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. Conseque....

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....

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