Hello, my iPSCs always seem to migrate to the center of my well despite shaking my 6-well plate (in an infinity sign shape) quite meticulously before I stick them in the incubator.
I always check them under the microscope and the spread of the clumps looks good, but when I check them the next day, they're always in the middle of the well.
This is concerning because I'm having to split them quite frequently and at higher ratios because there aren't that many cells, the colonies just happen to be coalescing in the middle and getting really large.
For extra info - I have been passaging my iPSCs with ReLeSR and have been obtaining iPSC clumps of a good size by pipetting the cells with a p1000 a couple of times before replating them.
Does anyone have any pointers? Is this a matrigel coating problem? A colleague suggested that I should let the cells settle down at room temp before I put them in the incubator to avoid this.
Thanks!
Hello Sowmya Theegalapalli
If you move the multi-well plate in a circular direction, it will make your cells come together in the center. The right way would be to move the plate two dimensionally in a figure eight or in all four directions, straight up and down and side to side. The bigger the growth surface, the better this approach works.
In addition to moving the 6-well plate in an infinity sign shape, you will obtain better results by not prefilling the well with media before adding the cells. Instead, you should prepare the cell suspension in a tube according to the number of cells needed. Resuspending the cell suspension frequently also helps. After having seeded the cells, you may keep the plate at room temperature for about 30 mins before placing the plate in the incubator (as rightly mentioned by your colleague). Let the cells sink down at the bottom at room temperature and when they are nicely spread around the plate, you may place the plate in the incubator.
Please note that as you walk with the muti-well plate to the incubator, the vibration caused by walking with the plate can make the cells concentrate in the center.
Best.
Hi,
Try to seed iPSC colonies in a high dilution. In the incubator, move the 6-well plate in all directions. Do not use circular movements.
Do not worry because the concentration of iPSC colonies in the center is not a problem and does not affect the characteristics of the iPSC.
Best,
Thanks Malcolm Nobre and Justyna Augustyniak! I used these techniques and the spread of my culture significantly improved. The only issue I faced was that there were a lot of dead cells in the culture, but I presume this was because of slight over-pipetting.
In general, would you conclude that if there is a "halo" of "light", or a "bright boundary" around your stem cell colonies, they are stressed? Curious.
Best,
Sowmya
- Badges
- Science topic