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 treatment involves maximal safe resection followed by radiotherapy and temozolomide chemotherapy⁵. Despite these interventions, GBM remains incurable due to its infiltrative nature and inherent resistance to therapies, creating an urgent need for novel treatment approaches.

Researchers commonly use human-derived tumor cell lines as models for studying cancer development and therapeutic mechanisms. However, classical glioma cell lines maintained in serum-containing medium and passaged extensively in vitro exhibit significant alterations in phenotypic characteristics and genetic profiles compared to patient tumors^6,7,8. Consequently, preclinical screening models based on these cell lines often fail to identify therapeutic targets and molecules that translate effectively to clinical settings. GBM treatment success is further complicated by tumor heterogeneity and plasticity⁹. This heterogeneity manifests not only in transcriptomic subtypes (e.g., proneural, mesenchymal) but also in the diverse developmental states of constituent cells¹⁰. GBM employs neurodevelopmental mechanisms and contains glioblastoma stem cell (GSC) subpopulations that drive tumor proliferation and exhibit enhanced resistance to radiotherapy and chemotherapy^11,12. Culturing patient-derived glioma cells in serum-free neural stem cell medium effectively preserves the mutation spectrum and gene expression patterns of the original tumor while maintaining partial GSC characteristics⁸.

Compared to neurosphere culture models, basement membrane matrix extract-assisted adherent culture promotes more stable and rapid cell growth, making it more suitable for chemical and genetic screening¹³. Using this approach, we established 50 patient-derived GBM cell lines and characterized them through whole-genome sequencing, transcriptomic sequencing, and FDA drug library screening to explore potential therapeutic strategies. This resource advances glioma research, with detailed results published in our previous studies¹⁴. Here, we describe the methodology used to develop patient-derived glioma cell lines (PDGCs).

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 processing

1. Sterilize scissors, tweezers, and other instruments by autoclaving.
2. Prepare PDGC culture medium by combining 47.5 mL of DMEM/F12, 500 µL of N2, 1 mL of B27, 500 µL of 100x Penicillin/Streptomycin, 250 µL of 100x glutamine substitute, 100 µL of 2.5 M HEPES, 100 µL of 30% Glucose, 50 µL of 50 mM β-mercaptoethanol, 1.5 mL of 1 µg/mL epidermal growth factor (EGF), and 1.5 mL of 1 µg/mL basic fibroblast growth factor (bFGF).
3. Prepare 1x Red Blood Cell Lysis Buffer (ACK) by dissolving 8.29 g of ammonium chloride, 1 g of potassium bicarbonate, and 37 mg of EDTA in water, and then diluting to 1 L.
4. Place sterilized instruments, PBS buffer, waste containers, centrifuge tubes, and other supplies in a biosafety cabinet and perform UV disinfection for 30 min.
5. Place liquid nitrogen in an insulated container.

2. Patient sample collection

NOTE: Conduct all tissue collection procedures under strict sterile conditions to prevent bacterial contamination of subsequent cell cultures. Maintain aseptic technique throughout the entire workflow.

1. Collect blood samples preoperatively and excise tumor tissue from the patient's brain. For large, highly infiltrative tumors where distinguishing the tumor from surrounding normal tissue is difficult, maximize peritumoral tissue resection while preserving critical functional brain areas. Separate approximately 500-2,000 mm³ of tumor core tissue and place into 15 mL centrifuge tubes containing 10 mL of hypothermic preservation medium.
2. Seal the sample tubes, place them in an ice box, and transport them rapidly to the laboratory for processing.
   NOTE: Ensure that the interval between sample collection and processing does not exceed 6 h at 4 °C. During sample collection, carefully exclude burnt tissue and areas of tumor necrosis. The surface of tissue incisions is often scorched by the electric knife used during surgery, which can compromise cell viability in culture.

3. Blood sample processing

1. Centrifuge blood sample tubes at 1,000 × g for 10 min and discard the upper plasma layer.
2. Transfer the remaining white blood cell and red blood cell layer to a 50 mL centrifuge tube. Add 10 mL of 1x ACK and incubate at room temperature for 5 min to lyse red blood cells.
3. Add 40 mL of PBS to terminate lysis, mix gently, and centrifuge at 1,000 × g for 10 min.
4. Discard the supernatant, add 3 mL of 1x ACK, and incubate at room temperature for 5 min for a second red blood cell lysis step.
5. Add 12 mL of PBS to terminate lysis, mix gently, and centrifuge at 1,000 × g for 10 min.
6. Discard the supernatant, resuspend the peripheral blood mononuclear cell (PBMC) pellet in 1 mL of PBS, transfer to a 1.5 mL centrifuge tube, and centrifuge at 510 × g for 10 min.
7. Discard the supernatant, freeze the PBMC pellet in liquid nitrogen, and store at -80 °C.

4. Tumor tissue processing

1. Remove tissue preservation solution from the sample tube and wash the tissue in PBS to remove visible blood clots and necrotic tissue. Perform a second PBS wash on the remaining tissue.
2. Cut out the most intact tissue portion and fix overnight in 4% paraformaldehyde (PFA) at 4 °C.
   NOTE: The size of the fixed tissue is approximately 10 mm. Skip this step if the sample is small.
3. Cut 2-4 small tissue pieces, transfer to a 1.5 mL centrifuge tube, freeze in liquid nitrogen, and store at -80 °C.
   NOTE: The size of small tissue pieces typically ranges from 5 to 10 mm, depending on the amount of DNA and RNA required for sequencing.
4. Place the remaining tumor tissue in a 1.5 mL centrifuge tube and mince into a fine paste using scissors while keeping on ice.
5. Add 1-3 mL of cell detachment solution and transfer the minced tissue to the digestion tube. Digest the tissue using a tissue dissociator at 37 °C for 16 min.
6. Add 3x volume of PBS to stop the digestion and transfer the cell suspension to a centrifuge tube with a 70 µm cell strainer. Gently grind the tissue through the strainer with a small rod to maximize cell collection. Centrifuge at 510 × g for 10 min and remove the supernatant.
7. Add 1-3 mL of 1x ACK and incubate at room temperature for 5 min to lyse red blood cells. Add 4x volume of PBS to stop the lysis, mix gently, and centrifuge at 510 × g for 10 min to remove the supernatant.
8. Resuspend cells in PDGC medium and seed part of the cell suspension in culture plates. Place the plates in a 37 °C hypoxic incubator. Freeze the remaining cells in culture medium containing 10% DMSO.
   NOTE: Typically, for tissue samples measuring approximately 10-20 mm, we seed about 1/3 to 1/10 of the cell suspension into a 6 cm dish with 3 mL of culture medium, and the remaining cells are cryopreserved in 2-4 vials, depending on the cell yield.
9. Coat plates by adding basement membrane matrix extract to ice-cold PBS at a 1:100 ratio and incubate plates at room temperature on a shaking incubator for 2 h. Discard the PBS before adding PDGC culture medium.
   NOTE: Perform all subsequent cell passage and culture using basement membrane matrix extract-coated plates.
10. After 2 days, transfer the cultured cells to basement membrane matrix extract-coated plates to enable cell adhesion and growth.

5. Cell culture and passage

1. Once cells adhere to culture plates, remove non-adherent cells and replace or supplement culture medium every 2-3 days.
   NOTE: The adhesion capacity varies among different cells. Even in the presence of basement membrane matrix extract, a small fraction of cells may remain non-adherent, such as BNI7-11, which requires continuous suspension culture. Additionally, some cells exhibit a semi-adherent growth pattern, such as BNI20. Therefore, careful observation of cell status is essential. For non-adherent cells, an increase in suspended cells can be noted. If the cell aggregates appear as dark, cotton-like structures and show no significant changes over time, they are typically incapable of long-term expansion.
2. When cells reach confluence, add cell dissociation solution to detach the cells. Terminate the dissociation with 3x PBS and centrifuge at 250 × g for 3 min. Passage the cells at a 1:2 to 1:4 ratio. Freeze the remaining cells in culture medium containing 10% DMSO.
3. Continue passaging for at least 10-20 generations to establish a stable cell line.
   1. Document cell morphology with images during each passage and record passage numbers. Clearly label the initial cell freezing with detailed information (e.g., passage numbers).
   2. Once the cell line stabilizes, collect 2-3 tubes of cell pellets (300,000-1,000,000 cells/tube) and store at -80 °C.

6. Subsequent experimental operations

1. For PFA-fixed samples, following overnight fixation, dehydrate samples through a graded series of ethanol solutions by sequential immersion in 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and two changes of 100% ethanol, each for 40 min. Subsequently, clear the samples in two changes of xylene, each for 40 min, and then infiltrate with two changes of paraffin solution, each for 40 min. Finally, embed the dehydrated and infiltrated samples in paraffin blocks for subsequent sectioning and staining¹⁵.
2. For PBMC pellets, tumor tissue, or tumor cell samples stored at -80 °C, extract DNA and RNA for various omics studies, including whole-genome sequencing, exome sequencing, bulk RNA sequencing, and single-cell RNA sequencing as needed.
3. Use stable PDGC cell lines for mouse xenograft model establishment, high-throughput drug screening, mechanism research, and other related experiments.
   1. To establish the intracranial orthotopic xenograft model of PDGCs, dissociate and count the cells, then resuspend them in a 1:1 mixture of basement membrane matrix extract and ice-cold PBS to a final concentration of 100,000 cells/µL. Inject 5 µL of the cell suspension stereotactically into the left striatum of 6-8-week-old immunodeficient mice using a microsyringe and a standard stereotaxic instrument. Set the injection coordinates relative to the bregma as follows: +0.5 mm anterior-posterior (AP), -1.9 mm medial-lateral (ML), and -3.6 mm dorsal-ventral (DV).
   2. Generate luciferase-expressing PDGCs and establish intracranial xenografts for bioluminescence imaging of tumor growth.
      1. Package the luciferase-tdTomato reporter gene into lentiviral particles, then infect PDGCs with the packaged lentivirus to establish stable expression¹⁶.
      2. After infection, sort tdTomato-positive cells by flow cytometry to obtain a stable cell population expressing luciferase.
      3. Prior to imaging, inject D-luciferin intraperitoneally at a dose of 10 µL/g body weight using a 15 mg/mL D-luciferin solution. Anesthetize the mice with isoflurane and wait for 10 min after D-luciferin injection. Place the mice into the bioluminescence imaging system. Set the exposure time to automatic mode during imaging¹⁷.

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