Open the bottle cap and add 500 mL of media including the inoculum. Place the bottle(s) in the FibraStage device, and place the entire system inside a standard CO2 incubator. Set temperature to 37C for mammalian cells and 28C for insect cells.
Adjust the FibraStage controls as per Table 3. Up (Rise) and Down Rate will adjust the speed of the platform, which creates gentle mixing of media to ensure cells have sufficient nutrients and oxygen. Increasing Top Hold Time allows nutrient exchange between media and disks and increases CO2 level in the culture. Increasing Bottom Hold Time will increase oxygen level in the culture, allowing sufficient time for oxygen transfer from air to cells and helps stabilize the pH.
It is important to note that increasing the Up Rate facilitates mixing the culture medium, but also increases shear stress to cells. Increasing the Bottom Hold time facilitates aeration efficiency, but also reduces the mixing frequency of culture medium. Longer Bottom-Hold times tend to promote initial cell propagation due to the concentration of cells, however, longer Bottom-Hold times may also restrict nutrients available to the cells.
After 2 - 3 hours of inoculation, reset the control parameters and Up/Down Hold Time as indicated in Table 4.
We have found that 90% of the tested cell lines will
become immobilized in the disks within approximately
30 45 minutes. During the run, monitor the substrate
and metabolite concentrations including glucose, glutamine,
ammonia and lactate once a day. Media and glucose