This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.
Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 °C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P < 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.
Low-grade gliomas (LGGs) are a group of relatively rare brain tumors that typically present as slow-growing and less aggressive compared to high-grade gliomas like glioblastoma. They can occur in both adults and children, with a slightly higher prevalence in adults. The exact prevalence varies by region and population, but LGGs account for approximately 15%-20% of all primary brain tumors1. Treatment strategies for LGGs often involve a combination of surgery, radiation therapy, and chemotherapy, aiming to maximize tumor resection while preserving neurological function. The management of LGGs can be complex, and the choice of therapy may depend on factors such as tumor location and molecular characteristics2. Advances in understanding the genetic and molecular underpinnings of LGGs have led to more targeted therapies, and ongoing research continues to refine treatment approaches.
Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM), on the other hand, is the most prevalent primary brain tumor found in adults, with an incidence rate between 3.19-4.17 cases per 100,000 person-years3. GBM causes symptoms such as headaches, seizures, focal neurological deficits, changes in personality, and increased intracranial pressure. The standard treatment for GBM involves debulking of the tumor, if feasible, followed by radiation therapy combined with Temozolomide4. Furthermore, combining Temozolomide and Lomustine may enhance the median overall survival rate in patients with O6-methylguanine-methyltransferase (MGMT)-promoter methylation5. However, despite these recent therapeutic approaches, GBM remains an incurable disease with poor prognosis, characterized by a patients' median overall survival rate of 16 months up to 20.9 months when Tumor Treating Fields (TTFields) is added3,6. Several immunotherapeutic approaches have been investigated in GBM but demonstrated limited efficacy in vivo. Moreover, clinical and preclinical limitations hinder therapeutic breakthroughs7. The establishment of a suitable and realistic ex vivo model has been challenging due to the inter-8 and intratumoral9 heterogeneity of GBM.
Conventional 2-dimensional (2D) patient cell lines represent homogeneous cell populations and are suitable for high-throughput drug screening. However, patient-derived and immortalized cell lines fail to mimic GBM adequately due to differences in growth conditions and deviations in genotypic and phenotypic features after multiple passages10,11,12.
On the other hand, 3D organoid models have recently emerged as promising systems that replicate the phenotypic and molecular heterogeneity of organ and various cancer types13,14,15,16,17,18. In the context of GBM, cerebral organoids have been genetically modified to simulate tumor-like characteristics16,17 or co-cultured with GSCs or spheroids to induce tumor cell infiltration18,19. While patient-derived GBM organoids cultured with Matrigel and EGF/bFGF exhibit GBM hallmarks such as stem cell heterogeneity and hypoxia20, it remains uncertain to what extent this model can represent the key molecular properties of patients' neoplasms.
Patient-derived GBM organoids (PDOs) are promising models that can maintain the predominant features of their analogous parental tumors, including histological characteristics, cellular diversity, gene expression, and mutational profiles. Additionally, they are rapidly infiltrated upon implantation into adult rodent brains, providing a realistic model for drug testing and personalized therapy21. However, manually dissecting tumor tissue to generate PDOs is time-consuming and costly. Therefore, an urgent need exists for a rapid method that can produce large numbers of PDOs, enabling comprehensive assessment of different therapeutic approaches holding promise for individualized drug testing. This study describes a new method for manufacturing PDOs directly from freshly dissected tumor tissue using an automatic tissue chopper. Furthermore, PDOs generated by this method were compared with manually dissected PDOs from the same patients in terms of PDO count, morphological features, apoptosis and proliferation of tumor cells.
This study presents a quick and efficient method for generating PDOs. GBM remains a challenging tumor to treat, often characterized by relapse and a high disease burden3,6. Innovative therapeutic approaches are urgently needed, as promising results observed in vitro often fail to demonstrate efficacy in vivo during phase-I trials. One of the reasons for this discrepancy could be the limited ability of patient-derived immortalized cell lines, grown in monolayer cultures, to reflect the complex cell-cell interactions and genetic properties of the parental tumor. Given the high inter- and intratumoral heterogeneity of GBM8,9, personalized targeted therapies are preferred and may hold promise for future applications. In contrast to 2D adherent cell lines, organoids have the ability to retain the properties of the parental tissue21, yet complex cell-cell interactions between the tumor and the normal brain are of paramount importance and could potentially be overlooked by this model. However, manual generation of PDOs is a time-consuming process, and tissue damage caused by squeezing with scalpels during cutting can hinder successful PDO growth. Therefore, an automated method was optimized using a tissue chopper to generate higher numbers of PDOs with reduced time and effort. Additionally, we demonstrated that overall proliferation and apoptosis rates did not differ between the two approaches.
The C approach is straightforward, easy to implement, and enables the generation of a larger number of PDOs (Figure 3). The rotation of the tissue between the second and third rounds of chopping was identified as a critical step in the protocol. At this stage, the tissue has already lost its integrity and can easily fall apart, resulting in larger pieces that require additional cutting or manual dissection under the microscope. While the automated chopper approach allows for a preset cutting size with greater accuracy, the manual approach lacks precision in determining the size of PDOs, leading to unevenly shaped and sized PDOs, which is a disadvantage for comparative drug screening (Figure 2). Nonetheless, with the proposed method, the standardization of cell numbers per PDO is not achieved, potentially posing a drawback for standardized drug screening protocols. The advantages and disadvantages of different organoid generation techniques18,19,20,24,25,26,27,28,29,30,31,32,33,34,35,36,
37,38,39,40,41,42 and their applications are summarized in Table 3.
GBM tissue can vary in consistency, ranging from tough (infiltration zone) to soft (necrotic core), which can pose challenges for the automated chopper approach. If the tissue is too tough, the chopper may squeeze and damage it, whereas too soft tissue might be squashed. The chosen tissue displayed distinctive attributes, including an intermediate level of firmness, sporadically featuring a pinkish-grayish coloration rather than manifesting brown or yellow discoloration. Tissue possessing a spongy and readily crumbly texture demonstrated superior preservation within the agarose blocks, whereas exceedingly delicate and liquified tumor tissue was omitted from the sampling procedure. However, the chopper approach enabled a successful generation of a higher number of PDOs compared to the manual approach, even with tissue of suboptimal consistency. The key solution is to maintain close interaction with the surgeon performing the tumor resection to process tissue from different areas of the tumor. In cases of suboptimal tissue consistency, manually reworking the tissue under the microscope was a helpful addition after chopping. To account for heterogeneity, the tumor tissue was initially divided into six segments, each subsequently halved for either the C or M approach. Within these six distinct sections, a substantial degree of heterogeneity is anticipated. Furthermore, even within the PDOs from the same section or well, the presence of distinct subpopulations is plausible.
As proof of concept, the proliferation and apoptosis data were reported from two patients with GBM and one patient with LGG, which show no significant differences between the two methods. The generation of PDOs is not limited to highly malignant brain tumors but can also be applied to LGGs. This study highlights that LGG seldom exhibit growth in 2D culture, making the development of an accurate model for their study highly valuable. This protocol aims to demonstrate the versatility of this approach in generating PDOs from GBM as well as LGG quickly and effectively.
Overall, PDOs could be utilized in the future for patient-oriented pre-therapeutic testing of targeted therapies in malignant brain tumors. Providing a quick and efficient method for individualized drug screening is crucial, as tumor progression occurs rapidly, and salvage treatment options are desperately needed. As a next step, the PDO model could be evaluated with various immunotherapeutic approaches to better mimic real treatment responses. In the future, PDOs could be utilized to draw sophisticated conclusions regarding the need for further exploration and evaluation of therapies in a clinical setting.
The authors have nothing to disclose.
This research was funded by the Interdisciplinary Center of Clinical Research (IZKF, B-450) Würzburg, Bavarian Center of Cancer Research (BZKF) and the publication supported by Open Access Publishing Fund of the University of Würzburg. We would like to thank Dagmar Hemmerich and Siglinde Kühnel, both Section Experimental Neurosurgery, Department of Neurosurgery, University Hospital Würzburg, for technical support. Figure 1 was created using www.biorender.com.
2-mercaptoethanol (1000x) | Gibco | 21985023 | |
30% formaldehyde methanol-free | Carl Roth | 4235.1 | Used in 4% concentration |
70% ethanol solution | For sterilisation | ||
Agarose tablets 0.5 g | Carl Roth | HP67.7 | |
Amphotericin B 250 µg/mL | Gibco | 15290018 | |
Anatomical forceps | Hartstein | N/A | |
Anatomical spatula | Hartstein | N/A | |
B-27 Supplement without vitamin A (50x) | Gibco | 12587010 | |
Biopsy cassette with cover | Resolab | 37001-b | |
Blades for McIlwain Tissue Chopper | Campden instruments | Model TC752-1 | |
CC3 antibody (Asp 175) | Cells signaling technology | 9661 | |
Disposable scalpel | Feather | 0200130015 | |
Distilled water | Gibco | 15230089 | To dilute the formaldehyd |
Dulbecco's Modified Eagle Serum Nutrient Mixture (DMEM) F-12 (1:1) (1x) | Gibco | 11330032 | Includes L-Glutamine and 15 mM HEPES |
Dulbecco's Phosphate Buffered Saline (PBS) | Sigma Life Sciences | D8537-500ML | Modified, without calcium, chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture |
eBioscience 1x RBC Lysis Buffer | Invitrogen | 433357 | |
Falcon tube 50 ml Cellstar | Greiner Bio-One | 227261 | |
GFAP antibody | Santa Cruz Biotechnology | sc33673 | |
Glass beaker | N/A | N/A | |
Glass petri dish | N/A | N/A | |
GlutaMAX (100x) | Gibco | 35050061 | |
Heracell 240i CO2 Incubator | Thermo scientific | 51032875 | |
Herasafe 2025 Biological Safety Cabinet | Thermo scientific | 5016643 | |
Hibernate-A | Gibco | A1247501 | |
Histoacryl glue | B. Braun surgical | 1050052 | |
Human Insulin, Solution | Santa Cruz Biotechnology | sc-360248 | |
Ice box | N/A | N/A | |
Ki67 antibody | Abcam | ab16667 | |
McIlwain Tissue Chopper | Cavey Laboratory Engineering | 51350V | |
Microscope Leica DMI 3000B, DMI 4000B, DMI 6000B | Leica | DMI6000B | For brightfield and immunofluorescence pictures |
Microscope stereozoom S9D | Leica | W841832 | For manual cutting and to organoids monitoring |
Microwave | Bosch | N/A | To heat the agarose solution |
Mounting plastic discs | Cavey Laboratory Engineering | 51354 | |
N-2 Supplement (100x) | Gibco | 17502048 | |
NEM Non-Essential Amino Acids (NEAA) (100x) | Gibco | 11140050 | |
Neurobasal (1x) | Gibco | 21103049 | |
Orbital shaking machine Rotamax120 | Heidolph | 10304491 | |
Penicilin Streptomycin | Gibco | 15140122 | |
Plastic petri dishes Cellstar | greiner bio-one | 628160 | n = 12 |
Single channel pipette 1000 µm | Eppendorf | 4924000010 | |
Single channel pipette 5000 µm | Eppendorf | EP3123000276 | |
Statistical Package for the Social Sciences (SPSS) version 23.0 | IBM | ||
Surgipath Paraplast | Leica | 39601006 | Embedding medium |
Ultra-low attachment Nucleon Sphera 6-well plate | Thermo Scientific | 174932 |