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%2,4,5.
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'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 (PDOs) are grown in a 3D culture system that mimics human development and organ regeneration in vitro. Generated from patients' 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.
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 incubator at 37 °C and 5% CO2 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
2. Harvesting organoids
3. Subculturing without digestion
NOTE: This method aims to increase the PDOs' 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.
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.
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.
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 (Figure 1, top row). In contrast, the single cell digested PDOs show hollow structures with a hollow core (Figure 1, bottom row). Figure 2 shows the Hematoxylin-Eosin (H&E) staining and immunohistochemistry (IHC) staining of paraffin-embedded EAC PDOs with compact and hollow structures. The pan-cytokeratin (Pan-CK) enables the identification of epithelial tumor cells18. The cytokeratin 7 (CK7) highlights the glandular differentiated tumor cells19. The compact structure (top row) predominantly exists in the undigested culture, while the hollow structure (bottom row) is dominant in the culture that underwent single cell digestion.
Figure 3 shows the immunofluorescence (IHC) staining of paired EAC tissue and PDOs with compact structure and hollow structure. The Ki67 highlights the cell populations with higher cellular proliferation20. The Ki67 (red) and Pan-CK (green) were similarly distributed among EAC primary tissue, EAC PDO compact structure, and EAC PDO hollow structure. Figure 4 shows the morphological characteristics of EAC PDOs on the first day of recovery from frozen stock with single cell-based cryopreservation (left) and undigested PDO-based cryopreservation (right).
Figure 5 summarizes a flow chart of the subculture process of EAC PDOs with and without single cell digestion. Briefly, a well growing EAC PDO is ready to be passaged. EAC PDOs were harvested and pelleted. For single cell digestion, PDOs were enzymatically digested for 5-10 min to get single cells, which were likely to grow into hollow structures that facilitate experiments requiring cell number control, uniform density, and size tracking. For undigested subculture, PDOs were split to gain more growing space without enzymatically disrupting, which were likely to grow into compact structures that facilitate histological analyses, quick expansion, and faster recovery from cryopreservation.
Figure 1: Morphological characteristics of EAC PDOs subculture with and without single cell digestion under a phase-contrast microscope. EAC PDOs grow to a certain density prior to subculture (left). Upon subculturing EAC PDOs without single cell digestion, PDOs gradually grow from hollow structures to compact structures (right, top row), whereas PDOs grown from single cells show predominantly hollow structures (right, bottom row). Pictures were taken with inverted light microscope using a 5x objective. Scale bar: 100 µm. Please click here to view a larger version of this figure.
Figure 2: Histological characteristics of EAC PDOs' compact structure and hollow structure. The H&E staining (left), Pan-CK staining (middle), and CK7 staining (right) of the compact structure (top row) and hollow structure (bottom row). Pictures were taken with inverted light microscope using a 20x objective. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 3: Immunohistochemistry staining of paired EAC tissue and PDOs. The immunofluorescence (IF) staining of paired EAC tissue (top row), compact structure (middle row) and hollow structure (bottom row) with Pan-CK (green), Ki67 (red), and DAPI (blue). Pictures were taken with inverted automated fluorescence microscope using a 20x objective. Scale bar: 50 µm. Please click here to view a larger version of this figure.
Figure 4: Morphological characteristics of EAC PDOs on the first day of recovery from frozen stock. Phase-contrast pictures of recultivation from single cell-based cryopreservation (left) and undigested PDO-based cryopreservation (right) on the first day of recovery. Pictures were taken with inverted light microscope using a 5x objective. Scale bar: 100 µm. Please click here to view a larger version of this figure.
Figure 5: The flow chart of the subculture process of EAC PDOs with and without single cell digestion. Please click here to view a larger version of this figure.
Stock | Final Concentration | 50 mL | |||
Basal medium (see Table 3) | 24 mL | ||||
Wnt-3A conditioned medium | 12 mL | ||||
R-Spondin1 conditioned medium from Cultrex R-Spondin Cells | 12 mL | ||||
N-2 | 100x | 1x | 500 μL | ||
B-27 | 50x | 1x | 1 mL | ||
N-Acetylcysteine | 0.5 M | 1 mM | 100 μL | ||
CHIR-99021 | 5 mM | 0.5 µM | 5 μL | ||
Recombinant human epidermal growth factor (EGF) | 100 µg/mL | 250 ng/mL | 125 μL | ||
A83-01 | 25 mM | 0.5 µM | 1 μL | ||
SB202190 | 10 mM | 1 µM | 5 μL | ||
Gastrin | 100 µM | 0.1 µM | 50 μL | ||
Nicotinamide | 1 M | 20 µM | 1 mL | ||
Gentamicin | 50 mg/mL | 10 µM | 5 μL | ||
Penicillin/Streptomycin | 100x | 1x | 500 μL | ||
Amphotericin B | 250 µg/mL | 0.60% | 300 μL | ||
Add freshly into well: | |||||
Noggin | 100 µg/mL | 50 μL | |||
Y-27632 | 10.5 mM | 50 μL | |||
Add when establishing new PDOs from primary tissue or recovering from frozen stocks | |||||
FGF-10a | 100 µg/mL | 100 ng/mL | 50 μL |
Table 1: Preparation of EAC PDO culture medium.
Soybean Trypsin Inhibitor (STI) | 12.5 mg |
Adjust to 50 mL with DPBS | |
Filter through 0.2 µm sterile filter |
Table 2: Preparation of Soybean Trypsin Inhibitor (STI) solution.
Reagent | Volume | Final concentration |
Advanced DMEM/F-12 | 48.2 mL | |
HEPES (1 M) | 500 µL | 10 mM |
L-Glutamine (100X) | 500 µL | 1X |
Penicillin-Streptomycin (100X) | 500 µL | 1X |
Amphotericin B | 300 µL | 0.60% |
Gentamicin (50 mg/mL) | 5 µL | 5 µg/mL |
Table 3: Preparation of basal medium.
Single cell digestion | |
Pros | Cons |
Cell number control | Fragile during embedding |
Viability check | Longer time needed between passages |
Applicable for e.g., Drug Screening, Flow Cytometry | Longer recovery time from frozen stocks |
Without single cell digestion | |
Pros | Cons |
Morphology is beneficial for histological analyses | Expansion of PDOs more in size than in number |
Higher stability in embedding process | Not applicable for analyses where single cell suspension is mandatory |
Quick recovery from frozen stocks | Lack of cell number control and size tracking |
Table 4: Pros and cons for subculturing EAC PDOs with and without single cell digestion.
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 frozen stocks and less compact morphology during the culture period. Experience indicates 2-3 weeks for single cell-based recultivation to reach applicable density for the subculture process. In contrast, frozen EAC PDOs without single cell digestion can reach the same size in a shorter period (about 1 week) after recultivation. One reason could be the extra stress from the trypsin digestion for a relatively long time (10 min). Therefore, it is recommended to preserve undigested EAC PDOs in a ratio of 1:1.5 (freezing two domes of undigested EAC PDOs and seeding back into three domes for the recultivation). In addition, using undigested EAC PDOs is recommended for quick expansion and histological characterization by IHC or IF staining due to the compact structure. The pros and cons of the two subculture methods are summarized in Table 4.
Several critical steps require attention in this protocol. Firstly, the plates for PDO culture need to be pre-warmed overnight in a 37 °C incubator to ensure the solidifying process of freshly seeded ECM gel domes. It is recommended to use a hot plate for keeping the plate at 37 °C while dealing with extended seeding duration. Secondly, low bind tubes are required during the subculture process to avoid significant PDO loss. To prevent ECM gel loss, tips with a wide bore opening can be pre-cooled in the -20 °C freezer before use. Here, the wide opening of the tips avoids the damage of PDO structures during the harvesting step. Next, it is recommended to incubate PDOs for 20 min on ice before the first centrifugation step, to ensure complete liquefying of the ECM gel. Note that the centrifuge needs to be set at 4 °C during centrifugation steps to keep residual ECM gel in the liquid state. In addition, for the single-cell method, it is recommended to thoroughly mix the PDOs after trypsin incubation using a normal 1,000 µL tip to break cell clumps before adding the STI, rather than directly filtering the cell suspension with cell strainers, to avoid cell loss.
Some modifications can be made in this protocol. The cell recovery solution can be replaced by ice-cold DPBS for dissolving the ECM gel in the harvesting step. However, experiences showed a better ability to dissolve the ECM gel using the cell recovery solution. Therefore, ice-cold DPBS is rather recommended only as an alternative backup method. If the laboratory is not equipped with a rotating incubator, EAC PDOs can be incubated with trypsin in a 37 °C water bath along with mixing by inverting the tube every 2-3 min. 10% DMSO with fetal bovine serum (FBS) can be used as an alternative for freezing medium to prepare frozen PDO stocks. However, a commercial freezing medium with lower or no serum is preferred due to a better PDO recovery.
Some limitations need to be addressed in this protocol. Since these methods have been tested only in EAC PDOs, the application of this protocol to other types of PDOs is not clear. Although procedures for passaging PDOs with and without single cell digestion are standardized for most organoid types21,22, there is still a need to attempt current protocols on other cancer types to ensure reproducibility. In addition, a 10 min 0.25% trypsin incubation may stress the cells during digestion; therefore, the incubation time could vary based on the pre-subculture PDO condition and the individual PDO diversity. During early attempts, it is suggested to set different trypsin incubation times for each EAC PDO.
In conclusion, this is the first protocol describing and discussing subculture and cryopreservation of EAC PDOs with and without single cell digestion. Subculturing EAC PDOs with single cell digestion is applicable for comparison experiments between groups while undigested EAC PDOs are beneficial for histological characterization, cryopreservation, and quick expansion. Here, the routine maintenance of EAC PDOs is standardized, providing a guide for researchers to choose appropriate methods for EAC organoid generation.
The authors have nothing to disclose.
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.
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 |