladhesion and proliferation were observed, and expressions of celladhesion proteins and cellmarkers were detected. RESULTS AND CONCLUSION:celladhesion was promoted when cells were cultured in Corning? cellBIND? Surface medium coated with poly-L-ornithine for 24 hours, and the cultured cells grew at the logarithmic phase. The cellproliferation was also enhanced, and the cells expressed celladhesion protein but not the cellmarkers of CD73, CD90, CD105. Corning? cellBIND? Surface medium was superior to Corning? polystyrene culture medium in the improvement of celladhesion and proliferation. Additional y, both of these two media showed no influence on cellphenotype. These findings indicate that Corning? cellBIND? Surface medium can promote celladhesion and proliferation, but shows no effects on cyclin and cellphenotype. This medium coated with poly-L-ornithine can further accelerate celladhesion and proliferation, and stably express cellphenotype of human umbilical cord mesenchymal stem cells.
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BACKGROUND:Many types of mammalian cells aggregate and display three-dimensional multicellular spheroids when they are in normal physiological conditions. In order to observe and explore cellular natural states, many researchers try to use spherical cellculture in vitro, a common three-dimensional culture pattern. OBJECTIVE:To use three different methods for spherical culture in vitro of adipose-derived stem cells and to observe their biological features. METHODS:Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes as wel as adipogenic and osteogenic differentiation potential assays. Three different methods of sphere cultures were used as fol ows:(1) ultra low attachment culture;(2) hanging-drop culture and (3) Eppendorf tube culture. The sphere formation was compared among above three methods. We used Imagej to calculate mean areas of these spheres. And we used Viability/Cytotoxicity Assay Kit for Animal Live&Dead cells to detect their vitality. RESULTS AND CONCLUSION:(1) Adipose-derived stem cells were confirmed by the analysis of the markers for cellphenotypes, CD29, CD44, CD59 were positive, as wel as adipogenic and osteogenic differentiation potential assays were positive. The conventional monolayer cultures of adipose-derived stem cells showed spindle and cloning growth within three passages. (2) Ultra low attachment culture, hanging-drop culture, Eppendorf tube culture al could elicit adipose-derived stem cells spherical growth. However, spherical size, shape and uniformity differed depending on cellnumbers, culture time and spherical culture methods. The ultra low attachment culture was comparatively difficult to control spherical shape and uniformity of adipose-derived stem cells. But hanging-drop culture and Eppendorf tube culture were able to form even cellspheres. (3) Spherical formation of adipose-derived stem cells using our three methods displayed good cellvitality.
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BACKGROUND:Under mitomycin C treatment, feeder cells appear to have restricted proliferation, but they are stil able to secret different cytokines. Non-mesenchymal stem cells from the bone marrow and secreted factors in plasma maintain the micro-environment suitable for the growth of mesenchymal stem cells that can improve the yield of mesenchymal stem cells. OBJECTIVE:To study the biological characteristics of C57 mouse bone marrow mesenchymal stem cells isolated using a whole bone marrow adherent culture technique. METHODS:Using the whole bone marrow adherent culture technique, purified and amplified C57 mouse bone marrow mesenchymal stem cells were harvested. cellproliferation kinetics, immune cellsurface markers, multiple differentiation potential and cellcycle were detected. RESULTS AND CONCLUSION:Using the whole bone marrow culture, mouse bone marrow mesenchymal stem cells were harvested and capable of adhering to the plastic culture vessel. The obtained cells expressed CD45, CD105 and Sca-1, but were negative for CD34, CD33 and C-kit. The doubling time was (57.11±1.5) hours. The cells could be induced to differentiate into osteoblasts, adipocytes and chondrocytes. The cellcycle analysis showed that 64%of cells were in G 0-G 1 phase. These indicates that C57 mouse bone marrow mesenchymal stem cells isolated using a whole bone marrow adherent culture technique have biological characteristics of mesenchymal stem cells.
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BACKGROUND:The tumor tissue engineering can build an integrated culture model to ful y simulate the in vivo microenvironment of tumor growth, which can be used to study tumor developmental dynamics and related treatment strategies. OBJECTIVE:To review the three-dimensional culture of tumor cells using tumor engineering technology. METHODS:PubMed database was retrieved for articles related to tumor engineering, three-dimensional culture of tumor cells, biological scaffold materials and tumor microenvironment published from January 1992 to March 2013. RESULTS AND CONCLUSION:Three-dimensional culture, because of its reproducible tissue and cellgrowth in vivo, has become an important platform for study of tumor resistance, invasiveness and tumor microenvironment. The three-dimensional culture has showed a trend to gradual y replace the flat culture technique in many fields, and provides a research platform which is very close to in vivo environment. In recent years, with the development of tumor engineering, a variety of new polymer materials have been used in the three-dimensional culture of tumor cells. Three-dimensional culture technology is becoming a hotspot in the field of tumor biology, in which, using a variety of methods and materials, the cells show a growth in the spatial manner to form a biological support or matrix similar to in vivo growth environment. Biomaterials have become the soil on which seed cells can grow wel , and plays an alternative to the extracellular matrix or the matrix of tissues and organs in the tumor engineering. Therefore, the three-dimensional cellculture has been widely used in cancer research, which has become a powerful tool to tumor drug resistance, angiogenesis, cel-cellinteraction, signal transduction, stem cells and other research.