Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population
Trascrizione
Differentiation of ESCs into neurons starts during a routine cell passage. Dissociate the ESCs as before, but include an additional plate for neuronal differentiation. Transfer 350 microliters of the cell suspension into a 10-centimeter low-attachment dish containing 25 milliliters of differentiation medium.
Place the dish on an orbital shaker set to 30 to 45 RPM inside a tissue culture incubator at 37 degrees Celsius and 5% carbon dioxide, and incubate for 48 hours. After 48 hours, use a 25-milliliter pipette to transfer the differentiating cell aggregates to a 50-milliliter conical tube.
Immediately after, add 25 milliliters of fresh differentiation medium to the Petri dish. Allow the aggregates to settle for two to five minutes, producing a visible pellet that is one to two millimeters deep. Carefully aspirate the medium, ignoring any single cells or small aggregates still in suspension, and use a P1000 pipette to transfer the cell pellet back to the Petri dish. Return the dish to the rotary shaker in the incubator. After a further 48 hours, repeat the media change procedure. This time, add 30 milliliters of differentiation medium supplemented with six micromolar all-trans retinoic acid.
The pellet formed will now be two to four millimeters deep. After incubating the cells as before, repeat the media change procedure with retinoic acid supplementation for the final time. The pellet will be four to eight millimeters deep at this point.
The following day, prepare the plating surfaces as outlined in the written portion of the protocol. On the afternoon of the day of plating, thaw 5 milliliters each of pre-aliquoted NPC trypsinization medium and 0.1% soybean trypsin inhibitor at 37 degrees Celsius. Then, use a 25-milliliter pipette to transfer differentiating aggregates from the culture plate to a 50-milliliter conical tube.
Allow the aggregates to settle for three to five minutes, and then carefully aspirate the medium without disturbing the pellet. Next, wash the pellet with 5 to 10 milliliters of PBS, and after aggregates have settled, aspirate the PBS and repeat the wash. After the second PBS wash, add 5 milliliters of NPC trypsinization medium to the pellet and incubate at 37 degrees Celsius for five minutes, gently flicking the tube two to three times during the incubation.
Next, add 5 milliliters of 0.1% soybean trypsin inhibitor to the cells and quickly mix by inverting. Then, gently triturate the cells 10 to 15 times with a 10-milliliter pipette until a relatively homogeneous cell suspension is produced. Slowly filter the cell suspension through a 40 or 70-micron cell strainer placed on top of a 50-milliliter conical tube.
Then, once all of the suspension has been filtered, add 1 milliliter of N2 medium to the filter to wash the remaining cells through the strainer. Transfer the cell suspension to a 15-milliliter conical tube, and centrifuge for six minutes at 200 times g.
After aspirating the medium, without disturbing the pellet, triturate in 2 milliliters of N2 medium, then add N2 medium to a total volume of 10 milliliters. Centrifuge for five minutes at 200 times g. Aspirate the media and repeat the pellet washing process, again, resuspending the pellet in 10 milliliters of N2 medium.
Remove an aliquot of the cells, and count with a hemocytometer, and then repeat the centrifugation. Resuspend the cells in N2 medium to a final concentration of 1x times 107 cells per milliliter, and plate at a density of 150,000 to 200,000 cells per centimeter squared in dishes prepared at DIV minus 1, and place in the tissue culture incubator at 37 degrees Celsius. Maintain ESNs according to the instructions in the written protocol.
