This video will show you how to passage PSCs with EDTA in Essential 8 Medium on vitronectin coated plates.
The following protocol uses a 6-well plate. If you're using another type of cell culture vessel, please refer to table 2 on the written protocol for volumes.
There are three major differences that you'll observe with cells cultured in Essential 8 Medium on Vitronectin compared to other feeder-free systems such as mTeSR and StemPro hESC SFM:
• Cells are typically passaged ~24 hours sooner than they would be with other feeder-free media.
• Passaging should take place when cells are at ~85% confluency. If cells are passaged when they are more than 85% confluent, the health of the cells and final cell yield may be compromised.
• Cells must be passaged in EDTA. Collagenase and Dispase are not recommended.
Cells will reach optimal confluency typically every four to five days.
You should also split cultures or passage cells when PSC colonies become too dense or too large or show increased differentiation.
The split ratio can vary, although it's generally between 1:2 and 1:4 for early passages and between 1:3 and 1:12 for established cultures. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted.
A good rule is to observe the last split ratio and adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy, and the colonies have enough space, split your cultures using the same ratio. If the colonies are overly dense and crowding, increase the ratio; if they are sparse, decrease the ratio.
Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It's not necessary to remove differentiated material prior to passaging. By propagating/splitting the cells, the overall health of the culture should improve throughout the early passages.
Prepare 0.5 mM EDTA by combining 50 ÎĽL of UltraPure 0.5 M EDTA, pH 8.0 with 50 mL of DPBS without Calcium and Magnesium. Filter, sterilize the solution. and store at room temperature.
Pre-warm complete Essential 8 Medium and VTN-N-coated culture vessels to room temperature.
Aspirate the spent medium from the vessel containing PSCs and rinse the vessel twice with DPBS without calcium and magnesium.
Add 0.5 mM EDTA in DPBS to the vessel containing PSCs. Swirl the vessel to coat the entire cell surface.
Incubate the vessel at room temperature for 5 to 8 minutes or at 37 degrees Celsius for 4 to 5 minutes. When the cells start to separate and round up, and the colonies appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel.
Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes.
Aspirate the EDTA solution, and add pre-warmed Essential 8 Medium to the vessel.
Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up. Avoid creating bubbles.
Try to work with no more than 1 to 3 wells at a time, and work quickly to remove cells after adding Essential 8 Medium to the well(s), which quickly neutralizes the initial effect of the EDTA. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to re-attach, and may be removed 3 wells at a time.
Collect cells in a 15mL conical tube. There may be obvious patches of cells that were not dislodged and left behind. Don't scrape the cells from the dish in an attempt to recover them.
Add an appropriate volume of pre-warmed Essential 8 Medium to each well of a vitronectin coated 6-well plate so that each well contains 2 mL of medium after the cell suspension has been added.
Move the vessel in several quick figure eight motions to disperse the cells across the surface of the vessels. Place the vessel gently into the 37 degrees Celsius, 5% CO2 incubator and incubate the cells overnight.
Feed the PSC cells with Essential 8 Medium beginning the second day after splitting and replace the spent medium daily.
It is normal to see cell debris and small colonies after passage.
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