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Simply perform your harvest as usual. One of the issues that many researchers have when using our system is that they are afraid to harvest out too many cells. The cartridge can be overgrown if you don't remove cells. After centrifugation your cell pellet should be between 1-3 mL or more (with the C5011 as much as 5 mL). Simply decant off the supernatant and re-suspend the cell pellet in 2 pellet volumes of fresh medium that has been diluted 10% with sterile distilled water. Leave it overnight at room temperature. The cells will consume the medium and also the hypotonicity will slightly squeeze the cells causing them to release any intracellularly sequestered antibody. This will get you an extra few milligrams every time you harvest.
Too few cells in relation to volume of media can result in lag phase. In the hollow fiber cartridge lag phase can be defined by the cells not consuming glucose but still show significant viability via the trypan exclusion assay. Always seed the cartridge with the recommended cell density. If this is not possible, adjust the volume of media down to keep the cells in culture from being too dilute.If the cells do go into lag phase you can either:
- reduce the volume in the reservoir bottle back down to 125 mL
- inoculate more cells
- wait a few days - sometimes the trick with hollow fiber cell culture is to know when to NOT do something.
To keep contamination at a minimum, clean the inside of the hood weekly, remembering to remove the working surface and clean underneath. Be sure the hood is validated on a regular basis. Never pour media, always pipette. Always wear a lab coat. Wait 60 seconds for the air currents to stabilize inside the incubator. Pull hair back and be sure to never have bare flesh inside the hood. Use plenty of alcohol and wipes but remember, it is not the application of alcohol that sterilizes, it is the evaporation. Good sterile technique will allow production from the cartridge for many months or more. We have produced a monoclonal antibody for over one year of continuous production.
New cell lines should always be quarantined until a mycoplasma screen has shown negative. The presence of mycoplasma contamination can be hard to detect without specific assays. The culture will just appear to be non-productive.
Fungizone and gentamycin when the bioreactor is seeded as a fail-safe against contamination. This works fine for HEK 293 cells, but not for CHO’s. In the bioreactor, the cells don’t die, but they just sit there and use very little glucose. Then we removed all but the Penn Strep and the cells are taking off. They are using close to 2g/day and would use more if I put a larger reservoir on them. I will get to 2L by early next week. So I don’t know which drug was the problem or if it was the combination. Dennis had received a Linked-In comment that CHO’s don’t like fungizone, but maybe we just overdid the antibiotics. Sorry for the confusion, I was sure it was something we were doing, it just took awhile for us to figure it out.
If you are experienced problems with reaching the bottom of a media bottle merely adjust the stainless steel tubing accordingly. If you are having problems sliding the tubing run the cap under warm water.
When the glucose level in the reservoir bottle reaches 2.5 grams per liter or so simply add 5 mls of 1N NaOH and a gram of glucose (dissolved in distilled water and sterile filtered or from a commercial source). This will neutralize the lactate and correct the pH and also add some carbon source. You should be able to get another 24-36 hours worth of culture from the medium and potentially double the concentration of conditioning factors in the reservoir bottle medium.
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