A Protocol for Explant Cultures of IDH1-mutant Diffuse Low-grade Gliomas
May 9th, 2025
IDH1-mutant diffuse low-grade gliomas are rare tumors that are difficult to culture. We introduce a simple, non-enzymatic explant method that preserves IDH1-mutant protein expression and sustains cultures for months. Importantly, this technique works with cryopreserved tumors, offering a valuable resource for glioma research.
In our lab, we focus on the rare type of brain tumors which is called diffuse low-grade gliomas. These gliomas will affect often young adults, and they are characterized by a mutation in the gene called IDH-1. The main advance in the field of gliomas are, first, the use of 3D models, like tumoroid or organoid, second, the use of imaging directly in the brain of mice transplanted with gliomas, and third, the use of special transcriptomic and single-cell RNA.
The main challenge we face when we work on IDH-1 mutant gliomas is the low proliferation of these cells and their difficulty to be maintained in culture, and so that we have derived and define a protocol to keep the cells alive, and for some of them, we have been able to derive a cell line, but not for all patients. By studying IDH-1 mutant cells in vitro, but also in vivo, we have found that these cells are quite plastic, and they can adopt two cells'fate, either being astrocyte-like or oval-like, and we have also found that nudge is playing a major role in this plasticity. With this new explant protocol, we have been able to maintain the IDH-1 mutant cells in vitro for weeks, but even more, for months, and we have shown that there are different type of tumoral cells, as we found in patient.
We have also found that there are some tumoral environment cells, like microglial cells, which make it possible to study interaction between tumor cells and their environment, so these models offer a more accurate representation of the biological diversity found in the tumors. To begin, prepare the workspace and arrange all the sterilized tools. Transfer the tumor resection onto ice to preserve tissue viability for culture.
Examine the tumor resection for size and heterogeneity. Select portions displaying a gray color with a soft or jelly-like texture, as these areas are rich in tumor cells. Transfer the selected tumor samples into a 1.5 milliliter microtube or cryovial and add one milliliter of media.
Using sterile scissors, chop the samples into small pieces measuring one to two cubic millimeters. Next, with sterile scissors, trim the end of a 1, 000 microliter pipette tip to create a wide opening approximately three to four millimeters in diameter. Pipette 100 to 200 microliters of the minced tissue fragments with a cut tip into wells of a plate.
Gently rehomogenize the suspension to ensure uniform distribution, as the smaller fragments tend to settle rapidly. Place the plate in an incubator set at 37 degrees Celsius with 5%carbon dioxide and 100%humidity. Replace the media daily if the tumor's metabolic activity causes rapid yellowing or if the fragment density requires it.
To begin, obtain the minced tumor tissue suspension. For the freezing medium preparation, add two milliliters of DMSO to eight milliliters of cell culture medium. Combine one volume of the minced tumor suspension with an equal volume of the prepared 20%DMSO freezing medium.
Carefully pipette the mixture up and down to ensure thorough blending. Rapidly transfer the cryovials into a cryogenic container to facilitate gradual temperature reduction, then incubate overnight at minus 80 degrees Celsius before transferring to liquid nitrogen for long-term storage. For thawing, remove the cryovials from liquid nitrogen and immediately place them in a 37 degrees Celsius water bath.
Warm them until approximately 80%of the cells have thawed. To remove the DMSO, quickly transfer the suspension into a 15 milliliter centrifuge tube containing five milliliters of prewarmed 1X PBS and centrifuge the tube at 300 G for five minutes. Remove the supernatant and repeat the PBS wash, followed by centrifugation.
Aspirate the supernatant, ensuring the pellet remains undisturbed. Resuspend the pellet in an appropriate amount of medium and transfer it into a precoated PDL Laminin 24-well plate. Incubate the plate at 37 degrees Celsius.
Cells exhibiting elongated, bipolar, or multipolar morphologies were observed growing from tumor explants attached to the well surface after a minimum culture duration of four weeks. Tumor-derived cells exhibited comparable morphology between fresh and cryo-preserved tumors across three cases, as shown by elongated and bipolar appearances forming intricate networks. Radial migration of the tumor-derived cell along the fiber was clearly observed.
Unsuccessful explant cultures showed non-adherent fragments and numerous gitter cells, characterized by autofluorescence and lipid content.
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Overview
IDH1-mutant diffuse low-grade gliomas are rare tumors that are challenging to culture. This study introduces a non-enzymatic explant method that maintains IDH1-mutant protein expression and supports long-term cultures.
Key Study Components
Area of Science
- Neuroscience
- Oncology
- Cell Biology
Background
- Diffuse low-grade gliomas primarily affect young adults.
- These tumors are characterized by mutations in the IDH1 gene.
- Maintaining IDH1-mutant glioma cells in culture is difficult due to low proliferation rates.
- Existing methods have limitations in sustaining these cells long-term.
Purpose of Study
- To develop a protocol that allows for the long-term culture of IDH1-mutant glioma cells.
- To explore the plasticity of these cells in vitro and in vivo.
- To provide a valuable resource for glioma research.
Methods Used
- Non-enzymatic explant culture technique.
- Use of cryopreserved tumors.
- In vitro and in vivo studies to assess cell behavior.
- Analysis of cell fate and plasticity.
Main Results
- The explant method successfully maintains IDH1-mutant cells for months.
- Different types of tumor cells were identified, reflecting patient samples.
- Cells exhibited plasticity, adopting astrocyte-like and oval-like fates.
- Nudge was found to play a significant role in this plasticity.
Conclusions
- The new explant protocol is effective for long-term culture of IDH1-mutant gliomas.
- This method enhances the understanding of glioma biology.
- It provides a platform for future glioma research and therapeutic development.
Frequently Asked Questions
Executive Industry Relevance
Establishing robust explant cultures of IDH1-mutant diffuse low-grade gliomas addresses a critical gap in disease-relevant model systems for early discovery and translational research. This protocol enables sustained in vitro maintenance of patient-derived tumor heterogeneity, supporting predictive confidence in target validation and mechanistic de-risking for glioma portfolios. The approach enhances the reliability of preclinical studies and facilitates more informed advancement decisions in neuro-oncology pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of oncogenic mechanisms in IDH1-mutant gliomas using patient-derived tissue.
- Supports functional target validation by preserving tumor cell diversity and microenvironmental interactions.
- Facilitates mechanistic de-risking by maintaining both proliferative and differentiated tumor cell populations.
- Improves predictive confidence for pathway and target selection in neuro-oncology programs.
Screening & Assay Development
- Provides a validated explant platform for quantitative drug screening and gene perturbation studies.
- Ensures reproducibility and standardization by maintaining IDH1-mutant protein expression and lineage markers.
- Enables videomicroscopy and phenotypic readouts in a physiologically relevant context.
- Supports scalable preparation of explant cultures from both fresh and cryopreserved tumors.
Translational & Preclinical Research
- Aligns with disease-relevant biology by preserving tumor heterogeneity and microenvironmental components.
- Facilitates continuity from discovery through preclinical validation by enabling long-term culture and cell line derivation.
- Supports risk-adjusted advancement decisions by providing a platform for functional and pharmacological studies.
- Enhances translational biomarker discovery through sustained expression of lineage and tumor markers.
Pipeline & Workflow Integration
This explant culture protocol integrates into the discovery-to-preclinical continuum, enabling early hypothesis testing, target validation, and downstream screening in IDH1-mutant glioma research.
- Discovery Biology: Supports hypothesis testing and pathway clarification by maintaining tumor cell plasticity and heterogeneity.
- Screening: Provides assay-ready explants for reproducible compound evaluation and quantitative phenotypic analysis.
- Analytics: Enables measurement of lineage marker expression and cell morphology for comparative studies.
- Translational Research: Maintains disease relevance and supports biomarker alignment for preclinical validation.
- Enterprise Reuse: Offers a reusable, standardized platform for diverse neuro-oncology R&D applications.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in glioma target validation.
- Operational Value: Delivers standardized, reproducible, and scalable explant cultures for cross-functional teams.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by enabling robust preclinical evaluation.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of neuro-oncology assets.
Implementation Considerations
- Requires expertise in tissue handling, sterile technique, and neuro-oncology model systems.
- Needs access to cell culture infrastructure, cryopreservation equipment, and imaging platforms.
- Demands cross-team standardization for reproducibility and data comparability.
- Adaptable to both fresh and cryopreserved tumor samples, supporting flexible workflows.
- Limited by the inherent low proliferation of IDH1-mutant glioma cells and variable cell line derivation success.
