How many passages can I expect to get out of my cells in long-term culture and on what days do you recommend splitting them? Also do you have any suggestions on how to increase the rate of expansion of cells?

Answer

Human pluripotent stem cells (both embryonic and induced pluripotent stem cells) can be successfully cultured and maintained for a very long time. Although they can be cultured for very long periods of time, most researchers and laboratories have guidelines as to how many passages they would use cells before they thaw a new vial (most guidelines suggest that scientists will use cells unto 50-75 passages before they will retire those cells and thaw a new vial). It is important to perform routine karyotype analysis to ensure that the cell line has not acquired an abnormal karyotype after long-term passaging.

There are no specific ways to increase the rate of expansion of cells. Most human pluripotent stem cell lines are routinely passaged on day 4 or day 5 and it is vital to keep the cells on a routine in order to avoid stress (passaging them too early or letting them overgrow will cause stress). Scientists have developed techniques to scale up the passaging by changing the split ratios and this is the optimal way to increase the yield. A good example is the use of EDTA as a dissociation agent when cells are cultured in a defined medium such as Essential 8 medium with a defined substrate such as Vitronectin. In this case, since the use of EDTA generates smaller colonies (compared to other dissociation agents such as collagenase and dispase) the passaging ratio can be adjusted to 1:8 1:10 or even 1:12.

We have attached the relevant protocols and a publication for culturing cells in Essential 8 medium on Vitronectin using EDTA.

Please find some links below which you may also find helpful:

Culturing PSCs in Essential 8™ Medium – http://tools.lifetechnologies.com/content/sfs/manuals/feeder_free_PSCs_in_essential8_medium.pdf

Frequently Asked Questions – Essential 8™ Medium and Vitronectin – http://tools.lifetechnologies.com/content/sfs/manuals/FAQ_Essen8_Medium_vitronectin_man.pdf

Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions

Authors: Wang Y, Chou BK, Dowey S, He C, Gerecht S, Cheng L, Journal: Stem Cell Res (2013) 11:1103-1116 – http://www.ncbi.nlm.nih.gov/pubmed?cmd=Retrieve&dopt=AbstractPlus&list_uids=23973800