<meta charset="utf8"></head><body>אורגנואידים של מלנומה שמקורם בחולה: חזית באימונותרפיה לסרטן

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	<li>Mirabelli, P., Coppola, L., Salvatore, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Cell+Lines+Are+Useful+Model+Systems+for+Medical+Research.">Cancer Cell Lines Are Useful Model Systems for Medical Research.</a> Cancers (Basel). 11 (8), 1098 (2019).</li><li>Shannon, A. E., Boos, C. E., Hummon, A. 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Y., 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=Conservation+of+copy+number+profiles+during+engraftment+and+passaging+of+patient-derived+cancer+xenografts.">Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts.</a> Nat Genet. 53 (1), 86 (2021).</li><li>Somasundaram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-infiltrating+mast+cells+are+associated+with+resistance+to+anti-PD-1+therapy.">Tumor-infiltrating mast cells are associated with resistance to anti-PD-1 therapy.</a> Nat Commun. 12 (1), 346 (2021).</li><li>Abdolahi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+(PDX)+models,+applications+and+challenges+in+cancer+research.">Patient-derived xenograft (PDX) models, applications and challenges in cancer research.</a> J Transl Med. 20 (1), 206 (2022).</li><li>Yang, S., 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=Organoids:+The+current+status+and+biomedical+applications.">Organoids: The current status and biomedical applications.</a> Med Comm. 4 (3), e274 (2023).</li><li>Minoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+cancer+organoids+as+a+functional+system+to+model+different+disease+stages+and+therapy+response.">Bladder cancer organoids as a functional system to model different disease stages and therapy response.</a> Nat Commun. 14, 2214 (2023).</li><li>Ou, L., 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=Patient-derived+melanoma+organoid+models+facilitate+the+assessment+of+immunotherapies.">Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.</a> eBioMedicine. 92, 104614 (2023).</li><li>Gr&#246;nholm, M., 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=Patient-derived+organoids+for+precision+cancer+immunotherapy.">Patient-derived organoids for precision cancer immunotherapy.</a> Cancer Res. 81 (12), 3149-3155 (2021).</li><li>Ou, L., 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=Preclinical+platforms+to+study+therapeutic+efficacy+of+human+&#947;&#948;+T+cells.">Preclinical platforms to study therapeutic efficacy of human &#947;&#948; T cells.</a> Clin Transl Med. 12 (6), e814 (2022).</li><li>Murine L-Wrn conditioned medium protocol. The University of Texas MD Anderson Cancer Center Available from: <a target="_blank" href="https://www.mdanderson.org/documents/Labs/Taniguchi%20Laboratory/Mouse%20CM.pdf">https://www.mdanderson.org/documents/Labs/Taniguchi%20Laboratory/Mouse%20CM.pdf</a> (2024)</li><li>Jee, J. H., 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=Development+of+collagen-based+3D+matrix+for+gastrointestinal+tract-derived+organoid+culture.">Development of collagen-based 3D matrix for gastrointestinal tract-derived organoid culture.</a> Stem Cells Int. 13 (2019), 8472712 (2019).</li><li>Pipkin, M. E., Rao, A., Lichtenheld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptional+control+of+the+perforin+locus.">The transcriptional control of the perforin locus.</a> Immunol Rev. 235 (1), 55 (2010).</li><li>Li, X., 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=Tim-3+suppresses+the+killing+effect+of+V&#947;9V&#948;2+cells+on+colon+cancer+cells+by+reducing+perforin+and+granzyme+B+expression.">Tim-3 suppresses the killing effect of V&#947;9V&#948;2 cells on colon cancer cells by reducing perforin and granzyme B expression.</a> Exp Cell Res. 386 (1), 111719 (2020).</li><li>Li, L. T., Jiang, G., Chen, Q., Zheng, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki67+is+a+promising+molecular+target+in+the+diagnosis+of+cancer+(Review).">Ki67 is a promising molecular target in the diagnosis of cancer (Review).</a> Mol Med Rep. 11 (3), 1566-1572 (2015).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Li, H. Y., Ren, K., Wang, C., Bu, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+organoid+research+progress+and+potential+applications.">Skin organoid research progress and potential applications.</a> International Journal of Dermatology and Venereology. 5 (2), 101-106 (2022).</li><li>Sun, L., 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+human+mucosal+melanoma+organoid+platform+for+modeling+tumor+heterogeneity+and+exploring+immunotherapy+combination+options.">A human mucosal melanoma organoid platform for modeling tumor heterogeneity and exploring immunotherapy combination options.</a> Sci Adv. 9 (43), (2023).</li><li>Vlachogiannis, G., 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=Patient-derived+organoids+model+treatment+response+of+metastatic+gastrointestinal+cancers.">Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.</a> Science. 359 (6378), 920-926 (2018).</li><li>Ganesh, K., 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+rectal+cancer+organoid+platform+to+study+individual+responses+to+chemoradiation.">A rectal cancer organoid platform to study individual responses to chemoradiation.</a> Nat Med. 25 (10), 1607-1614 (2019).</li><li>Vilgelm, A. E., 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=Fine-needle+aspiration-based+patient-derived+cancer+organoids.">Fine-needle aspiration-based patient-derived cancer organoids.</a> iScience. 23 (8), 101408 (2020).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mL Red Cap Internal Threaded Polypropylene Cryogenic Vial</td>
			<td>Corning</td>
			<td>431420</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm TC-treated Cell Culture Dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>105 mm Polystyrene Forceps Sterile</td>
			<td>Caplugs Evergreen&#160;</td>
			<td>222-1121-B1I</td>
			<td></td>
		</tr>
		<tr>
			<td>150 cm2 Cell Culture Flask</td>
			<td>Corning</td>
			<td>CLS430825</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12 (1x) Reduced Serum Medium (1:1)</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 510 anti-human Perforin Antibody</td>
			<td>BioLegend</td>
			<td>308120</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 605 anti-human Ki-67 Antibody</td>
			<td>BioLegend</td>
			<td>350522</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 anti-human CD366 (Tim-3) Antibody</td>
			<td>BioLegend</td>
			<td>345028</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 711 anti-human CD45 Antibody</td>
			<td>BioLegend</td>
			<td>304050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Inserts 0.4 &#181;m, 30 mm Diameter</td>
			<td>Millipore Sigma</td>
			<td>PIHP03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Staining Buffer</td>
			<td>BioLegend</td>
			<td>420201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning&#160;</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning&#160;</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type IV, powder</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Rat Collagen I</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Plate, PS, 48 wells, TC treated with lid, sterile</td>
			<td>Max Scientific&#160;</td>
			<td>07-6048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM - high glucose 4.5 mg/mL</td>
			<td>Corning</td>
			<td>MT10-0130CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I - Grade II</td>
			<td>Millipore Sigma</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, 1%&#160;</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, FBS</td>
			<td>Corning</td>
			<td>MT35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Annexin V Apoptosis Detection Kit I</td>
			<td>BD Biosciences&#160;</td>
			<td>556547</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Tocris</td>
			<td>1099</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)</td>
			<td>ThermoFisher</td>
			<td>10131035</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F12 Nutrient Mix</td>
			<td>ThermoFisher</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B (50 mg/mL)</td>
			<td>ThermoFisher</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN cell</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Phenol Free &#38; Growth Fact. Reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex PVDF syringe filter, 0.22 &#956;M</td>
			<td>Millipore Sigma</td>
			<td>SLGVR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc TripleFlask Treated Cell Culture Flask</td>
			<td>ThermoFisher</td>
			<td>132867</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine5 anti-human CD8a Antibody</td>
			<td>BioLegend</td>
			<td>301010</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Cy 7 Mouse anti-Human CD279 (PD-1)</td>
			<td>BD Biosciences&#160;</td>
			<td>561272</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-acidic</td>
			<td>Peprotech</td>
			<td>100-17A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate Solution (NaHCO3) (7.5%)</td>
			<td>Quality Biological&#160;</td>
			<td>118-085-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless Steel Surgical Blades no. 22</td>
			<td>Integra</td>
			<td>4-322</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express&#160;</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td></td>
		</tr>
	</tbody>
</table>

unveiling therapeutic opportunities with melanoma patient-derived organoid models

Conventional 2-D cell culture models are essential tools in cancer research for studying cancer progression and therapy1. These models not only allow for controlled experimental conditions to investigate the molecular and cellular mechanisms underlying cancer development and progression but also provide cost-effective and relatively rapid experimental results. Their usage, however, is restricted due to the limited cellular diversity in the tumor microenvironment (TME), which cannot be recapitulated in a coplanar nature of mono-culture models2. Additionally, cell culture models offer an oversimplified environment compared to the human body3. Thus, to better understand cancer progression and develop effective treatments, researchers have turned to innovative models that aim to recapitulate the TME in nature closely. Consequently, maintaining fidelity to native tumors is paramount in these models. Given the intricate nature of cancer, any significant deviation from the original tumor could introduce unforeseen variables that might confound meaningful conclusions about tumor behavior. 
While patient-derived xenografts (PDX) have emerged as a model system that allows for monitoring of tumor progression in vivo, some limitations question how well the developed tumor recapitulates human cancer biology4. For instance, melanoma TME contains tumor-infiltrating immune cells and stromal cells. When passaged as a xenograft, the non-cancer cells from the patient will be gradually lost, which can select adaptations that differ from those in patient tumors. While these limitations can be mitigated by performing limited passages of the PDX in vivo, the differences between the species can still pose a risk of genetic changes to the PDX that deviate from that of the native tumor5. Moreover, PDX models are typically established in immunocompromised mice to prevent rejection of human tissues. This limits the ability to study the role of the host immune system in cancer, especially in immunotherapeutic studies6. We and others have engrafted PDXs in "humanized" mice7,8. However, establishing PDX models in humanized mice is time-consuming and expensive. It can take several months to years to develop a single PDX model from a patient's tumor specimen, thus limiting the speed and scale of research. Despite these limitations, PDX models have been shown to accurately represent the biology of the TME, thus having been extensively used for the development of cancer therapeutics, personalized medicine, and immunotherapy, among others9. 
An alternative 3D culture technique that has emerged is the patient-derived organoid (PDO), where a freshly resected patient tumor is cultured to create models that maintain phenotypic heterogeneity, histoarchitecture, and intercellular communications10. Its ability to closely resemble native tumors has been previously demonstrated where cultured organoids from non-muscle invasive (NMIBC) and muscle-invasive bladder cancer (MIBC) successfully recapitulated key aspects of the parental tumors11. Due to their potential and advantages over other models, PDOs offer a wide variety of tailored applications that other models lack, including but not limited to the study and development of immune checkpoint inhibitors along with cellular and targeted therapies. For example, we have previously used melanoma patient-derived organoids (MPDOs) to study the ability of tumor-infiltrating lymphocytes (TILs) to be expanded by IL-2 and anti-PD1 antibodies (&#945;PD-1). Results showed that the TILs expanded by IL-2 and &#945;PD-1 not only had a higher number but could also successfully infiltrate MPDOs and kill melanoma cells with higher efficiency than TILs expanded using other methods12. Other groups have shown similar results where the usage of anti-PD-1 and/or anti-PD-L1 leads to TILs remaining functional in PDOs, which subsequently leads to tumor killing13. Additionally, our group has also used MPDOs to assess &#947;&#948; T-cell infiltration and killing ability14. PDOs' applicability spans a wide range of areas, including TIL expansion, cytotoxicity studies, and small molecule screening. All these applications highlight the potential and usability that this model confers. As such, we describe the detailed protocol for the culture of MPDOs.

<meta charset="utf8"></head><body>מחבר זרקור: שחזור מורכבות המלנומה עם אורגנואידים שמקורם במטופל להערכת אימונותרפיה

חשיפת הזדמנויות טיפוליות עם מודלים אורגנואידים שמקורם בחולה מלנומה

We develop and validate melanoma patient-derived organoids. It aims to accurately replicate the tumor microenvironment, study immune cell interactions, and assess therapeutic responses, particularly to immunotherapies. We're trying to advance understanding of tumor biology, improve drug development strategies, and potentially enable personalized cancer treatments.Accurately mimicking the complex tumor microenvironment, including stromal and immune cell interactions, remain difficult. Ensuring that the organoids retain the genetic and the phenotypic diversity of the original tumors is challenging. Developing consistent and scalable protocols for organoids culture and ensuring reproducibility across different samples in the studies is essential.A lot of times, oncology patients exhibit varied responses to treatments, necessitating diverse treatment strategies. Therefore, having the ability to grow these organoids derived from the patient's tumors enables us to conduct drug screenings which facilitate the development of these personalized treatment plans tailored to each patient.

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

unveiling-therapeutic-opportunities-with-melanoma-patient-derived

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Cancer Research

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

722 Views.  University of Pennsylvania. אנו מפתחים ומאמתים אורגנואידים שמקורם בחולה מלנומה. מטרתו לשכפל במדויק את המיקרו-סביבה של הגידול, לחקור אינטראקציות בין תאי מערכת החיסון ולהעריך תגובות טיפוליות, במיוחד לאימונותרפיה. אנו מנסים לקדם את ההבנה של הביולוגיה של הגידול, לשפר אסטרטגיות לפיתוח ...

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With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

Developing Organoid Models from Patient-Derived Melanoma Tissues for Enhanced Tumor Microenvironment Studies

To begin, prepare a coating solution containing growth factor reduced basement membrane matrix and organoid culture medium in a one to two ratio. Add 115 microliters of the coating solution to each well of a 48-well plate and place the plate at 37 degrees Celsius for 30 minutes in a humidified incubator. For the collagen method, add one milliliter of collagen gel to a 30 millimeter inner membrane insert, and let it solidify for 30 minutes at 37 degrees Celsius in a humidified incubator.For harvesting the cellular mixture, wash the freshly resected melanoma tissue in a 10 milliliter Petri dish containing washing medium that is placed over ice. Using sterile tweezers, transfer the tumor into a second dish with washing medium for the next wash over ice. Throughout the three rinsing cycles using sterile scissors and a blade, remove excess connective tissue, fat, and residual blood.Place the tumor in an empty Petri dish and mince it finely with sterile blades. Then transfer the mince tissue to a 50 milliliter conical tube containing 10 milliliters of the prepared digestion medium. Place the conical in a water bath at 37 degrees Celsius and vortex every five minutes.After 25 minutes, add 30 milliliters of the ADMEM/F12 medium containing 10%FBS to stop the digestion. Next, filter the digested cell suspension through a 70 micrometer nylon cell strainer and rinse the strainer with supplemented ADMEM/F12. Using a pipette, retrieve any remaining media at the bottom of the filter.Then pellet the cells at 300 x g for seven minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in the organoid culture medium. For culturing, seed 200, 000 cells in the pre-war matrigel-coated wells supplemented with 200 microliters of organoid culture medium.Alternatively, seed 1 million cells mixed with one milliliter of collagen gel on the pre-solidified collagen gel membrane insert placed in a six-well plate and add the organoid culture medium to the well. For passage of 150 to 200 micrometer organoids, transfer them into a 15 milliliter conical and wash them with Dulbecco's PBS. Centrifuge the mixture at 300 x g for seven minutes at four degrees celsius.After removing the supernatant, incubate the organoids with trypsin substitute and mix after every three minutes. To stop the digestion, add ADMEM/F12 with reduced serum medium containing 10%FBS. Centrifuge the mixture at 300 x g for seven minutes, and resuspend the palate in the organoid culture medium.Finally, seed the single cell suspension into a new matrigel or collagen-coated well at the same initial seeding density for incubation. Microscopy images of the organoids in late in matrigel on day two and day seven indicated that the size of organoids gradually increased. Additionally, there was no statistically significant distinction in alpha-beta-T-cell proportion between the parental tumors and the respective organoids cultured in either matrigel or collagen.