How many times can I passage my cells before the health suffers? Are there any products that can help extend the number of passages or time in culture?

Answer

Pluripotent stem cells, unlike primary cells, have an infinite lifespan. However, with increasing passage number there are a number of reports indicating chromosomal instability and differentiation bias that can result due to the stress associated with passaging methods and culture conditions. Therefore, we generally recommend that karyotype analysis of cultures be assessed every 10 passages and we ensure that a working bank of earlier passage PSCs be maintained as a safety stock. The most predominant large karyotypic abnormalities that have been shown to accumulate include gain of chromosomes 12 and 17 and chromosome X. There is a very nice review that was published early last year reviewing genome maintenance of pluripotent stem cells (J. Cell Biol. 204(2): 153-163).

One of the mechanisms outlined in this review which is responsible for some of the genomic instability is the build-up of reactive oxygen species (ROS) during stressful processes, which include passaging conditions, as well as recovery from cryopreservation. ROS can result in double stranded breaks which are particularly deleterious as during the repair process point mutations, as well as non-homologous end joining can result. Thermo Fisher Scientific recently launched a supplement which can be used in post-thaw recovery as well as for support of cells during passaging conditions-RevitaCell™ Supplement (Cat #A26445-01). NOTE: It should only be added to the culture medium for the first 24 hours post-thaw or post-split followed by feeding with unsupplemented medium for the remainder of time in culture. RevitaCell™ Supplement contains a specific ROCK inhibitor coupled with compounds having antioxidant and free radical scavenger properties. In general, addition of antioxidants to the culture medium has been shown to improve genome stability by reducing the ROS within the PSC during a range of processes (Stem Cell Reports 2:44-51, Scientific Reports 4:3779).