culturing chopper-processed tumor pieces for generating patient-derived 3d glioma organoids

Derivazione di organoidi di glioma 3D da tumori di pazienti trattati con chopper

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&#39; 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&#39; 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.

Autore in primo piano: Generazione potenziata di organoidi 3D derivati da pazienti per glioblastoma e glioma

Coltivazione di organoidi di glioma derivati da pazienti ex vivo utilizzando un tritatutto di tessuti

1 So our research focuses on<v>2 improving generation of patient derived organoids 3 for glioblastoma patients. 4 And our key questions here revolve around 5 whether this approach can efficiently 6 produce more organoids, reduce manufacturing time, 7 and at the same time maintain 8 the characteristics of the original tumor. 9 And we would use that for an effective drug 10 and immunotherapy screening in glioblastoma patients.11 The recent developments in the glioblastoma field<v>12 include advancement in understanding the genetic 13 and molecular aspects leading to 14 the recognition of new targets. 15 Additionally, there's a great growing emphasis 16 on using 3D organoids 17 to better replicate the complexity of tumors 18 and allow drug testing. 19 Currently, technologies like manual dissection, 20 enzymatic dissociation, organ-on-a-chip, 21 and bioprinting are being widely used to prepare organoids.22 On the other hand, 23 mechanical chopping provides 24 metabolically active brain slices in various studies, 25 but lacks the methodological studies 26 for organoid preparation. 27 Our research has introduced an innovative method 28 for generating patient derived 3D organoids, 29 using an automated chopping machine, 30 we demonstrated a nearly 70%reduction in manufacturing time 31 and a significantly higher organoids count 32 compared to the manual dissection, 33 offering a more efficient method for drug 34 and immunotherapy screening in glioblastoma patients. 35 The advancement of our study significantly accelerates<v>36 the organoid production process, 37 and at the same time is able 38 to maintain the main characteristics of the original tumors.39 And by reducing the time as well as the resources required 40 for organoid generation, 41 our approach enhances the feasibility of large scale drug 42 and immunotherapy screening for glioblastoma patients.

Coltura di pezzi tumorali processati con tritatutto per la generazione di organoidi di glioma 3D derivati dal paziente

culturing-chopper-processed-tumor-pieces-for-generating-patient

Research

JoVE Journal

Cancer Research

Culturing Chopper-Processed Tumor Pieces for Generating Patient-Derived 3D Glioma Organoids

260 Views. 1 Per iniziare, prelevare il tessuto tumorale2 dai pazienti e lavorarlo con un tritatutto. 3 Inclinare la capsula di Petri contenente il tumore tritato 4 verso l'alto fino a 45 gradi e rimuovere con cura 2,5 millilitri 5 del terreno H-GPSA. 6 Per lavare i pezzi di tumore, aggiungere due millilitri 7 di tampone di lisi dei globuli rossi e incubare in un agitatore orbitale 8 a bassa velocità.9 Quindi, eliminare completamente il tampone di lisi. 1...

Video: Coltura di pezzi tumorali processati con tritatutto per la generazione di organoidi di glioma 3D derivati dal paziente

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

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 &#176;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 &lt; 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.

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 ...

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Processing Tumors with Chopper to Obtain Patient-Derived Organoids

1 To begin, transfer the tumor tissue<v>2 immersed in 25 milliliters of hibernate A 3 into a sterilized glass petri dish 4 under a laminar flow cabinet. 5 Using a microscope, carefully eliminate 6 all the necrotic tissue. 7 Then use a scalpel 8 and tissue forceps to dissect the blood vessels out.9 Cut the tumor tissue into 10 one to two cubic centimeter pieces. 11 Transfer them into a plastic petri dish 12 containing three milliliters of H-GPSA medium, 13 and place the dish on ice. 14 To manually process the tumor, transfer the tumor piece 15 to a fresh glass petri dish.16 Using two scalpels, dissect the segment 17 into 0.5 milliliter sections under a microscope. 18 To mechanically process the tumor, first, 19 position the blade properly. 20 Then adjust the slice thickness to 0.45 to 0.50 millimeters, 21 and fix the table release knob to start mode.22 Cut the agaro block into a two centimeter-long cylinder 23 and glue it onto the circular plastic dish of the chopper. 24 Using a scalpel, create a pit in the agaro cylinder 25 and place the tumor tissue in the pit. 26 Then position the plastic disc onto the mounting disc 27 of the cutting table, and press the reset button 28 to start the chopping process.29 After the first round, rotate the mounting disc 30 by 90 degrees, and repeat again 31 to cut the tissue into rectangular pieces. 32 Remove the plastic disc. 33 And then using a tissue spatula, 34 rotate only the tumor tissue by 90 degrees.35 Return the plastic disc to the cutting table 36 and press reset to continue cutting. 37 Then using a five milliliter pipette, 38 transfer the processed material, 39 along with the medium, to the petri dish. 40 Place the dish immediately on ice.

Elaborazione dei tumori con Chopper per ottenere organoidi derivati dal paziente

1 To begin, obtain the tumor tissue<v>2 from the patients, and process it with a chopper. 3 Tilt the Petri dish containing chopped tumor 4 upwards to 45 degrees, and carefully remove 2.5 milliliters 5 of the H-GPSA medium. 6 To wash the tumor pieces, add two milliliters 7 of RBC lysis buffer, and incubate in an orbital shaker 8 at a slow speed.9 Then, discard the lysis buffer completely. 10 Next, add four milliliters of patient-derived 11 organoid medium to the tissue pieces, 12 and transfer the tissue chunks to an ultra low attachment 13 six well-plate. 14 Incubate the plate in an orbital shaker 15 for two to four weeks.16 Every two days, change half of the spent medium 17 with a pre-warmed fresh patient-derived organoid medium. 18 To prevent tissue hypoxia, 19 transfer the patient-derived organoids 20 to a sterile glass Petri dish 21 and observe the tissue morphology under a microscope. 22 Then, using a scalpel, cut the adhesive tissue.23 Patient-derived organoids obtained using the chopper method, 24 reached the desired rounded shape within one week 25 as compared to the manually chopped ones. 26 And the number of organoids formed 27 was significantly higher using this method.