I am working on H1 cells, I am sub-culturing in 10 cm dish coated with matrigel at 1:10 split ratio of confluent cells. I am having this frequent problem of cells not getting colonized on 3rd of splitting which is unexpected. I am depleting ROCK inhibitor on 2nd day. I would like to know the reason for stem cells not getting colonized. My hypothesis is that matri-gel is getting degraded. If it is the case, I would like to get some suggestions on matri-gel coating of plates. Attached are the images of my cells with A-unhealthy and B-healthy.
Hi,
it looks like it is a coating problem to me. I had similar looking iPSC, when I coated with vitronectin. The problem was that not the whole surface was coated, and at these non-covered surface the colonies rounded up because they did not attach and spread.
I would recommend checking the diluting ratio of matrigel. How do you do the coating? I do the dilution in DMEM-F12 (or Knock out-DMEM) and leave it overnight at RT. If not used immediately, the dish/plate could be stored at 4C. I would say if your coating is fine, you should see the matrigel structure--which I do not really see in none of the images. You could check the attachment to see what I mean by the matrigel structure.
Good luck.
Hi,
coating might be a problem here. As suggested above you may extend the coating time.
I assume that you use colonies for passaging so you might consider to get rid of ROCK completely. ROCK is only useful when passaging in single cells. In our hands ROCK-treated cells look extremely stressed compared to non-treated ones.
How do you treat the cells? Dispase or collagenase? In our hands enzymatic-assisted pasaging is less reliable than non-enzymatic. I would propose to use a non-enzymatic passaging solution (which is commercially avaibable or use the one mentioned within this answer). This has greatly improved our routine culture.
All the best
Article Scalable Passaging of Adherent Human Pluripotent Stem Cells
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