Pluripotent Stem Cell Culture – A Discussion about Successful Culture

By on April 29, 2014
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We recently finished our Ask the Expert discussion with Dr. Nirupama Shevde on Pluripotent Stem Cell Culture. This week we had several interesting questions and helpful suggestions from Dr. Shevde. General information topics included synthetic surfaces, recombinant proteins as ingredients in cell culture media, and reprogramming adult cells into iPS cells. More specific culture questions were asked about maintaining genome integrity, weaning cells from serum, passaging cells, splitting cells, and cell expansion.

Pluripotent stem cell (PSC) culture has evolved over the past decade, just as stem cell researcher needs have been changing and the market has been growing. Initial culture conditions for stem cells used FBS and mouse embryonic feeders (MEFs). The need for a more defined system brought about the introduction of KnockOut™ Serum Replacement (KSR) in 1997 for the culture of mouse embryonic stem cells (ESCs). Even today, KSR has remained an integral part of the feeder based workflow for both mouse and human PSC culture. The desire to move away from the variability and work required to maintain MEFs led to the development of feeder-free media systems. There are a number of options for feeder-free based media and matrices; StemPro® hESC SFM and Essential 8™ media are two robust options available and both work with Geltrex® and Vitronectin matrices. As choice of media systems for PSCs grows, understanding which media to use for a specific application becomes an important consideration.

This Ask the Expert Session was Sponsored by Life Technologies and hosted by Dr. Nirupama (Rupa) Shevde. Dr. Shevde is the Customer Training Manager in the Primary and Stem Cell Systems group at Life Technologies. She obtained her doctorate at Harvard University and her postdoctoral training at USCD and Ligand Pharmaceuticals in San Diego, CA. She has extensive experience in both mouse and human pluripotent stem cell research and served as a Senior Scientist and Director of Education at WiCell Research Institute and Morgridge Institute for Research since 2005 until July 2012. Under the scientific leadership of Dr. James Thomson, Rupa developed the Stem Cell Training Course, which has served over 800 scientists from 32 US states and 20 countries.  Most recently, Rupa was able to work in Dr. Thomson’s laboratory and gain expertise in the novel Essential 8™ Medium, Vitronectin (VTN-N) substrate and non-integrating episomal reprogramming technology developed by Dr. James Thomson and marketed by Life Technologies.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert – Dr. Nirupama Shevde on Pluripotent Stem Cell Culture.

Question:

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?

The 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

Question:

My lab has begun to try and reduce the amount of serum used in our pluripotent cell culture media. Do you have a method you would recommend for weaning the cells or a lowest serum amount you think would work?

The Answer:

I am assuming that you are working with human pluripotent stem cells and you use Knockout serum replacement (commonly known as KSR). A lot of scientists have moved away from KSR as it is not defined and includes components that are of animal origin. We have a defined medium called Essential 8 medium that is serum free and is widely used by scientists to culture human pluripotent stem cells. I have attached the necessary protocols and a publication for your review. We have designed a protocol that will allow your cells to be weaned off the serum in a gentle way by changing one variable at a time in the culture conditions, so as to not stress the cells too much. Essential 8 medium has 8 components and has been successfully used to culture both human embryonic stem cells and human induced pluripotent stem cells. Cells cultured in this medium have shown to retain pluripotency, ability for trilineage differentiation and maintain a normal karyotype for more than 50 passages. Essential 8 medium can be used with substrates such as Geltrex or vitronectin.

The links below may also be 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

Question:

We are having difficulty maintaining genome integrity in our ipsc in culture. We have seen several mutational changes when the cells are in culture for longer periods. Any suggestions on how to troubleshoot.

The Answer:

It is difficult to answer this question without having details of your culturing conditions. Following are the most common reasons for mutational changes and abnormal karyotypes in human pluripotent stem cell cultures-

  1. Overgrowth of cells- if cells are routinely overgrown and not passaged when they are in log phase (actively growing), the stress can cause mutations
  1. Use of a harsh dissociation agent- if a harsh dissociation agent such as trypsin is used for routine passaging and maintenance of human pluripotent stem cells, the cells get dissociated as single cells and will not be able to survive in cultures. IF they do survive, they will adapt under stress and have mutations
  1. In general any culture conditions that will induce inadvertent stress or selective pressure will result in mutations after long-term passaging
  1. Also, passaging the cells too soon (every two or three days) can induce stress
  1. I would suggest to check culture conditions, dissociation agents, dissociation methods, passaging frequency as possible stressors

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