Posted By Morgan Mathieu
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Successful Research Tips,
We Are Enzo!
Enzo Life Sciences now provides 25 years of experience in the supply of research kits, biochemicals and biologicals.
As Scientists Enabling Healthcare, Enzo Life Sciences realizes the value in providing relevant information to our customers working in the fields of Life Science and Drug Discovery. We are happy to provide simple but useful tips for improving daily tasks as well as the overall quality of your research. With this in mind, here is a list of tips for maintaining high standards in cell culture.
Cross-contamination is cell culture’s biggest enemy. Even the slightest contamination can ruin weeks of research. Fungi grow easily due to the warm and moist conditions of the incubator, so it is important to look after it and clean it regularly. Moreover, wipe clean media bottles, pipettes, and other pertinent materials with ethanol prior to placing them in the hood with your culture to avoid this frustration.
The fragility of the cell cultures cannot be overstated. Vigorous shaking, consistent vibrations, or continuous temperature fluctuation can impact the growth pattern in disadvantageous ways. Make sure that the incubator is level, with a uniform temperature profile and away from motorized instruments. Furthemore, handling multiple cell lines at any one time should be avoided as it might compromise the genotype and the phenotype of your cells. An STR profiling should also be done on a regular basis to guarantee or confirm the identity of the cell lines.
While thawing may seem like a rudimentary step, it is essential that it is done appropriately to avoid damage to the cells. Prolonged exposure to heat and centrifugation render the cells unable to be plated. Therefore, the cryovial should be placed in a 37°C water bath for approximately two minutes, and then diluted in conditioned medium to prevent immediate harm caused by the DMSO.
There are 3 phases of the cell culture process. The lag phase, the log phase, and the plateau phase represent low cell growth, high cell growth, and no cell growth, respectively. The most viable cells are healthy and quickly dividing and it occurs at about 70-80% confluence at the heart of the log phase.
Confluence refers to the proportion of adherent cells to uncovered surface area in the flask. Complete confluence means that 100% of the surface is covered in adherent cells. It is important to avoid this state because it represents an inability for the cells to continue growth. However, it is imperative to keep on using actively growing cells.
In cell culture, the media is a very important factor in governing product quality, yield, and cost. It is vital that the media be tailored to each culture to optimize the efficacy and results of the experiment. There are several options to choose from when deciding which way to develop your media. You can buy off-the-shelf, develop the media in lab, work with another company to develop more specific media, or a combination of any of these. Some factors to consider while choosing a development strategy include timeline, cost of materials, and stage of product development.
Liquid media from a manufacturer tends to yield higher quality results than dry powder media (DPM) solution. This can be attributed to the quality of water inside of the commercial media company’s laboratory. Due to the rapid bacterial growth in water, it is important to monitor endotoxins and other contaminants. The commercial companies have the resources to monitor and control the growth of bacteria, such as bacterial filters and ball milling. It is therefore more reliable to use liquid media made by a manufacturer.
Spent media analysis can help identifying the metabolism rate of vital media components including amino acids and vitamins. The dynamic metabolic profiles gathered from the cell growth phase and stationary production phase can be utilized to stoichiometrically balance basal and feed media, direct the focus of the media for a specific reaction, and alter the ratio of biomass formation to protein production.
Trypsin is a proteolytic enzyme that allows for the passage of adherent cells by digesting the proteins responsible for binding the cells to the vessel. If the cells remain exposed to trypsin for too long, it will begin to cleave cell surface proteins, which will affect the cell’s ability to function properly.
As a cell culture becomes exposed to antibiotics more frequently, strains with an innate resistance to the antibiotics will begin to develop. Such a phenomenon can mask underlying contamination, which could be extremely detrimental to the experiment.
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