First of all I'm informing you about a "new" way of culturing hESCs FEEDER-/CONDITIONED MEDIA FREE using a newly released Matrigel (Matrigel hESC-qualified Matrix) and media (mTeSR1). The mTeSR1 is fully defined and there is no requirement for adding anything (besides antibiotics if you want) NOT EVEN FGF. I have attached the protocols for both the Matrigel and the mTeSR1. Both products are available immediately at the core. We and Ji-Eun have already tested it and we are quite happy with it.
If you still prefer using MEFs for growing your cells there are aliquots in the freezer. Due to the contamination we have decided not to use the ones we make but instead we bought them from Global Stem. There are neo-resistant cells and also normal ones for everyday use. These cells are irradiated, of course, and tested for mycoplasma. One vial seeds two and a half 10 cm dishes. The NON-NEO MEFs are in the -170C freezer in tower 6, 4th box from the bottom. Regarding the NEO-MEFS -- please ask for them.
Another lab is fighting a serious mycoplasma contamination right now, most likely because of culturing cell lines which came from outside, without testing them. As a precaution NO cells from outside will be allowed in the clean area (these are the two rooms you are used to working in). If you have to work with cells from outside they need to be cultured in "The Dungeon" (this is the room Zoe and I used to work in) till they are tested for mycoplasma and any other contamination. The cells are allowed to be moved to the clean area ONLY with the ok from either Karl, Thomas or Zoe. It will take less than a few days to test them so that you can do your experiments asap.
In case you have some non-hESCs you want to culture in the Core, they are only allowed in the Dungeon and NEVER in the clean area!!!!!!!!!!!!!!!!!!! FYI the Dungeon is a fully set up cell culture room and therefore the same rules as for the clean area apply to it (NO ENTRY w/o GLOVES, BOOTIES, LAB COAT, ....). You find everything you need in front of the dungeon and please don't wear the same lab coat, gloves and booties you are wearing in the clean area!!!! Also DON'T bring any reagents (media, trypsin, ....) from the Dungeon to the clean area and vice versa. You need to have separate reagents for these two areas. The dungeon has a fridge and therefore you can store your stuff in there without walking back and forth to the cold room. We want to keep these two areas ABSOLUTELY separated so that we don't transfer any contamination from one room to the other.
Since the contamination has been going on for so long we need you to let us know when you are planning on thawing cells because we want to make sure that no contaminated cells were frozen down. Some of the contamination we had is really hard to see and spreads rather slowly. For now IT IS NOT ALLOWED to thaw cells of any kind in the clean area only in the dungeon. please let us know so that we can take a look at them and test them.
Karl Willert, Ph.D.
Tel: 858-822-3235