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