Neuroscience

Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in in vitro culture systems have demonstrated that the use of three-dimensional (3D) culture models could be more relevant for this purpose as compared to monolayer or two-dimensional (2D) models due to their resemblance to in vivo cancer pathology. High-throughput techniques such as RNA sequencing, microarray analyses and cloning could provide useful insights into the relevance of these systems to the native tissue. Previous studies have reported RNA extraction protocols needed for such applications. We have modified these protocols to suit the isolation of total RNA from monolayer and hydrogel cultures of astrocytoma established using basement membrane matrix, Geltrex^TM. We have used this method to demonstrate the differences in the expression of genes involved in autophagy, a process deregulated in many cancer types, in monolayer and hydrogel cultures using quantitative polymerase chain reaction (qPCR). This protocol can be adopted by the researchers who wish to understand the molecular basis of gene expression in hydrogel cultures of normal as well as cancer cell lines.

Glioblastoma multiforme (GBM) is a grade 4 astrocytoma tumor in central nervous system. Astrocytes can be isolated from human GBM. Study of astrocytes can provide insights about the formation, progression and recurrence of glioblastoma. For using isolated astrocytes, new studies can be designed in the fields of pharmacology, neuroscience and neurosurgery for glioblastoma treatment. This protocol describes the details for preparing high purity primary astrocytes from human GBM. Tumor tissue is disrupted using mechanical dissociation and chemical digestion in this protocol. 2 weeks after plating the cell suspension in culture, primary astrocytes are available for further subculturing and immunocytochemistry of S100-beta antigen.