This research was funded by the Interdisciplinary Center of Clinical Research (IZKF, B-450) W&#252;rzburg, Bavarian Center of Cancer Research (BZKF) and the publication supported by Open Access Publishing Fund of the University of W&#252;rzburg. We would like to thank Dagmar Hemmerich and Siglinde K&#252;hnel, both Section Experimental Neurosurgery, Department of Neurosurgery, University Hospital W&#252;rzburg, for technical support. Figure 1 was created using&#160;www.biorender.com.

All patients were treated at the Department of Neurosurgery, University Hospital W&#252;rzburg, Germany, after giving written informed consent in accordance with the declaration of Helsinki and as approved by the Institutional Review Board of the University of W&#252;rzburg (#22/20-me). Tumor tissue material from four GBM patients and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients (low grade glioma, LGG) (Table 1) was obtained from surgery and processed using the following protocol. The automated process of generating PDOs utilizing a tissue chopper is referred to as chopper (C) and the process of manually cutting the tissue with two scalpels under microscopic control as manual (M). Six equally sized sections (1-2 cm3) were dissected from the tumor sample, then each one was cut in half and processed homogenously using the two methods. Due to the aforementioned intratumoral heterogeneity, one 6-well plate for each approach was generated from each patient with each well representing PDOs from a different site within the original tumor. Both procedures took place under a laminar airflow cabinet and all used instruments were sterilized prior to use. The overview of the approach is illustrated in Figure 1.
1. Preparing agarose blocks (for the C-approach only, optional)
<ol>
	<li>Fill 50 mL of phosphate buffered saline (PBS) into a beaker, add one tablet of agarose (see Table of Materials), and mix well until suspended.</li>
	<li>Heat the mixture in the microwave for 30-40 s, while avoiding boiling. Then cool the mixture down until it reaches 47 &#176;C.</li>
	<li>Pour the agarose mixture into a sealed cylinder-shaped casting mold and avoid any bubble-formation. Immediately cool the cast using a frozen (-20 &#176;C) clamp or by placing it on dry ice for 30 min.</li>
</ol>
2. Processing the tumor material
<ol>
	<li>Prepare a box of ice to keep the tumor material cooled on the way from the operating room to the laboratory.</li>
	<li>Transfer the tumor tissue (4-5 cm&#179;) into a sterile 50 mL tube containing 25 mL of Hibernate A (see Table of Materials) covering the tumor and place the tube into the ice box.</li>
	<li>Under a laminar airflow cabinet, transfer the tumor material together with the Hibernate A into a sterilized glass Petri dish.</li>
	<li>Eliminate the necrotic tissue and dissect the blood vessels carefully using a scalpel and tissue forceps under microscopic control. Identify necrotic tissue by hemorrhagic areas exhibiting a brownish hue resulting from bleeding, or tissue exhibiting a paler or whiter appearance relative to the adjacent viable tissue. Pay attention not to squeeze or disrupt the tissue.</li>
	<li>Cut the tumor material into six pieces with an approximate size of 1-2 cm3. Distribute the pieces to plastic Petri dishes (n = 6) pre-filled with 3 mL of H-GPSA medium (Table 2), each22. Place the Petri dishes on ice.</li>
</ol>
3. Setting up the tissue chopper
<ol>
	<li>Position the blade as described in the manufacturers&#39; manual23.</li>
	<li>Adjust the slice thickness to 0.45-0.50 mm. Set the blade force to medium. Fix the table release knob to &#34;start&#34; mode.</li>
</ol>
4. Processing the tumor tissue pieces
<ol>
	<li>Processing tumor tissue with the chopper (C method)
	<ol>
		<li>Cut the agarose blocks into cylinders of 2 cm length and glue one of these cylinders onto the chopper circular plastic dish using the histoacryl glue (see Table of Materials).</li>
		<li>Create a deepening in the agarose cylinder using a scalpel, and then fit the tumor tissue from the first well (step 2.5) within this pit. 
		NOTE: The tumor material should be handled carefully and not be squeezed or pushed into the gap. The gap should be big enough to fit the tumor easily, but small enough to keep the tumor material stable during the cutting process. Steps 4.1.1. and 4.1.2. are optional.</li>
		<li>Position the plastic disc onto the mounting disc of the cutting table (see Table of Materials).</li>
		<li>Switch the chopper on and press the reset button. The chopper now starts cutting (first round). The machine stops automatically after the table reaches the end and both agarose and tumor tissue are cut into the desired diameter.</li>
		<li>Rotate the mounting disc by 90&#176;, and then adjust the releasing table knob to the starting mode.</li>
		<li>Press the reset button and let the machine cut the tissue again creating rectangular shaped tissue (second round).</li>
		<li>Remove the plastic disc with the processed material and carefully rotate only the tumor tissue by 90&#176; using tissue spatula.</li>
		<li>Place the plastic disc onto the cutting table, then adjust the releasing table knob to the starting mode and press the reset button for a final cutting round (third round).</li>
		<li>Switch the chopper off and remove the plastic disc. Clean the chopper and the blades.</li>
		<li>Using a 5 mL single channel pipette, aspirate the processed material along with the medium into the pipette and flush the suspension back into the dish.</li>
		<li>Repeat the previous step 2-3 times to separate the tissue properly.</li>
		<li>Place the Petri dish back onto ice and repeat steps (4.1.1-4.1.12.) with the other 5 dishes of each tumor.</li>
	</ol>
	</li>
	<li>Processing tumor tissue manually (M method)
	<ol>
		<li>Transfer the tumor tissue from the first plastic Petri dish (step 2.5) together with 3 mL of H-GPSA medium (Table 2) into a glass Petri dish. Dissect the segment manually under the microscope into sections of 0.5 mm using two scalpels.</li>
		<li>Transfer the dissected tissue back to its plastic Petri dish using a 2 mL pipette.</li>
		<li>Repeat steps (4.2.1.-4.2.3.) for the tumor sections in the other five Petri dishes (step 2.5).</li>
	</ol>
	</li>
</ol>
5. Washing the tumor tissue
<ol>
	<li>Tilt each Petri dish upwards to 45&#176; and wait for 30 s until the tumor chunks sink to the bottom of the dish.</li>
	<li>Aspirate 2.5 mL of the H-GPSA medium (Table 2) carefully using a 1 mL pipette and be aware not to take up any tumor tissue.</li>
	<li>Add 2 mL of RBCs lysis buffer (see Table of Materials) to each sample. The processed tumor pieces must be covered completely by lysis buffer.</li>
	<li>Place the 6 dishes on a laboratory orbital shaking machine on slow speed for 10 min.</li>
	<li>Aspirate 2 mL of the lysis buffer carefully to not take up any tumor tissue.</li>
	<li>Repeat the previous washing steps (step 5.1-5.5) twice using 2 mL of H-GPSA medium (Table 2) instead of lysis buffer each time.</li>
</ol>
6. Culturing the tumor tissue
<ol>
	<li>Aspirate the H-GPSA medium (Table 2) from each dish and replace it with 4 mL of PDO medium (Table 2).</li>
	<li>Transfer the tissue chunks of each dish to one respective well of an ultra-low attachment 6-well plate (see Table of Materials).</li>
	<li>Place the plate onto an orbital shaker inside an incubator and incubate at 37 &#176;C, 5% CO2 and 150 rpm for 2-4 weeks.</li>
	<li>Perform a half medium change every two days by aspirating 2 mL of medium from each well and replacing it with 2 mL of fresh PDO medium (Table 2) pre-warmed to 37 &#176;C.</li>
	<li>Observe the tissue under the microscope (morphology, growth, medium color) and cut growing PDOs (&#62;0.7 mm) or adhesive tissue to prevent tissue hypoxia.
	<ol>
		<li>To do so, transfer the PDOs from the ultra-low attachment well to a sterilized glass Petri dish and use a scalpel for cutting. Alternatively, adhesive PDOs can be resolved by aspirating them with a 1 mL pipette. Be careful not to squeeze the PDOs and handle them gently.</li>
	</ol>
	</li>
	<li>Evaluate PDO formation by counting the PDO every two days and thoroughly check for the desired round morphology (Figure 2).</li>
</ol>
7. Fixing and embedding the PDOs
<ol>
	<li>Fix two PDOs from each well of each patient with 4% formalin for 24 h after 6 weeks of culture.</li>
	<li>Immerse the fixed PDOs in neutrally buffered (sodium phosphate) formalin until embedding.</li>
	<li>Place each PDO into a cassette (see Table of Materials) for further processing.</li>
	<li>Initiate a dehydration process by immersing the cassette in the following solutions as mentioned in the NOTE below: 
	NOTE: 50% ethanol for 20 min, 70% ethanol for 20 min, 80% ethanol for 20 min, 96% ethanol for 20 min, 100% ethanol for 20 min, 100% ethanol for 30 min, 100% ethanol + chloroform (1:1 ratio) for 30 min, 100% ethanol + chloroform (1:1 ratio) for 30 minutes, Absolute chloroform for 30 min, Absolute chloroform for 30 min, Paraffin for 30 min, Paraffin for 30 min using the STP 120.</li>
	<li>Embed the dehydrated PDOs in paraffin wax at 58-60 &#176;C.</li>
	<li>Slice the embedded PDOs at 2.5 &#181;m thickness and mount them on slides for staining.</li>
</ol>
8. Immunofluorescence staining
<ol>
	<li>Mount the PDOs after 6 weeks of culture.</li>
	<li>Subsequently, perform double staining against glial fibrillary acidic protein (GFAP, dilution: 1:100) and the proliferation marker Ki67 (dilution: 1:1000) (see Table of Materials), as previously reported22.</li>
	<li>Similarly, evaluate apoptosis by double staining PDOs against GFAP and anti-caspase-3 (CC3, dilution: 1:400) (see Table of Materials).</li>
	<li>Capture images of the PDOs using a fluorescence microscope at 40x magnification.</li>
	<li>Analyze the images for GFAP-, Ki67-, and CC3-positive cells, as well as GFAP/Ki67 and GFAP/CC3 double-positive cells.</li>
	<li>Utilize the open-source program Fiji (ImageJ-win 32) for image analysis.</li>
</ol>
9. Evaluation and data analysis
<ol>
	<li>Capture daily microscopic bright field pictures during the first week of culture at standard settings and 5x magnification.</li>
	<li>Observe the morphological changes and track the maturing process in both manual and automated processing approaches.</li>
	<li>Conduct morphological analysis using the microscope in standard brightfield mode settings.</li>
	<li>Evaluate the PDO number and morphology during the first 4 weeks of culture in PDOs from all five patients.</li>
	<li>Obtain three readings of each PDO count from each patient to calculate the mean and standard error of the mean (SEM).</li>
	<li>Investigate proliferation and apoptosis in PDOs from three patients.</li>
	<li>Analyze the data using a commercially available statistical software package (see Table of Materials).</li>
	<li>Apply Students-T and Mann-Whitney U Tests to determine differences between the manual and automated PDO generation in terms of PDO count, proliferation, and apoptosis.</li>
</ol>

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+mammalian+cell+perfusion-culture+system+in+microfluidic+channels.">A novel 3D mammalian cell perfusion-culture system in microfluidic channels.</a> Lab Chip. 7 (3), 302-309 (2007).</li><li>Zhang, C., Zhao, Z., Abdul Rahim, N. A., van Noort, D., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+human-on-chip:+culturing+multiple+cell+types+on+a+chip+with+compartmentalized+microenvironments.">Towards a human-on-chip: culturing multiple cell types on a chip with compartmentalized microenvironments.</a> Lab Chip. 9 (22), 3185-3192 (2009).</li><li>Homan, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-enhanced+vascularization+and+maturation+of+kidney+organoids+in+vitro.">Flow-enhanced vascularization and maturation of kidney organoids in vitro.</a> Nat Methods. 16 (3), 255-262 (2019).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+organ-level+lung+functions+on+a+chip.">Reconstituting organ-level lung functions on a chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Kengla, C., Atala, A., Sang Jin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+of+organoids.">Bioprinting of organoids.</a> Essentials of 3D Biofabrication and Translation. , 271-282 (2015).</li></ol>

The authors have nothing to disclose.

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.

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.

<table><tbody><tr><td>2-mercaptoethanol (1000x)</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>30% formaldehyde methanol-free</td><td>Carl Roth</td><td>4235.1</td><td>Used in 4% concentration</td></tr><tr><td>70% ethanol solution</td><td></td><td></td><td>For sterilisation&amp;nbsp;</td></tr><tr><td>Agarose tablets 0.5 g</td><td>Carl Roth</td><td>HP67.7</td><td></td></tr><tr><td>Amphotericin B 250 &amp;micro;g/mL</td><td>Gibco</td><td>15290018</td><td></td></tr><tr><td>Anatomical forceps</td><td>&amp;nbsp;Hartstein</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Anatomical spatula</td><td>&amp;nbsp;Hartstein</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>B-27 Supplement without vitamin A (50x)</td><td>Gibco</td><td>12587010</td><td></td></tr><tr><td>Biopsy cassette with cover</td><td>Resolab</td><td>37001-b</td><td></td></tr><tr><td>Blades for McIlwain Tissue Chopper</td><td>Campden instruments</td><td>Model TC752-1</td><td></td></tr><tr><td>CC3 antibody (Asp 175)</td><td>Cells signaling technology</td><td>9661</td><td></td></tr><tr><td>Disposable scalpel</td><td>&amp;nbsp;Feather</td><td>&amp;nbsp;0200130015</td><td></td></tr><tr><td>Distilled water</td><td>Gibco</td><td>15230089</td><td>To dilute the formaldehyd</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Serum Nutrient Mixture (DMEM) F-12 (1:1) (1x)</td><td>Gibco</td><td>11330032</td><td>Includes L-Glutamine and 15 mM HEPES</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Sigma Life Sciences</td><td>D8537-500ML</td><td>Modified, without calcium, chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture</td></tr><tr><td>eBioscience 1x RBC Lysis Buffer</td><td>Invitrogen</td><td>433357</td><td></td></tr><tr><td>Falcon tube 50 ml Cellstar</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>GFAP antibody</td><td>Santa Cruz Biotechnology</td><td>sc33673</td><td></td></tr><tr><td>Glass beaker</td><td>&amp;nbsp;N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Glass petri dish</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Heracell 240i CO2 Incubator</td><td>Thermo scientific</td><td>51032875</td><td></td></tr><tr><td>Herasafe 2025 Biological Safety Cabinet</td><td>Thermo scientific</td><td>5016643</td><td></td></tr><tr><td>Hibernate-A</td><td>Gibco</td><td>A1247501</td><td></td></tr><tr><td>Histoacryl glue</td><td>B. Braun surgical</td><td>1050052</td><td></td></tr><tr><td>Human Insulin, Solution</td><td>Santa Cruz Biotechnology</td><td>sc-360248</td><td></td></tr><tr><td>Ice box</td><td>&amp;nbsp;N/A</td><td>&amp;nbsp;N/A</td><td></td></tr><tr><td>Ki67 antibody</td><td>Abcam</td><td>ab16667</td><td></td></tr><tr><td>McIlwain Tissue Chopper</td><td>Cavey Laboratory Engineering</td><td>51350V</td><td></td></tr><tr><td>Microscope Leica DMI 3000B, DMI 4000B, DMI 6000B</td><td>Leica</td><td>DMI6000B</td><td>For brightfield and immunofluorescence pictures</td></tr><tr><td>Microscope stereozoom S9D</td><td>Leica</td><td>W841832</td><td>For manual cutting and to organoids monitoring</td></tr><tr><td>Microwave</td><td>Bosch</td><td>N/A</td><td>To heat the agarose solution</td></tr><tr><td>Mounting plastic discs</td><td>Cavey Laboratory Engineering</td><td>51354</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>NEM Non-Essential Amino Acids (NEAA) (100x)</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Neurobasal (1x)</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>Orbital shaking machine Rotamax120</td><td>Heidolph</td><td>10304491</td><td></td></tr><tr><td>Penicilin Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Plastic petri dishes Cellstar</td><td>greiner bio-one</td><td>628160</td><td>n = 12</td></tr><tr><td>Single channel pipette 1000 &amp;micro;m</td><td>Eppendorf</td><td>4924000010</td><td></td></tr><tr><td>Single channel pipette 5000 &amp;micro;m</td><td>Eppendorf</td><td>EP3123000276</td><td></td></tr><tr><td>Statistical Package for the Social Sciences (SPSS) version 23.0</td><td>IBM</td><td></td><td></td></tr><tr><td>Surgipath Paraplast</td><td>Leica</td><td>39601006</td><td>Embedding medium</td></tr><tr><td>Ultra-low attachment Nucleon Sphera 6-well plate</td><td>Thermo Scientific</td><td>174932</td><td></td></tr></tbody></table>

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.

Four patients with GBM and one with LGG were included after&#160;pathological confirmation by an experienced neuropathologist (CMM). The majority of patients had an unmethylated MGMT promoter, and all GBM patients were IDH1 and IDH2 wild type (Table 1). On average, the manufacturing process lasted 88.8 min (+/- 6.3 min) in the C approach and 322 min (+/- 17.2 min) in the M approach. The overall success rate was 87% in the manual and 93% in the chopper approach after 4 weeks of culture (n = 5). Moreover, PDOs derived from the C group reached the desired rounded shape within 1 week and were mature enough to be used in in vitro experiments, while the PDOs of the M group mostly remained sharply edged and undefined (Figure 2). The tumor tissue processed with the C approach resulted in overall 281 PDOs (Mean per patient = 56 +/- 43) after the first week of culture, while 250 PDOs (Mean per patient= 50 +/- 41) developed with the M approach. During the second week of culture, the tissue of all five patients yielded higher PDO numbers when they were generated with the C approach (801; Mean per patient= 130 +/- 38) compared to the M approach (601; Mean per patient= 76 +/- 44; P = 0.018). During the third week of culture, the C approach accumulated overall 1105 PDOs from all patients (Mean per patient = 221 +/- 32) compared to 771 PDOs (Mean per patient= 155 +/- 34) in the M approach (P = 0.032). Furthermore, a total of 1195 PDOs (Mean per patient= 239 +/- 50) formed after four weeks of culture when generated with the C approach compared to 784 (Mean per patient= 157 +/- 36) utilizing the M approach (P &#60; 0.01). Therefore, the C method showed a significantly higher PDOs count starting from the second week (Figure 3). Furthermore, the relative fluctuations in PDO counts were evaluated to explore the dynamic trends between successive weeks. The analysis unveiled an impressive surge in PDO counts during the initial transition from the first to the second week in the C approach (265%), which was indicative of rapid progress. Subsequently, there was a lower rise in counts during the third week (75%), reflecting a temporary adjustment. In contrast, the M approach demonstrated a consistent and steady increase in PDO counts (92% in second week, respectively 67% in the third week), which contributed to a remarkable stability in counts during the fourth week. This consistent upward trend in PDO counts underscores the reliability and resilience of the C approach throughout the observation period.
Two GBM patients and one LGG patient were included for the analysis of astrocyte-numbers (GFAP) within PDOs, PDO cell proliferation (Ki67), and apoptosis (CC3). The determined astrocyte count revealed no significant differences between the two processing methods with an average of 43% in the C approach and 45% in the M approach (Figure 4 and Figure 5, Supplementary Figure 1 and Supplementary Figure 2). Similarly, the proliferation rates within the PDOs were comparable between the C (3%) and M approaches (1%). Only PDOs generated with the C approach from patient 5 displayed a proliferation rate of 26% compared to 1% with the M approach (P = 0.001; Figure 4C). Overall low apoptosis rates were detected in PDOs processed with the C approach (3%) compared to 2% from the M approach for all patients, which were not significantly different (Figure 5C). Furthermore, there was no significant difference between the two methods regarding the number of astrocytes undergoing apoptosis (Figure 5D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65952/65952fig01.jpg" /> 
Figure 1: Graphical overview of the patient-derived organoid (PDO) manufacturing process using an automated chopper versus a manual approach. The illustration depicts the various steps involved, including (A) sample collection, (B) tumor material dissection, (C) washing, and (D) incubation. <a href="https://www.jove.com/files/ftp_upload/65952/65952fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65952/65952fig02.jpg" /> 
Figure 2: PDO morphology during the first week of culture. Comparison of PDOs formation post-dissection using both the automated chopper and manual method. Scale bars = 1000 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65952/65952fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65952/65952fig03.jpg" /> 
Figure 3: PDO count during the first four weeks of culture. The x-axis displays the time in weeks (W), and the y-axis the number of PDOs of the C (blue) and M (red) approach (n = 5). Each data point represents the mean count, with error bars indicating the standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/65952/65952fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65952/65952fig04.jpg" /> 
Figure 4: Cell proliferation rates within PDOs. (A) Representative immunofluorescence images (n = 3) of GFAP-positive cells (green), Ki67-positive cells (red), and DAPI (blue) in PDOs from patients 3, 4 and 5 (Table 1). All PDOs were processed using the chopper (C) and manual (M) methods. Scale bars = 100 &#181;m. (B) Comparison of the two methods regarding the relative number of GFAP-positive cells, (C) Ki67-positive cells, and (D) Ki67/GFAP-double positive cells. Not significant results are indicated by &#34;ns&#34;. <a href="https://www.jove.com/files/ftp_upload/65952/65952fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65952/65952fig05.jpg" /> 
Figure 5: Apoptosis rates within PDOs. (A) Representative immunofluorescence images (n = 3) of GFAP-positive cells (green), CC3-positive cells (red), and DAPI (blue) in PDOs from patients 3, 4 and 5 (Table 1). All PDOs were processed using the chopper (C) and manual (M) methods. Scale bars = 100 &#181;m. (B) Comparison of the two methods regarding the relative number of GFAP-positive cells, (C) CC3-positive cells, and (D) CC3/GFAP-double positive cells. Not significant results are indicated by &#34;ns&#34;. <a href="https://www.jove.com/files/ftp_upload/65952/65952fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Patients&#39; characteristics and clinical parameters. GBM = glioblastoma, IDH-wild type, CNS WHO grade 4; LGG = low grade glioma; KPS = Karnofsky performance score; MGMT = O6-methylguanine-DNA methyltransferase; IDH1 = isocitrate dehydrogenase 1, IDH2 = isocitrate dehydrogenase 2, ATRX = &#945;-thalassemia/mental retardation, X-linked gene; M = morphology; CC = cell count; p = proliferation; A = apoptosis. <a href="https://www.jove.com/files/ftp_upload/65952/65952_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Medium compositions. H-GPSA = Hibernate A-Glutamax pencillin streptomycin amphotericin B. PDO = Patient-derived organoids&#160;DMEM: Dulbecco&#8217;s Modified Eagle&#8217;s Medium. NEAA: non-essential amino acids. Pen Strep: Penicillin/ Streptomycin <a href="https://www.jove.com/files/ftp_upload/65952/65952_Table 2_updated.zip" target="_blank">Please click here to download this Table.</a>
Table 3: Overview of the used techniques to generate cell culture models. <a href="https://www.jove.com/files/ftp_upload/65952/Table 3_updated.zip" target="_blank">Please click here to download this Table.</a>
Supplementary Figure 1: Individual channels of proliferation staining of PDOs. (A) DAPI (blue), (B) GFAP-positive cells (green), (C) Ki67-positive cells (red) and (D) overlay channel in PDOs from patients 3, 4 and 5. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65952/Supplementary figure1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Individual channels of apoptosis staining of PDOs. (A) DAPI (blue), (B) GFAP-positive cells (green), (C) CC3-positive cells (red) and (D) overlay channel in PDOs from patients 3, 4 and 5. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65952/Supplementary figure2.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma at JoVE.com

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

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.

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 ...

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1.7K Views.  University Hospital Würzburg. 1 Ons onderzoek richt zich dus op2 het verbeteren van het genereren van van patiënten afgeleide organoïden 3 voor glioblastoompatiënten. 4 En onze belangrijkste vragen hier draaien om 5 of deze aanpak efficiënt 6 meer organoïden kan produceren, de productietijd kan verkorten, 7 en tegelijkertijd 8 de kenmerken van de oorspronkelijke tumor kan behouden. 9 En we zouden dat gebruiken voor een effectief medicijn 10 en immunotherapiescreening bij glioblast...

Video: Auteur in de schijnwerpers: verbeterde generatie van van patiënten afgeleide 3D-organoïden voor glioblastoom en glioom

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Medicine

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Bioengineering

Generating and Maintaining Slice Cultures from Human Glioblastoma Tissue

Source: Parker, J. J., et al. <a href="https://app.jove.com/v/53557">A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs.</a> J. Vis. Exp. (2017).
The video demonstrates the generation and maintenance of organotypic slice cultures from glioblastoma tissue. The isolated tissue is embedded in an agarose matrix, and thin sections are obtained using a vibratome. These sections are then placed on permeable membrane inserts within a microplate and cultured in a slice culture maintenance media for long-term maintenance.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pre-slicing Preparation 

	...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating an Organoid Culture from a Brain Tumor Cell Suspension

Source: Sundar, S. J. et al.&#160; <a href="https://app.jove.com/v/63745">Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture.</a> J. Vis. Exp. (2022).
This video describes the preparation and cultivation of brain tumor organoids from single-cell suspension. Using a laminin-rich extracellular matrix as a scaffold and culture media for nutrients, cells are facilitated to multiply and self-organize within the organoid. Consequently, ex vivo organoids are generated, mimicking cellular diversity and the microenvironment of brain tumors.

1. Making organoids from a single-cell suspension

	In an ice bucket or cold block, place the laminin-rich extracellular matrix (lrECM) and a small ...

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

Samenvatting

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