I tried passaging iPSC using cell scraper for three cell types. One of them got attached and other two did not attach. Any idea why the other two did not attach. I use feeder free (matrigel coated dishes).
I am also thinking of alternative to cell scraping. Does anyone have experience using versene for passaging?
Hello Sahana T G
I suggest you switch over to versene because it is an enzyme-free method which is much quicker avoiding the use of enzyme neutralization step, and will also avoid the risk of contamination. Moreover, versene treatment will cause partial dissociation of cells to generate small aggregates which could survive better than individual cells. Also, versene is least damaging to the extracellular matrix.
Enzymatic methods like TrypLE or Accutase can also be used for passaging which I would not recommend because they are not consistent.
Thank you.
Thank you Malcolm Nobre
Can you please explain how do you use versene?
For instance, if I am using 10 cm dishes, I will first remove mTeSR (wash if there are many dead cells) and then I add 5-6 mL of versene and incubate for 3-4 min. Later, I will collect the cell suspension and then centrifuge.
Is this the right way?
Hi Sahana T G,
I am working with iPSCs (Human Episomal iPSCs, Gibco). This is the protocol I follow to dissociate and passage iPSCs using versene solution as per the Thermofisher product manual:
1. Remove the spent media
2. Wash the cells carefully with DPBS (without Calcium and Magnesium) twice
3. Add the appropriate volume of versene solution (5-6 ml for 10 cm dish)
4. Either incubate at 37 degrees for 4 to 5 minutes or keep it in room temperature 5 to 8 minutes, until you see holes in the colonies and cells rounding up.
5. Carefully remove versene solution ( If timed right no colonies would come out during aspiration)
6. Add fresh iPSC media and gently flush the cells. (Careful not to flush too much and break the colonies into single cells).
7. Collect the cells in a 15 ml or 50 ml centrifuge tube and plate them in new Vitronectin/GFR-M coated dishes.
(I skip step 7 and directly plate the cells)
Please find the Gibco Episomal hiPSC cell line manual for your reference:
https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fepisomal_hipsc_man.pdf&title=VXNlciBHdWlkZTogR2liY28gRXBpc29tYWwgaGlQU0MgTGluZQ==
Hello Sahana T G
The following is the protocol :
1.Remove mTeSR and wash with PBS (without Ca2+ and Mg2+) to remove the dead cells.
2. Rinse the dish with 5ml versene and aspirate it immediately without incubation. Do the washing two times. While washing add versene to the wall of the wells slowly.
3, Add 5ml versene again and incubate for 4-5 mins at room temperature.
4. After 5 mins aspirate the versene and add 5ml medium rapidly to wash and disperse the cells as quickly as possible. (This step has to be done as quickly as possible because cells may reattach to the plate).
5.Gently pipette out the cell suspension carefully taking care not to break the colonies and plate fresh.
Please note that there is no centrifugation step.
I hope this helps.
All the best.
Hello Sahana T G,
As an alternative, you can use 0.5mM EDTA (sterile/cell culture grade), as follows:
1. Gently rinse the cells with PBS (without Ca2+/Mg2+). Discard PBS.
2. Add 0.5mM EDTA for 5-8 mins at room temperature (you can observe the cells under a microscope; when they have rounded up somewhat, but before they detach by themselves, they are ready to go).
3. Very carefully remove the EDTA, e.g. with the plate tilted, using a 5ml stripette, making sure you don’t disturb the cells.
4. Rinse the cells off the bottom of the well, using fresh medium and transfer them to a new (coated) plate at the desired passage ratio. Important: Do NOT use a Gilson to do this, as it will likely break up the cell clusters. You do not want to end up with single cells! I use a gun with a 5ml stripette instead on my 6-well plates.
I hope this helps, although note that sometimes the best method is cell line dependent. Also, if you switch to a new method, the cells might need a few passages to get used to it, so in the meantime you may experience higher death rate than usual. Also note, my protocol is based on Vitronectin substrate, so incubation time may differ for Matrigel.
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