Stem cells have attracted a lot of attention in recent years because of their developmentally pluoripotent and lineage-independent characteristics. Stem cell derived cell types offer utilities in research and development opportunities for various industries (pharmaceuticals, cosmetics, etc) and are used widely in academia for research on cellular differentiation and diseases. Whether you are a stem cell culture enthusiast, specialist, or “immortalized-cells-only” scientist, there are a few important pointers that we’d like to share. We hope that they will help you with your cell culture in general and stem cells in particular. So, let’s get started!

What culture conditions should a scientist use? It depends largely on if you are culturing mouse or human stem cells.

For mouse ESCs: Most of the discussions (borderline on “debates”) center around the usage of fetal calf serum and a cytokine named leukemia inhibitory factor (LIF). There are two main conditions scientists use: “serum+LIF” vs. “2i+LIF”.  When culturing mESCs with serum, use DMEM (standard formula) and 10-15% FCS/FBS. If you are culturing mESCS using non-serum recipes (such as 2i),  try N2B27 medium, 3 μM CHIR992021 and 1 μM PD0325901 (with 100units/ml LIF).  Which recipe is better? Unfortunately, as we’ve found out, every manufactured LOT of FBS serum is slightly different in content and concentration of growth factors. Thus, in general, non-serum-mediated cultures yield higher homogenous cultures and reproduce results at significantly higher frequencies for scientists’ project needs.

For human iPSCs: The story is much simpler with culturing human iPSC colonies. The most popular media products come from Stem Cell Technology’s mTeSR1 family line. Recently, the company has expanded their mTeSR media to a variety of serum-free versions (“hurray”!). Alternatively, LIfeTech has popularized their Knockout DMEM/F-12 + KOSR + bFGF kits. More recently, LifeTech has introduced Essential8 and Essential8 Flex product line, to assist scientists with simplifying their cell cultures during the week and over the weekend.

To go “feeder-free” or not? What’s the best way to passage the colonies? We always prefer “feeder-free” cultures. Why? It is MUCH easier to passage the colonies, especially if you are new to stem cell cultures. However, “feeder-free” cultures require a base matrix, which can be costly if you are culturing hundreds of colonies at the same time. ES-grade Matrigel (by Corning) is an excellent choice for propagating iPSC colonies (humans) in a feeder-free manner. They work well with Accutase (a gentle chemical dissociant) for passaging the cells. Media changes should happen every 2~3 days, depending on the density of your colonies. Just check the color change (red to orange to yellow). Splitting ratios of 1-to-3 works best.

Your thoughts? Need more detailed protocol and/or help with other stem cell derived cell types? Drop us a line!

~Lilly of Tempo’s Support Team