Hello,

I did not work with this specific ESC line but if they have been reported to be feeder free, there are no reasons why you won't be able to use the 2i+LIF culture medium. My advice is to try. It should be fine.

Best wishes,

Christophe

Hello,

I also haven't worked with this cell line, but as Christophe said it should be OK. I would try it and I'm almost certain that it should work fine.

Cheers,

Daniel

Hi,

My only comment would be that if they have been feeder dependent before freezing down, it might be wise to bring them up on feeders for a couple of passages to establish them before moving to feeder free.

It depends on the cells, some cells have been known to be easy to get from feeders to non-feeders transition, some others fail if they have been frozen on feeders and thawed on feeder free environment, at least on the human embryonic stem cells I have been working with, could this be the case with mESCs as well?   

Kind Regards,

George

Most mES cells I have worked with have been transitioned to feeder free without problem. I agree with George though, if they were feeder dependent prior freezing, thaw them on feeder, you will get better recovery and then transition to feeder free.

Best,

Christian

Hi,

I have not worked with these cells per se but, I have used mESCs in the past. In my experience they work very well in feeder free conditions if you grow them on ECM such as, Collagen IV or I. Feel free to refer to our published manuscripts for more information on ECM use. Additionally, George makes a very good point of growing them initially on feeder layers and then moving them onto feeder free conditions. Good luck! 

Hi,

I work a lot with mES cells, and I always culture them on feeder cells, the supplement of LIF in the media is not enough to keep ES in the embryonic stage (inhibit differentiation) and healthy. the only case that I culture the mES cells w/o feeders is when I use them for genotyping purpose; Other than that, for instance, balstocysts injections, transfections or electroporations, feeders would not cause any problem (on the efficiency of your experiment). 

I hope that helps!

Good luck.

Foued

Hi,

I have not worked with that specific line either, but I have done quite some work with other lines. You have two options:

B27N2 media + 2i + LIF on gelatin coated plates. With this culture you will reach a more homogeneous and more "naive" population.

Also on gelatin coated dishes, but using MEF conditioned media + LIF. Basically use mES serum media on MEF for three days and then collect the media and filter it to get rid of floating cells, it is then ready to be used on ESC. You can also freeze it.

The two options will lead to different morphologies. B27N2+2i+LIF will look like more tight colonies, while the conditioned media ones will look more flat.

Good luck!

Javier

Hi

http://www.jove.com/video/832/from-mefs-to-matrigel-3-passaging-hescs-from-matrigel-onto-matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

Preparation of Conditioned Medium (CM)

1. Plate irradiated MEFs at 20,000 cells/cm2 in MEF medium as described in Culturing Human Embryonic Stem Cells with Mouse Embryonic Fibroblast Feeder Cells (McElroy and Reijo Pera 2008b), and culture overnight in a 37°C incubator supplied with 5% CO2.

Use either six-well tissue culture plates or 10-cm tissue culture dishes for plating MEFs for production of conditioned medium.

2. The next day, remove the MEF medium, wash cells twice with 1X PBS and replace PBS with KSR medium for hESCs (0.3 mL/cm2).

3. Collect the CM from the MEF culture dishes after 24 h and sterilize with a 0.2-μm filter after adding an additional 4 ng/mL bFGF.

CM can be stored at −20°C before adding bFGF for later use.

4. Add fresh KSR medium to the MEF feeder dishes, and collect CM every day.

The irradiated MEFs can be used for up to 1 wk to produce CM.

Preparation of Matrigel-Coated Plates

5. Slowly thaw the Matrigel stock solution on ice at 4°C for at least 2 h to avoid gel formation.

6. To prepare the working solution, dilute the Matrigel stock solution 1:15 in cold knockout DMEM. Coat each well of a six-well plate with 1 mL of Matrigel working solution.

7. Incubate the Matrigel-coated plates for 1-2 h at room temperature, or overnight at 4°C.

Passaging hESCs on Matrigel-Coated Plates

8. Aspirate the medium from the six-well tissue culture plate containing hESC colonies.

9. Wash once with 2 mL of 1X PBS (Ca2+/Mg2+-free) per well.

10. Replace the PBS with 1 mL of collagenase solution and incubate for 10-15 min at 37°C.

Incubation time will vary depending on the batch of collagenase solution and the hESC lines.

11. During incubation, wash the Matrigel-coated plate twice with 1X PBS (Ca2+/Mg2+-free), and add 2 mL of CM (from Step 3) per well.

12. Check the hESCs under the microscope.

Colonies should appear to have shrunken a bit, with the edges of the colonies starting to pull away from the plate, but should not be detached or floating in the medium. If the colonies have detached, collect the medium and centrifuge it to harvest the cells.

13. Remove the collagenase solution and wash twice with 1X PBS (Ca2+/Mg2+-free).

14. Add 1 mL per split of CM (e.g., add 3 mL of CM if the cells will be split at a 1:3 ratio).

We recommend a split ratio between 1:1 and 1:3, depending on ESC colony concentration.

15. Gently scrape the hESCs from the plate with a 5- or 10-mL pipette or cell scraper into the new medium, and transfer the contents of the wells into a 15-mL tube.

16. Dissociate the cells into small clusters (50-500 cells) by gentle pipetting. Do not reduce cells to a single cell suspension.

17. Plate 1 mL of CM containing cells into each well of the Matrigel-coated six-well plate.

18. Return the plate to the incubator. Tilt the plate several times both in the horizontal and vertical directions to spread out cell clumps evenly.

19. Feed hESCs with 2-2.5 mL of CM supplemented with 4 ng/mL bFGF per well every day.

20. Passage when cells are 80% confluent (see Fig. 1).

Previous Section 

REFERENCES

↵McElroy S.L.,

Reijo Pera R.A.

(2008a) Preparation of mouse fibroblast feeder cells for human embryonic stem cell culture. CSH Protocolsdoi:10.1101/pdb.prot5041, (this issue).

FREE Full Text

↵McElroy S.L.,

Reijo Pera R.A.

(2008b) Culturing human embryonic stem cells with mouse embryonic fibroblast feeder cells. CSH Protocolsdoi:10.1101/pdb.prot5042, (this issue).

FREE Full Text

↵Panula S.,

Reijo Pera R.A.

(2008) Preparation of human foreskin fibroblasts for human embryonic stem cell culture. CSH Protocols doi:10.1101/pdb.prot5043,(this issue).

FREE Full Text

Hi,

I started ESC culture feeders free at  my new job. The experts who taught me at the stem cell institute are teaching in a workshop this summer: http://cambioscience.com/2016/05/08/from-zygote-to-blastocyst/

Good luck