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In this video you will see how to use the Gibco PSC Cardiomyocyte Differentiation Kit to generate beating cardiomyocytes from pluripotent stem cells. By using this easy-to-use three-part media system, you can start from a small population of pluripotent stem cells and obtain a large number of beating cardiomyocytes. You can access the product insert and the quick reference protocol online.
A supplemental list of reagents used in this protocol can also be referenced here.
First, prepare a complete Essential 8 Medium. Find details for this preparation on the web link listed below.
Then prepare your substrate by coating a 12-well plate with a 1:100 Geltrex solution. And place in an incubator for at least one hour.
Next, prepare a cell recovery solution by adding 250 µm of RevitaCell supplement to 25 mL of Essential 8 Medium.
Let warm to room temperature.
Remove the Geltrex coated plate from the incubator and aspirate the plate.
Add 1 mL of cell recovery solution to each well.
Quickly thaw a vial of cryopreserved pluripotent stem cells in a 37˚C water bath until just a small ice crystal remains.
In the biosafety hood, transfer vial contents to a 15 mL tube and slowly add 10 mL cell recovery solution while swirling the tube.
Centrifuge the cells at a relative centrifugal force of 200 x g for 5 minutes.
After centrifugation, aspirate supernatant and gently flick the tube to dislodge cells.
Resuspend cells by adding 2 mL cell recovery solution dropwise.
Perform a viable cell count and calculate the cell density of the cell solution.
Seed plate with the cell solution to achieve 30 to 70% confluence within 3 to 4 days. The differentiation efficiency of PSCs into cardiomyocytes varies between different PSC lines.
A critical variable for the generation of a robust cardiomyocyte culture is the relative confluence at the onset of differentiation. The suggested confluence range at the time of induction is 30 to 70%, and it is strongly recommended that you perform a confluence range finding study to determine the seeding density needed to achieve the optimal confluence for your PSC line within 3 to 4 days of seeding.
Confirm attachment of cells the following day.
Refeed cells with pre-warmed Essential 8 Medium every day for 3 to 4 days to reach target confluency for your PSC line.
Once the plate has achieved target confluency, replace the medium with Cardiomyocyte Differentiation Medium A. Note that the addition of medium should be done slowly and along the sides of the well, as forceful addition of medium will disrupt the differentiation process.
Two days after Cardiomyocyte Differentiation Medium A induction, replace the medium with Cardiomyocyte Differentiation Medium B.
Two days later, replace the medium with Cardiomyocyte Maintenance Medium.
Continue to refeed cells with this medium every other day.
10 days after differentiation has begun using the PSC Cardiomyocyte Differentiation Kit you should observe beating syncytia of cardiomyocytes. These cells are now ready for further studies.
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