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>

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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><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>-20&amp;deg;C Freezer</td><td>Bosch</td><td></td><td>Economic</td></tr><tr><td>-80&amp;deg;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>CO&lt;sub&gt;2&lt;/sub&gt; 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 &amp;micro;L</td><td>Eppendorf</td><td>3123000063</td><td>Research Plus</td></tr><tr><td>Pipette 200 &amp;micro;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>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 mL tube</td><td>Sarstedt</td><td>62.554.502</td><td>Inc&amp;nbsp;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 &amp;micro;L</td><td>Starlab</td><td>E1011-8000</td><td>200 &amp;micro;L Graduated tip, wide orifice</td></tr><tr><td>Pipette tip 1000 &amp;micro;L</td><td>Starlab</td><td>E1011-9000</td><td>1000 &amp;micro;L Graduated tip, wide orifice</td></tr><tr><td>Pipette tip 1000 &amp;micro;L</td><td>Sarstedt</td><td>70.3050</td><td>Pipette tip 1000 &amp;micro;L</td></tr><tr><td>Sterile filter 0.2 &amp;micro;m</td><td>Sarstedt</td><td>83.1826.001</td><td>Filtropur 0.2 &amp;micro;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>&lt;strong&gt;Reagent/Chemical&lt;/strong&gt;</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&#039;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 &amp;micro;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&#039; 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. Het is een eenvoudig en gevisualiseerd protocol dat de behandeling van oesofageale adenocarcinoom organoïden in twee verschillende subculturele processen met en zonder eencellige spijsvertering aantoont. Het gestandaardiseerde protocol voor het subcultureren van EAC-organoïden in twee verschillende methoden kan onderzoekers ondersteunen bij het kiezen van geschikte technieken voor verschillende downstream-toepassingen in hun op EAC BOB gebaseerde experimenten. Bij het omgaan met BOB's is he...

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

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Medicine

Culture of Bladder Cancer Organoids as Precision Medicine Tools

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

Patient-derived organoids (PDOs) are a powerful tool in translational cancer research, reflecting both the genetic and phenotypic heterogeneity of the disease and response to personalized anti-cancer therapies. Here, a consolidated protocol to generate human primary bladder cancer PDOs in preparation for the evaluation of phenotypic analyses and drug responses is detailed.

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as ...

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

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

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

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

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

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

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

Immunology and Infection

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine. Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely. Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.

Experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures are described. Furthermore, the laboratory techniques to ensure consistent growth and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures are discussed.

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

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

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