Low adherence in thawed HUVEC - HUVEC won't attach after thawing (Jun/09/2010 )

Hi everyone,

My HUVEC aliquot was stored at -80 degrees for about a month in cryopreservation medium containing DMSO. When I thawed the HUVEC this week, I thawed them at 37 degrees and added them to prewarmed media. I gently pipetted to distribute the cells and then spun them down to wash off the cryopreservation medium. I removed the supernatant and resuspended the cells in culture medium and seeded them into 12 T25cm2 flasks.

By now I thought that most of the cells would have fully attached to the surface of the flask. However the majority of cells seem to be just floating around. They do not appear dead but just clumped together. The flasks I use are from Corning and are tissue-culture treated for attachment.

Given that 1. HUVEC are finicky, 2. I used a non-enzymatic trypsin replacement before freezing and 3. The number of cells in the aliquot was low to begin with,
why won't my cells attach?

I'd be grateful if someone could give some possible reasons. Thanks

Candace

candace on Jun 9 2010, 09:19 AM said:

In our experimental conditions, we immediately transfer cells after thawing into a 0.2% gelatin coated flask containing warm medium. Once the cells adhere firmly on the coated surface (usually within 30-60 min), we replace the medium with the fresh medium.

You can minimize cell injury (after thawing) by avoiding mixing and centrifugation steps.

good luck!

Thapa

Thapa on Jun 10 2010, 06:44 AM said:

Hi Thapa,

I have another aliquot of these cells so I will try your suggestion.

Thanks!
Candace

Hi Candace and Thapa.
I'm starting thawing HUVEC cells that were frozen two years ago.
I'm experimented several troubles in the process..
I'm using M199 medium 20X BFS + 1% ciprofloxacin
This is the protocol that i'm following:
I take the vial of frozen cells and transfer them fastly to a thermal bath at 37ºC.
When became thaw, I add 200 uL of M199 pre-warmed and transfer the content of the vial to the culture flask, pre-filled with 10 mL of M199.

Everytime that i did it i had a lot of dead cells, and today I have pre-coated two flask with gellatin, one with 1% and the other with 0,2%.
tomorrow I will check them and change the medium.....

I want to know if there's any mistake that i'm doing... could you please help me???
regards!!

Finally i found the best method for thawing Huvecs.

Previously, coat the culture flask with gelatin 0,2% for 30 minutes, then eliminate the excess.
Then, add 10 mL of warm M199 20X 1% ciprofloxacin.
I take the vial of cells from liquid nitrogen and transport to my lab in my hands (wearing gloves, of course)
Then I use my hands to "squeeze" the vial and warm it with my body heat. (dessinfect the vial with great amount of alcohol 70%)
When the ice is thawed and free from the walls, throw the ice in the flask, literally throw it.
Then incubate the cells at 37ºC for 24 hours, and change the media without washing them with PBS.
Wash them 3 times with PBS 3 days after that, and change the media.
It's been a week since the first time I followed this protocol and i have 75% of confluence.

Regards!