Isolation and Adherent Culture of Neural Stem Cells from Mouse Embryo / 华中科技大学学报(医学版)

Objective To develop a modified method of adherent culturing NSCs to replace the traditional suspension culturing method.Methods Neural stem cells(NSCs)were isolated from brain of fetal mouse at 15.5-day fetal age.In culture bottle which had been pre-coated with poly-L-ornithine hydrochloride/fibronectin(PO/FN),the cells were adherently cultured and passaged.The cell morphology was observed under inverted microscope.NSCs and their differentiated cells were identified by immunofluorescence.Results NSCs obtained in this study were successfully adherently cultured and expressed specific markers.Conclusion NSCs are successfully adherently cultured in this study.As compared with the traditional suspension culturing method,adherent culturing is simpler and has lower wastage of passage.It is easy to observe cell morphology and differentiation by this method.It is also helpful to culturing and storage of NSCs.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines / 대한해부학회지

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

Comparison of isolation ways of MSC and VEGF and SDF-1αconcentration in respective culture medium / 重庆医学

Objective To choose one protocol that can quickly ,safely and effectively provide amount enough of bone marrow derived mesenchymal stem cells(MSCs) for use of clinical or experimental test through comparison of their growth characteristics and growth factors levels in culture solution .Methods Cells extracted from bone marrow of C57BL/C mice respectively underwent two different isolation protocols :whole bone marrow adherent culture(WBMAC) or gradient density separation(GDS);characteris‐tic surface antigens of MSCs were identified by flow cytometry on cells isolated in different ways ;the distinct growth curve of pri‐mary stem cells cultured in vitro described their different proliferation rate;levels of vascular endothelial growth factor(VEGF) and stromal cell‐derived factor‐1α(SDF‐1α) in culture medium were detected by ELISA .Results Primary MSCs obtained by WBMAC proliferated at higher speed and exhibited shorter growth cycle than those separated by GDS ;on MSCs from both groups ,surface antigens CD29 were detected positively ,and antigens including CD31 ,CD34 and CD45 were assayed negatively ;concentration of VEGF and SDF‐1αin both two nutrient solution primarily keep at low levels ,comparatively ,level of VEGF and SDF‐1αin the di‐shes which contain MSCs by WBMAC was higher than the one in the dishes which contain MSCs by GDS .Conclusion MSCs ex‐tracted by WBMAC shows unimpaired cell function ,can build automatically more suitable microenviroment for their growth;this classic method was qualified for clinical and experimental use in a safe ,rapid ,effective way .