My iPS cells are growing into large floating clumps in E8 media. I am not too worried about the cells not adhering to matrigel coated plates since they are proliferating, but the clumps are very difficult to dissociate for cell splitting even with vigorous pipetting. Does anyone have suggestions on how to break apart these clumps without stressing the cells too much?
I would suggest Accutase (Thermo Fisher) as I have always got really good results with it. It is preferred over Trypsin or Tryple for the following reasons (from http://www.accutase.com/accutase.htm):
Can be used whenever gentle and efficient detachment of any adherent cell line is needed. Accutase is a direct replacement for trypsin.
Works extremely well on embryonic and neuronal stem cells; mono layers of stem cells can be grown after passaging with Accutase.
Preserves most epitopes for subsequent flow cytometry analysis.
Does not need to be neutralized when passaging adherent cells. The addition of more media after the cells are split dilutes Accutase so it is no longer able to detach cells.
Does not need to be aliquoted. A bottle is stable in the refrigerator for 2 months.
Good luck!
I would suggest Accutase (Thermo Fisher) as I have always got really good results with it. It is preferred over Trypsin or Tryple for the following reasons (from http://www.accutase.com/accutase.htm):
Can be used whenever gentle and efficient detachment of any adherent cell line is needed. Accutase is a direct replacement for trypsin.
Works extremely well on embryonic and neuronal stem cells; mono layers of stem cells can be grown after passaging with Accutase.
Preserves most epitopes for subsequent flow cytometry analysis.
Does not need to be neutralized when passaging adherent cells. The addition of more media after the cells are split dilutes Accutase so it is no longer able to detach cells.
Does not need to be aliquoted. A bottle is stable in the refrigerator for 2 months.
Good luck!
Hello
Yes i agree with Sayandip Mukherjee , this good suggest
Good Luck
Hi,
If you use the E8 Medium you should culture the iPS cells on Vitronectin, and passage the cells in clump with EDTA.
Good luck!
no ReLeSR anyone? I found that the problem with Accutase is the extreme level of disassociation, which can induce differentiation (iPS cells like to be close to each other). it is more gentle than Accutase. However, I was using mTeSR....the people I know that use E8 also use EDTA to pass them and it works well / costs less. Also, what Justyna said is also true :-)
good luck!
Hi,
Just to clarify, are your cells not sitting down onto matrigel at all or are they growing to a moderate size and then lifting off from the matrigel in clumps? In terms of passaging I've had the best results by using EDTA for 1-3 minutes, so the edges of the colony are starting to round, remove the EDTA without disturbing the colonies and gently wash with PBS before adding media and then gently wash cells off the plate using a 1ml stripette. spin at relatively low speed
Hi Nicole,
It seems odd that the cells aren't adhering to Matrigel as it is essentially extracellular matrix. Is this occurring with just one cell line or are you seeing this across a range of iPSC lines? My first thoughts are not enough- or uneven coating of Matrigel on the tissue culture plastic.
Pluripotent cells are a particularly sensitive cell type to grow - I would avoid at all costs any kind of harsh pipetting. To break up the clumps Accutase as mentioned by a few others is a good solution but also TrypLE (should give single cell suspension) and EDTA (will give small cell clusters) should well. I would avoid using Trypsin. We routinely use TrypLE in our labs to dissociate to single cell suspensions (requirement for our automated cell counter) and re-plate in ROCK inhibitor (only 24 hr) on Vitronectin-N (Life Tech). The purpose of our work is to test cell quality in routine manufacture so we've tested and validated this culture system extensively.
Happy to provide more detail on request.
Regards,
James
I agree with others. E8/matrigel or E8/vitronectin- routinely passage with EDTA. Use Accutase only when single cell suspensions are desired for electroporation or FACS sorting. Make sure to add Rock inhibitor after plating with Accutase. If cells are hard to lift off matrix, incubate longer at 37C. Don't try to break up clumps by pipetting harshly as this induces differentiation as I'm sure you know.
Alternatively you could try monolayer culturing using DEF-CS which uses TrypLE for routine passaging . This keeps your cells in single cells (no colonies) throughout the process and unlike other monolayer culture systems has been shown to keep karyotype stable.
Hey Nicole,
EDTA 0.5mM in PBS w/o ions. It is much more gentle on the cells (in my opinion) than accutase. I actually grow my PSCs in 2D and 3D systems and can use it for passing in both. I typically use ReLSR for clump passaging and EDTA for single cell passaging or setting up monolayer differentiation protocols. Im wondering why your cells are not attaching to coated plates though? Im agreeing with James that something doesnt seem right with your coating technique/protocol. I may suggest revisiting that to determine where the problem is.
Ive never used DEF-CS before, but sounds interesting Liz!
I would be worried your cells are not attaching. If they are forming spheres in suspension they will most likely be differentiating. The fact they are proliferating does not mean these are still pluripotent stem cells. Have you FACS'd or immuno stained for pluripotent stem cell markers?
On the issue of not attaching, I'd go back to an original vial of the frozen iPSCs and thaw onto MEFs or human laminin521. hlam521 is great for culturing difficult lines and if you know the cells thaw poorly, use some rock inhibitor at thawing for overnight attachment.
If you are culturing feeder-free then its preferable to passage your cells as small clumps, not single cell. Therefore EDTA or RelesR is perfect.
If your cells aren't acting like pluripotent stem cells then you're probably not culturing pluripotent stem cells.
Good luck!
Are you using hESC qualified matrigel? Are you sure your matrigel plates are coated correctly?
Thank you all for your comments. I am plating them on hES qualified matrigel plates and passing them in small clumps using EDTA. The plates may be getting 'older' now, but the cells have never adhered well, even after initial thawing and using ROCK inhibitor. And if I stain the cells, there are still some SSEA-4+ undifferentiated cells in my culture.
Hello,
This already happens with us, same thing.
Was a problem with E8 sample (bottle).
Did you already test this E8 with geltrex?
And test another new E8 sample with matrigel?
Maybe your problem could be with medium or matrix.
We use Rock (5-10 uM) to make passages.
Regards.
The floating clumps do not need to be corrected. The issue is the improper matrigel coating or the media or the incubation conditions. If your iPS cells are not attaching, then you need to correct THAT problem, not the resulting symptom of that problem.
Hi,
The iPSC cell passage methods depend on the culture method. The iPSC cells in the undifferentiated stage cultured in the E8 medium on vitronectin should be passage with EDTA. The iPSC culture on the feeder layer (MEFs) should be passaged with collagenase. The iPSC growth in the MEF-Conditioned Medium on Matrigel / Geltrex can be passaged with Accutase, Dispase or TrypLE.
Good luck!
I see you have started another barrage of shilling. The substrate has nothing to do with the OP's question about breaking up clumps.
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