Generating Tumor Organoid Cultures from the Lung Adenocarcinoma Cell Line

Summary

Here, we present a rapid, pure, and cost-effective method for establishing tumor organoids from the A549 cell line, suitable for preliminary tests and educational purposes.

Abstract

Here, we describe a protocol for generating Cell Line-Derived Tumor Organoids (CDTOs) from the A549 human lung adenocarcinoma cell line. The protocol involves embedding 2D-expanded A549 cells in Matrigel and maintaining them in 3D culture medium for long-term culture. Recommended seeding density of 500 cells/µL was determined to support consistent organoid formation. The resulting CDTOs were characterized by hematoxylin and eosin (H&E) staining and immunofluorescence (IF). The organoids maintained high expression of the lung adenocarcinoma markers Thyroid Transcription Factor 1 (TTF-1), adhesion protein E-Cadherin (ECAD), and cytoskeleton protein Keratin 7 (KRT7). Furthermore, tight junction protein Zona Occludens 1 (ZO-1) expression showed dysregulated polarity of tumor organoids. This protocol offers a technically straightforward, cost-effective, and purely tumorous organoid platform for lung adenocarcinoma research. Its simplicity and reproducibility also make it suitable for undergraduate laboratory teaching, where it can help students acquire fundamental 3D tumor organoid culture techniques within a limited lab schedule.

Introduction

Organoids are in vitro. self-organized three-dimensional (3D) structures that originate from stem cells and are capable of recapitulating biological complexities of real organs to a considerable extent¹. The cells constituting organoids may derive from induced pluripotent stem cells or tissue-derived cells, the latter including normal stem/progenitor cells, differentiated cells, and tumor cells². Compared with traditional two-dimensional culture systems and animal models, organoids can more closely mimic the physiological characteristics of the human body while offering greater experimental flexibility. Consequently, organoid technology has been widely applied in drug development³, personalized medicine⁴, and disease modeling⁵.

Numerous studies have established organoid models for a broad spectrum of cancers, including colorectal⁶, prostate⁷, pancreatic⁸, gastric⁹, hepatic¹⁰, biliary¹¹, breast¹², and neuroendocrine tumors. However, one of the major challenges in tumor organoid research lies in maintaining purity. For instance, a study on non-small cell lung cancer revealed that only 17% of the established organoids were purely tumorous, as tumor samples often contain a mixture of normal and malignant cells that are difficult to fully separate prior to culture¹³. This impurity issue can cause elimination of tumor cells by outgrowth of non-tumor cells, interfere with readouts of drug susceptibility tests, and lead to inaccurate molecular analyses, such as RT-qPCR or Western blot, where RNAs and proteins from tumor cells are diluted by uncontrollable proportions of contaminating cells.

To address this issue, researchers have explored the design of selective culture media that exploit the reduced dependency of tumor cells on certain growth factors, thereby inhibiting normal cell growth while promoting tumor cell expansion¹⁴. In addition, strategies such as cell sorting or monoclonal identification are being developed to construct pure tumor organoids.

This protocol describes the generation of Cell line-Derived Tumor Organoids (CDTOs) using the A549 human lung adenocarcinoma cell line, which has been reported to partially exhibit cancer stem cell-like properties, as well as enhanced clonogenicity, proliferative potential, and tumorigenicity in vitro.¹⁵. The resulting CDTOs maintained expression of lineage-specific biomarkers Thyroid Transcription Factor 1 (TTF-1)¹⁶, adhesion protein E-Cadherin (ECAD)¹⁷, and cytoskeleton protein Keratin 7 (KRT7)¹⁶ and exhibited physiological features characteristic of tumor organoids. This model is easy to establish and contains defined cancer cell components, making it suitable for researchers new to the tumor organoid field or those challenged by loss of tumor identity in primary tumor organoid cultures. Potential applications include preliminary drug susceptibility testing¹⁸, tumor microenvironment studies¹⁹, and educational training in 3D culture techniques.

Protocol

The room temperature in the lab is 22–24 °C and is referred to as RT. Using a swing-bucket centrifuge is recommended. The reagents and the equipment used are listed in the Table of Materials.

1. Establishment of primary A549 organoids

1. Preparation and A549 cell harvest
   1. Prepare a 6 cm dish containing A549 cells at 70% confluency. Prewarm TrypLE and PBS to 37 °C. Calculate the cell amount and Matrigel volume according to the experimental design and Table 1.
      1. Thaw required Matrigel aliquots with redundancy for at least two wells on ice for at least 60 min. Prepare polystyrene and TC-treated plates to allow the domes to stay hemispherical.
      2. Freshly prepare 2D medium (Dulbecco‘s Modified Eagle’s Medium containing 10% fetal bovine serum) and 3D medium (Human Lung Adenocarcinoma Organoid Medium). 3D medium can be generated from commercial kits or according to the works of Sachs, N. et al. from Hans Clever’s lab²⁰.
         NOTE: One 6 cm dish can usually yield about 5,000,000 cells after digestion. Reduce the number of input cells for the experimental design. Plates designed for confocal imaging using a glass bottom are not suitable for Matrigel to form a proper 3D shape.
   2. Aspirate the spent 2D medium and wash the cells once with 1 mL PBS to remove residual serum. Aspirate PBS, add 1 mL of prewarmed TrypLE, and incubate the plate at 37 °C for 4 min.
   3. Observe the cells under a microscope. When the cytoplasm retracts and the cells lose intercellular connections, add an equal volume (1 mL) of fresh 2D medium to terminate the enzymatic reaction.
   4. Gently pipette to detach and resuspend the adherent cells from the culture surface. Transfer the cell suspension into 1.5 mL centrifuge tubes and centrifuge at 300 × g. for 3 min at RT to pellet the cells.
   5. Discard the supernatant and resuspend the cell pellet in 1 mL 2D medium. Dilute 10 µL cell suspension to 100 µL and count cell numbers with a hemocytometer. Calculate the required volume according to step 1.1.1 and aspirate to a 1.5 mL centrifuge tube with pre-rinsed tips.
2. Seeding harvested cells into Matrigel
   1. Prepare ice and precool pipette tips in -20 °C fridge for 1 min and use low-retention tips if possible. Make sure no crystals remain in the Matrigel aliquots and that no bubbles form when the tip enters the solution.
   2. Centrifuge the tube containing harvested cells at 300 × g. at RT and discard the supernatant. Place the tube on ice. Use precooled pipette tips to slowly aspirate the required volume of Matrigel and add it to the pellet on ice. Resuspend using only the first stop of the pipette slowly on ice. Please do not leave ice for more than 10 s if observation is required.
   3. Use precooled pipette tips and keep the mixture on ice. Dispense one aliquot of the mixture into the center of each well. Slowly withdraw the pipette while dispensing, using only the first stop to avoid bubble formation.
      NOTE: The mixture should form a dome-shaped 3D structure in the center. Resuspend the mixture before proceeding to the next well. If possible, avoid using the edge wells and add PBS to these wells.
   4. Incubate the plate at 37 °C in a 5% CO₂ incubator for 40 min to allow gel polymerization. After the Matrigel has solidified, carefully add 3D medium along the well wall, and continue culturing at 37 °C.
      NOTE: Avoid generating bubbles during dispensing. If bubbles are generated, avoid using that portion of the mixture.
   5. During culture, replace the 3D medium every 3 days or whenever a color change (from pink to yellow) is observed. Examine organoid growth under an inverted microscope at each medium change, and document morphology photographically to monitor structural development.
      NOTE: Organoids should be dense and convoluted with diameters longer than 30 µm instead of disintegrated or fragmented as indicated by 5000 cells/µL (Figure 1D).

2. Fixation, embedding, and sectioning of organoids

1. Prepare organoid cultures at day 6–9. Remove the culture medium from the cell culture plate. Gently detach the organoids by pipetting with 1 mL of cold PBS, and transfer them into a 1.5 mL tube pre-rinsed with 5% BSA to prevent adhesion. Allow the organoids to settle naturally by gravity on ice and carefully remove the supernatant.
2. Add 1 mL cold PBS to resuspend the pellet. Allow the organoids to settle naturally by gravity for at least 5 min and carefully remove the supernatant. If some organoids fail to sediment, centrifuge briefly at 200 × g. for 1 min at 4 °C to collect them. This step will be performed multiple times and will be referred to as ‘small wash’.
3. Perform the small wash twice to remove residual Matrigel. If residual Matrigel persists, add a final concentration of 10 mM EDTA to cold PBS while washing.
   ​CAUTION: Transfer the samples to the fume hood. Add 4% paraformaldehyde (PFA) sufficient to cover the organoids, and fix on ice for 1 h in a fume hood. Discard the fixative and perform the small wash twice. Proceed with step 4.1 for IF staining.
4. After the organoids settle at the bottom of the tube, discard the supernatant and preheat the samples in a 70–75 °C metal bath for 5 min to avoid rapid agarose solidification during step 2.7.
5. Prepare 3% (w/v) agarose in 1× PBS, and heat the mixture in a microwave water bath until fully dissolved and transparent. Avoid boiling to prevent volume loss or changes in agarose concentration.
6. Rapidly resuspend the fixed organoids in 30–50 µL of 3% agarose, ensuring even distribution, and cool on ice for 20–30 min until solidified, avoiding generating bubbles.
7. Once the agarose-organoid mixture has solidified, carefully remove the agarose block from the tube using a needle and transfer it into a labeled tissue embedding cassette.
8. Sequentially immerse the cassette containing the agarose block in graded ethanol solutions (30%, 50%, 70% [may be stored overnight at 4 °C], 80%, 90%, 95%, 100%, and 100%) for dehydration. Incubate each step on a shaking platform at 20 rpm for 30 min.
9. Pre-melt paraffin wax blocks in an oven before use at 65 °C. Transfer the dehydrated agarose-embedded organoids into xylene for clearing. Immerse twice, each for 15–20 min, to remove residual ethanol.
10. Transfer the tissue cassettes sequentially into paraffin bath 1 for 45 min, and then into paraffin bath 2 for another 45 min.
11. Place the tissue cassettes and metal molds into the molten paraffin chamber of the embedding station. Remove the organoid or tissue sample from the cassette and position it in the center of the metal mold. Place the mold on a cold plate to fix the sample orientation.
12. Position the tissue cassette on top of the metal mold, fill with additional molten paraffin, and allow to cool on the cold plate for at least 60 min. Once the paraffin block is fully solidified, remove the metal mold, and store the paraffin-embedded organoid block at 4 °C until sectioning.
13. Section the paraffin-embedded organoid blocks using a microtome. Ensure that the thickness of each section is uniform to facilitate subsequent staining and imaging.
14. Allow the cut sections to float in a water bath maintained at 40 °C to allow complete expansion of the paraffin ribbons. Ensure that each section spreads out as a single, wrinkle-free layer; discard any folded or overlapping sections.
15. Mount the expanded sections onto adhesive microscope slides by immersing the slide at a slight angle beneath the section and lifting it vertically to capture the tissue. Label the slides with a pencil to preserve sample identification during subsequent processing.

3. Hematoxylin and Eosin (H&E) staining

1. Bake the slides containing tissue or organoid sections in a 60 °C oven for 1–2 h to ensure proper adhesion and remove residual paraffin.
2. Dewax the sections sequentially by immersing them in xylene I for 15 min, xylene II for 15 min, and then rehydrate through a graded ethanol series of 100%, 100%, 95%, 90%, 80% and 70%, each for 5 min. Then, rinse the slides in double-distilled water (ddH₂O) twice, each for 5 min.
3. Stain the sections with hematoxylin for 5 min, rinse in running tap water for 1–2 min, and then differentiate in 1% acid alcohol for 2 s. Wash again in tap water for 1–2 min, then bleach the nuclei in 1% ammonia water for 5 s, followed by a final rinse in tap water for 1–2 min.
4. Dehydrate the sections through a graded ethanol series of 70%, 80%, 90%, and 95%, each for 3 min, and then counterstain with eosin in an alcohol-based eosin solution for 10 min.
5. Rinse the sections twice in absolute ethanol, each for 5 min.
   NOTE: For alcohol-based eosin, ensure dehydration up to 95% ethanol prior to staining. For water-soluble eosin, perform staining before dehydration.
6. Clear the sections by immersing them twice in xylene, each for 5 min, and keep them in xylene until mounting.
7. Mount the slides immediately after removal from xylene using neutral resin mounting medium, carefully avoiding air bubbles.
8. Air-dry the mounted slides at RT for 30–60 min, then observe under a light microscope.
   NOTE: Immediate mounting after xylene clearing prevents oxidation and ensures optical clarity.

4. Whole-mount immunofluorescence staining

1. Continue from step 2.4. Add 1 mL blocking and permeabilization solution (5% BSA + 0.3% Triton X-100) to the pellet and incubate at RT for 60 min or at 4 °C as a stopping point for 1–2 days.
2. Dilute primary and secondary antibodies following manufacturer’s recommended ratio in the blocking and permeabilization solution. Small wash once and add 200 µL diluted primary antibodies to the pellet. Incubate at 4 °C overnight.
3. Perform the small wash 3 times and add 200 µL diluted secondary antibodies to the pellet. Incubate at RT for 60 min.
4. Next, perform the small wash 3 times. Add 15 µL mounting medium to resuspend the pellet and drop the medium containing organoids on a glass slide. Slowly apply a coverslip to the medium and seal the coverslip with nail polish. Do not manually compress the cover slip and allow the mounting medium to spread by surface tension.

Results

The general process of this protocol includes expanding 2D A549 cells into the exponential stage (Supplementary Figure 1A) and passage into 3D Matrigel to form organoids (Figure 1A). Upon encapsulation in Matrigel, cells proliferated and self-assembled, predominantly forming monoclonal organoids (Figure 1B). Its dense and convoluted morphology was uniform across passage (Supplementary Figure 1C). In comparison, most of these str...

Discussion

This protocol describes a method for generating A549 lung adenocarcinoma Cell line-Derived Tumor Organoids optimized for technical simplicity, reproducibility, and educational use. Three parameters critically determine success: initial cell state, medium formulation, and matrix composition. Additionally, culture duration is not recommended to exceed 14 days. Listed below are key parameters and troubleshooting.

Initial cell state
The cells were maintai...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Micro Centrifuge tube	Biofil	CFT003015
A549 [A-549]human non-small cell lung cancer cells	ZQXZbio	ZQ0003	STR: Amelogenin: X,Y; CSF1PO: 10,12; D13S317: 11; D16S539: 11,12; D5S818: 11; D7S820: 8,11; TH01: 8,9.3; TPOX: 8,11; vWA: 14. Mycoplasma was negetive when purchased was tested every 2 weeks. Passage number within 10 passages from the P1 aliquots of the purchased batch was used.
Advanced DMEM/F12	Gibco	12634010
Anti-E Cadherin antibody	Abcam	ab231303
Anti-Fade Mounting Medium	YEASEN	36307ES08
Anti-HIF-1 alpha antibody	Abcam	ab51608
Biological Safety Cabinet	Nuaire LABGARD	NU-540
Biological Tissue Embedding Machine	KEDEE	KD-BM
BSA	Sigma	A1933
Cell-Grade BSA	Sigma	A1933-100G
Centrifuge	Eppendorf	Centrifuge 5702 R	Swing bucket; Mild to low speed.
Centrifuge tube	Biofil	CFT920150
Citrate Antigen Retrieval Solution (pH 6.0)	ZSGB-BIO	ZLI-9065
Cryostage	KEDEE	KD-BL
Cytation 5 Cell Imaging Multimode  Reader	Biotek	Cytation 5
Cytokeratin 7 (KRT7) Rabbit mAb	Abclonal	A4357
DMEM	Gibco	11965092
DMSO	Solarbio	D8371
Donkey Serum	Solarbio	SL050
Dulbecco's Modified Eagle Medium	Gibco	11965118
Eclipse Ts2 Inverted Microscope	Nikon	Eclipse Ts2
Eosin Staining Solution (Alcohol-Soluble)	Servicebio	G1001
FBS	Nobimpex	B118-500
Forma Steri-Cycle i160 CO2 Incubator	Thermo Scientific	i60
Hematoxylin Bluing Solution	Servicebio	G1040
Hematoxylin Differentiation Solution	Servicebio	G1040
Hematoxylin Staining Solution	Servicebio	G1004
Hemocytometer	Solarbio	YA0810
Human Lung Adenocarcinoma Organoid Medium (3D medium)	PMO Bio	HC1001
Immunohistochemistry (IHC) Pen	ZSGB-BIO	ZLI-9305	Store in 4°C
Laser Confocal Microscope  FV3000	Olympus	FV3000	Usual parameter range: PMT Voltage: 500 ± 200 V; Laser Transmissivity: 5 ± 3%; Offset: 3 %
Manual Rotary Microtome	Leica	RM2235
Metal Bath	ALLSHENG	MK-3000
Microwave Oven	Galanz
Nail Polish	Lete
Neutral Balsam (Mounting Medium for Microscopy)	Beyotime	C0173
Organoid-Specific Extracellular Matrix Gel	PMO Bio	BM1001
PBS	WISENT	311-010-CL
Peroxidase Blocking Solution	Beyotime	P0100A
Pipettes	Eppendorf	312300063/312300020/312400083
Pipettes tips	Axygen	14-222-692/14-222-723/14-222-869	Consider using low retention tips if possible.
Three-Dimensional Shaker	SCILOGEX	SK-D3309-Pro	Low speed.
Tissue Flotation Water Bath	KEDEE	KD-P
Triton X-100	VWR	92046-34-9
Trypan Blue	Gibco	15250061
TryPLE	Gibco	12605028
TTF1 Recombinant Monoclonal Antibody	HUABIO	HA720067