ES cells grow like weeds and quickly need more space. Generally, cells are fed every day and split every other day. Cells should be split once confluent. If you can't split them right away and the medium is yellow, then at least feed them. Get back to them as quickly as possible. To prevent differentiation colonies need to be vigorously broken up into single cells when passaged.
-To passage a plate of cells, aspirate medium and wash once with an equal volume (or greater) of PBS. Apply enough trypsin to cover: approximately 0.7-1.0 ml for a 35 mm, 1.5-2.0 ml for a 60 mm, or 3.0-4.0 ml for a 100 mm.
We use a concentrated trypsin solution in Hank's balanced salt solution.
Trypsin-EDTA solution 1:250, .25% porcine trypsin, .02% EDTA-2Na, JRH Bioscience cat. no. 59228-78P, 500 ml
The 500 ml bottles are thawed at 37 degrees, then aliquotted 40 ml per 125 ml bottle and refrozen. Once an aliquot is thawed, store in the refrigerator for one week--this stuff is very cheap so don't be concerned with pitching some. I will note steps that require freshly thawed trypsin.
-Return plates to incubator for 5 min.
-Using a plugged pasteur pipet with a pasteur bulb, vigorously pipette each plate in order to break the colonies up. Swish all around the dish with a circular stream, avoiding bubbles as much as possible. The goal is an even suspension of single cells. ES cell colonies require vigorous pipetting in order to break them up--the high concentration trypsin and the pasteur pipets help this process, but ultimately it requires your patience and thoroughness. When first starting out examine the plates to see that the colonies are well broken up and mostly single cells. Usually the cells need to be drawn up at least 8 to 10 times to achieve this. When first starting out do only a few plates at a time. I do 6 plates at a time, maximum, since there are only 6 slots in the centrifuge.
-Transfer the suspension of cells to an equal volume of ES cell culture medium. Triturate (i.e. draw up and down) about three times to mix the suspension with the medium and stop the trypsinization. Cells can be left at this stage, for instance to queue up more tubes for spinning down.
-Centrifuge the cells in the clinical centrifuge on setting 4 for four minutes, then turn the knob up to setting 5 for one last minute. Stop the spin and retrieve tubes promptly. Cells are very unhappy after being spun down so keep the time spent in a mass to an absolute minimum.
-Aspirate medium and promptly break up pellets. Be persistent: do not leave large clumps.
-For routine splitting into 60 mm or 100 mm dishes, I prefer to add a bolus of concentrated cells to one side of a dish that is covered with medium. Cells are therefore resuspended in a convenient volume of medium--say 0.5 or 1.0 ml for each plate they'll be put into. Since the cells are coming out of a stressful spin, be gentle. Mix cells by running them along the side of the tube. When thoroughly resuspended, apply the cells to one side of the receiving dish and gently rock the plate back and forth so that a wave front goes from side to side. When first starting out, do only one plate at a time and keep the dish down on the metal table--eventually you can do three or four plates by rocking them gently in the air. Whatever you do, do not swirl the dishes as this concentrates all the cells in the middle.
-35 mm dishes (and below) are too small to rock back and forth--gently resuspend cells with their full volume of medium and apply to the dish
|
dish |
surface area, cm2 |
volume of medium, ml |
|
96-well |
~0.2 |
0.200-0.300 |
|
24-well |
2.0 |
0.500 |
|
35 mm |
9.5 |
1.5-2.0 |
|
60 mm |
21 |
3.5 |
|
100 mm |
56 |
10.0 |
-Incubate plates overnight and evaluate your technique the next morning. If there seem to be large numbers of clumps, or the dish is highly lopsided (cells weren't evenly distributed), then repeat the passaging onto fresh plates. If you were thorough when trypsinizing this is extremely rare. ES cells usually have a plating efficiency of ~20%. You can therefore expect a lot of floating material the next day.
-Feed the next day. Keep growth in culture to a minimum and freeze cells back.
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