Contamination is caused by many possible sources. Insufficient sealing to the matrix vessel or connectors, non-sterile components in contact with the culture medium, and inappropriate aseptic connection technique are among some of these. It is important that filling reagents or connecting tubing must be handled under sterile condition such as a laminar flow cabinet whenever possible.

If the setting parameters for the bioreactor are out of the optimal range, the product yield of the cell culture may have been compromised. Over-fluctuating pH or DO may also lead to poor yield of the product where lack of nutrients such as glucose may also indirectly cause the change of pH and DO.

Basically, once cells reach the growth plateau, the product harvest of secretory protein may last for a few weeks. Our harvest record is 4 months without breaking the bellow of BelloCell.

Yes, the serum-free medium is compatible to the cell culture in both BelloCell and TideCell systems. In an effort to eliminate potential risks associated with the introduction of adventitious agents from animal-derived components, EBE also provides a medium additive Super Plus to replace serum in the medium. 

Generally speaking, it is necessary to replace the old medium three days after the cell seeding (ensure that cells attach well and start to propagate). You might want to constantly inspect the appearance and turbidity of the medium by naked eyes, and measure the pH once changing the medium. However, if you prefer not to change the medium, add NaHCO₃ to adjust pH (till orange red). 

This highly depends on the customer experimental settings, such as the CO₂ introduction, components of the medium, number of cells you seed as well as their metabolic activity, growth factors’ activity, total working volume of your culture medium in BelloCell/TideCell bioreactors. A rough indicator could be the change in the color of the culture medium (if phenol red is included) due to pH change. The alternative is that change the medium when the glucose level drops below the desired threshold.

Yes, you can. The purpose to do triplicate measurement is to increase the precision, in order to monitor and control well the process. Once the process is stabilized and assured, the frequency of measurement can be decreased.

Virus aggregation has not been reported in TideCell or BelloCell system. The system functions by exposing and submerging the carriers periodically, in which the mechanism is very similar to that of the roller bottle. The major difference is that the cell growth is 3D pattern in the TideCell system. However, higher virus concentration during down-stream purification process may cause virus aggregation and sometimes users can add Tween20 or Tween80 to alleviate the aggregation problems.

Firstly, using proper cleaning agents as instructed to wash the probe thoroughly between and after use. Secondly, regular maintenance is important to extend the lifetime of the pH probe. Electrolyte (e.g. 3 M KCl), in which the end of the probe is submerged for storage, has to change regularly and to be topped up when storing the probe. Never store the probe dry or in distilled H₂O. Thirdly, be careful not to introduce any bubbles in the electrode, or the measurement will be unstable.

Usually the pH probe can tolerate 20~50 autoclave cycles. Contact your pH probe supplier for determining the best time to replace it.

Firstly, using proper cleaning reagents (e.g. ddH₂O) as instructed to wash the probe thoroughly. Do not use any agents containing alcohol. This could damage the sensor or lead to erroneous current. Secondly, prior to calibration, always visually inspect the membrane foil, which is supposed to be intact and clean. Thirdly, if you find jumpy readings, inability to do calibration, or low DO probe current, the membrane body might be required to replace. Lastly, for short term storage, fill the sensor with O₂ electrolyte; while for long-term storage (6 months), the probe should be stored dry.

For VERO cells, if seeding 1x10⁸ cells in one BelloCell bottle(100 ml matrix), it will take 10 days to reach maximum cell density (4x10⁹). If seeding 1.5x10¹¹ VERO cells in TideCell-100 matrix vessel (100 L matrix), it will take 6 days to reach the maximum.

You could observe CPE the same way as you observe cells by fixing the carriers with alcohol and stain with dyes. Different viruses will cause different CPE with different cells. Some viruses will cause clear CPE, and some won’t. For rabies, it was observed by fluorescence due to no CPE could be observed. Baculoviruses in Sf-9 insect cells will cause shrinking of nuclei. H5N1 influenza virus in MDCK will kill the cells and cells will detach from the carriers and the shape will become irregular.

For virus production process, we usually decide the end point by checking the glucose consumption rate but not checking the CPE, since CPE is a roughly evidence but glucose consumption rate is more precise to determine the time of harvest. It also depends on if the viruses are stable or not, and how long the post-infection period is. 

For instant measurement during run, you might try to use Virus Counter to count the free total virus particles before and after infection. If you are not required to monitor the real-time change, you could perform validation study by measuring the virus titer against time after infection. Take the medium sample right after infection, 10 mins, 30 mins, 60mins, 2 hours, etc. to measure the virus titer by conventional methods. 

CO₂ concentration is adjusted dynamically according to the pH situation. During the cell culture, the CO₂ will keep reducing when cell growth. If cells keep alive once infected by virus, the CO₂ concentration will be minimized. However, if cells dye, the pH will rise and it is required to increase CO₂ concentration.

1.    Healthy cells

2.    Infectious active virus

3.    Post-Infection temperature

4.    Multiplicity of Infection (MOI)

5.    Occasion and strategy of harvest