The authors would like to thank Kersi Pestonjamasp from the Moores Cancer Center imaging core facility for help with the microscopes UCSD Specialized Cancer Support Center P30 grant 2P30CA023100. This work was additionally supported by a JoVE publication grant (JRW), as well as generous gifts from the estate of Elisabeth and Ad Creemers, the Euske Family Foundation, the Gastrointestinal Cancer Research Fund, and the Peritoneal Metastasis Research Fund (AML).

The deidentification and acquisition of all tissues were performed under an IRB-approved protocol at the University of California, San Diego.
1. Preparation of human PMP tissues for tissue processing and culture
<ol>
	<li>Transport of tumor tissues and microdissection
	<ol>
		<li>Prepare the transport and culture media: complete 10% (v/v) Dulbecco&#39;s Modified Eagle Media (DMEM), 10% FBS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin (Pen Strep).</li>
		<li>Upon tissue arrival and according to an institutional IRB-approved protocol, transfer PMP tumor tissues into 35 dishes with a 6 cm diameter containing complete DMEM.</li>
		<li>Micro-dissect solid regions of the tumor from any liquefied mucin using a scalpel. While mucin nodules embedded within solid portions of the tissue can be cut, remove all liquified regions of the tumor. In addition to removing any liquified mucin, or highly mucinous tissue regions, remove any tissue that is difficult to cut with a scalpel. Cut tissue nodules into smaller pieces, roughly 1 cm3 in size. 
		NOTE: Obtaining tumor nodules cleaned of mucin and dense ECM will be essential for successful cutting using a vibratome. As quality control, tumor tissue that arrives within the research laboratory is first cut by a pathologist, which is subsequently distributed by the UCSD biorepository. Tissue donors who undergo palliative debulking of the omental cake are optimal cases for this protocol given a high disease burden, which enables more slice production from the increased mass of tissue availability for research. 
		CAUTION: Working with human tissues requires Biosafety Level 2 (BSL2) certification. Check with the institution for training on BSL2 procedures.</li>
	</ol>
	</li>
	<li>Agarose preparation and tissue embedding
	<ol>
		<li>Prepare a 5% solution of low melt agarose in PBS without FBS on the same day of tissue arrival. Pay close attention to the agarose solution as it tends to boil quickly. 
		NOTE: Low melt agarose is essential for embedding tissue pieces. High melt agarose will solidify at higher temperatures, reducing the viability of tissue slices if embedded. Additionally, FBS in the dissolving solution will cause precipitates to form.</li>
		<li>Place the tissue pieces obtained in 6 cm dishes without liquid. Place no more than 2-3 pieces per 6 cm dish so that they can be removed as agarose cubes without disturbing or touching other tissues.</li>
		<li>Add the liquid 5% agarose solution to 6 cm dishes containing the 1 cm3 tumor specimens. Add enough agarose to just cover the specimen. Once added, place the embedded tissues in a 4 &#176;C refrigerator for 30 min.</li>
	</ol>
	</li>
	<li>Mounting tissue specimens on the vibratome
	<ol>
		<li>Remove the solidified agar from the refrigerator and cut the agarose cubes slightly larger than the width of the tissue using a scalpel.</li>
		<li>Apply super glue to the vibratome stage and gently place the agarose cube onto the super glue. Allow 1-2 min for the glue to solidify. At this point, the agarose cube with tissue should be fixed to the stage.</li>
		<li>Fix the blade to the vibratome and set the desired thickness of the tissue section. For optimal downstream imaging using confocal microscopy, sections should be roughly 150 to 250 microns. Hit the Start button and continue to cycle through cutting of the agarose tissue block until the tissue is completely cut. 
		NOTE: If the tissue is not cutting, or dislodges from the agarose cube, consider embedding the tissue in a higher concentration agarose and trim off any additional tissue pieces that may be too dense to cut or sticks to the vibratome blade.</li>
	</ol>
	</li>
	<li>Culturing tissue slices on permeable inserts
	<ol>
		<li>Prepare a permeable insert plate for culture by adding 2 mL of complete DMEM media above and 3 mL below the permeable membrane.</li>
		<li>Gently lift the tissue slices out of the cutting chamber of the vibratome using a thin paintbrush and place two to four slices into permeable culture dishes containing complete DMEM media. After 24 h, aspirate the media using a pipette and replace the media with fresh culture media.</li>
		<li>Culture the slices for up to 7 days at 37 &#176;C/5% CO2, replacing the media every 24 h. At this time point, add controls for cell killing (bortezomib) and testable chemotherapies 5-fluoruracil (5-FU) to the culture media to perform pharmacological intervention.</li>
		<li>After 7 days of culturing, expect about a 15%-25% loss of viability compared to the day 0 time point (immediately after cutting) under non-drug treated conditions (complete DMEM). Measure the viability using live-dead viability dyes such as calcein AM (live) and propidium Iodide (dead).</li>
	</ol>
	</li>
</ol>
2. Confocal imaging of living human tissue slices
NOTE: Once the organotypic tumor slices have been prepared, it is essential to determine the tissue viability to perform an efficient and optimized downstream analysis. Given that tumor specimens are 150-250 &#181;m thick, confocal, or two-photon microscopy is highly recommended over wide-field microscopes for determining studies in situ. Flow cytometry can also be used for determining the viability and cellular populations (methods are below). However, the spatial resolution is lost during flow cytometry analysis, and viability is likely underestimated given the need to disassociate tumor slices by mechanical and chemical methods.
<ol>
	<li>Reagent preparation and experimental setup
	<ol>
		<li>Place tumor slices for confocal imaging analysis in a single well of a 12-well dish containing PBS with 1% FBS. For calcium imaging experiments, instead of PBS, incubate the slices in extracellular calcium solution prepared as follows: 125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 0.1% BSA, pH 7.4, 3 mM glucose, sterile filtered. Prepare this solution ahead of time and store it for up to 1 month at 4 &#176;C (see section 2.1.8).</li>
		<li>Following the concentrations and protocols recommended by the manufacturer, stain samples with imaging dyes (see Table of Materials) to determine the viability of tissue slices. Image for 10 min-1 h after adding the viability dye.</li>
		<li>After incubation, transfer the slices to an optically clear, glass bottom Petri dish containing PBS with 1% FBS to be used for imaging on a confocal imaging device. 
		NOTE: For confocal imaging, the Nikon A1R Confocal platform was used.</li>
		<li>Open the confocal imaging software. Begin imaging at 10x and select the XYZ imaging mode. Then, configure the acquisition settings as follows.
		<ol>
			<li>Turn on the following lasers: 488 nm and 561 nm. Adjust the gain to 100 with laser power at 1%. Adjust the laser power and gain as needed during image acquisition. Depending on the image quality desired, select 512 x 512 pixels or 1024 x 1024 pixels for higher resolution.</li>
		</ol>
		</li>
		<li>Select Remove Interlock, and then select Scan to initiate imaging.</li>
		<li>For calcium imaging, incubate tissue slices with Fluo-4-AM for 1 h protected from light. Follow the manufacture&#39;s protocol for dissolving Fluo-4-AM in DMSO. After 1 h, wash slices 3x with extracellular calcium solution and follow the imaging protocol in steps 2.1.4-2.1.5.</li>
		<li>For calcium imaging experiments using Fluo-4-AM, select XYZT and unselect the 561 nm laser to increase acquisition speed. Additionally, use the resonance scanner to further increase the speed of imaging.</li>
	</ol>
	</li>
</ol>
3. Disassociation of living slices for flow cytometry
NOTE: Disassociation of living tissue slices can be used for several downstream applications, including immunotyping, assessment of the viability, and interrogation of the changes to cell populations after pharmacological intervention. Steps should be taken to ensure that tissue quality and cell viability are maintained during the disassociation process.
<ol>
	<li>Cut a small piece from the end of a 1 mL pipette tip to widen the opening to allow vigorous mechanical dissociation by pipetting for 1 min.</li>
	<li>Incubate slices at 37 &#176;C with rotation for 5-15 min in 1 mL of digestion buffer consisting of high glucose DMEM, 10% gentle collagenase/hyaluronidase (GCH), 10% FBS, and 10% DNaseI (1 mg/mL stock). 
	NOTE: Batch-dependent changes of collagenase/hyaluronidase are often found.</li>
	<li>Perform vigorous disruption of the tissue using mechanical dissociation 2-3 times during incubation. Be sure to check digestion, by eye, every 5 min for evidence of complete digestion. If needed, stop the enzymatic digestion by proceeding to step 3.1.4.</li>
	<li>Once digested, pipette the slices and the digestion media on top of a 70 &#181;m filter placed on top of a 50 mL conical tube. Pick larger pieces using sterile forceps or a pipette.</li>
	<li>Using the blunt back end of a plastic 5 mL syringe, mash any larger undissociated slices and further wash with 4 mL of PBS containing 2% FBS.</li>
	<li>Take the disassociated cell supernatant in a 50 mL conical tube and spin at 300 x g for 5-10 min. Remove the supernatant and wash the pellet with 1 mL of PBS containing 2% FBS.</li>
	<li>To prepare the sample for flow cytometry, add the blocking buffer containing 50 &#181;L of human Fc-block and leave at room temperature for 15 min. Perform extracellular staining using the respective antibodies in 50 &#181;L of PBS containing 2% FBS. 
	&#8203;NOTE: There are many flow cytometry systems. Once the sample is prepared for flow cytometry, refer to the manufacturer&#39;s protocol for operation of the device.</li>
</ol>
4. Pharmacological intervention using living tissue slices for viability and proliferation analysis
NOTE: Once the organotypic tumor slices have been prepared, interventional approaches can be performed by using several methods, including drug testing, siRNA, as well as viral infection of living tissue slices. Here we will discuss drug testing, as well as downstream functional readouts, which include viability analysis using local proliferation.
<ol>
	<li>Prepare 10 mL of 10% v/v complete DMEM with 10% FBS, 2 mM L-Glutamine, 1% Pen Strep.</li>
	<li>Aliquot 5 mL of complete media for control and treatment conditions. To the remaining 5 mL of media, add bortezomib (2 &#956;M) to induce cell death in tumor slices as a positive control. 
	NOTE: Bortezomib, at high concentrations, is a cytotoxic drug that induces cell death. The other cytotoxic drugs may be used as positive control for cell killing.</li>
	<li>Using two to four tissue slices, which have been plated onto the permeable dishes, add the media prepared in step 4.2 to the dish, as well as any additional combination therapies to be used for downstream viability LIVE/DEAD analysis using confocal microscopy as described in step 2.1.2, or use a commercial luminescent viability analysis using sequentially matched slices. 
	NOTE: Sequential slice matching is essential for luminescent viability analysis, given that luminescent viability assay relies on cellular ATP content. As internal controls are lost during cell lysis, similar-sized slices are required since results are dependent on the starting number of viable cells (i.e., unbalanced slices will result in unbalanced results). If the user does not obtain sequential slices, luminescent viability analysis is not possible, and as such another viability assessment method will be required (flow cytometry or confocal imaging based live cell analysis).</li>
	<li>Leave tissue slices in culture containing complete DMEM for 2-5 days, changing media as well as bortezomib and 5-FU every other day.</li>
	<li>For luminescent viability analysis, add 500 &#181;L of PBS into a 12-well dish and transfer the sequentially matched tissue slices to the PBS solution using a paintbrush or use the suction of a 1 mL pipette gently to transfer these.</li>
	<li>Remove the PBS and add the luminescent viability analysis solution that was prepared. See the instruction manual for preparation of the reagent.</li>
	<li>Add 500 &#181;L of the luminescent viability solution per condition and incubate with a slow rotation on the shaker at room temperature for 30 min. Read luminescence using a luminescence plate reader.</li>
</ol>

<ol>
	<li>Bevan, K. E., Mohamed, F., Moran, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomyxoma+peritonei.">Pseudomyxoma peritonei.</a> World Journal of Gastrointestinal Oncology. 2 (1), 44-50 (2010).</li><li>Votanopoulos, K. I., 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=Appendiceal+cancer+patient-specific+tumor+organoid+model+for+predicting+chemotherapy+efficacy+prior+to+initiation+of+treatment:+A+feasibility+study.">Appendiceal cancer patient-specific tumor organoid model for predicting chemotherapy efficacy prior to initiation of treatment: A feasibility study.</a> Annals of Surgical Oncology. 26 (1), 139-147 (2019).</li><li>Holliday, D. 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=The+practicalities+of+using+tissue+slices+as+preclinical+organotypic+breast+cancer+models.">The practicalities of using tissue slices as preclinical organotypic breast cancer models.</a> Journal of Clinical Pathology. 66 (3), 253-255 (2013).</li><li>Koerfer, J., 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=Organotypic+slice+cultures+of+human+gastric+and+esophagogastric+junction+cancer.">Organotypic slice cultures of human gastric and esophagogastric junction cancer.</a> Cancer Medicine. 5 (7), 1444-1453 (2016).</li><li>Misra, 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=Ex+vivo+organotypic+culture+system+of+precision-cut+slices+of+human+pancreatic+ductal+adenocarcinoma.">Ex vivo organotypic culture system of precision-cut slices of human pancreatic ductal adenocarcinoma.</a> Scientific Reports. 9 (1), 2133 (2019).</li><li>Ohnishi, T., Matsumura, H., Izumoto, S., Hiraga, S., Hayakawa, T. <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+model+of+glioma+cell+invasion+using+organotypic+brain+slice+culture.">A novel model of glioma cell invasion using organotypic brain slice culture.</a> Cancer Research. 58 (14), 2935-2940 (1998).</li><li>Seidman, J. D., Elsayed, A. M., Sobin, L. H., Tavassoli, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+mucinous+tumors+of+the+ovary+and+appendix.+A+clinicopathologic+study+of+25+cases.">Association of mucinous tumors of the ovary and appendix. A clinicopathologic study of 25 cases.</a> The Amerian Journal of Surgical Pathology. 17 (1), 22-34 (1993).</li><li>Mizuta, 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=Pseudomyxoma+peritonei+accompanied+by+intraductal+papillary+mucinous+neoplasm+of+the+pancreas.">Pseudomyxoma peritonei accompanied by intraductal papillary mucinous neoplasm of the pancreas.</a> Pancreatology. 5 (4-5), 470-474 (2005).</li><li>Gong, Y., Wang, X., Zhu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomyxoma+peritonei+originating+from+transverse+colon+mucinous+adenocarcinoma:+A+case+report+and+literature+review.">Pseudomyxoma peritonei originating from transverse colon mucinous adenocarcinoma: A case report and literature review.</a> Gastroenterology Research and Practice. 2020, 5826214 (2020).</li><li>Fleten, K. 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=Experimental+treatment+of+mucinous+peritoneal+metastases+using+patient-derived+xenograft+models.">Experimental treatment of mucinous peritoneal metastases using patient-derived xenograft models.</a> Translational Oncology. 13 (8), 100793 (2020).</li><li>Kuracha, M. R., Thomas, P., Loggie, B. W., Govindarajan, V. <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+mouse+models+of+pseudomyxoma+peritonei+recapitulate+the+human+inflammatory+tumor+microenvironment.">Patient-derived xenograft mouse models of pseudomyxoma peritonei recapitulate the human inflammatory tumor microenvironment.</a> Cancer Medicine. 5 (4), 711-719 (2016).</li><li>Jiang, 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=Long-lived+pancreatic+ductal+adenocarcinoma+slice+cultures+enable+precise+study+of+the+immune+microenvironment.">Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment.</a> Oncoimmunology. 6 (7), 1333210 (2017).</li><li>Sundstrom, L., Morrison, B., Bradley, M., Pringle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+as+tools+for+functional+screening+in+the+CNS.">Organotypic cultures as tools for functional screening in the CNS.</a> Drug Discovery Today. 10 (14), 993-1000 (2005).</li><li>Liu, L., Yu, L., Li, Z., Li, W., Huang, W. <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+organoid+(PDO)+platforms+to+facilitate+clinical+decision+making.">Patient-derived organoid (PDO) platforms to facilitate clinical decision making.</a> Journal of Translational Medicine. 19 (1), 40 (2021).</li><li>Croft, C. L., Futch, H. S., Moore, B. D., Golde, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+to+model+neurodegenerative+proteinopathies.">Organotypic brain slice cultures to model neurodegenerative proteinopathies.</a> Molecular Neurodegeneration. 14 (1), 45 (2019).</li><li>Carr, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+in+the+pathology+of+peritoneal+surface+malignancy.">New insights in the pathology of peritoneal surface malignancy.</a> Journal of Gastrointestinal Oncology. 12, 216-229 (2021).</li><li>Votanopoulos, K. I., 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=Outcomes+of+repeat+cytoreductive+surgery+with+hyperthermic+intraperitoneal+chemotherapy+for+the+treatment+of+peritoneal+surface+malignancy.">Outcomes of repeat cytoreductive surgery with hyperthermic intraperitoneal chemotherapy for the treatment of peritoneal surface malignancy.</a> Journal of the American College of Surgeons. 215 (3), 412-417 (2012).</li><li>Weitz, J., 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=An+ex-vivo+organotypic+culture+platform+for+functional+interrogation+of+human+appendiceal+cancer+reveals+a+prominent+and+heterogenous+immunological+landscape.">An ex-vivo organotypic culture platform for functional interrogation of human appendiceal cancer reveals a prominent and heterogenous immunological landscape.</a> Clinical Cancer Research. 28 (21), 4793-4806 (2022).</li><li>Pitoulis, F. G., Watson, S. A., Perbellini, F., Terracciano, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+slices+come+to+age:+an+intermediate+complexity+in+vitro+cardiac+model+for+translational+research.">Myocardial slices come to age: an intermediate complexity in vitro cardiac model for translational research.</a> Cardiovascular Research. 116 (7), 1275-1287 (2020).</li><li>Habeler, W., Peschanski, M., Monville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+heart+slices+for+cell+transplantation+and+physiological+studies.">Organotypic heart slices for cell transplantation and physiological studies.</a> Organogenesis. 5 (2), 62-66 (2009).</li></ol>

The authors declare that they have no competing financial interests.

This manuscript describes a technique that can be used to culture, interrogate, and analyze human pseudomyxoma peritonei (PMP) tumor specimens. We have utilized numerous downstream functional assays to interrogate the tumor immune microenvironment and a platform for bench-to-bedside testing.
While the method is highly efficient in our hands, it will require some practice to cut tumor specimens using a vibratome. Namely, we encountered problems that were due to highly mucinous samples, as well ...

Pseudomyxoma peritonei (PMP) is rare syndrome with an incidence rate of 1 per million people per year1. Most PMP cases are caused by metastases from appendiceal neoplasms. Given that mice do not have a human-like appendix, modeling this type of cancer remains extremely challenging. While the primary disease is often curable by surgical resection, treatment options for metastatic disease are limited. Therefore, the rationale for developing this novel organotypic slice model is to study the pathobiology of PMP. To date, there are no appendiceal organoid models that can be perpetually cultured; however, a recent model was shown to be useful for the pharmacological testing of therapeutic agents and immunotherapy2. As such, we have adapted an organotypic slice culture system, which has been used in other types of human cancers, such as brain, breast, pancreas, lung, ovarian, and others3,4,5,6.
In addition to appendiceal neoplasms, PMP occasionally results from other tumor types, including ovarian cancers7, and in rare circumstances, intraductal papillary mucinous neoplasms8 and colon cancer9. Additionally, these tumors tend to grow slowly, with poor engraft rates in patient-derived xenograft (PDX) models10,11. Given these challenges, there is an unmet need to develop models to study this disease to begin to understand the pathobiology of PMP, and how these cancer cells: are recruited to the peritoneal surfaces, proliferate, and escape immune surveillance.
While cut from the systemic vascular circulation, tumor slices do contain cellular and acellular components, including the extracellular matrix, stromal cells, immune cells, cancer cells, endothelial cells, and nerves. This semi-intact microenvironment allows for the functional investigation of these cell types, which is uniquely advantageous compared to 3D organoid cultures, which consist only of cancer cells12. While organotypic slice cultures are advantageous in some respects, they are also inherently a low-throughput-based approach, compared to 3D organoids, which can be expanded, and are suitable for multiplexed investigational therapeutic drug screening13,14,15. In the case of PMP, there have been no reports documenting reliable establishment and perpetual passaging of PMP-derived organoids16. This is likely due to the slow growing nature of PMP-derived tumor cells, as well as the low number of malignant epithelial cells found within these mucinous tumors. Given the need to develop models to study PMP, organotypic slices are uniquely suited to study this disease. We present a protocol for preparing, imaging, and analyzing PMP from human specimens.

<table><tbody><tr><td>1 M CaCl2 solution</td><td>Sigma</td><td>21115</td><td></td></tr><tr><td>1 M HEPES solution</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>1 M MgCl2 solution&amp;nbsp;</td><td>Sigma</td><td>M1028</td><td></td></tr><tr><td>100 micron filter</td><td>ThermoFisher</td><td>22-363-549</td><td></td></tr><tr><td>22 x 40 glass coverslips</td><td>Daiggerbrand</td><td>G15972H</td><td></td></tr><tr><td>3 M KCl solution</td><td>Sigma</td><td>60135</td><td></td></tr><tr><td>5 M NaCl solution</td><td>Sigma</td><td>S5150</td><td></td></tr><tr><td>ATP&amp;gamma;S&amp;nbsp;</td><td>Tocris&amp;nbsp;</td><td>4080</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>Calcein-AM&amp;nbsp;</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>CD11b&amp;nbsp;</td><td>Biolegend</td><td>101228</td><td></td></tr><tr><td>CD206&amp;nbsp;</td><td>Biolegend</td><td>321140</td><td></td></tr><tr><td>CD3</td><td>Biolegend</td><td>555333</td><td></td></tr><tr><td>CD4&amp;nbsp;</td><td>Biolegend</td><td>357410</td><td></td></tr><tr><td>CD45&amp;nbsp;</td><td>Biolegend</td><td>304006</td><td></td></tr><tr><td>CD8&amp;nbsp;</td><td>Biolegend</td><td>344721</td><td></td></tr><tr><td>CellTiter-Glo&amp;nbsp;</td><td>Promega</td><td>G9681</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Thermo Fisher</td><td>11965084</td><td></td></tr><tr><td>DPBS&amp;nbsp;</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>ThermoFisher</td><td>16140071</td><td></td></tr><tr><td>Fc-block&amp;nbsp;</td><td>BD Biosciences</td><td>564220</td><td></td></tr><tr><td>Fluo-4</td><td>Thermo Fisher</td><td>F14201</td><td></td></tr><tr><td>Gentle Collagenase/Hyaluronidase&amp;nbsp;</td><td>Stem Cell</td><td>7912</td><td></td></tr><tr><td>Imaging Chamber</td><td>Warner Instruments</td><td>RC-26</td><td></td></tr><tr><td>Imaging Chamber Platform</td><td>Warner Instruments</td><td>PH-1</td><td></td></tr><tr><td>LD-Blue&amp;nbsp;</td><td>Biolegend</td><td>L23105</td><td></td></tr><tr><td>L-Glutamine 200 mM</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>LIVE/DEAD imaging dyes</td><td>Thermofisher</td><td>R37601</td><td></td></tr><tr><td>Nikon Ti microscope&amp;nbsp;</td><td>Nikon</td><td></td><td>Includes: A1R hybrid confocal scanner including a high-resolution (4096x4096) scanner, LU4 four-laser AOTF unit with 405, 488, 561, and 647 lasers, Plan Apo 10 (NA 0.8), 20X (NA 0.9) dry objectives.&amp;nbsp;</td></tr><tr><td>Peristaltic pump&amp;nbsp;</td><td>Isamtec</td><td>ISM832C</td><td></td></tr><tr><td>Propidium Iodide</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Vacuum silicone grease</td><td>Sigma</td><td>Z273554-1EA</td><td></td></tr></tbody></table>

culture and imaging of ex vivo organotypic pseudomyxoma peritonei tumor slices from resected human tumor specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

In short, human tumor specimens from PMP are obtained under an IRB-approved protocol. The tissue is prepared, micro-dissected, and solidified in an agarose mold to be cut using a vibratome (Figure 1A; Video 1). Once cut, tissue slices are placed and cultured on permeable insert membranes (Figure 1B), which can be utilized for imaging assays in situ, as well as for cellular and functional interrogation using flow cytometry, confocal imag...

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Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

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Research

JoVE Journal

Cancer Research

1.7K Views.  University of California. Dit onderzoek toont een nieuwe benadering van de studie van appendiceale kankertherapieën en pathobiologie in een preklinische setting. Deze methoden kunnen breder toepasbaar zijn op de studie van andere maligniteiten. Appendiceale kanker is moeilijk te bestuderen gezien de lage incidentie bij afwezigheid van een appendix bij muizen voor ziektemodellering.Deze techniek maakt de studie van celinteracties binnen de micro-omgeving van de tumor mogelijk. Deze methode kan worden toegepast ...

Video: Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicine

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

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 ...

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

Immunology and Infection

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate ...

Lung-tumor Tissue Explant Culture: A Procedure to Generate and Culture Precision-cut Lung Tumor Slices

Source: Nagaraj, A. S., et al. <a href="https://www.jove.com/v/58569/establishment-analysis-tumor-slice-explants-as-prerequisite-for">Establishment and Analysis of Tumor Slice Explants as a Prerequisite for Diagnostic Testing.</a>&#160;J. Vis. Exp.&#160;(2018)
Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. This video describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. The protocol describes the methodology to generate and culture precision-cut lung tumor slices for drug response studies.

Source: Nagaraj, A. S., et al. Establishment and Analysis of Tumor Slice Explants as a Prerequisite for Diagnostic Testing.&#160;J. Vis. Exp.&#160;(2018)
...

JoVE Encyclopedia of Experiments

Lung Cancer

Ex vivo study

Formalin-Fixed Paraffin Embedded (FFPE) Tissue Preservation: A Method for Studying Tissue Morphology

Source: Nagaraj A.S., et al., <a href="https://www.jove.com/v/58569/establishment-analysis-tumor-slice-explants-as-prerequisite-for">Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing.</a> J. Vis. Exp. (2018)
The following video describes how to preserve morphology of a tissue with formalin fixation and paraffin embedding.

Source: Nagaraj A.S., et al., Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing. J. Vis. Exp. (2018)
The ...

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Source: Chadwick, E. J. et al. <a href="https://app.jove.com/v/53304">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> J. Vis. Exp. (2015).
This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plating the Slices onto the ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

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