The development of transgenic and gene knockout technology has provided an effective tool for the analysis of gene function. Critical to this has been the ability to isolate and culture murine embryonic stem (ES) cells in vitro. Derived from the inner cell mass of early mouse embryos, ES cells contribute to all tissues including germline tissue. Efficient procedures for the in vitro culture and maintenance of mouse pluripotent ES cells have been crucial to the success of gene targeting experiments. We offer a wide range of mouse ES qualified products providing researchers with convenient and cost effective solutions for the reliable culture of mouse ES cells including ESGRO mLIF supplement, primary mouse embryonic fibroblasts, mouse ES cell lines, stem cell screened FBS and proprietary serum-free media.
The cell culture protocol described here includes the in vitro culture of mouse ES cells in serum-containing media using mouse embryonic fibroblasts and ESGRO mLIF. It should be noted that the protocols included in this manual are intended to serve as a guide only, and optimization of culture protocols is encouraged to ensure success.
Plan out a flow chart from day 1 to day 16–19. Note, depending upon the growth of the ES cells, the days may have to be shifted. Electroporation, screening, picking and preparation of DNA will take 2–3 weeks, including weekends.
EmbryoMax® Primary Mouse Embryo Fibroblasts (PMEF) feeder cells are supplied as frozen vials containing 5–6 x 106 cells per vial at passage 3 (2–3 population doublings per passage). It is recommended that PMEF feeder cells be plated one day prior to plating ES cells, which guarantees approximately 95% confluence of the PMEF cells. If ES cells are plated earlier than one day after PMEF plating, there may be some small gaps in the feeder layer. Although plating ES cells when gaps are present may not have any detrimental effects on the ES cells, it is not recommended.
Figure 4.A) PMEF feeder cells at the correct density B)PMEF feeder cells at too low density C)PMEF feeder cells at too high density
ESGRO® supplement is a special formulation of mouse LIF protein. Unlike regular LIF, which is sold by weight, each lot of ESGRO® supplement is sold based on its biological activity. The benefits of ESGRO® mLIF medium supplement include consistent inhibition of mouse ES cell differentiation, no batch-to-batch variation and the increased ability to grow mouse ES cells in feeder-free conditions.
Figure 5A & B.ES Cell Culture without PMEF Feeder Cells
Prior to selection, it is recommended that a kill curve should be determined for each ES cell line in order to determine the exact drug concentration to be used. The following selection regimes can be used as a guide:
Depending upon the cell line and the media used, ES cell colonies are generally ready for picking 5–10 days after electroporation. The most suitable colonies to select are those that appear rounded or oval in shape, with a phase contrast bright edge and often a dark necrotic center. Differentiated colonies are flat and often surrounded by fibroblast like cells that form cobblestone-like structures. These cells should be avoided when selecting cells. ES cells can be picked onto either gelatinized plates or a PMEF feeder cell layer depending upon the ES cell line used. If gelatinized plates are preferred, please disregard the use of feeder cells as described in the procedure below.
The two most common problems encountered when generating knockout mice are (1) ES cell differentiation, and (2) the inability to generate chimeras once a targeted ES cell clone has been established. In Table 25.1 are a number of common causes of ES cell differentiation and recommendations on how to help prevent differentiation from occurring. The extent of ES cell differentiation can be determined by examining the morphology of the ES cell colonies, or more thoroughly assessed using EMD Millipore’s ES Cell Characterization Kit or Alkaline Phosphatase Detection Kit. These kits contain monoclonal antibodies to ES cell markers and reagents for Alkaline Phosphatase detection that permit a discrimination of pluripotent and differentiated ES cells. Also included in Table 25.2 are a number of reasons that often cause the slow growth of ES cells. Table 25.3 contains possible causes for the lack of generating chimeras.