Currently I am dealing with Calu-6 cell line . I want to keep the cells in T225cm2 flask with 2.8 million/T225cm2 with medium volume of 20mL/flask. I want to know what will be the rate of Gaseous exchange and growth rate of the cells seeded
Please help me !!!!
Hi Anuj,
The thumb rule for medium volume is 0.2 -0.5 ml/cm2. if you beyond 0.5 ml/cm2 gaseous exchange rate would be hindered because the bottom area is constant and increasing the media volume will increase the height of medium column.This might cause less gaseus diffusion to the adherent cells. I guess 20 ml is too low for T225 cm2. You should use atleast 45 ml of medium.
Thanks
Hi Anuj,
The thumb rule for medium volume is 0.2 -0.5 ml/cm2. if you beyond 0.5 ml/cm2 gaseous exchange rate would be hindered because the bottom area is constant and increasing the media volume will increase the height of medium column.This might cause less gaseus diffusion to the adherent cells. I guess 20 ml is too low for T225 cm2. You should use atleast 45 ml of medium.
Thanks
Dear Anuj,
Regarding the generalized answer of your question yes, there is a relationship between medium volume, gaseous exchange and cell growth during cell culture of cancer cells. Maintaining a proper number of cells and volume of the medium is necessary because cells secrete various cell signaling molecules in the cell culture medium. These secreted molecules help in cell to cell cross-talk, cell signaling and stimulate growth. Most of the cases without a minimum number of cells the cell secreted molecules will be less (diluted) and therefore cells will not get sufficient stimulation for growth.
Good answers. Just keep in mind that fast growing cells need fresh media to be added sometimes to prevent them from getting stressed out. Generally, adding 1/3 the amount of fresh medium after 2-3 days of cells are growing may be needed for fast growing cells. Or of course, splitting more often.
The above comments are all relevant to your problem. The medium depth is critical for gas exchange to maintain O2 levels and PH (CO2 levels) at the level where your cells are growing. It is not clear why you are using such large flasks. If you wish to build large numbers of your cell line, you may want to look at culture of your cells attached to beads (e.g., agarose or other) kept in suspension in spinner bottles. You can get much larger yields and grow your cells at a higher density (i.e., cells/ml medium) and then either change media frequently or have continuous removal and addition of a fraction of hte medium to maintain nutrient levels. You would have to collect and trypsinize your beads every so often so as to maintain sufficient bead surface available for continued cell number increases (growth). There is a considerable literature on these techniques and even some books.
Of course, the primary question is what are you seeking to learn from these cells? The culture conditions can be very important too the responses of your cells (e.g., monolayer vs 3-D culture). We have shown that diluted media (20% medium, 80% saline) allow cells to grow with the same rate and cell cycle times and distributions, until nutrients are exhausted, but during that growth period, the cells become flatter, changing the shape of their nucleus, the number of neighbors each chromosome has, and hence the frequency of exchange aberrations and radiosensitivity of the irradiated cells. Cell:cell communication can also be critical, as in the Radiation induced Bystander Effect, in which neighbors of irradiated cells can be sterilized just as if they themselves had had a photon or particle traverse them. Cell production is one thing, but the nature of your scientific question should determine what culture method is most appropriate.
Good Luck.
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