Culture conditions defining glioblastoma cells behavior: what is the impact for novel discoveries?

Abstract

In cancer research, the use of established cell lines has gradually been replaced by primary cell cultures due to their better representation of in vivo cancer cell behaviors. However, a major challenge with primary culture involves the finding of growth conditions that minimize alterations in the biological state of the cells. To ensure reproducibility and translational potentials for research findings, culture conditions need to be chosen so that the cell population in culture best mimics tumor cells in vivo. Glioblastoma (GBM) is one of the most aggressive and heterogeneous tumor types and the GBM research field would certainly benefit from culture conditions that could maintain the original plethora of phenotype of the cells. Here, we review culture media and supplementation options for GBM cultures, the rationale behind their use, and how much those choices affect drug-screening outcomes. We provide an overview of 120 papers that use primary GBM cultures and discuss the current predominant conditions. We also show important primary research data indicating that "mis-cultured" glioma cells can acquire unnatural drug sensitivity, which would have devastating effects for clinical translations. Finally, we propose the concurrent test of four culture conditions to minimize the loss of cell coverage in culture.

Keywords: culture conditions; culture media; drug discovery; glioblastoma; heterogeneity.

Figure 1. Timeline of important milestones in cell culture and GBM cell culture (reference numbers for milestones can be found in Supplementary File 1)

Figure 2. Literature review of growth conditions used in GBM primary cultures

Venn diagram of the most used culture media (in black) and the respective supplementation factors (different colors) used in GBM primary cultures. NS: non-specified basal medium. Ko: DMEM/F12 knockout.

Figure 3. The main players in the evolution of a cancer

The cell that undergoes the first mutation event is considered the cell of mutation, which may or may not undergo transformation to originate a tumor. The cell of origin is the specific cell type that is capable of undergoing transformation that generates the tumor mass. The tumor mass is very heterogeneous, with more differentiated (darker blue colors) and less differentiated cell (lighter blue colors), and it is not clear which cells are responsible for establishing metastasis or new tumors in animal models. To maintain the heterogeneity of clinically relevant tumors, either an “all-inclusive” condition or more than one condition must be used separately.

Figure 4. Culture conditions can interfere with response to drugs

(A) Mouse glioma was dissociated, purified through immunopanning and cultured in OPC media (containing PDGF as growth factor) and NSC media (containing EGF/FGF-2 as growth factors). Wild type (wt) OPCs were purified through immunopanning and used as control. PDGFR expression, naturally present in wt OPCs, is reduced when cells are cultured in NSC media, and EGFR starts to be expressed in an active (phosphorylated) form. (B) EGFR inhibitors Gefitinib and AG-1478 reduce cell viability of treated cells only when they are cultured in NSC media (NmA– NSC media accustomed cells), and not in OPC media accustomed cells (OmA). pEGFR = phosphorylated EGFR, tEGFR = total EGFR. Values were normalized based on a non-treated control (vehicle–DMSO-only). (C) A similar response is seen in a human GBM primary cell line accustomed to both NSC media and OPC media and treated with Gefitinib. Samples were normalized based on non-treated controls. **p ≤ 0.01; ****p ≤ 0.0001.

Figure 5. Schematic representation of media choice options available for GBM primary culture

To select all cell types possible, one can grow GBM cells in NSC media (with EGF and FGF-2 as growth factors), in PDGF-enriched media (a.k.a. OPC media), in astrocyte specific media (HBEGF as the main growth factor) or in Serum-enriched media. Other possibilities might also be considered.