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Titration is defined as a common laboratory method
Contaminants are most commonly of biological nature and can include bacteria, fungi, viruses, and parasites. It is important to limit biological contaminants since they can alter the phenotype and genotype of the cultured cell line through competition for nutrients, synthesis of alkaline, acidic or toxic by-products, and the potential interference of viral components with the cell culture genome.
Cell culture refers to laboratory methods that enable the growth of eukaryotic or prokaryotic cells in physiological conditions. Its origin can be found in the early 20th century when it was introduced to study tissue growth and maturation, virus biology and vaccine development, the role of genes in disease and health, and the use of large-scale hybrid cell lines to generate biopharmaceuticals.
The experimental applications of cultured cells are as diverse as the cell types that can be grown in vitro. In a clinical context, however, 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. One of the advantages of using cell culture for these applications is the feasibility to manipulate genes and molecular pathways.
Furthermore, the homogeneity of clonal cell populations or specific cell types and well-defined culture systems removes interfering genetic or environmental variables, and therefore allows for data generation of high reproducibility and consistency that cannot be warranted when studying whole organ systems.
Aseptic Cell Culture Practices
Indeed, microbiological infections represent the main problem for the maintenance of cells in vitro. Infectious agents such as bacteria are toxic for eukaryotic cells and ultimately lead to cell death. Furthermore, even low levels of contamination can result in abnormal results and lead to wrong scientific interpretations.
By adhering to several techniques that ensure asepsis in the cell culture lab, researchers can reduce the frequency and extent of contaminations and diminish loss of cells, resources, and time. This can be achieved by eliminating the entry of microorganisms into the cell culture through contaminated equipment, media, cell culture components, incubators, work surfaces, and defect or opened cell culture vessels.
Given that atmospheric air is laden with microparticles of potentially infectious nature, the biosafety cabinet is the most crucial piece of equipment to restrict nonsterile aerosols and airborne components from contaminating cultured cells.
Biosafety cabinet should be decontaminated with an antifungal detergent (e.g., 5% Trigene) followed by 70% ethanol. All equipment entering the biosafety cabinet also needs to be sprayed and wiped with 70% ethanol.
Disposable gloves sprayed with 70% ethanol and lab coats can further reduce the introduction of contaminants carried by hair, skin cells, or dust.
Commercially sourced media and supplementary cell culture products are generally supplied in sterile condition. In addition, filter-sterilizing allows for the generation of cell culture media that are based on nonsterile culture reagents, while autoclaving is conventionally used to sterilize equipment in contact with cultured cells. The filter-sterilization of liquids can be achieved by forcing the liquid through a 0.22 μM polyethersulfone low-binding filter system using a vacuum pump. The addition of antibiotics (e.g., Penicillin/Streptomycin) further limits the risk of bacterial growth in media bottles after opening and in cell culture vessels.
Contaminants are most commonly of biological nature and can include bacteria, fungi, viruses, and parasites. It is important to limit biological contaminants since they can alter the phenotype and genotype of the cultured cell line through competition for nutrients, synthesis of alkaline, acidic or toxic by-products, and the potential interference of viral components with the cell culture genome. Other contaminants may include the introduction of undesired chemicals impurities (e.g., plasticizers in cell culture vessels) or other cell types cocultured in the lab.
Cell cultures affected by bacterial contamination generally appear turbid in appearance. Furthermore, the high metabolic rates of bacteria can modify the pH of the culture media and thus change the color of phenol red to yellow. While bacteria may be detected as small particles at low microscope magnification, their distinct shapes are generally detected at higher magnification. While bacterial strains such as E. coli can therefore be uncovered quite easily due to their size (~2 μM) and flagella-induced mobility, other strains such as Mycoplasma are smaller in size (<1 μM), immobile, and therefore not as easily detectable. As a result, Mycoplasma infections can go unnoticed for a longer time and usually only become apparent through declining quality of the cultured cells. This can manifest as reduced cell proliferation and cell death. In order to monitor cell cultures for potential infections with Mycoplasma, it is advisable to routinely test cultures for their presence using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), or immunostaining.
Yeast budding cells appear ovoid in shape, can grow to approximately 4 μm of size and are therefore easily detected at low microscope magnifications. Moulds are additional members of the fungi kingdom that can be found in cell cultures. Their growth is marked by the production of multicellular, highly connected, thin filaments (hyphae).
The presence of viral contaminants can be challenging to confirm but generally relies on PCR, ELISA, immunocytochemistry, or electron microscopy.
Viruses are infectious agents that rely on host cells for their own replication. Owing to their limited size of up to 300 nm and their intracellular lifecycle, they are not visible in generic light microscopy and very difficult to detect. While some viruses may induce morphological changes in the cultured cells (cytopathic effects), other species may integrate into the cellular genome and alter the phenotype of the investigated cell line.
Viruses can enter cell cultures, for example, through the use of animal-derived cell culture products such as trypsin or fetal bovine serum and are a serious health concern for laboratory workers. The presence of viral contaminants can be challenging to confirm but generally relies on PCR, ELISA, immunocytochemistry, or electron microscopy.
Abdos Labtech offers range of cell culture consumables to become the partner in your journey to seed, expand, grow & store your cell culture starting from cell culture flasks, plates, dishes, bio-filtration system to cryo-vials for cryopreservation purposes.
For more information. Go to our website link given below: https://www.abdoslifesciences.com/
Source: Abdos Life Science
Author: Saurabh Joshi
Deputy Manager- Product