Low-cost media formulation for culture of brain tumor spheroids (neurospheres)

Abstract

Recent studies have found that the biological features of primary tumors are faithfully recapitulated when a patient's tumor is processed and then maintained as a 3-D spheroid in specialized cell culture media. However, a major drawback for maintenance and routine passage of primary tumors as spheroids has been the high cost of custom-formulated media compared to regular serum-supplemented media. Here we report the formulation of a cost-effective, serum-free medium in which high-grade primary brain tumor (glioblastoma) explants can be established and maintained as spheroids. Based on DMEM, this formulation requires only supplementation with several amino acids, vitamins, synthetic EGF, and bFGF, with most of the cost being associated with the growth factors. A simple addition of BSA (fraction V) obviated the need for numerous other components (or human serum) commonly used in the specialized commercial media formulations.

Figure 1. Low-power microscopy (40×) images of spheroids (neurospheres) generated using the low-cost (HMSF) media formulation

(A) U87-MG glioma in regular DMEM supplemented with 10% FBS; (B) U87-MG glioma after passaging in HMSF for two weeks, to induce spheroid formation; (C) human glioma spheroids established from a grade IV glioma (glioblastoma multiforme) after maintenance and passage in HMSF for six months; (D) spheroids established from a primary tumor tissue (grade III astrocytoma) after four weeks of passage; and (E) after two months of passage.

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

(A) Invasive capacity of primary glioma spheroids versus monolayer cultures. Assays were carried out in Boyden chambers essentially as described before (9). Matrigel was diluted 1:3 in Optimem-1 media (Life Technologies) supplemented with 4% FBS. Glioma single cell suspensions were prepared by treating with Accutase and 4 × 10⁴ cells plated per well-insert in 0.5 mL of Optimem-1 media as above. 0.75 mL of DMEM/F12 media supplemented with 10% FBS was placed in bottom wells as the chemo-attractant. Twenty-four hours post-plating, the well-inserts were placed in a new set of wells containing 4 μg/mL Calcein AM (Caymen Chemical, MI) in 0.5 mL PBS. The bottom surface of wells was read at 517 nm (494 nm excitation). P-values (± SD, Student's t-test) were: GBM1, 0.049; GBM2, 0.347; GBM3, 0.068. (B) Proliferative rates of glioma spheroids in NeuroCult versus HMSF media. Three to ten cell spheroids maintained in NeuroCult or HMSF media were plated at approximately 1.2 × 10⁶ cells per well in 60 mm diameter plates in 5 mL of each medium, respectively. The media were changed every three days for two weeks. Single cell suspensions were prepared from each spheroid culture and plated in 6 well plates pre-coated in poly L-lysine (10 μg/mL) and allowed to attach overnight in Optimem-1 media supplemented with 4% FBS. MTT assays were carried out on the cells as described previously (8). P-values (± SD, Student's t-test) were: GBM1, 0.959; GBM2, 0.008; GBM3, <0.001. (C) CD133 expression in glioma spheroids versus monolayer cultures. Single cell suspensions were prepared using Accutase and washed in Optimem-1 media supplemented with 4% FBS. The cells were suspended in PBS supplemented with 0.2% BSA and 2 mM EDTA at 10⁷ cells per 80 μl. Twenty μl of FcR blocking reagent and 10 μl of phycoerythrin coupled CD133 antibody (Miltenyi Biotech, CA) were added to each sample and incubated in the dark for 30 min at room temperature. The cells were then washed (2 mL) and resuspended in the same PBS buffer as above and analyzed in a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). FlowJo ver. 9.3.2 software (TreeStar, Inc., Ashland, OR) was used for analysis.