Do any of you know why this is happening with the Matrigel?
I am an undergrad working with Matrigel (Cat. 354234). Currently, I have been having issues, and I am having trouble understanding why. I have tried to think about what I am doing wrong. I have tried different things but it hasn't helped. During the fall semester, I was using aliquots another member of the lab made. The aliquots were perfectly fine and while there were issues caused by me, I stopped doing those.
Then in January I had to order another vial, thaw, and aliquot it. This went perfectly fine and worked. I used up the aliquots and repeated what I did. This one did not work out great. The image shows how the droplet looks and when it looks like this the cells do not grow. If they do, they do not grow to the size they were growing before.
I thought the aliquot partially polymerized or thawed wrong and used another one. It went okay, and each aliquot was hit or miss.
I repeated this process with another vial, it went worse. I thought maybe I was thawing the vial wrong, so I repeated the thawing process with Organoid Matrigel (basically the same type but tested for qualities involved in making organoids and more $$$). This one thawed perfectly fine and didn't have the background issue.
By now I am ordering my 4th bottle of Matrigel and don't trust my own judgement. I asked another graduate student whether she thought the Matrigel was too viscous even for a gel, she agreed and just recommended pipetting even slower. The paint image is to show how it looked.
It's been a year and the member of the lab who worked with organoids has returned as a grad student after finishing her rotations. I would thaw the matrigel vials in ice, in the cold room, overnight, placing it there around or past 5 pm. She had me do the same but in the lab's fridge. That did not help. When aliquoting I tried to show how it just seemed too viscous even for Matrigel. It also had a clump in it. She said it is a gel and to just break the clump. When trying to show the background issue she said she didn't think it would affect the cell growth. Its been over a week since then and the organoids barely grew and multiple droplets broke including the ones she made.
Now why do I have a fixation on viscosity. I believe that the matrigel has been too viscous making it incredibly easy to introduce incredibly tiny air bubbles into the gel and that is the background issue. Part of the reason why I think this is because I was horrible in my technique during the fall and had the matrigel just thaw at room temp while breaking it (Ik it was horrid) but I didn't see this issue instead only seeing very reasonable partial polymerization in the droplets.
The only other major change between when it was working and not working is that I was asked to make organoids for a different type of experiment which led me to keep the 1:1 mix of matrigel to cells/media on ice while I prep another vial as there are two different cells. Before that, the matrigel would thaw on ice, be mixed with media and then immediately used.
Thank you for any help!