Maintaining Serum-Free Hybridoma Culture: 7 Day Run

Sometimes, it is very tricky to cultivate hybridoma cells in serum-free conditions. There are many options in terms of serum-free hybridoma cell culture media and supplements. However, one of the challenges that we face is the sustained cell growth and viability for more than 7 days in the absence of serum. What are the factors that we can control in order to accomplish such a task?

To increase the total number of cells achieved at the end of the run, we need to feed the cells throughout the run to maintain high cell viability. Feeding is advised rather than initial supplementation in order to maintain similar specific productivity of the cells. If you are supplementing basic media with FBS replacement supplements (as this is generally cheaper than commercially available serum-free media), we have found that DMEM/F12 is optimal to support serum-free conditions with additional supplementation as described below. If you must use DMEM, then using the high glucose DMEM with sodium pyruvate will also be helpful.

  1. Supplement with Glutamine to have a starting glutamine concentration of 6-8 mM. This will provide an additional Energy source along with Glucose.
  2. Glucose will be consumed throughout the run. Consider additional glucose feed.
  3. Additional amino acid supplementation may improve cell densities.