you can coat some additional plates with gelatin, plate your cell suspension out and let it sit for 10-20min. The MEFs will adhere quickly and the hESCs clumps will stay in the solution. Take off the hESC containing supernatant and plate them on your new feeders. Just be careful not to sheer-stress the colonies to much when pipetting them.

Tobi

Hi Tobias, thanks for your answer, I'll try it!!!

do you use any other particular method for the passaging?

Hi Vicente,

What Tobias has suggested is the easiest way to get rid of MEFs from ESC culture and it works quite well.  If you still not happy with the purity of your ESC cutlure, you may opt for a feeder free culture for one or two passages. but then this gets a bit expensive with having to use LIF or other similar factors.

Jose

HII Vicente,

I would like to tell you what exactly i am doing for the same.. i prefer CTK treatment for  passaging ES cells on MEF feeder layer which are plated on gelatin coated dish.  

Hello 

I use these protocol .

1. Aspirate media from 6-well plate.

2. Add 1 ml of collagenase IV per well and incubate at 37oC for 3-5 minutes until

the edges of the colonies start to curl. Accutase can also be used (only 1 min

incubation).

Longer incubation is not advisable. However, a badly differentiated

human ES cell culture can be incubated with collagenase IV for up to 30

minutes. Undifferentiated human ES cell colonies will detach, leaving

behind feeders and any spontaneously differentiated cells on the original

flask.

3. Gently roll the colonies off with glass beads.

4. Add 5 ml of DMEM and gently triturate. Transfer cell suspension to a 10 ml

centrifuge tube.

5. Spin at 800rpm for 3 min at RT.

6. Aspirate supernatant, leaving human ES cell pellet intact.

7. Remove medium from the fresh well of MEF feeders.

8. Gently flick human ES cell tube to disperse pellet.

9. Gently resuspend human ES cell pellet in an appropriate volume of human ES

medium (e.g. 3 ml for a 1:3 split into three wells of a 6-well plate (at 2 ml per

well). Distribute between wells of feeders.

Add 2 ml human ES media per well for the first day of passaging.

Gradually increase the volume of human ES media to ensure sufficient

medium to support the growth of cells.

10. Carefully place the 6-well plate into CO2 incubator. After placing the plate down,

swirl the plate carefully to ensure an even distribution of cells across the flask.

11. Human ES cells are to be fed with fresh medium daily, if possible. Cells are

grown until they reach 80-90% confluency before they are ready to be split again.

Cells from 1 well on a 6-well plate typically yield 1.5- 2 million cells.

not :  for preparation Collagenase IV solution (1mg/ml)

1. Weigh out 50 mg of collagenase type IV (Invitrogen, 7104019)

2. Solubilize lyophilized powder in 50 ml DMEM/F12

3. Filter-sterilize with a 0.2 micron cellulose acetate filter.

Store at 4oC. Use within 2 weeks.

Good luck