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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 approaches^7,8,9. 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 environment^10,11,12. 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 treatment^13,14. 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.

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Protokół

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

1. Pre-warm a 12-well plate by placing it into a 37 °C CO₂ incubator overnight before subculture to ensure complete warming of the plate. If available, use empty wells from a plate with the current PDO culture.
   NOTE: Continuous storage of 1-2 fresh plates at 37 °C is recommended for flexible subculture planning.
2. Pre-cool 1,000 µL and 200 µL tips with a wide orifice at -20 °C (continuous storage recommended). Pre-cool centrifuge at 4 °C.
3. Set up the temperature of the rotating incubator to 37 °C (if single cell digestion is performed).
4. Incubate an appropriate volume of ECM gel for 1 h on ice to liquefy. Place cell recovery solution on ice.

2. Harvesting organoids

1. Remove the plate with growing PDOs from the CO₂incubator.
2. Aspirate old medium using a vacuum pump.
   NOTE: Avoid touching the domes.
3. Add an appropriate volume of ice-cold cell recovery solution (500 µL/dome) into the well.
4. Disintegrate the ECM gel by pipetting up and down several times to fragment ECM gel domes into small pieces using 1,000 µL tips with a wide orifice.
5. Combine the mixture of PDO, ECM gel and cell recovery solution from a maximum of two wells (six domes) and transfer it into a 5 mL low bind tube (use a second tube in case more wells are used for subculture).
   NOTE: Optionally, if ECM gel was not dissolved completely, add an additional 1.5 mL of cell recovery solution to the mixture of PDO, ECM gel, and cell recovery solution.
6. Incubate the tube containing the mixture in step 2.5 on ice for 20 min, mix every 5 min by inverting the tube five times to ensure the liquefaction of the ECM gel.
7. Centrifuge at 500 x g for 4 min at 4° C.
8. If there is a visible and stable pellet after centrifugation, proceed with step 2.10. Otherwise, continue with step 2.9.
9. If there is no visible pellet and the PDOs still seem to be stuck in a gel phase, carefully remove the supernatant with a vacuum pump until the phase containing ECM gel-PDO-Solution is reached and add 3 mL of ice-cold cell recovery solution.
   1. Invert the tube a few times and incubate on ice for another 10 min. Mix by inverting the tube from time to time.
   2. Centrifuge at 500 x g for 4 min at 4 °C and continue with step 2.10.
10. Discard the supernatant carefully using a vacuum pump or a 1,000 µL pipette. Try to remove the supernatant as much as possible.
    NOTE: Due to the low bind surface of the tube, the pellet will not be as stable as usual.
11. Store the PDO pellet on ice and proceed with step 3 (without digestion) or step 4 (with single cell digestion) depending on the different purposes.

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.

1. Remove pre-cooled 200 µL and 1,000 µL tips with a wide orifice from the -20 °C freezer and place them onto a clean bench.
2. Resuspend the pellet from step 2.11 in ECM gel using pre-cooled 1,000 µL tips. Mix by pipetting up and down about 10 times to make sure PDOs are not clumping and are evenly distributed in the ECM gel.
   NOTE: Use 50 µL ECM gel/dome. Always calculate for one dome more than required (e.g., for nine domes (i.e., three wells), resuspend the pellet in 500 µL of liquid ECM gel (450 + 50 µL extra). Try to avoid producing bubbles during resuspension!
3. Remove the pre-warmed 12-well plate from the incubator right before seeding the domes.
4. Seed domes containing 50 µL ECM gel into the warm plate (three domes/well). Avoid pipetting bubbles into the ECM gel domes.
5. Put the plate back into the 37 °C and 5% CO₂incubator and incubate for 20-30 min to solidify the ECM gel.
6. Add pre-warmed PDO medium (Table 1) carefully without disturbing the domes.
7. Culture the PDOs for 7-14 days until the required density and morphology occur.

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.

1. Prepare digestion medium by mixing 2 mL of 0.25% Trypsin-EDTA and 20 µL DNase I (for digestion of three domes).
2. Resuspend the pellet from step 2.11 with an appropriate volume of pre-warmed 0.25% Trypsin-EDTA + DNase I and mix it about 10 times by pipetting up and down using a 1,000 µL pipette (use normal 1,000 µL tips).
3. Incubate for 10 min at 37 °C in a rotating incubator with a rotation speed of a minimum 28 rpm.
4. Prepare a 15 mL tube containing 6 mL of Soybean Trypsin Inhibitor (STI, Table 2) solution (per 2 mL of 0.25% Trypsin-EDTA).
5. After digestion, mix the digested PDOs thoroughly a few times with a 1,000 µL pipette to disrupt the PDOs.
6. Transfer the digested PDOs to the 15 mL tube containing STI solution to stop the digestion process.
7. Centrifuge at 500 x g for 4 min at 4 °C. Discard the supernatant carefully using a vacuum pump or a 1,000 µL pipette. Resuspend the pellet in 1 mL of basal medium (Table 3).
8. Determine cell concentration and viability using an automated cell counter or a Hemocytometer.
9. Seed digested PDO into a 12-well plate with 2 x 10⁴ cells per dome.
   1. Calculate the cell number according to the domes planned for seeding and transfer them to a fresh 1.5 mL low bind tube.
      NOTE: Calculate for one dome more (+ 2 x 10⁴ cells extra). For example, for seeding three domes into one well, take 8 x 10⁴ (2 x 10⁴* 3 + 2 x 10⁴ extra) cells.
   2. Centrifuge at 500 x g for 4 min at 4 °C.
   3. In case there is no visible pellet, remember the orientation of the tube inside the centrifuge to know where the pellet is located.
   4. Carefully discard the supernatant using a 1,000 µL pipette. Remove the supernatant as much as possible without disturbing the pellet.
   5. Add appropriate volume of ECM gel to the pellet using a 1,000 µL pipette with pre-cooled 1,000 µL wide orifice tip (50 µL/dome + 50 µL extra).
   6. Follow steps 3.3-3.7.

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.

1. Cryopreservation of the undigested PDOs.
   1. Start cryopreservation process with the pellet from step 2.8. Use 500 µL of cold freezing medium to resuspend the pellet and transfer it to a cryogenic vial.
      NOTE: Store two domes per vial.
   2. Freeze PDOs overnight in a -80 °C freezer using an appropriate cell freezing container.
2. Cryopreservation of the single cell digested PDOs
   1. After harvesting and digesting PDOs, start cryopreservation from step 4.8.
   2. For storing one cryogenic vial, transfer 4-5 x 10⁵ cells into a fresh 1.5 mL low bind tube.
      NOTE: Store three domes/vial.
   3. Centrifuge at 500 x g for 4 min at 4 °C. Discard the supernatant carefully using a 1,000 µL pipette. Remove the supernatant as much as possible without disturbing the pellet.
   4. Resuspend the pellet in an appropriate volume of freezing medium (500 µL/vial) and transfer it to a cryogenic vial.
   5. Freeze PDOs overnight in a -80 °C freezer using an appropriate cell freezing container and transfer them into a -150 °C freezer or liquid nitrogen for long-term storage.

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Wyniki

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 (

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Dyskusje

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 ...

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Materiały

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