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- 저자 : Andrew W. Boyd (검색결과 1 건)
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Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines
Maryam Rahman,Karina Reyner,Loic Deleyrolle,Sebastien Millette,Hassan Azari,Bryan W. Day,Brett W. Stringer,Andrew W. Boyd,Terrance G. Johns,Vincent Blot,Rohit Duggal,Brent A. Reynolds 대한해부학회 2015 Anatomy & Cell Biology Vol.48 No.1
Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques forbrain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer usinglaminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growthand expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons weremade using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrixanalysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtuallyidentical (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) revealedno statistical difference between the sphere and attachment methods. Several different methods were used to determine thenumbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningfulvariance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differencesbetween the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibitingGBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization ofhuman GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grownusing sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options forexpanding primary high-grade gliomas in tissue culture.
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