Please show us the cell morphology? Culture embryonic bodies? embryoid bodies?

I'm not sure how you came up with your 80:1 ratio, but you need to find out the protein concentration of your particular lot of matrigel and adjust accordingly so that you always are using the appropriate and uniform amount of matrigel in each plate.

For comparison, I routinely use 1mg per 100mm plate for hES and hiPS cultures.

Are you having trouble with the morphology of hESc colonies or of embryoid bodies?

If the latter, you should not be attempting to culture them on matrigel at all, as they should not be attached to the plate.

Here are the images of hESCs that abovementioned. I don't know whether these kind of morphology are good or not, because previously i got better embryoid bodies than this sample.

According to the Matrigel coating composition (80:1), i follow the protocol from the company of the matrigel product that i use (here is the link www.stemcell.so/images/upload/pdf/5189c7ede1618.pdf )

First, could we please address the terminology you are using? Those are photos of ES cell colonies, not embryoid bodies. Are you trying to make embryoid bodies from these colonies or are you calling these embryoid bodies? Please clarify.

The background area suggests that your matrigel is not in good shape--either plated too long or mishandled before plating. Are you keeping the matrigel cold while you are plating it? How old was the matrigel coating when you added the stem cells to the plate? I would speculate that your colony morphology will improve after you get them onto a better matrigel coating.

Generally, i want the ESCs formation like this attached picture (with good embryoid bodies), but last time my sample during 1 week culture ends with those pictures that i had shown in my previous answer. I guess the problem comes from the matrigel coating, but i'm not not really sure. That's why i'm asking others opinion. Fyi, I'm always keeping the matrigel solution in cold condition after it is fully thawed, then right after i make mixture (Fresh DMEM F12 w/o FBS with matrigel solution in ratio abovementioned), i directly coat onto the ESCs corning dish with that mixture and incubate following the protocol (1.5h in incubator 37oC 5% CO2, then 1h in RT, then i seal with parafilm and storage in refrigerator 4oC), and also the matrigel-coated plate that i used is less than 1 week storage period.

Hi,

1. Embryoid bodies are 3D aggregates of ESC or iPSC cells cultured in suspension on the plastic without the Matrigel with the medium without bFGF. Embryoid bodies are formed to improve differentiation potential of ESC or iPSC cells.

2. Morphology of ESC colonies present on all your photography looks good.

On the first two photography, you present smaller colonies of ESC (2-3 days after passage), while at the last photo you show the oldest ESC colony (5 or more days after passage or colonies which was passage as a large fragments of earlier ESC colonies). The ESC cells colonies during growth change their shape. After 5 days or longer, the cells in colonies are more close together and the shape of colonies are more round and "symmetrical" especially if they are a cultured in the low density.

3. The shape of ESC colonies depended on the medium composition, for example, the ESC growth on the MEFs creates more compact colonies.

4. On the two first photography, the quality of Matrigel is poor, "black dots" around the colonies are present. At the last photo, are visible some ESC colonies which died.

Your ESCs colonies at photo 1 and 2 will look like in the photo number 3 after a few days.

Good luck!

"Generally, i want the ESCs formation like this attached picture (with good embryoid bodies),..."

There are no embryoid bodies in that photo. Embryoid bodies are formed in suspension, not in attachment culture. That is a photo of a stem cell colony.

Once you have the matrigel suspension in the plate, you can immediately store the sealed plate in the refrigerator. After removing the plate from the fridge, it can be used after 30 minutes in the 37C incubator.

You might want to try a different lot of matrigel. The one you are using might have been mis-handled before you recieved it.