Hi, Tommy. I suggest to change coating material and concentration of trypsin. If you don't mind, how to use matrigel instead of gelatin? In my experience, matrigel coating showed better result. Also, 0.25% trypsin concentraion may be too strong for your cells. When I generate iPSC, my cells died under 0.25% trypsin. Maybe 0.25% trypsin is enough for some cell lines in the reference and labs but not my experiment. If it is your first experiment, I recommend to use 0.05% trypsin-EDTA. ESC is sensitive cell.

The protocol I am using is as follows

1. add 100 ul of 0,25% trypsin EDTA solution to each well of 24 well culture plate

2. Incubate 3-5 min (depending on cell line)

3. Aspirate trypsin edta using a micropipette

3. Add 1000ul growth medium and gently pipette up and down to make a single cell suspension.

Hi Tommy,

 I don't know what you call a conventional medium for ES, there are people who grow them in defined, serum-free media, and in this case your trypsin is killing the cells. Many people, including myself, use serum-containing medium (10 or 15%), and with these media trypsin concentration is not that critical, although Seungmin is absolutely right, these cells are very sensitive. They rely upon cadherins to attach to each other, and this is extremely important for their survival. If your trypsin chew extra cellular cadherin ends, they will die. 

I would suggest to shed your cells in parallel without 2i, just with LIF (double the concentration, to be on the safe side), and see if the survival will improve.

Also, I would suggest to seed defined cell densities, as you don't know what's happening. Try 30 000 cell per square cm, it's quite high density for the ESm they should grow in 2-3 days. Normally I seed them at 10 000 cells per square cm, with 2i+LIF.  They proliferate very well, I get 3 or more million cells per P6 well.

Good luck!

Geni

I agree with Holger. I recommend using Accutase instead of trypsin for dissociating feeder-free mESC cultures.

Also, I would recommend to plate at least 0.5 10exp6 mESCs onto a new 6 well. Splitting 1/8 might be too much.

Hello Tommy,

How long do you wait for the cells to be 80% confluent if you have a lot of death sicne the beginning then? I would try to seed them at higher density in order to get them happier (like Holger suggested). If after two days you can pass them it means they should feel better and then go into a bigger ratio of passage.

When you speak about classical mES media with LIF and 2i, do you use serum or KSR... it can have a certain impact as well on your culture. I would suggest you to run some tests with different serum before going into further experiments. 

Let us know if anything worked ;)!

Cheers

Thank you for all of your generous suggestion.  I will try to use lower concentration of trypsin and seed cells with defined cell number.

To Seungmin ,Holger and Tim, I am using 0.25% trypsin in EDTA, I have bought TrypLE express and will try to detach cells with it in diluted form. Hope it will cause minimal harm to the cells

To Evguenia and Fleur, I am using ESQ-FBS containing medium supplemented with 2i and LIF. Our lab do have Knock-Out serum replacement but since the mESC isolation and initial expansion by my lab mate were using ESQ-FBS, I just follow the recipe she provided.

Prior to passage, I averagely count around 3Mil cells or more per 6-well. So with a split ration of 1:8, the estimated cell density seeded is around 40,000 cell/sq.cm. With many cell death at last the number of cells adhered on culture dish can be as low as ~30,000 cell/6-well. Lucky that the cell survived can expand into very nice-looking colonies. It takes just less than 1 week to be needed to passage again.

In fact, the EB culture I did (1 day before asking this question) finally have EBs showing up, at a very small size (~100um), probably derived from single mESC. During the period I changed the medium every 2 days and remove debris by very low speed centrifuge.

Thanks for all of your help! :)