Hi everyone,
I am trying to adapt V6.5 mouse embryonic stem cells to feeder- and serum-free conditions. There was no problem getting rid of the feeders, cells grow nicely on 0.1% gelatin-coated plates. What I do next to the cells that grow on gelatin-coated plates in ES-DMEM (see below):
- detach cells with Accutase (I tried TrypLE as well) for 2-3 minutes
- resuspend in serum-free medium (I tried resuspending in PBS too)
- spin @350g for 5 min
- aspirate supernatant
- resuspend in serum-free medium
- plate (1:3 subculturing ratio)
Cells grow fast (medium replacement every day) and look nice, do not differentiate, but when I try to passage them as described above (usually two days after), they clump and do not attach.
My questions are: why is this happening, especially after nice growth in feeder- and serum-free conditions after the first passage? Does anyone have a detailed working protocol for successful adaptation of V6.5 (or any other) mESCs to serum-free conditions?
ES-DMEM:
- KnockOut DMEM (Thermo, 10829018) 25 ml
- ES Cell Qualified FBS (ATCC, SCRR-30-2020) 4.5 ml
- MEM NEAA (100X; Thermo, 11140050) 300 ul
- Penicillin-Streptomycin-Glutamine (100X; Thermo, 10378016) 300 ul
- 2-Mercaptoethanol (from 100 mM) 30 ul
- CHIR99021 in Solution (10 mM; Stemgent, 04-0004-02) 9 ul
- PD0325901 in Solution (10 mM; Stemgent, 04-0006-02) 3 ul
- ESGRO LIF (10 million units/mL; Sigma, ESG1107) 3 ul
Serum-free:
- KnockOut DMEM/F-12 (Thermo, 12660012) 14.85 ml
- Neurobasal Medium (Thermo, 21103049) 14.55 ml
- N-2 Supplement (100X; Thermo, 17502048) 0.3 ml
- B-27 Supplement (50X; Thermo, 17504044) 0.6 ml
- Penicillin-Streptomycin-Glutamine (100X; Thermo, 10378016) 300 ul
- 2-Mercaptoethanol (from 100 mM) 30 ul
- CHIR99021 in Solution (10 mM; Stemgent, 04-0004-02) 9 ul
- PD0325901 in Solution (10 mM; Stemgent, 04-0006-02) 3 ul
- ESGRO LIF (10 million units/mL; Sigma, ESG1107) 3 ul
Please let me know if any additional details might clarify the question.
Thanks in advance!
Hi Adam, thanks for the reply! Unfortunately for us it is absolutely critical to have serum-free conditions. I guess what strikes me the most is that this is happening already after one passage, and not after a continuous growth for weeks or months on serum-free medium. To me it tells that there is probably something very obvious that I am doing wrong (and I am not very experienced with mouse ESCs to see what exactly)...
Hi,
In order to run a free culture serum, change FBS on ES cell qualified serum replacement.
Grow ES cells in Mouse ES Cell Basal Medium that has been supplemented with the following components:
1. 0.1 mM 2-mercaptoethanol
2. 1,000 U/mL mouse leukemia inhibitory factor (LIF)
3. 10% to 15% ES-Cell Qualified FBS or an ES cell qualified serum replacement
Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C.
The change of medium should be gradual.
80%MeidumA + 20%MediumB
50%MediumA + 50%MediumB
20%MediumA+80%MediumB
100%MediumB
Good luck!
Hi Justyna, thanks for the suggestion. Gradual adaptation worked for me!
I will just leave it here in case someone else has a similar problem:
After one more attempt, direct adaptation worked as well. I figured that gentle detachment with Accutase (cells were treated with Accutase for less than a minute at room temperature, after which Accutase was aspirated, and cells were washed off the surface with and resuspended in growth medium) and coating with gelatin at room temperature for 30 minutes (I used to coat plates for ~1 day at 37C) were the changes I needed to make. I have also noticed that the cells grown in the freshly prepared defined medium grew much better than the cells in defined medium that was 6 days old.
Hi, I think you may need to use some serum to inactivate the Accutase or Tryple. ESCs growing without MEFs (especially under serum-free conditions) can become very sensitive to enzymatic dissociation. I work with v6.5 under the same serum-free conditions you describe here (N2-B27, 2i, LIF). I always treat with Tryple for a short period of time (2-3 minutes) at room temperature, I never incubate them at 37C. As soon as I see the cells start to dissociate under the microscope I immediately add medium containing serum to stop the trypsin. After that I spin the cells down and resuspend in Serum free medium. If serum absolutely cannot be used for your experiments I'd suggest maybe using tissue culture BSA.
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