This work was supported by Ko&#776;ln Fortune Program/Faculty of Medicine, University of Cologne. We thank the technical assistance from Susanne Neiss, Michaela Heitmann, and Anke Wienand-Dorweiler. Ningbo Fan was financially supported by Guangzhou Elite Scholarship Council (GESC). The authors thank Dr. Joshua D&#39;Rozario for his assistance in linguistic editing.

An established and well-growing PDO culture represents the basis for a successful subculture and cryopreservation described in this protocol. Here, EAC PDOs were generated from EAC patients&#39; primary tumor tissue using the protocol described by Karakasheva T. A. et al17. EAC tissues were collected from biobank under the approval of BioMaSOTA (approved by the Ethics Committee of the University of Cologne, ID: 13-091).
NOTE: EAC PDOs have been cultured in a humidified incubator at 37 &#176;C and 5% CO2&#160;using a PDO culture medium (Table 1). In the following steps, two methods of the subculture are described in detail. A 12-well plate is recommended for subculturing the PDOs with a seeding density of three extracellular matrix (ECM) gel domes per well, as it allows flexible use of each well and appropriate quantity of PDOs for different purposes. An aseptic technique is compulsory while handling the PDOs.
1. Preparations in advance
<ol>
	<li>Pre-warm a 12-well plate by placing it into a 37 &#176;C CO2&#160;incubator overnight before subculture to ensure complete warming of the plate. If available, use empty wells from a plate with the current PDO culture. 
	NOTE: Continuous storage of 1-2 fresh plates at 37 &#176;C is recommended for flexible subculture planning.</li>
	<li>Pre-cool 1,000 &#181;L and 200 &#181;L tips with a wide orifice at -20 &#176;C (continuous storage recommended). Pre-cool centrifuge at 4 &#176;C.</li>
	<li>Set up the temperature of the rotating incubator to 37 &#176;C (if single cell digestion is performed).</li>
	<li>Incubate an appropriate volume of ECM gel for 1 h on ice to liquefy. Place cell recovery solution on ice.</li>
</ol>
2. Harvesting organoids
<ol>
	<li>Remove the plate with growing PDOs from the CO2incubator.</li>
	<li>Aspirate old medium using a vacuum pump. 
	NOTE: Avoid touching the domes.</li>
	<li>Add an appropriate volume of ice-cold cell recovery solution (500 &#181;L/dome) into the well.</li>
	<li>Disintegrate the ECM gel by pipetting up and down several times to fragment ECM gel domes into small pieces using 1,000 &#181;L tips with a wide orifice.</li>
	<li>Combine the mixture of PDO, ECM gel and cell recovery solution from a maximum of two wells (six domes) and transfer it into a 5 mL low bind tube (use a second tube in case more wells are used for subculture). 
	NOTE: Optionally, if ECM gel was not dissolved completely, add an additional 1.5 mL of cell recovery solution to the mixture of PDO, ECM gel, and cell recovery solution.</li>
	<li>Incubate the tube containing the mixture in step 2.5 on ice for 20 min, mix every 5 min by inverting the tube five times to ensure the liquefaction of the ECM gel.</li>
	<li>Centrifuge at 500 x g for 4 min at 4&#176; C.</li>
	<li>If there is a visible and stable pellet after centrifugation, proceed with step 2.10. Otherwise, continue with step 2.9.</li>
	<li>If there is no visible pellet and the PDOs still seem to be stuck in a gel phase, carefully remove the supernatant with a vacuum pump until the phase containing ECM gel-PDO-Solution is reached and add 3 mL of ice-cold cell recovery solution.
	<ol>
		<li>Invert the tube a few times and incubate on ice for another 10 min. Mix by inverting the tube from time to time.</li>
		<li>Centrifuge at 500 x g for 4 min at 4 &#176;C and continue with step 2.10.</li>
	</ol>
	</li>
	<li>Discard the supernatant carefully using a vacuum pump or a 1,000 &#181;L pipette. Try to remove the supernatant as much as possible. 
	NOTE: Due to the low bind surface of the tube, the pellet will not be as stable as usual.</li>
	<li>Store the PDO pellet on ice and proceed with step 3 (without digestion) or step 4 (with single cell digestion) depending on the different purposes.</li>
</ol>
3. Subculturing without digestion
NOTE: This method aims to increase the PDOs&#39; size and density. The larger size and higher density facilitate the embedding process, histological characterization, and PDO expansion. Depending on the PDO split ratios (based on the density of PDOs, a ratio between 1:3 and 1:6 is recommended), resuspend the pellet from step 2.8 in an appropriate volume of liquid ECM gel.
<ol>
	<li>Remove pre-cooled 200 &#181;L and 1,000 &#181;L tips with a wide orifice from the -20 &#176;C freezer and place them onto a clean bench.</li>
	<li>Resuspend the pellet from step 2.11 in ECM gel using pre-cooled 1,000 &#181;L tips. Mix by pipetting up and down about 10 times to make sure PDOs are not clumping and are evenly distributed in the ECM gel. 
	NOTE: Use 50 &#181;L ECM gel/dome. Always calculate for one dome more than required (e.g., for nine domes (i.e., three wells), resuspend the pellet in 500 &#181;L of liquid ECM gel (450 + 50 &#181;L extra). Try to avoid producing bubbles during resuspension!</li>
	<li>Remove the pre-warmed 12-well plate from the incubator right before seeding the domes.</li>
	<li>Seed domes containing 50 &#181;L ECM gel into the warm plate (three domes/well). Avoid pipetting bubbles into the ECM gel domes.</li>
	<li>Put the plate back into the 37 &#176;C and 5% CO2incubator and incubate for 20-30 min to solidify the ECM gel.</li>
	<li>Add pre-warmed PDO medium (Table 1) carefully without disturbing the domes.</li>
	<li>Culture the PDOs for 7-14 days until the required density and morphology occur.</li>
</ol>
4. Subculturing with single cell digestion
NOTE: The following steps aim to increase the number of PDOs per dome. The single cell digestion facilitates cell number control and PDO expansion.
<ol>
	<li>Prepare digestion medium by mixing 2 mL of 0.25% Trypsin-EDTA and 20 &#181;L DNase I (for digestion of three domes).</li>
	<li>Resuspend the pellet from step 2.11 with an appropriate volume of pre-warmed 0.25% Trypsin-EDTA + DNase I and mix it about 10 times by pipetting up and down using a 1,000 &#181;L pipette (use normal 1,000 &#181;L tips).</li>
	<li>Incubate for 10 min at 37 &#176;C in a rotating&#160;incubator with a rotation speed of a minimum 28 rpm.</li>
	<li>Prepare a 15 mL tube containing 6 mL of Soybean Trypsin Inhibitor (STI, Table 2) solution (per 2 mL of 0.25% Trypsin-EDTA).</li>
	<li>After digestion, mix the digested PDOs thoroughly a few times with a 1,000 &#181;L pipette to disrupt the PDOs.</li>
	<li>Transfer the digested PDOs to the 15 mL tube containing STI solution to stop the digestion process.</li>
	<li>Centrifuge at 500 x g for 4 min at 4 &#176;C. Discard the supernatant carefully using a vacuum pump or a 1,000 &#181;L pipette. Resuspend the pellet in 1 mL of basal medium (Table 3).</li>
	<li>Determine cell concentration and viability using an automated cell counter or a Hemocytometer.</li>
	<li>Seed digested PDO into a 12-well plate with 2 x 104 cells per dome.
	<ol>
		<li>Calculate the cell number according to the domes planned for seeding and transfer them to a fresh 1.5 mL low bind tube. 
		NOTE: Calculate for one dome more (+ 2 x 104 cells extra). For example, for seeding three domes into one well, take 8 x 104 (2 x 104* 3 + 2 x 104 extra) cells.</li>
		<li>Centrifuge at 500 x g for 4 min at 4 &#176;C.</li>
		<li>In case there is no visible pellet, remember the orientation of the tube inside the centrifuge to know where the pellet is located.</li>
		<li>Carefully discard the supernatant using a 1,000 &#181;L pipette. Remove the supernatant as much as possible without disturbing the pellet.</li>
		<li>Add appropriate volume of ECM gel to the pellet using a 1,000 &#181;L pipette with pre-cooled 1,000 &#181;L wide orifice tip (50 &#181;L/dome + 50 &#181;L extra).</li>
		<li>Follow steps 3.3-3.7.</li>
	</ol>
	</li>
</ol>
5. Cryopreservation of the digested and undigested PDOs
NOTE: Single cell digested and undigested PDOs are suitable for the preparation of frozen backup stocks. Note that re-cultivated PDOs from the single cell frozen stocks require a longer time to recover and to reach a certain size.
<ol>
	<li>Cryopreservation of the undigested PDOs.
	<ol>
		<li>Start cryopreservation process with the pellet from step 2.8. Use 500 &#181;L of cold freezing medium to resuspend the pellet and transfer it to a cryogenic vial. 
		NOTE: Store two domes per vial.</li>
		<li>Freeze PDOs overnight in a -80 &#176;C freezer using an appropriate cell freezing container.</li>
	</ol>
	</li>
	<li>Cryopreservation of the single cell digested PDOs
	<ol>
		<li>After harvesting and digesting PDOs, start cryopreservation from step 4.8.</li>
		<li>For storing one cryogenic vial, transfer 4-5 x 105 cells into a fresh 1.5 mL low bind tube. 
		NOTE: Store three domes/vial.</li>
		<li>Centrifuge at 500 x g for 4 min at 4 &#176;C. Discard the supernatant carefully using a 1,000 &#181;L pipette. Remove the supernatant as much as possible without disturbing the pellet.</li>
		<li>Resuspend the pellet in an appropriate volume of freezing medium (500 &#181;L/vial) and transfer it to a cryogenic vial.</li>
		<li>Freeze PDOs overnight in a -80 &#176;C freezer using an appropriate cell freezing container and transfer them into a -150 &#176;C freezer or liquid nitrogen for long-term storage.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Sung, 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=Global+cancer+statistics+2020:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 71 (3), 209-249 (2021).</li><li>Coleman, H. G., Xie, S. -. H., Lagergren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+esophageal+adenocarcinoma.">The epidemiology of esophageal adenocarcinoma.</a> Gastroenterology. 154 (2), 390-405 (2018).</li><li>Rumgay, 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=International+trends+in+esophageal+squamous+cell+carcinoma+and+adenocarcinoma+incidence.">International trends in esophageal squamous cell carcinoma and adenocarcinoma incidence.</a> The American Journal of Gastroenterology. 116 (5), 1072-1076 (2021).</li><li>Qian, 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=Clinical+characteristics,+prognosis,+and+nomogram+for+esophageal+cancer+based+on+adenosquamous+carcinoma:+a+seer+database+analysis.">Clinical characteristics, prognosis, and nomogram for esophageal cancer based on adenosquamous carcinoma: a seer database analysis.</a> Frontiers in Oncology. 11, 603349 (2021).</li><li>Lagergren, J., Smyth, E., Cunningham, D., Lagergren, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oesophageal+cancer.">Oesophageal cancer.</a> Lancet. 390 (10110), 2383-2396 (2017).</li><li>Rockett, J. C., Larkin, K., Darnton, S. J., Morris, A. G., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+newly+established+oesophageal+carcinoma+cell+lines:+phenotypic+and+immunological+characterization.">Five newly established oesophageal carcinoma cell lines: phenotypic and immunological characterization.</a> British Journal of Cancer. 75 (2), 258-263 (1997).</li><li>Hashimoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+COX2+and+p53+in+rat+esophageal+cancer+induced+by+reflux+of+duodenal+contents.">Expression of COX2 and p53 in rat esophageal cancer induced by reflux of duodenal contents.</a> ISRN Gastroenterology. 2012, 1-5 (2012).</li><li>Quante, 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=Bile+acid+and+inflammation+activate+gastric+cardia+stem+cells+in+a+mouse+model+of+barrett-like+metaplasia.">Bile acid and inflammation activate gastric cardia stem cells in a mouse model of barrett-like metaplasia.</a> Cancer Cell. 21 (1), 36-51 (2012).</li><li>Kapoor, H., Lohani, K. R., Lee, T. H., Agrawal, D. K., Mittal, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Barrett's+esophagus+and+esophageal+adenocarcinoma-past,+present,+and+future.">Animal models of Barrett's esophagus and esophageal adenocarcinoma-past, present, and future.</a> Clinical and Translational Science. 8 (6), 841-847 (2015).</li><li>Lan, T., Xue, X., Dunmall, L. C., Miao, J., Wang, Y. <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:+a+developing+tool+for+screening+biomarkers+and+potential+therapeutic+targets+for+human+esophageal+cancers.">Patient-derived xenograft: a developing tool for screening biomarkers and potential therapeutic targets for human esophageal cancers.</a> Aging. 13 (8), 12273-12293 (2021).</li><li>Liu, D. S. 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=APR-246+potently+inhibits+tumour+growth+and+overcomes+chemoresistance+in+preclinical+models+of+oesophageal+adenocarcinoma.">APR-246 potently inhibits tumour growth and overcomes chemoresistance in preclinical models of oesophageal adenocarcinoma.</a> Gut. 64 (10), 1506-1516 (2015).</li><li>Ebbing, E. 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=Esophageal+adenocarcinoma+cells+and+xenograft+tumors+exposed+to+Erb-b2+receptor+tyrosine+kinase+2+and+3+inhibitors+activate+transforming+growth+factor+beta+signaling,+which+induces+epithelial+to+mesenchymal+transition.">Esophageal adenocarcinoma cells and xenograft tumors exposed to Erb-b2 receptor tyrosine kinase 2 and 3 inhibitors activate transforming growth factor beta signaling, which induces epithelial to mesenchymal transition.</a> Gastroenterology. 153 (1), 63-76 (2017).</li><li>Simian, M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids:+A+historical+perspective+of+thinking+in+three+dimensions.">Organoids: A historical perspective of thinking in three dimensions.</a> The Journal of Cell Biology. 216 (1), 31-40 (2017).</li><li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nature Reviews Cancer. 18 (7), 407-418 (2018).</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=Organoid+cultures+recapitulate+esophageal+adenocarcinoma+heterogeneity+providing+a+model+for+clonality+studies+and+precision+therapeutics.">Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics.</a> Nature Communications. 9, 2983 (2018).</li><li>Ebbing, E. 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=Stromal-derived+interleukin+6+drives+epithelial-to-mesenchymal+transition+and+therapy+resistance+in+esophageal+adenocarcinoma.">Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (6), 2237-2242 (2019).</li><li>Karakasheva, T. 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=Generation+and+characterization+of+patient-derived+head+and+neck,+oral,+and+esophageal+cancer+organoids.">Generation and characterization of patient-derived head and neck, oral, and esophageal cancer organoids.</a> Current Protocols in Stem Cell Biology. 53 (1), 109 (2020).</li><li>Ord&#243;&#241;ez, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+immunohistochemical+epithelial+markers:+a+review.">Broad-spectrum immunohistochemical epithelial markers: a review.</a> Human Pathology. 44 (7), 1195-1215 (2013).</li><li>Maniar, K. P., Umpires, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokeratin+7+(CK7,+K7).">Cytokeratin 7 (CK7, K7).</a> Pathology Outlines.com website. , (2021).</li><li>Sun, X., Kaufman, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki-67:+more+than+a+proliferation+marker.">Ki-67: more than a proliferation marker.</a> Chromosoma. 127 (2), 175-186 (2018).</li><li>Driehuis, E., Kretzschmar, K., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+patient-derived+cancer+organoids+for+drug-screening+applications.">Establishment of patient-derived cancer organoids for drug-screening applications.</a> Nature Protocols. 15 (10), 3380-3409 (2020).</li><li>Sachs, N., 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-term+expanding+human+airway+organoids+for+disease+modeling.">Long-term expanding human airway organoids for disease modeling.</a> The EMBO Journal. 38 (4), 100300 (2019).</li></ol>

The authors declare no conflicts of interest in this work.

In this protocol, two different subculture and cryopreservation methods of EAC PDOs are described, i.e, with and without single cell digestion. Several studies recommended passaging EAC PDOs with single cell digestion15,17, which is beneficial to most experiments that require cell number control, uniform density, and a hollow structure that facilitates size tracking. However, the single cell-based method is characterized by slower growth after recultivation from ...

Esophageal cancer (EC) is the tenth most common and the sixth leading cause of death from cancer worldwide1. Esophageal adenocarcinoma (EAC) is one of the major histologic subtypes of EC and mainly occurs in western countries2. In the recent decade, the EAC incidence has significantly increased in many developed countries, including Germany3. Due to the aggressiveness of cancer and the lack of symptoms during the early stage of tumor development, the overall prognosis in EAC patients is poor, showing a 5-year survival rate of about 20%2,4,5.
Since the late twentieth century, several models have been established for the biomedical research of EAC. The classic human EAC cell lines that were established in the 1990s6, extend our knowledge of EAC tumor biology, tumor genetics as well as anti-tumor strategies, and are commonly used in EAC research. Besides, some research groups have successfully developed animal models of EAC or Barrett&#39;s esophagus by exposing the animals to known risk factors such as gastroesophageal reflux through surgical or inflammatory approaches7,8,9. In addition, patient-derived xenograft (PDX) models that engraft EAC primary cancer tissues subcutaneously or orthotopically into immunodeficient mice, were developed to simulate human EAC tumor biological behavior and tumor environment10,11,12. However, despite these models improving clinical applications and our understanding of molecular mechanisms behind EAC tumorigenesis and progression, there is still a major challenge to extrapolate results from these research models to humans.
Patient-derived tumor organoids&#160;(PDOs) are grown in a 3D culture system that mimics human development and organ regeneration in vitro. Generated from patients&#39; primary tissue, PDOs recapitulate the molecular and phenotypic characteristics of the human tumor and have shown promising applications in drug development and personalized cancer treatment13,14. By comparing ten cases of EAC PDOs with their paired tumor tissue, EAC PDOs are reported to share similar histopathological features and genomic landscape with the primary tumor, retain intra-tumor heterogeneity and facilitate efficient drug screening in vitro15. EAC PDOs were also used in studying the interaction of EAC tumor cells with patient-derived cancer-associated fibroblasts (CAFs), indicating a powerful application in the field of tumor microenvironment research16. Unfortunately, there have been limited protocols available for developing and propagating EAC PDOs. Here, two different methods are described for subculturing and preserving EAC PDOs in detail: with and without single cell digestion. The standardized methods for maintenance of EAC PDOs and their applications can support researchers to choose appropriate methods for different purposes in their EAC PDO research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-20&#176;C Freezer</td>
			<td>Bosch</td>
			<td></td>
			<td>Economic</td>
		</tr>
		<tr>
			<td>-80&#176;C Freezer</td>
			<td>Panasonic</td>
			<td></td>
			<td>MDF DU500VH-PE</td>
		</tr>
		<tr>
			<td>Automated Cell counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAX1000</td>
			<td>Countess II</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet Class II</td>
			<td>Thermo Scientific</td>
			<td>51022482</td>
			<td>Herasafe KS12</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Heraeus</td>
			<td>75003060</td>
			<td>Megafuge 1.0R</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50116048</td>
			<td>Heracell 150i</td>
		</tr>
		<tr>
			<td>Inverted automated fluorescence microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>IX83</td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DMIL LED Fluo</td>
		</tr>
		<tr>
			<td>Pipette 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td>Research Plus</td>
		</tr>
		<tr>
			<td>Pipette 200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000039</td>
			<td>Research Plus</td>
		</tr>
		<tr>
			<td>Rotating Incubator</td>
			<td>Scientific Industries, sc.</td>
			<td>SI-1200</td>
			<td>Enviro-genie</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Eppendorf</td>
			<td>5355 000.011</td>
			<td>Thermomixer Comfort</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vacuubrand</td>
			<td>20727200</td>
			<td>BVC control</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Medingen</td>
			<td>p2725</td>
			<td>W22</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td>Inc&#160;Screw cap tube PP 15 mL</td>
		</tr>
		<tr>
			<td>Cryo vial 2 mL</td>
			<td>Sarstedt</td>
			<td>72.379</td>
			<td>CryoPure 2.0 mL tube</td>
		</tr>
		<tr>
			<td>Low bind tube 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.706.600</td>
			<td>Micro tube 1.5 mL protein LB</td>
		</tr>
		<tr>
			<td>Low bind tube 5 mL</td>
			<td>Eppendorf</td>
			<td>0030 108.302</td>
			<td>Protein LoBind Tube 5.0 mL</td>
		</tr>
		<tr>
			<td>Pipette tip 200 &#181;L</td>
			<td>Starlab</td>
			<td>E1011-8000</td>
			<td>200 &#181;L Graduated tip, wide orifice</td>
		</tr>
		<tr>
			<td>Pipette tip 1000 &#181;L</td>
			<td>Starlab</td>
			<td>E1011-9000</td>
			<td>1000 &#181;L Graduated tip, wide orifice</td>
		</tr>
		<tr>
			<td>Pipette tip 1000 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.3050</td>
			<td>Pipette tip 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Sterile filter 0.2 &#181;m</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td>Filtropur 0.2 &#181;m sterile filter</td>
		</tr>
		<tr>
			<td>Tissue culture plate</td>
			<td>Sarstedt</td>
			<td>83.3921</td>
			<td>12 well-plate</td>
		</tr>
		<tr>
			<td>Reagent/Chemical</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290026</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>MedChemExpress</td>
			<td>HY-10182/CS-0181</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I grade II, from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular matrix (ECM) gel: Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-10a</td>
			<td>Peprotech</td>
			<td>100-26-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing medium: Recovery Cell Freezing Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>12648010</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin</td>
			<td>Sigma</td>
			<td>G9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin-25 (25 mg/ 500 &#181;L)</td>
			<td>PromoCell</td>
			<td>C-36030</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10C-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 10,000 U/ mL (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin1 conditioned medium from Cultrex R-Spondin Cells</td>
			<td>Biotechne</td>
			<td>3710-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>MedChemExpress</td>
			<td>152121-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from Glycine max (soybean)</td>
			<td>Sigma-Aldrich</td>
			<td>93620-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25 %), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt-3A conditioned medium</td>
			<td>Wnt-3A expressing cell line was kindly provided by Prof. Hans Clevers&#39; group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td></td>
		</tr>
	</tbody>
</table>

subculture and cryopreservation of esophageal adenocarcinoma organoids: pros and cons for single cell digestion

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in esophageal adenocarcinoma (EAC). Patient-derived organoids (PDOs) have recently emerged as a remarkable preclinical model in a variety of cancers. However, there are still limited protocols available for developing EAC PDOs. Once the PDOs are established, the propagation and cryopreservation are essential for further downstream analyses. Here, two different methods have been standardized for EAC PDOs subculture and cryopreservation, i.e., with and without single cell digestion. Both methods can reliably obtain appropriate cell viability and are applicable for a diverse experimental setup. The current study demonstrated that subculturing EAC PDOs with single cell digestion is suitable for most experiments requiring cell number control, uniform density, and a hollow structure that facilitates size tracking. However, the single cell-based method shows slower growth in culture as well as after re-cultivation from frozen stocks. Besides, subculturing with single cell digestion is characterized by forming hollow structures with a hollow core. In contrast, processing EAC PDOs without single cell digestion is favorable for cryopreservation, expansion, and histological characterization. In this protocol, the advantages and disadvantages of subculturing and cryopreservation of EAC PDOs with and without single cell digestion are described to enable researchers to choose an appropriate method to process and investigate their organoids.

This protocol presents the procedures including subculture and cryopreservation of EAC PDOs with and without single cell digestion.
Figure 1 shows representative phase-contrast pictures of the two different subculture strategies. EAC PDOs reached appropriate density for subculturing (Figure 1, left). Subculturing without single cell digestion takes less time to reach comparable density and mainly leads to compact structures (<strong c...

Watch this Scientific Journal Video about Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion at JoVE.com

Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

This protocol describes the methods of subculture and cryopreservation of esophageal adenocarcinoma organoids with and without single cell digestion to enable researchers to choose appropriate strategies based on their experimental design.

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in ...

subculture-cryopreservation-esophageal-adenocarcinoma-organoids-pros

Research

JoVE Journal

Cancer Research

3.7K Views.  University Hospital Cologne. This protocol describes the methods of subculture and cryopreservation of esophageal adenocarcinoma organoids with and without single cell digestion to enable researchers to choose appropriate strategies based on their experimental design.

Video: Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...