Cancer Cell Lines Culture Protocol and FAQs

Cancer cell lines are obtained from tumor tissue or cancer cells by in vitro culture, and these cells are able to proliferate indefinitely. Cell lines provide an opportunity to study the characteristics of cancer cells in a controlled environment. Commonly used cancer cell lines include HeLa (cervical cancer), MCF-7 (breast cancer), A549 (lung cancer), etc. These cell lines play an important role in basic research, drug development and biomedical research.

The Principle of Cancer Cell Lines Culture

Simulate the in vivo environment in vitro to support the growth and reproduction of cancer cells. Key elements include:

1 Medium : Cell culture requires a medium containing nutrients, such as DMEM (Dulbecco 's Modified Eagle Medium) or RPMI-164. The medium is usually rich in glucose, amino acids, vitamins, minerals, and serum (such as fetal bovine serum) to provide the cells with the required nutrients.
2 Growth conditions : The cells were usually cultured in a thermostatic incubator at 37 ° C and 5 % CO2. These conditions mimic the environment in the human body to support cell proliferation and function maintenance.
3 Sterile operation : cell culture needs to be carried out under sterile conditions to avoid microbial contamination. Sterile techniques include the use of ultra-clean benches, autoclaves, filters and other equipment.
4 Cell passage : Since the cell will reach the growth bottleneck after a period of culture, it must be passaged to maintain cell activity. Cell passage usually involves digestion, dilution and reseeding into a new culture dish.

The Steps of Cancer Cell Lines Culture

Preparation

Ensure that all equipment and reagents are sterile, including culture flasks, pipettes, media, etc. Before the start of the experiment, clean the ultra-clean bench and perform ultraviolet disinfection.

Medium Preparation

Appropriate medium was prepared according to the needs of the cell line and preheated in a 37 ° C water bath. Add an appropriate amount of serum and antibiotics (if necessary), mix well and set aside.

Cell Resuscitation

The frozen cell bank was removed from liquid nitrogen and quickly thawed in a 37 ° C water bath. The thawed cell suspension was transferred to a centrifuge tube and diluted with fresh medium. The cryoprotectant was removed by centrifugation at low speed (about 100 rpm) for 5 minutes.

Cell Culture

After centrifugation, the supernatant was discarded, and an appropriate amount of fresh medium was added to re-suspend the cells. The cell suspension was transferred to a culture flask and placed in a culture chamber (37 ° C, 5 % CO2). The medium was changed every 24-48 hours, depending on the cell type.

Cell Passage

When the cells reach 80-90 % fusion, passage is required. Remove the old medium and wash the cells with PBS buffer. Add an appropriate amount of trypsin digestion solution and incubate at 37 ° C for about 2-5 minutes. Observe whether the cells fall off from the culture bottle wall, tap the bottle wall to help the cells fall off. The fresh medium was added to terminate the digestion, and the cell suspension was transferred to a centrifuge tube. The cells were centrifuged at low speed and the supernatant was discarded. The cells were resuspended in fresh medium, diluted as needed, and then seeded into new culture flasks for further culture.

Cell Counting and Storage

Cells can be counted using trypan blue staining. For long-term preservation, cells can be frozen in the presence of cryoprotectants (such as DMSO) and stored in liquid nitrogen.

Figure 1. Schema for the Cultivation of Human Lung Cancer Cells.

Advantages of cancer cell line culture

Infinite Proliferation Ability

A prominent feature of cancer cell lines is their ability to proliferate indefinitely. Users can obtain a large number of cells by repeated passages, which provides great convenience for the repeatability of experiments and long-term research.

Stability

Compared with primary cell culture, cell lines have higher genetic stability. Although some genetic variations may occur during long-term culture, most cell lines still perform well in terms of stability and are suitable for long-term use and comparative studies.

Controllable Experimental Conditions

Under laboratory conditions, researchers can accurately control the environmental factors of cell growth, such as temperature, pH, nutrients, etc. This controllability helps to carry out refined research and analyze the effects of specific variables on cells.

Standardization

Since many cell lines are widely used standard models worldwide, the results of the study have good comparability and repeatability. In addition, standardized cell lines also facilitate cooperation and data sharing between different laboratories.

Economy

Compared with animal experiments, cell line culture costs less and does not involve ethical issues. This makes the cell line an ideal choice for many preliminary screening experiments.

Applications of cancer cell line culture

Drug Screening and Development

Cancer cell lines are important tools for drug screening. By testing the effects of compounds in these cells, the anti-tumor activity, toxicity and mechanism of action of the drugs can be preliminarily evaluated. The application of high-throughput screening technology makes it possible to evaluate a large number of compounds in a short time, thus accelerating the process of new drug discovery.

Molecular Mechanism Research

Cell lines are widely used to study the molecular mechanisms of cancer, including signal transduction pathways, gene expression regulation, cell cycle and apoptosis. Through gene editing techniques (such as CRISPR-Cas9), researchers can manipulate specific genes to further explore their role in cancer development.

Drug Resistance Studies

Cell lines can be used to simulate and study the development of drug resistance. Through long-term exposure to chemotherapeutic drugs, cell lines may obtain drug-resistant phenotypes, and researchers can analyze their mechanisms and find ways to overcome drug resistance.

Personalized Medicine

Cancer cell lines can represent specific types or stages of cancer, so they are used to develop treatment strategies for specific cancers. At the same time, organoid technology and patient-derived xenograft models developed in recent years have further promoted the realization of personalized medicine.

Virus Research and Vaccine Development

Some cancers, such as cervical cancer, are closely related to viral infection. Cell lines also play a key role in studying the mechanism of viral carcinogenesis and developing corresponding vaccines.

Biomarker Discovery

By analyzing the genome, transcriptome and proteome of cancer cell lines, scientists can identify potential biomarkers for early detection, prognosis and treatment monitoring of cancer.

Alpha Lifetech provides cancer cell line culture assay, which has advanced cancer cell line culture detection technology and provides cancer cell line culture detection services to support scientific research and drug development.

FAQ

1. In the process of cell culture, bacterial, fungal or mycoplasma contamination can lead to experimental failure.

Solutions : (1) Sterile operation : Ensure that all operations are performed under sterile conditions, including the use of disinfected utensils and reagents. CO2 concentration and temperature were monitored regularly in the cell incubator. (2) Filtration and disinfection : Use a filter to filter the culture medium and regularly clean and disinfect the ultra-clean bench and culture equipment. (3) Mycoplasma detection : regular use of specific detection kits to check mycoplasma contamination, and timely treatment of contaminated cell lines.
2. Cell growth is slow or unable to proliferate

Solutions : (1) Ingredients of the medium : Check the ingredients of the medium to ensure that all necessary nutrients and serum are at the appropriate concentration. (2) Environmental conditions : Confirm whether the culture temperature, CO2 concentration and humidity are appropriate. Normally, cells need to be cultured at 37 ° C and 5 % CO2. (3) Passage frequency : avoid excessive passage, frequent passage may lead to poor cell status, follow the recommended passage ratio of the cell line.
3. Cell morphological changes, such as rounding, shedding, etc., may indicate poor health.

Solutions : (1) Observe cell density : control the appropriate cell density, too high or too low density will affect cell morphology. (2) Regulating the use of trypsin : too long digestion time may lead to cell damage, careful control of digestion time, timely suspension of digestion and medium neutralization. (3) Cell health examination : Cell morphology was observed regularly by microscope, and active staining such as trypan blue staining was performed if necessary to check cell viability.
4. The results were inconsistent between different experiments, even when the same experiment was repeated.

Solutions : (1) Standardized operation : ensure that each experiment using the same steps and conditions, including culture medium, cell density, passage ratio, etc. (2) Detailed recording : Detailed recording of the conditions and steps of each experiment, including any anomalies, for subsequent analysis and correction. (3) Batch effect : Note the possible effects of different batches of media and reagents, and using reagents from the same batch can reduce variables.
5. After long-term culture, cells may lose their original phenotypic characteristics.

Solutions : (1) Limit the number of passages : Keep within the recommended number of passages, and try to use the cells with initial low passages for key experiments. (2) Genotyping : Genotyping of cell lines was performed regularly to confirm that they maintained their original characteristics, such as STR (short tandem repeat) analysis. (3) Cryopreservation : Keep multiple low-passage generations of cryopreservation stock so that it can be re-thawed and amplified when features are lost.

reference

[1] Noel V, Berry MD. Culture of Adherent Cancer Cell Lines. Methods Mol Biol. 2022;2508:19-29. doi:10.1007/978-1-0716-2376-3_3

[2] Longjohn MN, Phan HD, Christian SL. Culturing Suspension Cancer Cell Lines. Methods Mol Biol. 2022;2508:9-17. doi:10.1007/978-1-0716-2376-3_2

[3] Tutty MA, Holmes S, Prina-Mello A. Cancer Cell Culture: The Basics and Two-Dimensional Cultures. Methods Mol Biol. 2023;2645:3-40. doi:10.1007/978-1-0716-3056-3_1

[4] Oie HK, Russell EK, Carney DN, Gazdar AF. Cell culture methods for the establishment of the NCI series of lung cancer cell lines. J Cell Biochem Suppl. 1996;24:24-31. doi:10.1002/jcb.240630504