Abstract
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.
Keywords: Glioblastoma multiforme, Astrocyte, S100-beta antigen, Primary cell culture, Malignant brain tumor, Astrocytoma tumor
Background
Astrocytes are glial cells that provide structural and nutritional support for brain neurons. The cell cycle of astrocytes seems to be disrupted in astrocytoma brain tumors. The World Health Organisation (WHO) has classified astrocytomas into four grades according to their malignancy. Glioblastoma multiforme (GBM, grade IV) is the most malignant form of astrocytoma. Glioblastoma is characterized by the invasive cells with the rapid proliferation rate and angiogenesis. Prognosis is poor for patients with glioblastoma. Current therapeutic approaches including surgery, chemo-therapy and radiation don’t have good effects on the treatment of suffering patients. Median survival time for patients is about one year after treatment (Stuup et al., 2005; Wen and Kesari, 2008). So many researchers focus on the study of evaluating the physiological function and apoptosis of glioblastoma cells in order to detect more effective treatment methods. Here we present a method for isolation of high purity primary astrocyte from human glioblastoma specimen without fibroblast contamination (Hashemi et al., 2016).
Materials and Reagents
Equipment
Procedure
Data analysis
Confirmation of cultured primary astrocytes was done by detection of marker S100-beta via fluorescence immunocytochemical analysis. This analysis demonstrated that isolated cells from glioblastoma expressed S100-beta antigen. As a result, this protocol is efficient to isolate a purified population of astrocytes. Test results are obtained from three independent experiments.
Notes
Recipes
Acknowledgments
This study was supported by grant 20035 from School of Advanced Technologies in Medicine and grant 19925 from Brain and Spinal Cord Injury Research Center, Tehran University of Medical Sciences. The protocol was adopted from Hashemi et al. (2016).
References
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