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

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 .......

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 digestion^15,17, 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 .......

Materials

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