ES cell culture


Readers are referred to a number of books that offer excellent descriptions of ES cell culture and protocols:


Gene targeting: a practical approach (2000). 2nd Edition (A.L. Joyner, editor), Oxford University Press.


Manipulating the mouse embryo (2003). 3rd edition. A. Nagy, M. Gertsenstein, K. Vintersten and R. Behringer. Cold Spring Harbor Press, Cold Spring Harbor, NY.


Guides to techniques in mouse development (2010). Methods in Enzymology volumes 476 & 477 (P.M. Wassarman and P.M. Soriano, eds.)


Teratocarcinomas and embryonic stem cells: a practical approach (1987). E.J. Robertson, editor. IRL Press. This book is out of print but has excellent chapters on ES cell derivation and generation of chimeras.


In our experience, important variables in culturing ES cells include using low passage cells, testing serum lots for highest ES cell plating efficiency, and using feeder cells such as STO SNL 76/7 cells or mouse embryonic fibroblasts (MEFs). The following points concerning preparation of media should be considered. Use either glass containers in which there is absolutely no trace of detergents or preferably disposable tissue culture ware. Use freshly drawn Milli-Q-purified (Millipore) water to make DMEM (high glucose, no pyruvate; we use GIBCO No. 12100-046). Buffer DMEM with 2.2 g/liter of sodium bicarbonate, not 3.7 g/liter as recommended by the manufacturer. This will allow proper pH equilibration in a 5% CO2 incubator. The osmolality of the completed medium (without serum) should be tested for each batch and should be approximately 290-300 mmol/kg. Once medium has been stored for 1 month, it is necessary to replenish the glutamine component to a final concentration of 2 mM from a 100 x stock (e.g., GIBCO No. 25030).


Plasmid DNAs should be prepared by standard techniques, such as Quiagen columns. The plasmid DNA is linearized by digestion with a restriction enzyme. After complete digestion, the DNA is purified by organic extraction and ethanol precipitation. Cleaned DNA is dissolved in sterile TE [10 mM Tris-Cl (pH 7.4), 1 mM EDTA (pH 8.0) at about 1 mg/ml.


Embryonic stem cells (such as AK7) are grown on mitomycin-inactivated or irradiated feeders. As feeders we use SNL 76/7cells, available from Allan Bradley, Sanger Centre, Cambridge, England (abradley@sanger.ac.uk). An alternative is to use MEFs. Feeders cells must be mitogenically arrested with Mitomycin C (10 μg/ml final concentration) or irradiated (6000 Rads total irradiation) before plating (5 x 104 cells/cm2 for STO cells; 105 cells/cm2 for MEFs). Feeder plates can be used within a period of a few weeks. The ES cells are cultured until just subconfluent, at which time they are trypsinized and counted. We use 0.25% trypsin with EDTA (GIBCO 25200-072) and leave it on the cells for 5 minutes at 37C before trituration to the level of single cells. Approximately 107 cells are transferred to a 15-ml culture tube, pelleted gently (250g for 5 min at room temperature), and suspended in 0.8 ml of phosphate-buffered saline (PBS). Twenty five micrograms of linear DNA is placed into an electroporation cuvette, the cells are added, and the combination is mixed gently by pipette. The cells should be electroporated as soon as possible at room temperature. Optimal conditions for electroporation should be determined empirically; we use a Bio-Rad (Richmond, CA) Gene Pulser unit set at 230 mV and equipped with a capacitance extender set at 500 ~mF. Time constants should not deviate greatly between experiments; our values range between 5 and 7 msec. Unusual time constants can indicate improperly made PBS, an incorrect number of cells, electroporation failure, or other problems. Treated cells are transferred immediately to one or several 100-mm culture dishes with feeders, according to the number of colonies anticipated. A large amount of cell death resulting in dramatically increased viscosity is normal, and so the cuvette should be rinsed with medium to ensure transfer of all viable cells.


The cells are cultured overnight to allow attachment and recovery. About 20 hr after electroporation, the medium is replaced with medium used for selection. In the case of neo selection, the medium is supplemented with (200 μg/ml active ingredient) of G418 (Geneticin, GIBCO, No. 860-1811). This concentration of G418 is suitable for use with vectors containing the E182D mutation in the neo gene. Cells are kept under selection for 10 to 12 days. The medium should be changed every day for the first 5 or 6 days and when needed thereafter. For rapidly growing ES cells, the medium should be replaced whenever an obvious change in color of the medium to a golden yellow is observed.


G418-resistant colonies are visible macroscopically after 6 or 7 days of culture and should be harvested after 10 days. At this point, various procedures can be followed, depending on the mutagenic strategy employed. ES cell clones grow in tight, bulging clumps. This morphology makes it easy to transfer cells by mouth pipette. Well-separated colonies are identified under magnification using a stereomicroscope, and clumps of cells are drawn up into a small pipette. We use a tissue culture hood equipped with a recessed dissection microscope, to maintain sterility while providing sufficient magnification, but the cells can also be picked under a stereoscope on the bench. Glass capillaries (~1.0 mm) are pulled by hand into pipettes, the tip of which is made by making a 90º break either by rolling the pipette on the edge of a sharpening stone or by using a diamond tip . Experience is the best guide for determining the optimal width of pipette to use (e.g., 100-200 μm). The medium is removed from the plate and the cells washed once with PBS and kept in a second wash of PBS for subsequent manipulations. The following technique should be performed quickly to ensure cell viability. Part (if using PCR) or all (if using Southerns or if the cells need to be expanded) of a ES colony is removed by applying gentle suction by mouth. If the colony is to be expanded, the cells are transferred in a minimal volume into a small (~10 μl) drop of trypsin. After about 5 min at 37C, the clump of cells is broken up by adding an equal volume of medium and pipetting up and down with a small tip on an Eppendorf brand micropipettor (0.5-10 μl). The homogeneous suspension of cells is transferred into a 4 well dish (10-mm diameter, e.g., Nunc 176740) with feeders and fresh, warm medium. When expanding the colony from this point onward, it is important to maintain a relatively high cell density. This usually means passaging all of the cells from the 4-well dish into a 35-mm dish and then into a 100-mm dish every 3-4 days.

Icahn
School of     Medicine at
Mount
Sinaihttp://www.mssm.edu