Preparation of ES Cells for Microinjection

Provide the core with a vial of frozen ES cells and any special instructions on how to expand them.  If the core facility has generated the cells in a transfection experiment it performed then it will expand and inject them according to standard procedures.  Investigators may also expand their own cells and provide a healthy single-cell suspension of them to the core facility on the day of injection. The cells should be plated the day before microinjection using healthy confluent cultures as the source (e.g., thaw the vial of cells several days in advance, trypsinize and replate them at least once but, of course, try to avoid extended culture). On the day prior to injection, you should plate several dilutions (e.g., 1:2, 1:3, 1:4) and you may also want to plate the cells with and without feeder cells (using gelatin-coated plastic for the latter). A convenient option is to use a 6-well plate with different plating dilutions and conditions in each well.

On the day of injection, change the medium 2 hours before trypsinization. Choose the well that has the nicest looking culture and trypsinize these as follows:

·         Wash the well with PBS;

·         Add tryspin for 5-7 minutes;

·         Add medium THEN pipet (using a short-form pasteur pipet or a 5ml pipet) to get single cells (but avoid pipeting so vigorously that you start killing lots of cells);

·         Add more medium and pellet the cells by centrifugation;

·         Remove the supernatant fluid and add a small amount (e.g., 0.5-1ml) of fresh medium (avoid old alkaline medium);

·         Pipet briefly but effectively to resuspend the cells, and place the suspension in a freezing vial or an eppendorf microcentrifuge tube on ice (keeping the cells on ice helps to prevent clumping).

It would be wise to double-check with the core facility to confirm details about preparing the cells and when they should be trypsinized on the day of injection.  Try to avoid letting the cells sit on ice for any longer than necessary (ideally, they should be brought to the core facility just before they are ready to be microinjected). Avoid anything that might reduce the viability of the cells – e.g., pipetting too much, or having the cells in very pink medium, or failing to feed the cultures 2 hours before trypsinization.  Avoid an overabundance of feeder cells in the cell preparation – i.e., have the cultures at a good healthy density.  If possible, it makes sense to have two or three ES cell lines ready to go on the day of injection – that way if one of the cultures does not look good, you will have a fall-back.  Also, there are times when we recover larger numbers of embryos than normal – if this happens, and if the injections are going well, you may be able to get two or more clones injected on the same day.

Note: it is the responsibility of the investigator to ensure that the cell suspension will be ready on time. The success of the microinjection experiment depends critically on the health of the ES cell suspension (assuming that the ES cells are otherwise proficient at colonizing the germline).