Optimized Workflow for Isolation and Long-term Culture of Patient-derived Glioma Cells Retaining Original Tumor Characteristics

Summary

This study presents a standardized protocol for establishing patient-derived glioma cell lines (PDGCs), ensuring their genetic and phenotypic fidelity. The basement membrane matrix extract-assisted adherent culture system enhances cell growth and homogeneity, making these models valuable for drug screening and GBM research.

Abstract

Glioblastoma (GBM) is the most aggressive primary brain tumor, characterized by high heterogeneity and resistance to standard therapies. Traditional glioma cell lines often fail to retain patient-specific genetic and phenotypic characteristics, limiting their translational relevance. To address this, we had established 50 patient-derived glioma cell lines (PDGCs) using a serum-free neural stem cell culture system. This protocol outlines the collection, processing, and long-term culture of tumor samples obtained from GBM patients, ensuring the preservation of key molecular features. Our methodology includes enzymatic tissue dissociation, red blood cell lysis, and basement membrane matrix extract-assisted adherent culture, which improves cell viability and facilitates high-throughput drug screening. Characterization of PDGCs via whole-genome and transcriptomic sequencing and immunofluorescence staining confirms their retention of common GBM genetic alterations and neural stem cell and progenitor markers. Additionally, these cells exhibit tumorigenic potential in xenograft models. By optimizing culture conditions, this protocol provides a standardized framework for generating biologically relevant GBM models to support therapeutic discovery and mechanistic studies.

Introduction

Glioma, the most common primary intracranial tumor, originates from neural stem or progenitor cells that have undergone genetic mutations¹. The World Health Organization classifies gliomas into grades 1-4 based on pathological and molecular characteristics, ranging from low grade to high grade². Glioblastoma (GBM), a grade 4 glioma, represents approximately 57% of all gliomas and 48% of all primary malignant central nervous system tumors³. GBM patients face a poor prognosis, with a median survival time of less than 2 years and a 5-year survival rate of only 5.4%⁴. Standard ....

Protocol

This protocol utilizes patient-derived glioma tissue and blood samples obtained through hospital collaborations. All sampling procedures and experimental protocols have received approval from the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University (KY 2020-093-02 and KY 2014-021-02). Patients provide signed informed consent prior to surgery, and all identity information undergoes anonymization before samples are transferred to the laboratory. Researchers must secure approval from their institutional ethics committee and obtain patient consent before implementing this protocol.

1. Preparation before sample processi....

Results

Figure 1 provides a comprehensive workflow overview of the protocol. Patient samples include peripheral blood and tumor core tissue. Blood samples undergo centrifugation to remove plasma followed by two ACK lysis rounds to obtain PBMCs that serve as controls for sequencing analyses. Tumor samples are PBS-washed to remove blood clots and necrotic areas before being processed into small fragments. Multiple tissue fragments are cryopreserved for future DNA/RNA extraction and sequencing, while a.......

Discussion

GBM's heterogeneity presents significant treatment challenges. Conventional GBM cell lines often develop homogeneous genetic backgrounds and lose patient tumor characteristics after extended culture in serum-containing medium, limiting their translational value. Optimizing GBM culture conditions and standardizing patient sample processing workflows are essential for maximizing limited clinical resources in research. A comprehensive GBM cell model system will enhance our understanding of GBM heterogeneity and improve ther.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70 μm cell strainer	Falcon	352350
Accutase cell detachment solution	BioLegend	423201
Ammonium chloride	Aladdin	60-92-4
Anti- Sox2 antibody	Merckmillipore	AB5603
Antifade Mounting Medium with DAPI	Beyotime	P0131
Anti-Nestin antibody	NOVUS	NB100-1604
B27 without vitamin supplemet	Gibco	12587010
BD FACSAria Fusion Flow Cytometer	BD Biosciences	/
Cell culture dish	NEST	705001
D-Luciferin, Potassium Salt	Invitrogen	L2916
DMEM/F12	Gibco	C11330500BT
DMSO	Sinopharm	30072418
EDTA	BBI Life Sciences	A600107-0500
Glucose	Sangon Biotech	A50991-0500
GlutaMAX	Gibco	35050-061	glutamine substitute
Hematoxylin-Eosin staining Kit	Solarbio	G1120
HEPES	Beyotime	ST090
Human bFGF	Origene	TP750002
Human EGF	Novoprotein	C029-500μg
HypoThermosol FRS	Biolife Solutions	101104	hypothermic preservation medium
Isoflurane	RWD Life Science	R510-22-10
IVIS Lumina XR III	PerkinElmer	/	bioluminescence imaging system
Matrigel	Corning	356231	basement membrane matrix extract
Microsyringe	Hamilton	80300
N2 Supplement	Gibco	17502048
Nunclon Sphera 6-Well Plate	Thermo Scientific	174932	ultra-low attachment plates
Paraformaldehyde	Sangon Biotech	A500684-0500
PBS buffer	Solarbio	P1010-100*2L
Penicillin/Streptomycin	Solarbio	P8420/S8290
Potassium bicarbonate	Sangon Biotech	A501195-0500
RWD tissue dissociator	RWD Life Science	DSC-410
Standard stereotaxic instrument	RWD Life Science	68801
Tissue sample processing tube	RWD Life Science	SCT-100
β-mercaptoethanol	Sigma	M3148-100mL