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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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<....

Protocol

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 .......

Representative Results

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 (

Discussion

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 .......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Equipment
-20°C FreezerBoschEconomic
-80°C FreezerPanasonicMDF DU500VH-PE
Automated Cell counterThermo FisherAMQAX1000Countess II
Biological Safety Cabinet Class IIThermo Scientific51022482Herasafe KS12
CentrifugeHeraeus75003060Megafuge 1.0R
CO2 IncubatorThermo Scientific50116048Heracell 150i
Inverted automated fluorescence microscopeOlympusIX83
Inverted light microscopeLeicaDMIL LED Fluo
Pipette 1000 µLEppendorf3123000063Research Plus
Pipette 200 µLEppendorf3123000039Research Plus
Rotating IncubatorScientific Industries, sc.SI-1200Enviro-genie
ShakerEppendorf5355 000.011Thermomixer Comfort
Vacuum pumpVacuubrand20727200BVC control
WaterbathMedingenp2725W22
Material
15 mL tubeSarstedt62.554.502Inc Screw cap tube PP 15 mL
Cryo vial 2 mLSarstedt72.379CryoPure 2.0 mL tube
Low bind tube 1.5 mLSarstedt72.706.600Micro tube 1.5 mL protein LB
Low bind tube 5 mLEppendorf0030 108.302Protein LoBind Tube 5.0 mL
Pipette tip 200 µLStarlabE1011-8000200 µL Graduated tip, wide orifice
Pipette tip 1000 µLStarlabE1011-90001000 µL Graduated tip, wide orifice
Pipette tip 1000 µLSarstedt70.3050Pipette tip 1000 µL
Sterile filter 0.2 µmSarstedt83.1826.001Filtropur 0.2 µm sterile filter
Tissue culture plateSarstedt83.392112 well-plate
Reagent/Chemical
A83-01Tocris2939
Advanced DMEM/F-12Thermo Fisher Scientific12634010
Amphotericin BThermo Fisher Scientific15290026
B-27Thermo Fisher Scientific17504001
Cell Recovery SolutionCorning354253
CHIR-99021MedChemExpressHY-10182/CS-0181
DNase I grade II, from bovine pancreasSigma-Aldrich10104159001
Dulbecco's phosphate-buffered saline (DPBS)Thermo Fisher Scientific14190094
Extracellular matrix (ECM) gel: Matrigel Growth Factor Reduced (GFR) Basement Membrane MatrixCorning356231
FGF-10aPeprotech100-26-100
Freezing medium: Recovery Cell Freezing MediumThermo Fisher Scientific12648010
GastrinSigmaG9020
Gentamicin-25 (25 mg/ 500 µL)PromoCellC-36030
HEPES (1 M)Thermo Fisher Scientific15630080
L-Glutamine 200 mM (100X)Thermo Fisher Scientific25030024
N-2Thermo Fisher Scientific17502-048
N-AcetylcysteineSigmaA9165
NicotinamideSigmaN0636-100
NogginPeprotech120-10C-50
Penicillin-Streptomycin 10,000 U/ mL (100X)Thermo Fisher Scientific15140122
Recombinant human epidermal growth factor (EGF)PeprotechAF-100-15
R-Spondin1 conditioned medium from Cultrex R-Spondin CellsBiotechne3710-001-01
SB202190MedChemExpress152121-30-7
Trypsin inhibitor from Glycine max (soybean)Sigma-Aldrich93620-1G
Trypsin-EDTA (0.25 %), phenol redThermo Fisher Scientific25200056
Wnt-3A conditioned mediumWnt-3A expressing cell line was kindly provided by Prof. Hans Clevers' group
Y-27632SigmaY0503

References

  1. Sung, H., et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 71 (3), 209-249 (2021).
  2. Coleman, H. G., Xie, S. -. H., Lagergren, J.

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