The studies from 7 or 8 days old mice were used to prepare the spermatogonial stem cells. The isolation and purification of spermatogonial stem cells were done by means of the Percoll discontinual density gradient con...The studies from 7 or 8 days old mice were used to prepare the spermatogonial stem cells. The isolation and purification of spermatogonial stem cells were done by means of the Percoll discontinual density gradient concentration. The cells of the 3rd band were collected and cultured in vitro in DMEM supplemented with 2.5% or 10% fetal bovine serum （FBS）. The results showed that with the feeder layer and 2.5% serum, the spermatogonial stem cells could proliferate, differentiate last more than 4 months.展开更多
3D （Three-dimensional） Caco-2 spheroids closely recapitulating in vivo physiological organization of intestinal epithelial cells, provide an excellent in vitro model system to study their pathophysiology and their r...3D （Three-dimensional） Caco-2 spheroids closely recapitulating in vivo physiological organization of intestinal epithelial cells, provide an excellent in vitro model system to study their pathophysiology and their response to stressful stimuli. The objective of this technical note is to provide optimized in vitro experimental protocols for culturing 3D Caco-2 spheroids and for analyzing their cell growth features. An optimized 3D Caco-2 spheroid culturing technique based on a new configuration of the culture medium is provided A methodological approach to determine the distribution of the cell cycle phases in disaggregated Caco-2 spheroids by using cytofluorimetric analysis is also described. The optimized culturing protocol favors 3D Caco-2 spheroid differentiation process, as evaluated by the number of well-differentiated spheroids with a single hollow lumen. The cytofluorimetric analysis allows rapid collection of cell cycle phase data from high numbers of spheroid samples, thus, permitting to estimate their growth dynamics in a relatively short time. The optimized technical approaches described here can be applied in systematic manner to a variety of research activities utilizing 3D Caco-2 spheroids. Ease of use, time and economic saving advantages deriving from these protocols further highlight their potential.展开更多
Objective： To explore the electrophysiological proper- ties of differentiation of rat bone marrow-derived stromal stem cells （rBMSCs） to neuron-like cells in vitro by edaravone, a new type of free radical scavenger...Objective： To explore the electrophysiological proper- ties of differentiation of rat bone marrow-derived stromal stem cells （rBMSCs） to neuron-like cells in vitro by edaravone, a new type of free radical scavenger. Methods： Stromal stem cells were separated from rat bone marrow with Ficoll-Paque reagent and expanded in different culture medium in vitro, rBMSCs were induced by edaravone containing serum-free L-DMEM. Morphologic observation and Western blot analysis including the expression of Nav1.6, Kvl.2, Kv1.3, Cav1.2 were performed, and whole patch-clamp technique was used. Results： Cyton contraction and long processes were shown in differentiated stromal stem cells. Navl.6, Kvl.2, Kv 1.3 and Cav 1.2 were expressed in both differentiated and undifferentiated ceils. However, the expression of channel proteins in differentiated cells was up-regulated. Consistently, their resting potential and outward currents were also enhanced in the differentiated cells, which was especially significant in the outward rectifier potassium current. Conclusion： In vitro, neuron-like cells derived from rBMSCs, induced by edaravone, possess electrophysiological properties of neurons.展开更多
Purpose： To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods： Primary osteoblasts, bone marrow-derived mesenchymal stem cells （BMSCs, cultured in ost...Purpose： To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods： Primary osteoblasts, bone marrow-derived mesenchymal stem cells （BMSCs, cultured in osteoinductive medium） and RAW264.7 cells cultured in osteoclast inductive medium were all subjected to a 1000μstrain （μs） at 1 Hz cyclic mechanical stretch for 30 min （twice a day）. Results： After mechanical stimulation, the alkaline phosphatase （ALP） activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the mRNA levels of ALP and collagen type I increased. Additionally, extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant acid phosphatase-positive multinucleated cells, matrix metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium solvency all decreased. Conclusion： The results demonstrated that 1000 μs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.展开更多
文摘The studies from 7 or 8 days old mice were used to prepare the spermatogonial stem cells. The isolation and purification of spermatogonial stem cells were done by means of the Percoll discontinual density gradient concentration. The cells of the 3rd band were collected and cultured in vitro in DMEM supplemented with 2.5% or 10% fetal bovine serum （FBS）. The results showed that with the feeder layer and 2.5% serum, the spermatogonial stem cells could proliferate, differentiate last more than 4 months.
文摘3D （Three-dimensional） Caco-2 spheroids closely recapitulating in vivo physiological organization of intestinal epithelial cells, provide an excellent in vitro model system to study their pathophysiology and their response to stressful stimuli. The objective of this technical note is to provide optimized in vitro experimental protocols for culturing 3D Caco-2 spheroids and for analyzing their cell growth features. An optimized 3D Caco-2 spheroid culturing technique based on a new configuration of the culture medium is provided A methodological approach to determine the distribution of the cell cycle phases in disaggregated Caco-2 spheroids by using cytofluorimetric analysis is also described. The optimized culturing protocol favors 3D Caco-2 spheroid differentiation process, as evaluated by the number of well-differentiated spheroids with a single hollow lumen. The cytofluorimetric analysis allows rapid collection of cell cycle phase data from high numbers of spheroid samples, thus, permitting to estimate their growth dynamics in a relatively short time. The optimized technical approaches described here can be applied in systematic manner to a variety of research activities utilizing 3D Caco-2 spheroids. Ease of use, time and economic saving advantages deriving from these protocols further highlight their potential.
基金The work was supported by Natural Science Foundation of Guangdong Province （No.06028967, 8452402301001081 ）.
文摘Objective： To explore the electrophysiological proper- ties of differentiation of rat bone marrow-derived stromal stem cells （rBMSCs） to neuron-like cells in vitro by edaravone, a new type of free radical scavenger. Methods： Stromal stem cells were separated from rat bone marrow with Ficoll-Paque reagent and expanded in different culture medium in vitro, rBMSCs were induced by edaravone containing serum-free L-DMEM. Morphologic observation and Western blot analysis including the expression of Nav1.6, Kvl.2, Kv1.3, Cav1.2 were performed, and whole patch-clamp technique was used. Results： Cyton contraction and long processes were shown in differentiated stromal stem cells. Navl.6, Kvl.2, Kv 1.3 and Cav 1.2 were expressed in both differentiated and undifferentiated ceils. However, the expression of channel proteins in differentiated cells was up-regulated. Consistently, their resting potential and outward currents were also enhanced in the differentiated cells, which was especially significant in the outward rectifier potassium current. Conclusion： In vitro, neuron-like cells derived from rBMSCs, induced by edaravone, possess electrophysiological properties of neurons.
基金This work was financially supported by the National Natural Science Foundation of China （No.11372351, No.31370942, No.81160223）, and Scientific Research Foundation of Guangxi Higher Education （No.KY2015LX241）.
文摘Purpose： To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods： Primary osteoblasts, bone marrow-derived mesenchymal stem cells （BMSCs, cultured in osteoinductive medium） and RAW264.7 cells cultured in osteoclast inductive medium were all subjected to a 1000μstrain （μs） at 1 Hz cyclic mechanical stretch for 30 min （twice a day）. Results： After mechanical stimulation, the alkaline phosphatase （ALP） activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the mRNA levels of ALP and collagen type I increased. Additionally, extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant acid phosphatase-positive multinucleated cells, matrix metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium solvency all decreased. Conclusion： The results demonstrated that 1000 μs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.
