Pluripotent stem cell (PSC) culture


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.

Join Dr. Nirupama (Rupa) Shevde, customer training manager at Thermo Fisher Scientific, to ask your questions regarding the culture of pluripotent stem cells. Questions can range from best culture options and technologies for deriving pluripotent stem cells to how to adapt cells to a new media and everything in between.

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Question 1

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?

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 … Continued

Question 2

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.

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 … Continued

Question 3

What alternative methods exist for reprogramming adult cells into iPS cells, excluding methods that introduce foreign genetic material into the host cells? If there are any, what are the major limitations of these methods?

We currently offer several reprogramming products that are considered non-integrating, for a complete list of reprogramming options available, visit CytoTune –iPS Sendai Reprogramming Kits – The CytoTune™-iPS Reprogramming Systems use vectors based on replication-incompetent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into … Continued

Question 4

How important is finding a reliable and economical source of recombinant proteins for the defined animal free culture of iPSCs?

Stem cell scientists use a variety of media systems based on their specific research needs. All of media systems are of the highest quality and are routinely purchased by scientists. Many publications also present data that is generated using these media systems. Since the needs are diverse, there are a lot of media systems spanning … Continued

Question 5

As a student working on a startup project, I would like to learn more about the current needs and problems of researchers that are using iPS cells. I could use as much help as I can get. Thanks!

It’s a bit hard to make recommendations for your start-up without having a lot more information and specifics about what you are trying to accomplish within your research. Here are some resources that may help you learn more. Good luck in your start-up endeavor! Life Technologies resource – Stem cell resources – includes protocols, webinars, … Continued

Question 6

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?

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 … Continued

Question 7

We are looking for a synthetic surface for our iPSCs. Do you have any suggestions and what should we look for when evaluating?

The most commonly used and commercially available synthetic substrate is Synthemax by Corning. This substrate has been used along with Essential 8 medium by many scientists. These are the following evaluations: 1. Test for pluripotency after culturing cells for a long period of time (50 passages or more). 2. Test for the ability to form … Continued