Name: subculture and cryopreservation of esophageal adenocarcinoma organoids: pros and cons for single cell digestion
Uploaded: 2022-07-06T12:40:00
Description: Watch this Scientific Journal Video about Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion at JoVE.com

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 ...

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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. 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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. 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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 ...

It is an easy and visualized protocol that demonstrate handling of esophageal adenocarcinoma organoids in two different subculture process with and without single-cell digestion. The standardized protocol for subculturing EAC organoids in two different methods can support researchers in choosing appropriate techniques for different downstream applications in their EAC PDO-based experiments. While dealing with PDOs, it is essential to pay attention to the details in each steps.A team-based SOP is crucial to the quality of PODs. Demonstrating the procedure will be one of our PhD student Ning Bo Fan and Lisa Raatz, the laboratory's technique assistant. Prewarm a 12-well plate by overnight incubation at 37 degrees Celsius using carbon dioxide.If available, use empty wells from a plate with the patient-derived organoid culture. Incubate an appropriate volume of ECM gel for one hour on ice to liquefy. Place cell recovery solution on ice.Remove the plate with growing PDOs from the incubator. Aspirate old medium using a vacuum pump. Add 500 microliters per dome of ice cold cell recovery solution into the well.Disintegrate the ECM gel by pipetting up and down several times to fragment ECM gel domes into small pieces using 1, 000 microliter tips with a wide orifice. Combine the PDO, ECM gel, and cell recovery solution from a maximum of two wells and transfer it into a five milliliter low bind tube. Incubate this tube on ice for 20 minutes.Mix every five minutes by inverting the tube. Centrifuge the tube at a speed of 500 times G for four minutes at four degrees Celsius. If there is no visible pellet, carefully remove the supernatant with a vacuum pump until the phase containing ECM gel PDO solution is reached and add three milliliters of ice cold cell recovery solution.Invert the tube a few times and incubate on ice for another 10 minutes. Centrifuge at 500 times G for four minutes at four degrees Celsius. Discard the supernatant carefully using a vacuum pump or a 1, 000 microliter pipette.Try to remove the supernatant as much as possible. Store the PDO pellet on ice. Remove pre-cooled 201, 000 microliter tips with a wide orifice from the minus 20 degrees Celsius freezer and place them onto a clean bench.Calculate 50 microliters of ECM gel per dome planned for seeding, plus 50 microliters extra. Then resuspend the pellet in ECM gel using a pre-cooled 1, 000 microliter tip and mix by pipetting up and down about 10 times. Remove the pre-warmed 12-well plate from the incubator before seeding the domes.Seed the domes containing 50 microliters of ECM gel into the warm plate. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 20 to 30 minutes to solidify the ECM gel. Add pre-warmed PDO medium carefully without disturbing the domes.Monitor seeded PDOs under the microscope. Culture the PDOs for 7 to 14 days until the required density and morphology occur. Prepare digestion medium by mixing two milliliters of 0.25%trypsin EDTA and 20 microliters DNAase I to digest PDOs harvested from two wells.Resuspend the previously prepared pellet in an appropriate volume of pre-warmed 0.25%trypsin EDTA and DNAase I and mix it about 10 times by pipetting up and down using a 1, 000 microliter pipette. Incubate for 10 minutes at 37 degrees Celsius in a rotating incubator with a rotation speed of a minimum of 28 RPM. Prepare a 15 milliliter tube containing six milliliters of soybean trypsin inhibitor solution.After digestion, mix the digested PDOs thoroughly a few times with a 1, 000 microliter pipette to disrupt the PDOs. Transfer the digested PDOs to the 15 milliliter tube containing SDI solution to stop the digestion. Centrifuge at 500 times G for four minutes at four degrees Celsius.Discard the supernatant carefully using a vacuum pump or a 1, 000 microliter pipette. Resuspend the pellet in one milliliter of basal medium. Determine cell concentration and viability using an automated cell counter or a hemocytometer.Calculate the cell number according to the domes planned for seeding, plus one dome extra, and transfer them to a fresh 1.5 milliliter low bind tube. Centrifuge at 500 times G for four minutes at four degrees Celsius. Carefully discard the supernatant using a 1, 000 microliter pipette.Add appropriate volume of ECM gel to the pellet using a pre-cooled 1, 000 microliter wide orifice tip. Mix by pipetting up and down about 10 times. Seed the digested PDOs into a 12-well plate as demonstrated previously.Start cryopreservation process of undigested PDOs with previously prepared pellet. Use 500 microliters of cold freezing medium to resuspend the pellet from two domes and transfer it to a cryogenic vial. Freeze PDOs overnight in the freezer using an appropriate cell freezing container.Transfer the frozen PDOs into a minus 150 degrees Celsius freezer. For cryopreservation of digested PDOs, transfer cells into a fresh 1.5 milliliter low bind tube. Centrifuge at 500 times G for four minutes at four degrees Celsius.Discard the supernatant carefully using a 1, 000 microliter pipette. Resuspend the pellet in 500 microliters freezing medium and transfer it to a cryogenic vial. Freeze PDOs overnight in a minus 80 degrees Celsius freezer using an appropriate cell freezing container and transfer them into a minus 150 degrees Celsius freezer for long-term storage.Subculturing without single-cell digestion takes less time to reach comparable density and mainly leads to compact structures. In contrast, the single-cell digested PDOs show structures with a hollow core. The figure shows the hematoxylin eosin staining and immunohistochemistry staining of paraffin-embedded EAC PDOs with compact and hollow structures.The pancytokeratin enables the identification of epithelial tumor cells. The cytokeratin 7 highlights the glandular differentiated tumor cells. The compact structure predominantly exists in the undigested culture, while the hollow structure is dominant in the culture that underwent single-cell digestion.The Ki67 highlights the cell populations with higher cellular proliferation. The Ki67 in red and PanCK in green were similarly distributed among EAC primary tissue, EAC PDO compact structure and EAC PDO hollow structure. The figure shows morphological characteristics of EAC PDOs on the first day of recovery from frozen stock with single-cell-based cryopreservation and undigested PDO-based cryopreservation.We recommend making a clear subculture plan in advance. Temperature control and gentle pipetting are essential for the correct handling of ECM gels when subculturing PDOs. Our protocol provides researchers the substantial to maintain their EAC PDOs according to their study purpose for downstream analysis such as drug screening assay, flow cytometry, or histological analysis.We hope that our work could help organoid researchers to make fast and easy decisions for the storage and subculture of organoids, particularly for robust outcomes of patient-derived materials in translational research.

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