Recovery of glioblastoma cells derived from patients after a decade of cryopreservation for drug response studies

Abstract

Patient-derived glioblastoma cells (GBMs) are essential for drug screening and personalized medicine. Limited patient sample availability requires the use of long-term cryopreserved GBM cells. However, current methods for recovering GBM cells after an extended period of cryopreservation are generally inefficient, resulting in poor survival of these cells. Therefore, this study sought to optimize a culture procedure to improve the recovery and growth of patient-derived GBM cells after long-term cryostorage. We show that the use of Matrigel with an increased percentage of fetal bovine serum significantly improves the viability and subsequent expansion of GBM cells, which were cryopreserved for more than 10 years, compared to standard culture methods. The enhanced growth is associated with an increase in the expression of YAP and TLR4, the key regulators of cell proliferation, in these GBM cells. These recovered GBM cells were successfully used for chemotherapeutic drug testing, specifically temozolomide (TMZ), in 2D and 3D spheroid culture systems. We believe that our optimized protocol offers a valuable tool to increase the availability of patient-derived GBM cells by allowing efficient recovery of long-term cryopreserved GBM cells for research and personalized drug testing.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-22908-0.

Introduction

Glioblastoma (GBM) is an aggressive brain cancer originating from glial cell astrocytes that infiltrates adjacent brain tissue and causes severe headaches, vomiting, seizures, and cognitive and behavioral impairments in patients^1–3. Because, these symptoms are not specific to GBM, most patients are initially misdiagnosed and only receive proper treatment when the cancer become too advance to be completely eradicated by surgery, resulting in the high recurrence and very poor survival rate of the patients.

GBM also exhibits a high degree of intertumoral and intratumoral heterogeneity, which plays an important role in its resistance to treatment^4,5. Furthermore, a highly complex tumor microenvironment in GBM, consisting of GBM cells, GBM stem cells, cancer stromal cells, and immune cells, also contributes to the aggressive behavior and therapeutic resistance of GBM^6,7. The blood-brain barrier (BBB), which prevents the penetration of most therapeutic agents, also limits treatment options^8,9. Due to these limitations, the median survival time of a glioblastoma patient is only 12–18 months, one of the lowest among cancer patients. Of these, only 25% of patients survive more than one year and only 5% of patients survive more than five years³. Given the challenges in treating GBM, recent studies have focused on developing new therapies to improve patient outcomes. For example, cold atmospheric plasma has been shown to enhance the efficacy of the standard GBM drug, Temozolomide (TMZ), in both in vitro and in vivo studies¹⁰. In addition to exploring novel treatment strategies, the use of diverse GBM models—including patient-derived cells, 3D culture systems, organoids, and animal models—is essential for strengthening research findings and improving clinical relevance^11,12.

Although patient-derived GBM cells have been an invaluable tool for discovering new therapeutic targets, testing new drugs, and choosing the most effective treatments in personalized GBM therapies, the limited amount of tissue sample and the inefficient derivation of primary GBM cells from resected tumors necessitates the use of immortalized GBM cell lines or long-term cryopreserved primary GBM cells in most studies^4,13.

However, recovery of long-term cryopreserved primary GBM cells is generally poor and usually takes weeks to months to revive the cells. Because the researches focusing on the optimization of reviving glioblastoma after cryopreservation are limited, this study aims to develop a more efficient protocol for recovering and subsequent expansion of the long-term cryopreserved patient-derived glioblastoma cells.

Using an optimized combination of extracellular matrix and an increase in the percentage of fetal bovine serum (FBS), we are able to significantly increase the viability and proliferative capacity of GBM cells after long-term cryopreservation compared to the standard procedure. Increased growth is associated with an increase in the expression of YAP and toll-like receptor 4 (TLR4), the key regulators of cell proliferation, in these GBM cells. These recovered GBM cells were successfully used for the test of chemotherapeutic drug temozolomide, in 2D and 3D spheroid culture systems (Supplementary Fig. 1).

Materials and methods

Standard medium for GBM cell culture

GBM cells were cultured in GBM culture medium, Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 100 IU/ml penicillin, and 100 µg/mL streptomycin (Servicebio, Wuhan, China), in a humidified incubator under normoxic condition (21% O₂) with 5% CO₂ at 37 °C. The medium was replaced every 3 days and cells were subcultured by incubating with 0.05% trypsin-EDTA (Life Technologies; USA) when their density reached 80% confluence.

Optimizing the culture conditions to revive patient-derived GBM cells after long-term cryopreservation

Four patient-derived primary GBM cell lines (Si-GBM4, Si-GBM10, Si-GBM15, and Si-GBM16) were obtained from different patients and had been cryopreserved in liquid nitrogen for more than ten years were thawed and cultured in high glucose DMEM supplemented with 20% FBS in tissue culture plate coated with 0.3 mg/ml Matrigel^®. The culture was carried out in a humidified incubator under normoxic condition (21% O₂) with 5% CO₂ at 37 °C. The medium was replaced every 3 days and the cells were subcultured by incubation with 0.05% trypsin-EDTA when their density reached 80% confluence. All patient cell lines were used at passages no later than fifteen to maintain optimal cellular characteristics and to ensure experimental comparability, all cell lines were used at the same passage or, at most, within a one- to two-passage difference. The general feature of GBM cells used in this study were shown in Supplementary Table 1.

Three-Dimensional (3D) spheroid formation using scaffold-free method

GBM cells were trypsinized into a single cell suspension and seeded into an individual well of AggreWell™ − 800 plate (STEMCELL Technologies, Canada) at a density of 1 × 10⁶ cells/well. The plate was then centrifuged at 160 g for 7 min and incubated overnight at 37 °C in a humidified incubator under normoxic condition with 5% CO₂ to form spheroids. The resulting spheroids were collected, transferred to an individual well of 6-well plate containing 2 ml of GBM culture medium, and placed on the orbital shaker platform that rotated at 120 rpm in a humidified incubator under normoxic condition with 5% CO₂ for another 48 h to increase the size of the spheroids for further experiments.

3D spheroid migration assay

For the 3D spheroid migration assay, a single GBM spheroid was placed in an individual well of a 96-well flat bottom place containing 100 µL GBM culture medium and cultured for 72 h. The migration capacity of GBM cells was assessed at 24, 48, and 72 h of culture. The migration area was calculated by subtracting the spheroid area at 0 h from the area measured at each time point. The migration rate was determined by dividing the migration area at each time point by the duration of culture (hours).

Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, incubated with 1x Triton X-100 in 10% FBS in Phosphate buffered saline (PBS) solution for 15 min, blocked with 10% FBS in PBS for 1 h, and incubated with the appropriate primary antibodies against human SOX2 (Sigma-Aldrich, USA), and NESTIN (Sigma-Aldrich, USA) at 4 °C overnight. After incubation, cells were washed three times with PBS and incubated with the appropriate secondary antibody conjugated with fluorescence dyes at room temperature for 1 h in the dark. The cells were then washed with PBS, counterstained with 1:1000 Hoechst 33,342, (Sigma-Aldrich) at room temperature for 5 min in the dark, and observed by fluorescence microscopy. The antibodies and their appropriate dilution were listed in Supplementary Table 2.

Transcription analysis

The total RNA was isolated from experimental cells before reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time polymerase chain reaction (PCR) was performed using Realtime PCR Master Mix (Applied Biosystems, USA). Real-time qRT-PCR was performed using Real-Time PCR Master Mix (Applied Biosystems) using a CFX384 Real-Time PCR System (Bio-Rad Laboratories, CA, USA). The primers used in the present study are listed in Supplementary Table 3.

Flow cytometry analysis

Cells were dissociated into single cells using Trypsin (Gibco) and fixed with 2% PFA (paraformaldehyde) for 20 min. The fixed cells were then incubated with PE/Cyanine7 anti-human CD133 antibody, and APC-conjugated anti-human CD44 antibody for 30 min prior to flow cytometry analysis.

Chemotherapeutic drug treatment and cell viability assay

1 × 10⁵ GBM cells were seeded in an individual well of a 6-well plate, treated with various concentrations of Temozolomide (TMZ), range from 0 to 400 µM, for 72 h, and harvested by trypsinization. Then 10 µL of cell suspension was collected and mixed with 10 µL Trypan blue. Cell viability was determined by a hemocytometer under a light microscope.

Determination of cell viability by the cell counting kit 8 assay

10 µL of cell counting kit 8 reagent (CCK-8) was added to cells cultured in 100 µL of GBM culture medium with or without TMZ chemotherapeutic drug supplementation and incubated at 37 °C for 3 h. The activity of CCK-8 was determined by a microplate reader with an absorbance of 450 nm.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0. Data were analyzed using bidirectional analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons to evaluate significant differences between experimental groups. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

Long-term cryopreservation reduces survival and growth of patient-derived primary GBM cells

Glioma cell lines and patient-derived primary GBM cells are usually cultured in DMEM supplemented with FBS^5,14–16. In this study, we revived four different patient-derived primary GBM cells, Si-GBM4, Si-GBM10, Si-GBM15, and Si-GBM16, that had been frozen in liquid nitrogen for 10 years using the standard freezing protocol at a density of 2 × 10⁶ cells/vial. After being thawed, cells were cultured in DMEM + 10% FBS^8,17 and determined for their efficiency of revival and confluence. In this study, the cell revival is defined as the ability of thawed cells to restore full vitality, exhibiting normal proliferation rates and morphology, while the confluence is defined as the stage when adherent cells completely cover the surface of the culture container. In our experiment, we consider 80–90% confluence as the optimal threshold for passaging cells to prevent overcrowding, which could lead to cell death. Result showed that only Si-GBM10 could be efficiently revived and expanded to full confluence on culture day 7 while Si-GBM4, Si-GBM15, and Si-GBM16 proliferated much slower and could not be expanded to full confluence even after 21 days of culture (Fig. 1). The results show that DMEM + 10% FBS could not efficiently revive patient-derived primary GBM cells after long-term cryopreservation and should be further optimized.

Fig. 1.

GBM cell lines Si-GBM4, Si-GBM10, Si-GBM15 and Si-GBM16 were cultured in standard DMEM medium supplemented with 10%FBS. The pictures were taken at 4x magnification. scale bar: 200 μm.

A combination of matrigel with 20%FBS significantly improves the survival and growth of long-term cryopreserved patient-derived GBM cells

To enhance the recovery of long-term cryopreserved patient-derived GBM cells by optimizing the ECM (extra cellular matrix) and percentage of FBS, we subjected the thawed GBM cells to 2 culture conditions, DMEM + 20% FBS and DMEM + 20% FBS + Matrigel (MTG), and compared the results with those cultured in standard medium, DMEM + 10% FBS. The result shows that Si-GBM4, Si-GBM10 and Si-GBM16 cells cultured with DMEM + 20% FBS + Matrigel generate a significantly higher number of cells on culture day 7 after thawing, compared to their counterparts that were cultured with DMEM + 10% FBS (Figs. 2 and 3). Although DMEM + 20% FBS alone also significantly increased the number of Si-GBM4, Si-GBM10, and Si-GBM16 cells, its effects are significantly less than those of DMEM + 20% FBS + Matrigel (Figs. 2 and 3). Unlike other patient-derived GBM cells, DMEM + 20% FBS + Matrigel did not increase the number of Si-GBM15 cells compared to the control as determined by physical appearance and total cell count (Figs. 2 and 3).

Fig. 2.

The effect of Matrigel and 20% FBS on the growth of four patient-derived GBM cells. Si-GBM4 (A), Si-GBM10 (B), Si-GBM15 (C), Si-GBM16 (D) were cultured in DMEM supplemented with 10% FBS, 20% FBS, and 20% + Matrigel (MTG). The pictures were taken at 4x magnification, scale bar: 200 μm.

Fig. 3.

Growth of frozen thawed GBM cells after cell culture in a different culture system and determined for total cells (A) and relative change in cell number compared to 10% FBS condition (B). Data are presented as mean ± SEM. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Increased FBS concentration and incorporation of matrigel significantly enhances GBM cell growth by increasing YAP and TLR4 signaling

To study the underlying mechanism by which our culture conditions improve the survival and proliferation of revived GBM cells, we performed a comparative analysis of the expression levels of YAP and TLR4, which are known regulators of cell proliferation and apoptosis, under these conditions. The results showed that 20% fetal bovine serum (FBS) significantly increased YAP (Fig. 4A and B, and 4D) and TLR4 (Fig. 4E and F, and 4H) levels in Si-GBM4, Si-GBM10, and Si-GBM16 cells compared to those cultured in DMEM supplemented with 10% FBS. Although a combination of 20% FBS with Matrigel (MTG) did not further increase the YAP level in Si-GBM4 and Si-GBM16 cells compared to those treated with 20% FBS alone (Fig. 4A and D), it significantly increased the level of TLR4 in Si-GBM4 and Si-GBM10 cells compared to those treated with 20% FBS alone (Figs. 4E–F). Consistent with the results of the proliferation assays indicating that 20% FBS and 20% FBS + MTG did not increase the proliferation of revived Si-GBM15 cells, these conditions also did not increase YAP (Fig. 4C) and TLR4 (Fig. 4G) levels in Si-GBM15 cells compared to those cultured in DMEM supplemented with 10% FBS. In Si-GBM16 cells, while the level of TLR4 in those treated with 20% FBS + MTG is marginally higher compared to those treated with 20% FBS alone, the TLR4 levels in both conditions remain significantly higher than those treated with 10% FBS (Fig. 4H). This observation is consistent with the results of proliferation assays, which demonstrate that 20% FBS + MTG significantly increases the proliferation of revived Si-GBM16 cells compared to those treated with 10% FBS. Based on these observations, it is possible that the positive effects of 20% FBS or 20% FBS + MTG on the growth of Si-GBM4, Si-GBM10, and Si-GBM16 cells could be mediated, at least in part, by increased levels of YAP and TLR4 in these Si-GBM cells under these two culture conditions. However, due to the difference in YAP and TLR4 levels between various GBM cells subjected to these two culture conditions, it remains possible that the observed effects of 20% FBS or 20% FBS + MTG in these GBM cells could also be mediated by other signaling, in addition to these two effector proteins.

Fig. 4.

Transcriptional analysis of YAP (AD) and TLR4 (EH) in Si-GBM cell lines after culture under different culture conditions. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

A combination of 20% FBS and matrigel did not affect the expression of GBM markers in most long-term cryopreserved patient-derived GBM cells

Next, we determined whether the revived patient-derived primary GBM cells maintain their characteristics after being cultured with DMEM + 20% FBS + Matrigel, by determining the expression of GBM markers in these cells. The results show that Si-GBM4, Si-GBM10, and Si-GBM16 cells strongly and homogenously expressed the typical GBM marker NESTIN, SOX2, CD44, and CD133 (Figs. 5A-B). It should be noted that although revived Si-GBM15 expressed NESTIN, SOX2, and CD44, they express a lower level of CD133 than other patient-derived primary GBM cells. The lower expression of CD133 in Si-GBM15 cells was determined by the lower percentage of CD133 + Si-GBM15 cells based on cell counts in flow cytometry data (Fig. 5B). CD133 is a well-established marker of glioblastoma stem-like cells (GSCs), a subpopulation characterized by its ability to self-renew, differentiate, and initiate tumors. In particular, CD133⁺ GBM cells have been reported to exhibit a higher proliferative capacity compared to CD133⁻ cells¹⁸. The lower percentage of CD133⁺ population in revived Si-GBM15 cells compared to other Si-GBM cells is associated with their lower proliferative capacity under our culture conditions (as shown by total cell count in Fig. 3). Although the percentage of CD133⁺ cells observed in Si-GBM10 cells was approximately 10% lower than that of Si-GBM4 and Si-GBM16, the level of CD133⁺ cells in revived Si-GBM10 cells was much higher than those found in Si-GBM15 cells. Our other experiments also show that the revived Si-GBM10 cells, although exhibited slightly lower percentage of CD133⁺ population, have the same levels of survival and growth, compared to Si-GBM4 and Si-GBM16, which have higher percentages of CD133⁺ cells. These results suggest that a combination of 20% FBS and Matrigel did not affect the expression of GBM markers in most long-term cryopreserved patient-derived GBM cells. Although reduced CD133 expression was observed in certain cell lines, this likely reflects intrinsic differences among the cell lines themselves, rather than being a result of the culture conditions.

Fig. 5.

Characterization of frozen-thawed Si-GBM cell lines after recovery. Expression of the stem cell markers NESTIN, SOX2 and Hoechst determined by immunofluorescent staining (A). Expression of the GBM cell markers CD44 and CD133 determined by flow cytometry (B). The numbers indicate percentage of positive cells of each marker. The pictures were taken at 10x magnification, scale bar: 100 μm.

Patient-derived GBM cells revived by a combination of 20% FBS and matrigel could be used for in vitro drug testing

To determine whether the revived patient-derived GBM cells can be used to test the effectiveness of the chemotherapeutic drug, the four patient-derived GBM cells were treated with various concentrations of Temozolomide (TMZ), a standard chemotherapeutic drug for GBM treatment, for 72 h. Result shows that Temozolomide (TMZ) treatment reduced the viability of all four patient-derived GBM cell lines in a dose-dependent manner, with minimal variation in the half-maximal inhibitory concentration (IC₅₀; indicated by the red dotted line) among them (Figs. 6A–D). By contrast, the U-251MG cell line demonstrated a markedly higher IC₅₀, as shown in Figs. 6E–F. These results demonstrated that long-term cryopreserved GBM cells can be used as a model to study the chemosensitivity to TMZ. This result also suggests that long-term cryopreserved patient-derived GBM cells could be effectively revived by cultured with DMEM + 20% FBS + Matrigel and used for personalized drug tests.

Fig. 6.

Chemotherapeutic responsiveness of long-term cryopreserved patient-derived GBM cells and U-251MG (A-E) after treatment with various concentrations of temozolomide. IC₅₀ values (mean ± SD) of Temozolomide (TMZ) in patient-derived GBM cell lines and U-251MG included as a control (F). Analysis of variance (ANOVA), values with asterisk *** are statistically different at probability values of p < 0.001.

Distinct characteristic of patient-derived GBM cells on their ability to form spheroids

In addition to a monolayer culture, a 3D spheroid culture has been considered to better mimic cellular activity and complexity and provide a much more accurate representation of GBM behavior and drug response in vivo. Therefore, we assessed the ability of revived patient-derived GBM cells to form tumor spheroids by culturing them in AggreWell™. The spheroids were then transferred to individual wells of a 6-well plate containing 2 mL of GBM culture medium and placed on an orbital shaker platform for over 48 h. The number of spheroids and their viability were observed at the 48-hour time point.

While Si-GBM4, Si-GBM10, and Si-GBM15 cells were capable of forming spheroids, their spheroid-forming efficiency differed significantly (Fig. 7A). Among these, Si-GBM10 cells produced fewer spheroids (Fig. 7B) with reduced viability (Fig. 7C) compared to Si-GBM4 and Si-GBM15 cells. Notably, Si-GBM16 cells, despite exhibiting robust growth in monolayer culture, failed to generate any spheroids (Figs. 7A–B). Based on these findings, Si-GBM10 and Si-GBM16 cells were excluded from subsequent experiments.

Fig. 7.

Spheroid formation capacity of each patient-derived GBM cells. Morphology of the spheroids (A), number of spheroids (B), and viability of the spheroids (C) derived from four patient-derived GBM cells as determined by CCK8 assay. The pictures were taken at 4x magnification, scale bar: 200 μm.

Patient-derived GBM cells revived by a combination of 20% FBS and matrigel maintain their migration capacity

To determine whether GBM spheroids maintain their migration capacity, an important property of GBM cells, spheroids derived from Si-GBM4 and Si-GBM15 cells were subjected to a 3D migration assay. These two cases were selected because they were capable of forming spheroids while still maintaining viability compared to the others. The results show that the revived Si-GBM4 and Si-GBM15 cells maintained their migration capacity, determined by an increase in both the migration area and the migration rate in a time-dependent manner (Figs. 8A-C). These results suggest that long-term cryopreserved patient-derived GBM cells maintain their migration capacity after being revived by culturing in DMEM + 20% FBS + Matrigel.

Fig. 8.

Migration of spheroids derived from Si-GBM4 and Si-GBM15. The area of migration, defined as the region between the white demarcated line (migration front) and the red demarcated line (spheroid boundary), reflects the cells’ migratory capacity from the spheroid (A). The migration area (B) and the migration rate (C) were determined at 24, 48 and 72 h. Data are presented as mean ± SEM. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. The pictures were taken at 4x magnification, scale bar: 200 μm.

GBM spheroids generated from revived patient-derived GBM cells can be used for in vitro drug testing

Next, we determine whether the 3D spheroid can be used to test the effectiveness of the chemotherapeutic drug, by treating the 3D spheroids derived from Si-GBM4 and Si-GBM15 cells with various concentrations of TMZ for 72 h. The result shows that TMZ markedly suppressed the migration of 3D spheroids from Si-GBM4 and Si-GBM15 in a dose-dependent manner (Figs. 9A–B). Consistent with these findings, CCK-8 assays performed after 72 h of treatment demonstrated a pronounced, dose-responsive reduction in cell viability (Fig. 9C). These results suggest that long-term cryopreserved patient-derived GBM cells revived by cultured with DMEM + 20% FBS + Matrigel could be induced to form a spheroid and used for personalized drug tests.

Fig. 9.

Migration of spheroids derived from Si-GBM4 and Si-GBM15 was determined after treatment with different concentrations of TMZ for 72 h. The area of migration, defined as the region between the migration front (white demarcated line) and the spheroid boundary (red demarcated line), reflects the cells’ migratory capacity from the spheroid (A). Migration area was calculated at 72 h (B). Viable cells determined by the CCK8 assay after being treated with TMZ for 72 h (C). Data are presented as mean ± SEM. Statistical significance was determined using two-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, ***P < 0.001. The pictures were taken at 4x magnification, scale bar: 200 μm.

Discussion

Although patient-derived GBM cells are an invaluable tool to investigate carcinogenic mechanisms and develop new treatments, but still facing some challenges¹⁹. Due to the limited availability of tissue samples and the inefficient derivation of primary GBM cells from resected tumors, most researchers use immortalized GBM cell lines or long-term cryopreserved primary GBM cells in their studies^20,21. However, most long-term cryopreserved GBM cells generally have a low survival rate after reviving, making it difficult to use for further study.

Our study showed that increasing the concentration of FBS from 10% to 20% and coating the culture surface with Matrigel significantly improve the survival and subsequent expansion of four long-term cryopreserved patient-derived GBM cells. The 4 patient-derived GBM cells (Supplementary Table 1), which exhibit different morphologies, maintained their distinct characteristics and highly expressed all typical GBM markers, NESTIN, SOX2, CD44, and CD133, after being revived by a combination of 20% FBS and Matrigel. Increased serum concentration and incorporation of ECM have been shown to activate cell growth by activating YAP and increasing TLR signaling. Among many factors presented in FBS, lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) have been shown to inhibit the Hippo pathway kinases Lats1/2 through G12/13-coupled receptors, resulting in the activation of YAP and TAZ transcription co-activators, which promote stemness and proliferation of cells including GBM^22,23. Consistent with this, our results show that a combination of 20% FBS and Matrigel increases YAP and TLR4 levels in these cells. Unlike other patient-derived GBM cells, a combination of 20% FBS and Matrigel did not increase YAP activity and TLR4 signaling in Si-GBM15 cells and could result in its failure to improve survival and subsequent expansion of these particular GBM cells. It has also been shown that ECM could activate TLR4 signaling in the context of tumor progression²⁴. However, it has also been suggested that FBS itself does not directly activate the TLR4 receptor. However, FBS contains trace amounts of lipopolysaccharide (LPS) which is known to be a TLR4 agonist. Therefore, when using FBS in cell culture medium, it can activate TLR4 resulting in the activation of cell proliferation^25,26.

It is known that ECM, growth factors, and nutrients play a crucial role in the survival, proliferation, and differentiation of various cancer cells, including GBMs^6–8. GBM have been shown to actively remodel their surrounding ECM and transform it into a permissive environment that promotes their growth, invasion, and neovascularization^27,28. Therefore, precoating the culture surface with Matrigel could promote GBM cell survival and growth. Consistent with this, our results show that a combination of 20% FBS with Matrigel, whose ECM composition is similar to that of GBM matrixes¹⁰, significantly improves the survival and subsequent expansion of four long-term cryopreserved patient-derived GBM cells compared to those revived with 20% FBS alone.

Unlike other protocols for spheroid formation, the spheroids in our study were formed in Aggrewell^® plate without the use of Matrigel or other scaffolds, which allows us to control the size of spheroids and reduces the variations between subsequent drug-testing experiments. It is interesting to note that GBM spheroids in this experiment were established using a scaffold-free method, therefore, it is possible that those GBM cells that failed to form spheroid in this experiment might be able to form a proper spheroid when co-cultured with scaffold or ECM.

Additionally, most long-term cryopreserved patient-derived GBM cells also maintain their spheroid formation capacity after being revived by a combination of 20% FBS and Matrigel that could subsequently be used for drug testing. Unlike other patient-derived GBM cells, Si-GBM16 cells, which grew very well in monolayer culture, did not form any spheroid. These results suggest distinct characteristic of each GBM cell line. Although GBM spheroids have been used to track the evolution of GBM, elucidate the resistance mechanisms, and identify potential drug targets^29,30, it has been showed that there are variations in the ability to form spheroid among patient-derived GBM cells^31,32. Consistent with this, our results show that some particular GBM cells, Si-GBM16, which grew very well in monolayer culture, could not form any spheroid, suggesting that the ability of GBM cells to grow in a monolayer culture might not always be correlated with their ability to generate spheroids. The difference in spheroid formation capacity between different patient-derived GBM cells could be due to genetic alterations that affect cell adhesion pathways or other factors related to the specific cell type and culture conditions^33,34.

Conclusion

In conclusion, we believe that our optimized protocol offers a valuable tool for increasing the availability of patient-derived GBM cells by allowing efficient recovery of long-term cryopreserved GBM cells for future research and personalized drug testing.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

WK: Formal analysis, Investigation, Methodology, Writing—review & editing. CL: Conceptualization, formal analysis, funding acquisition, writing original draft. PK: Conceptualization, formal analysis, writing—review & editing. SS: Conceptualization, providing GBM samples and chemical reagents. SI: Supervision, providing equipments and chemical reagents. All authors reviewed the manuscript.

Funding

This research project is supported by Mahidol University, [grant number R016633028 (fund 3)].

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics declarations

The patient-derived glioblastoma cells used in this study were originally collected more than 10 years ago by a previous research group, with informed consent obtained at the time of collection. As all patients have since passed away and the original investigators are now retired, re-contact with legal guardians was not possible. In recognition of the scientific value of these long-term cryopreserved samples, we sought approval from the institutional ethics committee, which reviewed the circumstances and granted authorization for their use in the present study. In addition, this manuscript does not contain any information or images that could lead to identification of the participant. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (COA no. S0 531/2024 and the date of approval is July 8, 2024).