Anat Cell Biol.  2015 Mar;48(1):25-35. 10.5115/acb.2015.48.1.25.

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

• Rahman M
• Reyner K
• Deleyrolle L
• Millette S
• Azari H
• Day BW
• Stringer B
• Boyd AW
• Johns TG
• Blot V
• Duggal R
• Reynolds B

• KMID: 2046072
• DOI: http://doi.org/10.5115/acb.2015.48.1.25

Abstract

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.

Keyword

Adherent culture; Neoplastic stem cells; Neurosphere

MeSH Terms

Animals
Apoptosis
Brain
Caspase 3
Culture Techniques
Glioblastoma
Glioma*
Humans
Laminin
Neoplastic Stem Cells
Stem Cells*
Tics
Tubulin
Caspase 3
Laminin
Tubulin

Figure

• 

  Fig. 1 The number of cells obtained in vitro over multiple passages of glioblastoma cell lines grown in neurosphere assay (NSA) and adherent culture conditions (n=4 each group). The fold expansion over time in the two different conditions was not statistically different (P=0.24).

• 

  Fig. 2 The geometric mean of markers of proliferation markers nestin (n=4, P=0.52), Ki67 (n=3, P=0.22), and MCM2 (n=7, P=0.51). The mean and standard error are shown in the graphs. NSA, neurosphere assay.

• 

  Fig. 3 The geometric mean of markers of differentiation including glial fibrillary acidic protein (GFAP) (n=6, P=0.44) and beta tubulin III (n=6, P=0.18) of laminin-grown cells compared to neurosphere assay (NSA) grown cells by flow cytometry. The mean and standard error are shown in the graphs.

• 

  Fig. 4 (A-D) The geometric mean of protein expression in various glioblastoma cell lines in laminin (L) and neurosphere assay (N) conditions.

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  Fig. 5 Graph depicting mean geometric mean values of protein expression in laminin versus neurosphere assay (NSA) grown glioblastoma cells (n=3). EGFR, epidermal growth factor receptor. *Statistically significant.

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  Fig. 6 Percent of cells positive for trypan blue denoting cell death at the time of trypsinization of neurospheres or cells grown on laminin (n=9). Standard error is shown with the error bars (P=0.7). NSA, neurosphere assay.

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  Fig. 7 Summary of apoptosis markers including propidium iodide (PI) (n=4), annexin V (n=4), activated caspase 3 (n=6), 4',6-diamidino-2-phenylindole (DAPI) (n=7), and 1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide (DiIC) (n=5) by flow cytometry depicting percent of cells positive in neurosphere assay (NSA) and laminin grown glioblastoma cells. P-value, not significant for all comparisons. The mean and standard error are shown in the graph.

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  Fig. 8 Kaplan-Meier demonstrating similar survival of non-obese diabetic/severe combined immunodeficient mice after implantation of glioblastoma cells grown in either neurosphere assay (NSA) and laminin culture conditions (n=5 in each group). Mice were euthanized once tumors reached 1.5 cm in any dimension.

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