Animal cell culture is a technique used to culture animal cells outside the tissue or organ from which they are extracted. Animal cell culture is done in a specially designed laboratory, sterile condition and environmental factors like temperature, pressure and gases are controlled in such a way that it mimics the in vivo environment. Animal cell are cultured mainly by two methods, such as suspension culture and anchorage dependant culture. But both type of culture method have got some issues. This article talks about the issues related to suspension culture method.
1. Animal cells cultured using suspension culture techniques can be easily damaged by small percentage of shear forces that are developed by vigorous shaking of the culture vessel. Animal cells cultured in serum-free media are more sensitive to sheer forces, and small difference in mixing speed, like from 30 to 50 rounds per minute can lead to major damages, and this will induce some growth problems in the cultured animal cells.
2. If animal cells are cultured using shaker flasks, it is advised to start with 75 to 150 RPM on an orbital shaker with 30 to 40 percent of medium volume of the normal flask capacity, for example one liter medium in a three liter flask.
3. The requirement of mixing rate of suspension culture can be reduced by using baffled spinner and shake flasks. For example some cell types, such as insect cells require high level of oxygen and also vigorous mixing of the media may benefit in growth of the cells. It is recommended that the optimum stirring or mixing conditions are first determined empirically.
4. To start with first lowest speed that has the capacity to give even cell distribution from top to the bottom of the flask is chosen. However, to get optimum animal cell culture, higher speeds may be necessary, in this condition shearing damage from this higher speed can be reduced by increasing the viscosity of the media by adding 1 to 2% of carboxymethylcellulose, BSA (100µg/ml) or Pluronic F-68 (0.1%) to the medium, this alteration in the medium is discovered by Mather in 1998. This viscosity alteration plays an important role during animal cell culture in reduced serum or serum free medium.
5. Avoid Cell Clumping and Sticking: some types of animal cells when cultured in suspension method tend to form large clumps. These clumps settle at the bottom of the flask or may attach to the sides of the flak and this will result in lower cell viability and also cell growth. Using calcium free medium such as Joklik's MEM; S-MEM will reduce cell clumping, as calcium plays important role in cell to cell attachment. Also coating the surface of suspension flasks with siliconizing solutions such as Sigmacote, AquaSil, and Siliclad before sterilization will also reduce formation of animal cell clumps on the surface of the suspension flasks.
6. Use of Appropriate Seedling Densities: Suspension culture of animal cells requires correct initial seedling density, it is always better to add too many cells than too few. To start with a seeding density of 1x105 to 5 x105 cell/ml; or even higher concentration is recommended when cells are cultured in serum-free media. An alternative method such as taking half the normal volume of the medium in shake flask or spinner will automatically reduces the required number of cells to reach the optimum seeding density by 50%. After cells start to divide activily may be after 24 to 48 hours, additional medium can be added to bring the flask to its final operating volume.
7. Avoid Overheating Cultures: suspension flask cultures placed on magnetic stirrer or shaker may overheat by transfer of excess heat from the motors to the flasks. Sometimes transfer of excess heat from the stirrer to the suspension flask can be reduced by elevating the flask a few millimetre above the stirrer surface, as this allows the air circulation. Also using specially designed suspension flasks to withstand the corrosive atmosphere can be used during stirrers or shakers used in humidified carbon dioxide incubator. Using shakers in incubators may prevent cell from attaching to the culture vessels, as they produce vibrations.
These are some of the issues related to the animal cell suspension culture method. By doing some research in the field of cell culture techniques and by designing cell specific media may help in solving some of the issues, hence will help in cell growth and development of animal cells in the laboratory condition.
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