Cell Culture: Definition, Applications, Protocol, And Contamination

Cell Culture Definition

Cell culture refers to the process of growing cells from an animal, plant, or microorganism in a controlled environment outside of their natural habitat. This technique is used in various fields such as medicine, biotechnology, and research to study cell behavior and develop new treatments.

Cell Culture Applications

Cell culture is the process of growing cells in a controlled environment outside their natural environment.

The applications of cell culture are diverse and include experimental model systems in basic and medical sciences, toxicity testing, drug screening and development, cancer research, virology, genetic engineering, gene therapy, stem cell therapy, and cell-based manufacturing.

In a clinical context, cell culture is most commonly linked to creating model systems that study basic cell biology, replicate disease mechanisms, or investigate the toxicity of novel drug compounds.

Cell culture offers certain advantages over whole organisms such as the ability to control environmental factors (e.g., temperature, osmotic pressure, oxygen, and carbon dioxide tension) very accurately.

However, highly skilled personnel are required to perform techniques using strict asepsis techniques because animal cells grow slower than many common contaminants (e.g., bacteria, viruses, and fungi).

Cell culture is used for the generation of genetically engineered proteins, antibodies, hormones, and biopharmaceuticals that can be isolated and used therapeutically.

Human cancer cell lines are used as models to assess novel chemotherapeutic drug screening or interactions between cancer cells and the immune system. Additionally, vaccine production requires the culture of infected cells to obtain virus proteins.

Cutting-edge research in regenerative medicine is using molecular biology techniques with cultured stem cells. Organ and tissue culture such as liver, kidney, or pancreas aims to replace damaged cells and tissues in patients suffering from severe insufficiencies or organ failure.

Cell Culture Protocol

Cell culture protocols are essential for maintaining healthy and viable cells in vitro. The basic guidelines for cell culture include subculturing, thawing, and freezing of cells in culture. However, it is important to note that cell culture conditions vary for each cell type.

Deviating from the culture conditions required for a particular cell type can lead to the expression of aberrant phenotypes or complete failure of the cell culture. Therefore, it is recommended to familiarize yourself with your cell line of interest and closely follow the instructions provided with each product used in experiments.

The first step in cell culture is to ensure sterility by undertaking all cell cultures in a microbiological safety cabinet using aseptic techniques. Most cell lines can be grown using DMEM or RPMI culture media with 10% FBS, 2 mM glutamine, and antibiotics if required.

Culture media and supplements should be sterile, and sterile reagents should be purchased when possible. Cells should be checked microscopically daily to monitor health, growth rates, and confluency (% surface area covered with a cell monolayer).

It is standard procedure to keep a record of the number of cell passages for all cell lines. For primary cell lines with a finite lifetime, it helps to monitor their viability and plan experiments before they reach senescence and cease dividing. For immortalized cells, it helps to monitor their passage number as they may undergo genetic changes over time.

Passaging cells involves detaching adherent cells, and collecting and washing suspension cells before resuspending them in a fresh medium or freezing medium depending on the experiment’s requirements.

Cryopreservation involves preparing a freezing medium supplemented with cryoprotectants such as DMSO or glycerol that prevent ice crystal formation from water present within cells that would destroy their integrity.

Good tissue culture facilities are designed to maintain sterility during experiments. Ideally work should be conducted in a single-use facility separated into an area reserved for handling newly received material (quarantine area) and an area for material known to be free of contaminants (main tissue culture facility).

Cell Culture Contamination

Cell culture contamination is a common problem encountered in cell culture laboratories, and it can have serious consequences.

Contamination can be caused by plasticizers, detergents, bacteria, molds, yeasts, viruses, mycoplasma, and cross-contamination by other cell lines. It is impossible to eliminate contamination entirely; however, it is possible to reduce its frequency and seriousness by gaining a thorough understanding of its sources and following good aseptic techniques.

There are several types of biological contamination. Microbial contamination is the most common type of contamination.

Cross-contamination of many cell lines with HeLa and other fast-growing cell lines is also a clearly-established problem with serious consequences. Obtaining cell lines from reputable cell banks and periodically checking the characteristics of the cells can help prevent cross-contamination.

If contamination is detected in a sample or flask, it is best practice to discard the sample or flask and start again. Autoclaving can eliminate contaminants. If contamination occurs frequently (more than once a week), additional steps should be taken to prevent it from happening again.

Antibiotics should only be used as a last resort because they might cross-react with cells and interfere with cellular processes under investigation. Best practices for reducing viral contamination include limiting the number of biological sources (suppliers, animals) from which cells are obtained.

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