MEFs not attaching - (Nov/19/2008 )
Hi fellow labbers,
So, I have just moved to this new institute and I am the first to setup (mES and hESC )stem cell works in this lab.
Having gone through the hectic and time consuming process of gathering the right reagents and cell lines etc. I am very excited to do the first experiments concerning hESC differentiation.
Despite having 2-3 years of experience with cell culture, my first few weeks under the hood have been anything but a nightmare 
-> infections and contaminations (never even seen it before I moved to this new place)
and now, the simple MEF cells would not grow.
The problem:
Upon thawing from liquid nitrogen, the inactivated MEFs didnt stick to the Falcon 6-well plate at all, they were all still floating the next morning.
The active MEFS, upon thawing, very few stick to the surface, the rest were still floating the next morning.
It is somewhat embarrasing and much more discouraging, even after almost 2 months, I cant get the cell culture started well
Can people speculate as to what might be the problem?
Many thanks 
Sam
PS: This being my first post, HI!
Upon thawing from liquid nitrogen, the inactivated MEFs didnt stick to the Falcon 6-well plate at all, they were all still floating the next morning.
The active MEFS, upon thawing, very few stick to the surface, the rest were still floating the next morning.
What type of cryopreservation media are you using? Do you know what % of your cells are alive after you thaw them? Do you give the cells a wash-rinse-spindown before plating them? Sometimes, if the cryoprotectant is not washed away, the residual might interfere with plating.
Do you put gelatin on the bottom of the 6-well plates the day before you plate your MEF's or do you just plate them? I add gelatin the day before and then remove the excess right before I plate the inactivated cells; I've never heard not using gelatin, so I don't know if you don't and that might be a problem.
How much antibiotic/antifungal do you add to your media? I'm sure you know from previous cell culture that too much is bad and can negatively affect your cells, and with inactivated MEF's they are already sort of impaired.
When you plate your active MEF's, do you plate directly into the dish you will use to isolate your hESC's into or do you plate into a different culture flask, then inactivate and then re-plate? When I thaw my feeder cells I grow them for about 3 days then inactivate 5 hrs with mito-C then plate on a dish that has had gelatin in it and they plate fine.
even 15 minutes of gelatin plating is enough (keep in incubator) for MEFs to attach well. Without gelatin it doesn't work. As you are setting up newly, For more
MEFs & Gelatin,
Human Embryonic Stem Cell Methods, and
Mouse ES Cell Methods.
So, MEFs are now growing but very slowly after having gone through massive cell death upon thawing.
It feels to me that the whole tissue culture needs a full scale cleaning etc... a new issue comes up everyday:
The below photo are bacteria, right? Grrrr, contamination. This is in a dish on which I thawed a vial of primary keratinocytes, given to me by some other lab.
This is driving me crazy, I am having hard time even to speculate that it might be my cell culture skills that is causing the infection etc.... :S I still dont think its me.
As a comparison, I thawed a vial of cells that I froze a while ago, It thawed perfectly well.
So, I dont know it is so depressing.
Also, I am being made extra paranoid, someone asked me what are these dots in my human foreskin fibroblast culture? (see below)
Any guesses?
Many thanks.
Sam
Upon thawing from liquid nitrogen, the inactivated MEFs didnt stick to the Falcon 6-well plate at all, they were all still floating the next morning.
The active MEFS, upon thawing, very few stick to the surface, the rest were still floating the next morning.
What type of cryopreservation media are you using? Do you know what % of your cells are alive after you thaw them? Do you give the cells a wash-rinse-spindown before plating them? Sometimes, if the cryoprotectant is not washed away, the residual might interfere with plating.
Do you put gelatin on the bottom of the 6-well plates the day before you plate your MEF's or do you just plate them? I add gelatin the day before and then remove the excess right before I plate the inactivated cells; I've never heard not using gelatin, so I don't know if you don't and that might be a problem.
How much antibiotic/antifungal do you add to your media? I'm sure you know from previous cell culture that too much is bad and can negatively affect your cells, and with inactivated MEF's they are already sort of impaired.
When you plate your active MEF's, do you plate directly into the dish you will use to isolate your hESC's into or do you plate into a different culture flask, then inactivate and then re-plate? When I thaw my feeder cells I grow them for about 3 days then inactivate 5 hrs with mito-C then plate on a dish that has had gelatin in it and they plate fine.
Cryopreservation media: since I am starting new in this lab, I have to obtain various cell lines for as many different ppl around the institute.
So, I havent froze them, just obtain a vial from the core facility.
This is how I thaw:
1) when the vial contents are slushy, add warmed media, aspirate and collect. repeat washing untill all the via contents are collected in 20ml media.
2) spin 1400rpm
3)aspirate supernatnat, replace with pbs
4) spin and aspirate after
5)resuspend in appropriate media for plating
Antibiotic: pen/strep 1%
I thawed active MEFs in a 6 well plate, need to expand them first to create my own stock.
RE: Gelatin:I did gelatinize another 6-well plate i.e. O/N with geltin in incubator and thawed another vial of inactive MEFs, 90% died :S and I just found out today that the core had screwed up the freezing process for that particular batch :S
But another problem: I also thawed a vial of WT mESCs on gelatin, majority died