Calu-3 Cell Line

Calu-3 cells constitute a human-derived airway epithelial cell line representing non-small cell lung carcinoma. They are widely used in biomedical research that includes studying lung cancer biology, respiratory diseases, host-pathogen interactions, and drug transport in the airway. Moreover, they are employed to develop treatments for several respiratory disorders.

This article will provide you with comprehensive knowledge about the Calu-3 cell line. Here, you will learn:

Origin and general characteristics of the Calu-3 cells
Calu-3 cell line: Culturing information
Advantages & disadvantages of Calu-3 cells
Applications of Calu-3 cell line in research
Calu-3 cells: Research publications
Resources for Calu-3 cells: Protocols, Videos, and More

1. Origin and general characteristics of the Calu-3 cells

The primary information you require about a cell line is its origin and general attributes. It will help you decide its use in your research work. This section will assist you in learning this imperative information about the Calu-3 cell line. It will include: What is a CALU-3 cell line? What is the morphology of Calu-3 cells? What is the origin of Calu-3 cells?

Calu-3 cells were obtained from the pleural effusion (metastatic site) of a Caucasian male (25 years old) with adenocarcinoma of the lung. The cell line was established in 1975 by Jorgen Fogh and Germain Trempe from the Memorial Sloan Kettering Cancer Center.
Calu-3 cells possess an epithelial-like morphology.
The Calu-3 cell size ranges from 8-9 to 20 microns in diameter.
They harbor mutations in K-RAS (G13D), TP53, and CDKN2A genes and express wild-type EGFR.

A549 Vs Calu-3

A549 and Calu-3 cells are human lung adenocarcinoma cell lines but have distinct characteristics. The primary difference between Calu-3 and A549 cell models is the thickness of the mucus layer. Calu-3 cells form a thinner mucus layer, thus modeling the proximal airway epithelium. On the other hand, A549 cells lack this characteristic and are more suitable for representing the physiological structure of the distal respiratory tract [1].

Visualization of the bronchi in bronchial asthma. Asthma is a lung disease in which the airways are constricted swollen and blocked by excess mucus, leading to typical symptoms such as shortness of breath.

2. Calu-3 cell line: Culturing information

This section will help you know the key points for culturing the Calu-3 cell line. Here we will discuss: What is Calu-3 cells doubling time? What is Calu-3 cells medium? What is the Calu-3 cell culture protocol? How do you culture Calu-3 cells?

Key Points for Culturing Calu-3 Cells

Doubling Time:	The Calu-3 cells doubling time is approximately 35 hours.
Adherent or in Suspension:	Calu-3 is an adherent lung adenocarcinoma cell line.
Split Ratio:	The subcultivation ratio for the Calu-3 cell line is 1:2 to 1:4. For subculturing; cells are rinsed with 1 x phosphate buffer saline (PBS) and incubated with Accutase (passaging solution) at an ambient temperature for almost 10 minutes. After that, fresh cell media is added, and detached cells are centrifuged. The cell pellet is resuspended carefully, and cells are dispensed into the flask containing fresh culture media for growth.
Growth Medium:	EMEM media containing 10% FBS, 2 mM L-Glutamine, 1.5 g/L NaHCO3, EBSS, 1 mM Sodium pyruvate, and NEAA is used to culture Calu-3 cells. The Calu-3 cells medium should be replaced 2 to 3 times per week.
Growth Conditions:	Calu-3 cells are grown in a humidified incubator at 37°C temperature and 5% CO2 supply.
Storage:	Frozen cells should be stored in the vapor phase of liquid nitrogen or at below -150 °C temperature to protect the viability of cells for the longer term.
Freezing Process and Medium:	CM-1 or CM-ACF freezing media is mainly used for freezing the Calu-3 lung cell line. To preserve cell viability and prevent cells from any shock, the cells undergo a slow freezing process that allows only a 1 °C per minute decrease in temperature.
Thawing Process:	Cells are thawed by placing a vial in a water bath pre-set at 37 °C for around 1 minute or until a small ice clump is left. Fresh culture media is added, and cells are centrifuged to remove freezing media components. Afterwards, the cell pellet is resuspended, and cells are poured into a new flask containing a growth medium.
Biosafety Level:	Biosafety level 1 laboratory settings are essential for handling the Calu-3 cell line.

Calu-3 cells at different passages, highlighting the typical islet-like adherent clusters.

3. Advantages & disadvantages of Calu-3 cells

Like other human cell lines, Calu-3 cells have their own set of advantages and disadvantages. Herein, we will discuss a few important ones.

Advantages

In vitro airway epithelial model:
In respiratory research, Calu-3 cells serve as an efficient in vitro model for airway epithelium. They reflect the attributes of the human airway lining, enabling investigations into drug transport, host-pathogen interaction studies, and mucin production.
Polarization:
Calu-3 cells form polarized monolayers, making them widely used for investigating drug transport and host-pathogen interactions in a more realistic context.

Disadvantages

Cancer cell line:
Calu-3 cells were obtained from lung adenocarcinoma, so it's important to note that they may not fully represent healthy lung tissue. Researchers should consider this when using them as a model in studies.

4. Applications of Calu-3 cell line in research

Calu-3 offers several applications in biomedical research. This section of the article will shed light on a few most promising ones.

Respiratory disease studies: Calu-3 lung cells are used to investigate various respiratory diseases or disorders, for instance, cystic fibrosis, asthma, and chronic obstructive pulmonary disease (COPD). A study by Chiara Papi and colleagues investigated the impact of the anti-miR-101-3p peptide-nucleic acid (PNA) in targeting the miRNA-101-3p using the Calu 3 cystic fibrosis in vitro model. The study found that PNA treatment tends to increase the expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, suggesting a potential therapeutic strategy for cystic fibrosis and related disorders [2].
Drug development: Calu-3 cells serve as a model for testing and developing drugs for several respiratory conditions. Besides, these cells are also employed in studying drug transport across the airway epithelium. For instance, research conducted in 2021 explored the anti-viral activity of Andrographis paniculata plant extract and its bioactive andrographolide on the Calu-3 cells sars-cov-2 infection [3].
Host-Pathogen Interactions: Calu-3 cells are ideal for studying pathogens' interaction with the airway epithelium, aiding in understanding respiratory infections such as SARS-CoV-2. For example, Byoung Kwon Park and colleagues examined the Calu-3 and Vero cell responses and virus production in response to SARS-CoV-2 infection [4].

5. Calu-3 cells: Research publications

The following are some exciting and frequently cited research studies featuring the Calu-3 cells:

Carbon monoxide releasing molecule-2 suppresses proliferation, migration, invasion, and promotes apoptosis in non-small cell lung cancer Calu-3 cells

This study was published in the European Review for Medical and Pharmacological Sciences in 2018. The research proposed that carbon monoxide-releasing molecules-2 (CORM-2) promote non-small cell lung cancer cells (Calu-3) apoptosis and suppress their proliferation, migration, and invasion.

Calu-3 epithelial cells exhibit different immune and epithelial barrier responses from freshly isolated primary nasal epithelial cells in vitro

This research in the Clinical and Translational Allergy (2018) compared immune and epithelial barrier responses of the Calu-3 cell line and freshly cultured primary nasal epithelial cells.

Quinine inhibits infection of human cell lines with SARS-CoV-2

This article in Viruses (2021) proposed quinine as a potential treatment for SARS-CoV-2 infection by assessing its effect on different virus-infected cell lines such as Calu-3.

High D-glucose levels induce ACE2 expression via GLUT1 in human airway epithelial cell line Calu-3

This research in BMC Molecular and Cell Biology (2022) proposed that high D-glucose encourages Calu-3 cells ACE2 expression via regulation of the GLUT1 gene.

Dolosigranulum pigrum Modulates Immunity against SARS-CoV-2 in Respiratory Epithelial Cells

This article in Pathogens (2021) explored the immunomodulatory effects of Dolosigranulum pigrum 040417 in lung epithelial cells. Besides it investigated the potential of this immunobiotic bacterium in protecting against SARS-CoV-2 infection.

6. Resources for Calu-3 cells: Protocols, Videos, and More

There are many online resources available on Calu-3 cells that comprise information related to cell culturing and transfection.

Calu-3 cells transfection protocol: This resource will provide vital knowledge about Calu-3 cell transfection.
Calu-3 transfection: This video tutorial is a step-by-step guide to learning the in vitro Calu-3 transfection protocol.

The Calu-3 cell culture protocol is mentioned here.

Calu-3 cells: This document encompasses information regarding Calu-3 cells medium and subculturing or passaging protocol.

References

Wiese-Rischke, C., R.S. Murkar, and H. Walles, Biological Models of the Lower Human Airways-Challenges and Special Requirements of Human 3D Barrier Models for Biomedical Research. Pharmaceutics, 2021. 13(12).
Fabbri, E., et al., Treatment of human airway epithelial Calu-3 cells with a peptide-nucleic acid (PNA) targeting the microRNA miR-101-3p is associated with increased expression of the cystic fibrosis Transmembrane Conductance Regulator () gene. European Journal of Medicinal Chemistry, 2021. 209: p. 112876.
Sa-Ngiamsuntorn, K., et al., Anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component andrographolide in human lung epithelial cells and cytotoxicity evaluation in major organ cell representatives. Journal of natural products, 2021. 84(4): p. 1261-1270.
Park, B.K., et al., Differential Signaling and Virus Production in Calu-3 Cells and Vero Cells upon SARS-CoV-2 Infection. Biomol Ther (Seoul), 2021. 29(3): p. 273-281.