Animal cell culture is a comparatively new technique used to culture animal cells outside its original place that is tissue or organ. Animal cell culture is performed in a laboratory, specially designed for this purpose. Animal cell culture is done in a sterile condition and also under controlled environmental conditions such as temperature, oxygen concentration, pressure and much more. Animal cell culture can be done by two methods, namely attachment-dependent animal cell culture and suspension animal cell culture. Both the ways has got some issues. But current article talks about the issues related to the attachment-dependent animal cell culture method.
1. Appropriate Seeding Densities: using appropriate initial seeding density is very important to get a proper cell culture when growing attachment or anchorage dependent animal cells. It is advised to add little more than the required amount of cells, and then the amount of cells can be lowered.
2. Quick animal cell attachment: attachment of the animal cells is in most of the times a serious issue or problem, especially when cells are cultured in reduced medium or serum-free medium. To solve this problem prewarming the culture medium used for cell seeding and also pregassing culture vessels will help in the maintenance of correct pH of the medium and this intern will facilitate the quick attachment of the cultured animal cells.
Pregassing of the large animal cell culture vessels is also recommended and advised to done with filtered medical grade 5% carbon dioxide and remaining 95% air mixture. This also facilitates the quick attachment of cultured animal cells.
3. Roller bottles should be rotated slowly: The constant rotation or movement of the medium throughout the roller bottle will make it difficult for cells to quickly attach and proliferate in the roller bottle, when compared to other animal cell culture vessels such as flasks or dishes. Therefore to encourage the quick attachment of cells cultured in roller bottle, very low initial rotation is recommended such as 0.5 to 1 revolution per minute. It is also recommended that until the cells are attached to the roller bottle slower rotation per minute such as 0.1 to 0.4 RPM. The constant rotation of the medium induces more stressful environment to the cultured cell when compared with other stationary culturing vessels.
4. Maintain the optimal ratio of animal cell to the medium is very important to get a good animal cell growth and proliferation. To obtain a good growth of animal cells it is recommended to start with 0.2 to 0.3 ml of the culture medium for every square centimetre of culture vessel growth surface area. For example if the culture vessel growth surface area is 25 cm2 flask then 5 to 7.5 ml of growth medium is recommended. Using more amount of culture medium also reduces the need of frequent changing of the medium, but due to increased static nature of the environment will also decreases the oxygen diffusion to the animal cells.
5. In some instances gassing the animal cell culture will also increase the cells viability and also cell proliferation. But this principle is not practical on large number of vessels such as roller bottle, flasks and also dishes. But this can be used in CellSTACK chambers.
6. Subculturing at appropriate time is also very important in order to keep the cultured animal cells viable and actively growing and also healthy. Subculturing of some kind of animal cells is also difficult. For example epithelial-like cells will form strong cell to cell bond and making it much difficult to subculture them.
7. During subculturing of animal cells, cells must be extracted very gently.
8. Over exposure of animal cells to harsh dissociating agents used during subculturing may reduce the viability of these cells and also make it difficult for harvested cells to reattach to the surface. This problem occurs when tying to harvest animal cells from too many vessels. Therefore it is advised to harvest cells from a few dishes at a time.
9. If animal cells are cultured in a serum free medium, then the inactivated form of dissociating agents are used to harvest the cells.
10. Harvested cells should be kept in chilled condition until they are ready for subculturing. This will help in maintaining the viability of animal cells and also will reduce the cell clumping.
These are some of the issues related to the animal cell anchorage dependent culture method. By doing some research in the field of cell culture techniques may help in solving some of the issues.
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