To maintain the human embryonic stem cell culture, the culture media must be changed every 24 hours. To do this, start with an embryonic stem cell media stock bottle stored at four degrees Celsius. Take out the amount of solution needed, which is usually about 20 milliliters per six.
Well plate and place that amount in a 50 milliliter Falcon tube and warm the tube with contents to 37 degrees Celsius in a water bath. Once the media is warmed, add the FGF, which has been stored at four degrees Celsius to a final concentration of 10 nanograms per mil. Be sure to remember to put the BFGF stock back at four degrees Celsius immediately after use.
Be sure the BFGF in the human amox stem cell culture media is thoroughly mixed. Next, remove all of the media from each well leaving about 500 microliters per well. Next, add three milliliters of fresh media back to each well of a six well plate and put the plate back in the incubator.
Now that we've shown you how to maintain human embryonic stem cells ONMs, we'll next show you how to transfer them to matrigel. To split human embryonic stem cells grown ONMs to matrigel, we start with a Cofluent six well plate of stem cells on mes, which can be split one to two or one to three onto matrigel. Six well plates.
This will allow the wells to become confluent again four to five days after splitting. When splitting human embryonic stem cells onto matrigel, use meth condition media to maintain pluripotency meth conditioned media is made in advance. This is done by collecting the media, which is supplemented with five nanograms per mil BFGF from a growing meth culture every day for seven days, embryonic stem cell media is used to replace the removed meth conditioned media.
Every day. Meth conditioned media should be used for up to one month after it is collected and stored frozen. The day before human embryonic stem cell splitting, we move matrigel aliquots in EOR tubes from minus 20 degrees Celsius to four degrees to thaw overnight.
One aliquot contains two milligrams of matri gel, which is the amount needed for one six. Well plate. On the day of splitting, take one vial of the thawed matri gel.
For each six, well plate and mix each vial with six milliliters of cold D-M-E-M-F 12 media. Remember to keep this on ice. Add one milliliter of the solution to each well of a six well plate.
Then swirl the plate to distribute matrigel on the surface and incubate the matrigel covered plate at room temperature for at least one hour before splitting the human embryonic stem cells. Before splitting the human embryonic stem cells, wash the stem cells on MES once with one XPBS pH 7.4. Next, add one milliliter of warm collagenase four at one milligram per mil to each well for the wells you are splitting, and then incubate the plate at 37 degrees Celsius for five to 10 minutes.
After incubation, add one milliliter of embryonic stem cell media without BFGF To each, well use a one milliliter pipette to blow the stem cells off the plate. Collect all the media containing clumps of stem cells and dead mes into a 15 milliliter falcon tube. Let big clumps of cells settle for five minutes so that the stem cell clumps form a pellet at the bottom of the tube while the mes remain suspended in the snat.
Once the cells seem to have settled, discard the supernatant and wash the cells by resus suspending the embryonic stem cell pellet. Using embryonic stem cell media without BFGF next centrifuge at room temperature at 200 times G for five minutes. While the cells are spinning, we can prepare the matrigel plates for stem cell plating by washing the plates with room temperature D-M-E-M-F 12 at one mil per well, and adding 2.5 milliliters of conditioned media supplemented with 10 nanograms per mil.
BFGF per well after centrifugation. Resus, suspend the pellet in an appropriate volume of meth conditioned media supplemented with 10 nanograms per mil. BFGF, pipette the cell suspension up and down several times until the clumps become uniform and small in size.
Next, aliquot the suspension at 0.5 milliliters per well on the matrigel plate to achieve three mils per well as the final volume. For matrigel, we use a higher colony density than for usual splitting on maths once the cells have been added. Place the plate in a 37 degree Celsius tissue culture incubator.
To maintain the human embryonic stem cells on matrigel meth condition. Media supplemented with 10 nanograms per mil. BFGF is changed every 24 hours for up to four to five days.
Human embryonic stem cells are usually contaminated with residual amounts of MEFs after the first splitting on matrigel, the second splitting from matrigel to Matrigel will completely remove any traces of feeder cells contaminating the stem cell culture.
