Hi
I am trying . to grow and expand mouse embryonic fibroblast (MEF) from ATCC (SCRC-1008). I have worked with human cell lines, but this was my first time using the mouse cell line. I followed the protocol and discussed with a colleague who has a lot of experience working with MEF and other mouse cell line. The purpose is to use these cells for the feeder layer for mouse embryonic stem cells. But the cells didn't attach to the T75 flask (viability was around 80%). I asked my colleague, and he told me that he sometimes has a problem with flasks, so use the gelatin-coated one. I transferred the cells the next day to the gelatin-coated flask, but they didn't adhere, and these are round. I suspect that when I thawed the cells were at the dying stage. The media was ok and didn't change color, and cells are still round. I would like to know if there is a reason for that or any other precautions /tips that I should follow next time?
Thank you.
Hi, I work with fibroblasts as well, so as you mention, many things could be happening here.
First, indeed it could be that from the very beginning, the cells you used didn't thaw well and were somehow affected, be careful in this step. Second, how much serum do you use in your maintenance medium? Or have you made sure the media you use has the correct pH? You know this is critical for certain cells, as they are quite sensitive! And last, if you have doubts about your culture flasks, many of them are coated with collagen, fibronectin for fastidious cells, you can also try a layer of poly lysine!
Lady López Thank you for the answer. I used DMEM(1X) + GLUTAMAX, ref no. 31966-021 (https://www.thermofisher.com/order/catalog/product/31966021) media. The pH for this media is from 6.8 to 7.2 and, I also added Pen/Strep and non-essential amino acids as well. For the protocol, I was guided by a colleague who has a lot of experience in MEF and other mouse cell lines, as mentioned earlier. Since I was doing it for the first time I asked a colleague help to watch over all the steps. So I am confident that I didn't make a mistake in thawing and later steps. I can try the polylysine coating next time. Can you share with me the MEF cell kine you use and your SOPs?