iPSCs generally need something like an anti-apoptotic when they are being passaged/cultured, atleast in the early stages after passaging. Try adding Rock inhibitor to the media that you are using. It might help. Though, I'm not sure why they are forming such dense clumps on geltrex.

Krishnamurthy Ravikumar I added ROCK inhibitor Y27632 after thawing and for around 24h after every passage I have done so far.

iPSCs react differently to different combinations of matrix and media. TESR-E8 is a tricky medium, not easy to use. If you can, switch to StemFit.

Geltrex is also not tested very much by many researchers. Companies nowadays offer all kinds of "new" and "super" media and matrixes but you should be cautious. iMatrix will work better, I think.

Clumps like these look very bad to me. You are getting extremely heterogeneous populations which is not good. You definitely need to change the matrix-media combination. If you cannot switch TESR-E8, try changing your coating to Vitronectin. That may fix the problem.

I agree with Yulia Panina 's comment. iPSCs react to different matrices and media differently. I usually use matrigel for the culture. You could try that out. It works well with TESR media. It is also correct that Geltrex is relatively new and not well-used by the community.

Hi,

1. Geltrex is the same as Matrigel;

2. This type of Media is dedicated to the rh-Vitronectin, and EDTA passaging

3. Your colonies are in very poor condition;

a) they are differentiated,

b) this what you see its EB stuck to other colonies; EB is up on the other ips cells colonies; they shoul be removed;

4. The cell line must to be cleared;

5. Single colony with good morpholog can be manually passage to cleared cell line and obtained cell line from single iPS colony;

6. The density of iPS colonies is too high; the single colonies should be visible;

7. Passage shoul be done at density 1:6 or 1:12;

8. The passage should be done when iPS colonies growth independently and have 80% of confluences.

Good luck!

Hi Justyna Augustyniak

Thank you for your advice - that's very helpful. There are some colonies that don't have these EB formations. How do I passage those separately to the rest? Normally we use TrypLE to just lift all the cells for passaging - how do I go about selecting individual colonies?

Hi,

On this page you will find a lot of information about iPS cells you need.

https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/culturing-puripotent-stem-cells-essential-8-medium.html#5

Good luck!

Hi,

You can also watch the movie.

https://www.youtube.com/watch?v=DeGpN2cpbR4

Good luck!

Thanks everyone.

It's interesting - that Thermo protocol removes the EDTA and then dislodges the cells by washing it with E8. The protocol I've been following has me dislodging the cells while still in TrypLE and then spinning them down after the addition of media. Is this difference down to the fact that Thermo are using a different dissociation reagent, or is there a rationale to not spinning iPSC?

Hi,

Type of passaging depend from:

- type of hiPSC experiments;

- medium (medium with FBS, SR or without FBS, SR);

- condition (feeder layer (MEFs), feeder free conditions);

- ECM (gelatin, vitronectin, laminin, fibronectin, Matrigel / Geltrex, collagen etc.)

- single cells, colony passage;

- dissociation reagent: EDTA, TrypLE, Trypsin, Acuase, Dispase, Collagenase IV,

- method of dissociation: enzymatic, non-enzymatic, mechanical (by colonies cutting or scraping).

There are certain rules for using a particular passage type. For example: Trypsin is inactivated by presents in the medium serum so is use usualy to passage cell growth in media with serum or serum replacement. EDTA, collagenase IV should be use to the passage hiPSC in colony. Long incubation time with EDTA dissociate hiPSC colonies to the single cells. Collagenase IV is use to the passaging hiPSC growth on MEFs on gelatin in ESC medium with FBS or SR. Tryple is gently Trypsin version dissociated hiPSC colonies to the single cells in medium also without serum. Enzymatic dissociation reagent should be remove by centrifugation (protease can lysed cells). EDTA is non enzymatic gently methods of hiPSC cell dissociation, and does not have to be remove by centrifugation. EDTA is the often use method of hiPSC growth on the rh-Vitronectin in E8 type media (Gibco or Stem Cells Technologies), but it not work good with Matrigel and Media contain FBS or SR. Trypsin should be use with PBS while EDTA with dPBS without calcium and magnesium. Trypsin is often use in the passaging hiPSC on Matrigel/Geltrex growth in conditioned medium contain FBS or SR as a single cells. hiPSC colonies are rather cutting at the clonal selection stage, while can be scraping in ESC type medium, because cells are more vital in the media contain except bFGF also serum.

Good luck!

Hi,

You have several options for cleaning the iPSC line.

The iPS cell line cleaning strategy depends on the number (%) of differentiated cells.

1. Manual scrapping of differentiated iPSC colonies

Select (highlight) the differentiated iPSC colonies on the bottom of the plate well. Scrape the every single differentiated iPSC colonies with the pipette tips. Wash plate well with medium 2 times to removed floating differentiated iPS cells colonies.

Repeat remove the differentiated iPS cells colonies before every passaging.

2. Passaging Human iPS Cells Using EZ-LiFT™ Reagent,

https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-culture/ez-lift-human-ips-cell-passaging.html

3. ReLeSR™ Enzyme-free human ES and iPS cell selection and passaging reagent, https://www.stemcell.com/relesr.html

4. Clonal selection of undifferentiated single iPSC colonies

Select (highlight) the undifferentiated iPSC colonies on the bottom of the plate well. Scrape the every single undifferentiated iPSC colonies with the pipette tips. Catch the single undifferentiated iPSC colonies and transfer them to a new medium with ROCK inhibitor and the plate coated with the ECM.

Good luck!

Geltrex is hardly "new." It has been used extensively for years and I have found it to be much more consistent than other commercially available matrigels. However, far too many people seem to be clueless about how to properly handle matrigel. How are you preparing your Geltrex coated plates? How much Geltrex are you putting in each plate? It should be distributed by protein mass per unit of area, and not by some random dilution ratio.

iPSC colonies should be passaged long before confluency. How many days are you going between passages?

The best way for you to rescue this line is to mechanically dissociate undifferentiated colonies.

Hi Ron Hrstka

I have attached the protocol that I use for coating plates with Geltrex.

As for colony passaging, generally I was passaging them anywhere from 5-7 days after plating. The cells seemed slow to grow, and preferred to form the horrid clumps seen in the original post images, as opposed to growing outwards into larger colonies. I'm not sure what i'm doing to cause them to behave this way - i'd really like to isolate the issue before I go wasting expensive cells.

Thanks for the recommendation of mechanical dissociation - that seems to be the general consensus. I will do my best to excise any differentiated portions of the culture.