Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

Abstract

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…Moreover, these cells injected orthotopically in immunocompromised mice can form cancers with a histological architecture similar to that of the human tumor [202]. In subsequent years, scientists have succeeded in obtaining neurospheres containing cancer stem cells from ependymomas, medulloblastomas, and primitive neuroectodermal tumors and from high grade gliomas (grade III anaplastic astrocytomas and grade IV glioblastomas) but not from lower grade gliomas [203][204][205][206]. Detailed protocols for obtaining them are now available in the literature [200,204,[207][208][209].…”

The biological properties of cancer stem cells in malignant gliomas in tissue culture: a review

“…Therefore, a quantitative indicator of neurosphere formation in culture should be considered as credible only at initially low seeding density of tumor cells (<1•10 5 cells/cm 2 ) [31]. Despite these limitations, the method of neurospheres culture is considered to be a highly informative way to study the biology of malignant gliomas CSCs.…”

“…Without a common set of CSCs markers Hasselbach L. A. et al [31] proposed the use of cells derived from the sorting of normal CSCs-enriched cultures. GBM cells, which were selected according to the ability to form neurospheres in culture medium with growth factors, expressed NSCs markers.…”

“…Hasselbach L. A. et al [31] optimized a protocol of enzymatic dissociation of GBM tumor tissue on more than 100 samples. In long-term GBM cultures the efficiency in neurospheres obtaining was more than 40 %.…”

“…In another series of studies Hasselbach L. A. et al [31] compared primary GBM cells cultured in either neurosphere medium with added growth factors, or in conventional nutrient media with 10 % FBS. Isolation of CSCs from dissociated cells of GBM tumors was performed using cell sorting based on the expression of CSCs markers CD133 [21,45].…”