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Low-cost media formulation for culture of brain tumor spheroids (neurospheres)
 
Michael D. Monterey, Nicholas J. Szerlip, and Saroj P. Mathupala
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Figure 1.  Low-power microscopy (40×) images of spheroids (neurospheres) generated using the low-cost (HMSF) media formulation. (Click to enlarge)




To compare the characteristics of glioma cells established and maintained as either spheroids or as monolayer cultures, we utilized 3 primary glioblastoma cell isolates: (i) GBM1, initially established as spheroids in commercial (NeuroCult) media and then maintained in HMSF media (as spheroids) or in DMEM supplemented with 10% FBS (monolayer culture) for 12 months, (ii) GBM2, a recent tumor explant, directly established as a monolayer culture in DMEM supplemented with 10% FBS and maintained in the same medium for 2 months, and (iii) GBM3, another recent tumor explant, but established and maintained as spheroids in HMSF media for 2 months.

We then placed each isolate in HMSF or 10% FBS supplemented DMEM for 2 additional months, to maintain (GBM1 and 3) or establish (GBM2) spheroids, or to establish (GBM 3) or maintain (GBM 1 and 2) monolayer cultures, respectively. Invasive capacity of each cell culture was examined using Matrigel-based invasion assays (Figure 2A). In separate experiments, each isolate was also placed in HMSF or commercial medium (NeuroCult), again for two additional months and allowed to maintain (GBM1 and 3) or form spheroids (GBM2). Proliferative capacity was measured for each isolate (Figure 2B). We also assessed CD133 expression for each GBM isolate using flow cytometry (Figure 2C). CD133, a known marker for tumor hypoxia, is the most commonly used marker to identify the “stemness” of glioblastoma explants (7).




Figure 2.  Phenotypic characteristics of primary glioma explants maintained as spheroids in HMSF or commercial neurosphere media, or as monolayer cultures in standard 10% FBS containing media. (Click to enlarge)




While the invasion assays indicated slightly higher (but statistically significant) penetrance of Matrigel by cells maintained as spheroids, no clear difference in proliferation rates was observed for spheroids maintained in commercial versus HMSF medium. Markedly higher expression of CD133 was observed in GBM1 spheroids over GBM1 cells maintained as monolayers. GBM2, initially established as a monolayer culture, either lost or did not harbor CD133 expression; GBM2 did not gain CD133 expression upon spheroid formation either. However, the GBM3 cell isolates that were established and then maintained as spheroids from inception displayed a small, but statistically significant, population of CD133 positive cells that diminished upon monolayer culture. Thus, our studies indicate that “stemness” is selected for and maintained, upon prolonged culture of primary glioblastoma explants as spheroids in HMSF medium.

BSA fraction V is the only “non-standard” component in our HMSF media formulation that may show batch-to-batch variability. However, similar variation will be present in donor human sera that are provided as separate supplements in most commercial neurosphere culture media. To date, we have tested four batches of BSA, three from Sigma Chemical Co., and one from Life Technologies, finding no variance in spheroid formation capabilities or in growth properties (data not shown).

In summary, our media formulation and protocols represent cost-effective and reproducible methods for the initiation, propagation, and maintenance of primary human glioma cultures as spheroids that maintain the phenotypic characteristics of the original tumor, allowing for more applicable clinical extrapolation.

Acknowledgments

Research support for the corresponding author was provided by a grant from the National Cancer Institute/National Institute of Health (CA 116257), the Fund for Medical Research and Education (FMRE), Wayne State University, and a gift from the Marvin E. Klein, M.D., Charitable Trust. The authors wish to thank Eric Van Buren and Jessica Back from the Cytometry Resources Core (supported in part by NIH Center grant CA022453) at Wayne State University for the flow analyses. This paper is subject to the NIH Public Access Policy.

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Saroj P. Mathupala, Department of Neurological Surgery & Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI. E-mail: [email protected]

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