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BACKGROUND:Fetal bovine serum as nutritional support is often used in the traditional cel culture. Consequently, a host of potential problems such as the spread of disease and immunological reactions exist. To find a suitable fetal bovine serum substitute and to establish a culture system of human bone marrow stromal stem cels in vitro which has been standardized, safe and efficient has just started. OBJECTIVE:To investigate the effects of different serums on proliferation of bone marrow stromal stem celsin vitro. METHODS:Bone marrow stromal stem cels were obtained from adult bone marrow, which were cultured in DMEM containing 10% AB serum, 10% autologous serum, or 10% fetal bovine serum. Cels at passage 3 were used in this study. RESULTS AND CONCLUSION:The cel confluence in the AB serum group was earlier than that in the fetal bovine serum group and autologous serum group. Human bone marrow stromal stem cels maintained the phenotypes of bone marrow stem cels in three serums detected by flow cytometry. AB serum group showed the highest fluorescence intensity and the most efficiency of cel proliferation which examined by the AlamarBlue assay. Apoptosis rate was < 5% in al the three groups, and cels grew wel in these serums. Alkaline phosphatase, calcium nodules and oil red O staining showed that the cels maintained the osteogenesis and adipogenesis capacity in the three groups. AB serum was found to have a better effect on proliferation capability of cels than fetal bovine serum and autologous serum. Taken together, AB serum is expected to be a substitute of fetal bovine serum to build anin vitro culture system of adult bone marrow stromal stem cels that accord with the clinical requirements of bone tissue engineering.
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BACKGROUND:Poultry mesenchymal stem cels are a particular subset of pluripotent adult stem cels derived from the mesoderm, which have great application prospects because of their strong proliferation and multi-directional differentiation potential. OBJECTIVE:To review the source, separation, purification, culture and differentiation of poultry mesenchymal stem cels, and to provide the theoretical foundation and experimental basis for the further research and application of poultry mesenchymal stem cels. METHODS:PubMed and CNKI databases were searched by the first author using key words of “mesenchymal stem cels, poultry, chicken, isolation, culture, differentiation” in English and in Chinese, respectively, to retrieve relevant articles published from 1990 to 2014. Literatures addressing induced poultry mesenchymal stem cels were included, and 42 articles were chosen for further analysis eventualy. RESULTS AND CONCLUSION: Poultry mesenchymal stem cels have great application prospects in the aspects of establishingin vitro model of poultry cels, studying poultry disease pathogenesis, animal nutrition and meat quality control. Its origin source is wide and easy to obtain. Isolation, purification, culture and biological characteristics of mesenchymal stem cels from different tissues are different. But, the study on poultry mesenchymal stem cels is stil in the exploration process, and there are many technical problems to be solved.
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BACKGROUND:At present, a lot of research about culture methods for umbilical cord mesenchymal stem cels, but not for the waste of primary system. OBJECTIVE:To explore the best culture method of human umbilical cord mesenchymal stem celsin vitro. METHODS:Human umbilical cord mesenchymal stem cels were prepared by tissue explants method, recorded as initial culture group. The centrifugal fluid and tissue of the primary culture flask were centrifuged and divided into three groups for secondary culture: tissue group, mixed group and pure liquid group. Cel morphology, time for cel acquisition, and yield of primary cels in the four groups were observed; the cel growth curve was analyzed by MTT assay; and cel cycle and phenotype were detected by flow cytometry. RESULTS AND CONCLUSION: The average time for cel acquisition in the initial culture group, tissue group, mixed group and pure liquid group were (15.00±0.45), (7.0±0.3), (8.00±0.25) and (8.00±0.25) days, respectively. The number of cels at first generation was (4.0±0.5)×105, (9.0±0.55)×105, (15.0±0.2)×105 and (7.0±0.33)×105 markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cels can be obtained rapidly and largely through the secondary culture to the primary culture system. T75 culture bottle, respectively. Under the inverted microscope, cels in the four groups were fusiform-like adherent cels, which were in paralel or circinate arrangement. Growth curve, proliferative activity, surface markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cells can be obtained rapidly and largely through the secondary culture to the primary culture system.
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Objective To investigate the effects of resveratrol on the first and double oxygen-glucose deprivation (OGD) primary cortical neuron silent information regulator 1 (SIRT1), AMP-activated protein kinase (AMPK) activity and ATP content, and its possible neuroprotective mechanism. Methods Cortical neurons were taken from the embryos of 18-day Wistar rats. An in vitro repeated ischemia model was induced by the double OGD after the success of primary culture. Trypan blue stalning was used to detect the cel survival rate. Western blot was used to detect the SIRT1 and phospho-AMPK expression. Deacetylase fluorescence assay was used to detect the SIRT1 activity. Bioluminescence assay was used to detect the ATP content. Results Compared with the control group, resveratrol (0. 5 μmol/L) preconditioning significantly increased the survival rates after the single and double OGD (al P < 0. 001), ATP content (al P = 0. 004), SIRT1 activity (single: P = 0. 001; double: P = 0. 002), and the expression levels of SIRT1 (single: P = 0. 029; double: P = 0. 023) and phospho-AMPK (al P = 0. 001). Conclusions Resveratrol has the neuroprotective effect for the first and double OGD cortical neurons. Its mechanism may be associated with upregulating the SIRT1/AMPK signaling pathways and decreasing the energy requirements.
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BACKGROUND:Previous studies have mainly focused on costal cartilage, articular cartilage, nasal septal cartilage, and auricular cartilage, but in vitro culture of human vertebral endplate cartilage is stil rarely reported. OBJECTIVE: To discuss the feasibility of culture of vertebral endplate chondrocytes from type I neurofibromatosis associated with scoliosis patientsin vitro and to study the biological characters of the chondrocytes. METHODS: Through two-step enzymatic digestion and tissue culture, the chondrocytes from the vertebral endplate of seven type I neurofibromatosis patients isolated and cultured in monolayer and passaged to observe the changes of cel morphology under inverted phase contrast microscope. Colagen type II expression was detected by immunocytochemistry to identify whether the cels had chondrocyte characters. The growth kinetics was detected by using MTT colorimetric assay to draw the growth curve of passage 2 chondrocytes. RESULTS AND CONCLUSION:A few chondrocytes crawled from the cartilage after 2 weeks culture and cels were passaged at 3 weeks. Along with passage going on, the phenotype of chondrocytes was changed from polygonal, round, triangle, and irregular shapes to fusiform. The colagen type II expression in passage 2 cels was positive by immunohistochemical staining. MTT test showed the growth curve of the passage 2 chondrocytes presented a transverse “S”. Cels were found logarithmic growth at days 4-7, reached platform stage at days 8-13, and decreased at day 14. It is an effective and simple procedure by two-step enzymatic digestion and tissue explant method to culture vertebral endplate chondrocytes with high purity and good viability from type I neurofibromatosis patients associated with scoliosisin vitro. Passage 2 chondrocytes from the vertebral endplate exhibit the best viability at days 4-7, which can be used as targets for research of pathogenesis of type I neurofibromatosis with atrophic scoliosis.
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BACKGROUND:How to get a lot of stable and dynamic human umbilical cord mesenchymal stem cels is stil a difficulty. OBJECTIVE:To investigate the best culture way of human umbilical cord mesenchymal stem cels by comparing tissue explant, enzyme digestion, enzymolysis methods. METHODS:Ten fresh umbilical cords were isolated to human umbilical cord mesenchymal stem cels by using tissue explant, enzyme digestion, and enzymolysis methods, respectively. Hereafter, we compared cels harvested using three culture methods in terms of the time for the primary cels to creep, successful rates of cel-culturing, and curves of cellgrowth. Surface markers of cels were detected by flow cytometry and multiple differentiations of cels were detected. RESULTS AND CONCLUSION: The time for the primary cels to creep out in the enzymolysis and enzyme digestion groups had no significant difference, but both of them were significantly shorter than that in the tissue explant group (P < 0.01). The successful rate of primary cellculture in the enzymolysis group was significantly higher than that in the other two groups. The cellproliferations of three groups had no significant difference. The cellsurface markers and differentiation ability of the third generation of human umbilical cord mesenchymal stem cels cultured by enzymolysis method met the characteristics of mesenchymal stem cels. In conclusion, the enzymolysis method can shorten the time for primary human umbilical cord mesenchymal stem cels to creep out and increase the successful rates of primary human umbilical cord mesenchymal stem cels.