- Feeding is an essential step to maintain cells in a healthy state in culture. Feeding of a culture is a process of replacing the old consumed medium with the fresh growth medium. The purpose is to provide fresh nutrients/growth factors and remove excreted by-products of cell metabolism, degraded ingredients of culture medium, and dead cells.
- Cells in culture consume nutrients and excrete metabolic wastes in order to survive and grow. These exchanges happen with the surrounding environment i.e., the culture medium. This leads to a change in the composition of the medium and after some time it is no longer physiological. For example, the growth of mammalian cells results in a drop in the pH of the medium due to the secretion of lactic acid by mammalian cells.
- The absence of feeding can cause nutrients deprived state and excess of cell metabolites (toxic to cells at high concentration), leading to growth retardation and cell death.
- The rate by which the composition of medium changes depends on the growth rate and metabolic activities of cells which in turn influence the feeding schedule. Cell lines with fast-dividing rate/high metabolic activities require feeding more regularly than the cell lines with slow dividing/low metabolic activities. Generally, cultures need to be fed on the second or third day, depending on the seeding densities and cell growth properties.
- The process of removing the medium from the adherent culture is relatively easy and involves no risk of losing the cells along with the medium as cells are adherent to the culture dish. The only associated risk factor is the drying of cells if the whole process of changing medium is not quick. Some floating and mitotic cells may be lost during this process which constitutes a negligible amount of the whole culture.
- A PBS wash can be given during changing the medium to remove dead cells that are often found sticking to the healthy adherent cell layer. Dead cells can be due to many reasons including cell death during subculturing (trypsinization).
- Some cultures are loosely adherent to the substratum. Such a culture must be fed with caution as there is a high risk of losing cells during the process. While changing the medium, precautionary the medium and PBS wash can be stored in a centrifuge tube until you make sure that there is no significant loss of cells in the culture dish. In case of loss of cells from the culture dish, cells can be recovered from the medium and PBS wash by centrifugation.
- Feeding suspension culture is a bit tricky as cells float in the medium. Ideally, one must collect the cells by centrifugation to remove the consumed medium and then resuspend them in the fresh culture medium. However, in practice, researchers either add sufficient amounts of fresh medium or replace a portion of consumed medium with the fresh medium without caring about losing cells in this process.
- Adding fresh medium without removing the old consumed medium, although supply fresh nutrients, unable to remove by-products of cell metabolism. Both nutrients and by-products are diluted in this process, which depends on how much fresh medium is added. Since cell metabolites remain in the culture medium, the medium quickly turns unphysiological although the nutrients are still present in diluted form. In addition, culture vessel maximum capacity to accommodate culture medium is also a factor in this case which limits the addition of fresh culture medium.
- The other method which involves replacing a portion of medium (often 1/2, 2/3, or 3/4 medium) with the fresh medium is very convenient and most commonly used to feed suspension culture. In this method often a culture dish is tilted for a short time to allow cells to settle at the tilted side of the dish due to high density. Then the medium is carefully removed by pipetting and a fresh medium is added. This method results in the loss of some cells.
Was this post helpful?
Let us know if you liked the post. That’s the only way we can improve.
