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Expansion of targeted ES clones
Day 0
If the targeted clone was frozen from a 24 well tissue culture dish, then it needs to be thawed and plated into a 6 well dish for expansion. Using an ongoing MEF flask, take up the MEF cells with Trypsin / EDTA, and irradiate them with 3000 rads. Place the inactivated MEFs at a concentration of 2 x 105 cells per well in a 6 well tissue culture dish. You will need 1 well for each targeted clone you are thawing.
Day 1
Exchange the MEF media in the prepared 6 well dish to ES media (3.0 ml / well). Take the frozen vial containing the targeted clone of interest and thaw quickly in a 37oC water bath. Pipette all the cells onto the prepared well of the 6 well dish. Pipette gently to evenly disperse cells.
Day 2
Exchange the ES media from the wells to fresh ES media (4.0 ml / well). You should have 50-100 healthy shiny small colonies in your well. Prepare three T-25 flasks/targeted clone of inactivated MEFs from ongoing MEF flasks, plating them at a concentration of 5 x 105 MEFs / T-25 flask.
Day 3
Today, if you do not have 50-100 healthy colonies in your well, then you are not yet ready to expand these cells to a T-25. In this case, you need to disaggregate your 6-well again with Trypsin/EDTA to further expand your clones. To accomplish this, aspirate off old media, add 2 ml PBS, rinse and aspirate, add 0.5 ml Trypsin/EDTA and place in the incubator for 10-15 minutes. Next, add 1 ml ES media to your trypsinized clones, pipette up and down with a 1000ul barrier tip to further disaggregate them into single cells, then add 3 ml ES media and gently pipette up and down to evenly disperse cells. Leave in the incubator overnight.
For those wells which are ready for expansion do the following: Aspirate the old ES media from the well of the 6 well dish. Rinse the well X 1 with PBS (2 ml), aspirate. Next add 0.5 ml Trypsin / EDTA to the well and incubate for 10-15 minutes. Pipette the cells up and down with a 1000ul barrier tip and view the cells in the microscope. If they look like single cells or small groups of cells add 1.0 ml ES media. Pipette the cells up and down with a 1000 ul barrier tip. Add 1.0 ml of disaggregated cells to the prepared T-25 inactivated MEF flask that has been freshly fed with 4 ml ES media. Add 3 ml ES media to the remaining 0.5 ml of cells in the 6 well dish. Allow these cells to grow to confluence for later isolation of DNA to be used as a second confirmation of your homologous recombinant using Southern analysis.
Day 4
Exchange the ES media for fresh ES media in your 6 well dish or your T-25.
Day 5, 6, 7
For the 6 well dish that has not yet been expanded to a T-25 you will be able to do this today. Follow the above procedure. For those cells which have already been expanded to a T-25, they should again be expanded as follows: Aspirate old media and rinse flask with 2 ml PBS, add 1.5 ml Trypsin/EDTAand incubate for 10-15 minutes. Pipette the cells up and down and view in the inverted scope. If the cells are single cells, add 2.5 ml ES media, pipette up and down and add 2 ml of cells to each of 2 T-25 flasks which have been freshly fed with 3 ml ES media. The next day (Day 6) feed these cells with fresh ES media. If you are going to use these expanded cells for injection or a second electroporation, you will freeze three fourths of the cells in the 2 T-25's and expand the other one fourth. If you are just expanding them for a future use then you will freeze all the cells in the 2 T-25's into 4 freezing vials. To freeze your cells proceed as follows (Day 7):
Remove old media from flask and rinse X 1 with PBS (2ml). Add 1.5 ml Trypsin / EDTA to the flask and place in the incubator for 10 minutes. Look at the cells in the microscope and see if they are single cells or large clusters of cells. If they are still in large clusters, pipette them up and down with a 5.0 ml pipette and again look at them under the microscope. If they are now mostly single cells, add 3.5 ml ES media, pipette up and down and place all the cells in a 15 ml centrifuge tube. If you are expanding them further pipette 2.5 ml of the cell suspension in a new inactivated MEF prepared T-25 flask. Pool the cells from the other T-25 flask with the first and bring the final volume up to 10 ml. Spin the cells gently at 1000 RPM for 5 minutes at 10oC. Resuspend the pellet in 2.0 ml ES media (if freezing down 4 vials) and place 0.5 ml cells in a Nunc 1.8 ml cryovial. Next add 0.5 ml 2 X freezing media to each vial and freeze in the -70oC freezer.
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