Hi, try to pick the undifferentiated colonies under the microsope, tripsiinize them and plate again.

sorry, no simple method....

Be brave and come the WE to watch your clones and pass them if necessary. Remember that good culture conditions helps a lot to maintain a both pluripotency and normal caryotype in your ES cells.

Good luck!

You can plate them for 20 min on non-gelatinized dishes, which will remove mot differentiated cells, which will adhere much faster than ES-cells.

Another option of course will be, to grow your cells in 2i-media. After about one week all differentiated cells will have disappeared.

Hope this helps.

Regards

Stefan

First you can not treat the cells like any old cancer cell line. They must be split every two days and fed every day. There is no over the weekend in ESC culture. They are like your children. Ae you growing them on feeders? What media are you using? How many passages are they? The all affect growth rates. If the cells are growing fast and are hi passage they likely have transformed and are not good anymore.

Simple Protocol To Remove Differentiated Cells

To remove the differentiated cells you can try a light trypsin treatment. Treat with trypsin for 1-2 minutes and tap the plate by holding the edges and taping it flat on to a hard surface, carefully not to vigorously or you will have a mess. Leave the lid on incase. The ESC will float up as colonies and single cells. Then remove the media and add to a new plate and continue to let the trypsin digest the colonies back into single cells for 3-4 minutes. Stop the trypsin reaction with media , pipette the break up the colonies and spin the cells to acheive a pellet. then plate on to another tissue culture plate but with no feeders or gelatin. Place the plate into the incubator for 20 minutes. Then remove from the incubator and remove the media and plate the media on to a fresh plate with feeders. This media will have your ESC. Being gentile when moving the media as the differentiated cells will have just begun to stick to the plastic but he ESC will not.

Why protocol are you using? The chapters on ESC culture from manipulating the mouse embryo is an excellent protocol.I can up load a copy of the book chapter if you need it.

Best

Brian

There is no easy way out. You will have to distinguish between the differentiated and undfferentiated cells under the microscope and pick up the undifferentiated cells to be plated in fresh plate. But this is not a very good situation as the differentiated cells already release factors which post a higher risk to your undifferentiated cells to start differentiating in culture. Hence, if possible do not leave your dishes unattended over the weekend or take care to passage them on friday so that they do not differentiate.

Different time of tripsinization. Trypsin 30 seconds with warm trypin at low contentation i.e. 0,25% max and help detachment od ESC with some soft pipetting

Cleaning the cultures is really very difficult. By picking up undifferentiated colonies you may be selecting for cells that are already abnormal. If you can afford it, the best is to start anew. The most critical step is to assure that you don't have any clumps - cells should be in a single cell suspension. Another critical parameter is the quality of the FBS. Make sure you are using only ES-qualified FBS. You can find a very useful protocol on the site of Stem Cell Technologies. Good luck,

Nelli

Thankyou to all respondents! I had wondered whether differentiated cells might be able to be removed via differential adhesion - I will try this in future. I am currently culturing on MEFs, with ES-qualified FCS (15% in the media) from Life Technologies. I do appreciate that the cells cannot be abandoned over the weekend - they are fed every day including weekends and typically split Mon/Wed/Fri. Really I just need to gain more experience with these lines and prepare a few extra MEF plates on Friday so that the cells can be split on the weekend if needed (though I'm keen to get the ratios right so this can be avoided).

Have you considered growing the cells on E-cad-Fc matrix? Our lab has developed this chimaric protein for the selection and lineage specific differentiation of mES/iPS. Growing ES cells on E-cad-Fc with the addition of LIF can maintain the pluripotent state for 20 or more passages. You can check out this paper: http://www.sciencedirect.com/science/article/pii/S0142961210014845

Best of luck.

Well you don't have to: differentiated cells tend to die out in 1 ~ passages.

You also can use EDTA 0,5 M to split the cells; with human ESC this method is really nice because the EDTA only picks up ESC colonies while the diff cells remain attached. In case you want to try it: you have to remove the media, make a quick wash with 0.5M EDTA and then put again warm 0,5M EDTA during approx. 5 min, check the cells under microscope, then remove the EDTA and add media to pick up the ESC in a 15ml falcon tube. You could need dis-aggregate the cells by pipetting more than it is necessary with human ESC (where you have to avoid a single cell suspension). I hope it is useful for you.

Hi Rae - ditch some of the clones - you'll spend all your time growing cells, not using them! If you do use 3i (or even 2i) give them a couple of passages in LIF/serum before use, residual i's will affect differentiation!

Cheers!

First, passing with good single cells suspension (no clumps) is key. For one well of 6well-plate, 50-70% confluence culture, I wash with 2ml 0.05% Trypsin/EDTA then treat with 2ml 0.25% Trypsin at 37 degree for 4 mins. Pipet gently but throughly. P2000 is your good friend if you have one. Examine under microscope. Add 4ml mES media (with 15% serum). I usually pass through cell strainer here. Count cells, I usually plate 0.5-2x10^5 per well in 2ml mES media with 2i ( 1uM PD184352 and 3uM CHIR99021, Ref as Susanne mentioned). For differentiation experiments, I will change to half conc. of 2i mES media the next day and w/o 2i the day before differentiation. Good luck.

I agree with Eva Garcia. If you have a few good colonies, you should manually pick them up using a dissecting scope and a 20uL micropipette. Alternatively, if you have a very mixed culture, (good & bad colonies) you should try EDTA. True hPSCs have a lower adhesion coefficient than progenitor cells, and somatic cells have pretty high adhesion coeff. so the 'good' hPSC colonies should preferentially lift up after treatment, leaving the diff. cells behind. Hope this helps!!!

I guess I am 5 years late to answer, but the simple way these days is to use ReLeSR from stem cell technologies. Just add it to your cells, incubate for up to 10min, then the stem cell colonies will detach from the differentiated cells...

Ahmad, I think you left out a step. The ReLeSR is added to the cells, then is removed within one minute, and only then the plates are incubated.

The kind of matrix that you used is equally important as having a good cell culture medium. The cell culture matrix that you use will make the world of difference. There are cell culture matrices available on the market that allow you to culture human and animal pluripotent cells as single cells without the need to add apoptosis inhibitors and ROCKi (human) or LIF (mouse). Biolaminin 521 is a natural ECM protein (laminin protein) that is expressed around the pluripotent cells in the developing embryo. The natural support from the matrix makes the cells feel right at home and they migrate, survive and rapidly proliferate on this matrix. Get away from the tedious colony passaging protocol. The Biolaminin 521 culture method is as easy as culturing cancer cell lines. No more manual selection. No more risk of spontaneous differentiation. Unlike colony cultures, the cells grow as a nice homogenous monolayer where the cells have equal access to medium and matrix. Anyone can do it. You will be up and running in no time and in the long run, you will save both time and money. http://www.biolamina.com/laminin-ln-521-stem-cell-matrix