源自人多形性胶质母细胞瘤标本的原发性星形细胞培养物的制备

Mansoureh  Hashemi; Mahmoudreza   Hadjighassem

doi:10.21769/BioProtoc.2241

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Peer-reviewed

Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen

源自人多形性胶质母细胞瘤标本的原发性星形细胞培养物的制备

MH Mansoureh Hashemi email

MH Mahmoudreza Hadjighassem

发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2241 浏览次数: 10784

评审: Oneil G. BhalalaSébastien GillotinEmmanuelle Berret

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Cover of Molecular Neurobiology, featuring study using the protocol.

Oct 2016

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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 (多形性胶质母细胞瘤)

Astrocyte (星形胶质细胞)

S100-beta antigen (S100-β抗原)

Primary cell culture (原代细胞培养)

Malignant brain tumor (恶性脑肿瘤)

Astrocytoma tumor (星形细胞瘤)

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

15 ml centrifuge tubes (Corning, Falcon^®, catalog number: 352096 )
Petri dish culture (Nest Biotechnology, catalog number: 704001 )
No. 10 scalpel blade surgical tool (BD, catalog number: 371610 )
Cell strainer sieve (Corning, Falcon^®, catalog number: 352340 )
50 ml centrifuge tubes (Corning, Falcon^®, catalog number: 352070 )
T25 flask culture (Nest Biotechnology, catalog number: 707003 )
1 ml microtube (Nest Biotechnology, catalog number: 613111 )
24-well plates of polystyrene with high clarity (Nest Biotechnology, catalog number: 702001 )
5 ml pipette (Nest Biotechnology, catalog number: 326001 )
Glioblastoma multiforme sample (Human)
Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 24020117 )
Antibiotic-antimycotic (Thermo Fisher Scientific, Gibco^TM, catalog number: 15240062 )
0.25% trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200056 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 )
Trypan blue solution 0.4% (Sigma-Aldrich, catalog number: T8154 )
Primary antibody of rabbit anti-S100-beta (Sigma-Aldrich, catalog number: S2644 )
Primary antibody of rabbit anti-fibronectin (Abcam, catalog number: ab23751 )
FITC-conjugated goat anti-rabbit (Abcam, catalog number: ab6717 )
Dulbecco’s modified Eagle medium/F12 (DMEM/F12) (Thermo Fisher Scientific, Gibco^TM, catalog number: 31331028 )
Phosphate buffer saline (PBS) (tablet) (Thermo Fisher Scientific, Gibco^TM, catalog number: 18912014 )
Paraformaldehyde (powder) (Sigma-Aldrich, catalog number: P6148 )
Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
Bovine serum albumin (powder) (Sigma-Aldrich, catalog number: A2058 )
Culture medium (see Recipes)
Phosphate buffer saline (PBS) (see Recipes)
4% paraformaldehyde (see Recipes)
0.2% Triton X-100 (see Recipes)
10% bovine serum albumin (see Recipes)

Equipment

Ventilation hood (VISION SCIENTIFIC, model: VS-7120LV )
37 °C water bath (Memmert, model: WNB14 )
Centrifuge machine (Hettich Lab Technology, model: Universal 320R )
Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
CO₂ cell culture incubator (Memmert, model: INC108 T2T3 )
Inverted fluorescence microscope (Optika, model: XDS-2FL )
Small forceps surgical tools (Fine Science Tools, catalog number: 11050-10 )

Procedure

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如何引用

Hashemi, M. and Hadjighassem, M. (2017). Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen. Bio-protocol 7(8): e2241. DOI: 10.21769/BioProtoc.2241.