PluriStem 129/S6 Murine ES cells

This page covers the indirect co-culture of embryonic stem cells with embryonic fibroblasts.

The cell culture protocols described in this instructional manual include the in vitro culture of murine ES cells using EmbryoMax products along with ESGRO® mLIF medium supplement, as well as feeder-free and serum-free ES cell culture using the ESGRO Complete™ line of products. Also included in this instructional manual are: methods for serum-free neuronal differentiation of mouse ES cells, iPS cell generation using STEMCCA™ lentivirus kits, cre-recombinase mediated excision of STEMCCA™ genes from iPS cells, derivation and rescue of new and existing ES cell lines using RESGRO™ Culture Medium and ESGRO Complete™ system.

"As transgenic mouse core laboratories know well, mouse models are a powerful means of determining the function of mammalian genes in the context of whole organisms—especially since over 95% of the mouse genome is similar to the human genome. However, you may not know that: 1. Laboratory mouse strains are descended from ancestors in modern-day Pakistan. 2. The first inbred strain of lab mouse was created in 1909. It was named DBA for its coat color: dilute brown non-agouti. 3. There is a mutant mouse actually named“Bad Hair Day” (Bhrd). Someone get this little guy a stylist! 4. Male mice, when treated with anticancer drugs, have been shown to pass on DNA instability to their offspring. 5. The founder of Jackson Laboratories, C. C. Little, once had lunch with Walt Disney to request that an animated film be created linking Mickey Mouse and the laboratory mouse. The request was denied."

Traditionally, culture and expansion of mouse embryonic stem cells (mESC) is performed using several single-layer T-flasks or Petri dishes. Harvesting and analyzing stem cells from multiple culture vessels is a repetitive and time-intensive process that consumes valuable incubator space. Here, we report a method for expanding mESCs using a new multilayer cell culture flask. The Millicell HY multilayer culture flask makes expansion and harvesting of mESCs faster and easier than traditional culture methods. The method for using the new flasks is linearly scalable per unit surface area. Therefore, no reoptimization of procedure is required – culture conditions and media volume are identical, regardless of whether culture is performed in traditional single-layer devices or in a Millicell HY culture flask. Resulting morphology, cell recovery, viability, and pluripotency of mESCs were not significantly different between multilayer flask culture and single-layer flask culture methods.