I froze some HUVECs in 10% DMSO and 90% FBS over a year ago. When defrosting I placed in a water bath until mostly thawed, then added warmed EGM-2 to cryotube before transferring to a 15 mL Falcon with several mL of warmed medium. I then spun down and resuspended and placed in a coated T-25 (which I could culture fresh primary HUVEC on) but no cells attached. How do other people freeze down and thaw HUVEC?
This is the (detailed) protocol that we use in our lab on a daily basis. We use Mr. Frosty freezing containers (Thermo Scientific) to gently freeze cells in the -80°C freezers. Frozen cells are then transferred to liquid nitrogen for storage.
Freezing mix
• Heat-inactivated FCS (we use FCS from Hyclone) containing 10% DMSO (tissue culture grade, sterile).
• Keep sterile at 4°C.
Freezing
• Pre-cool Mr. Frosty freezing container at 4°C.
• Pre-cool freezing media on ice. Will need ~1 ml per vial.
• Equilibrate 50 ml tube with HUVEC growth media (HGM: in our case EGM-2 from Lonza supplemented with FCS to 18% final concentration FCS) to RT.
• Aspirate media, rinse 1x RT PBS, aspirate and then trypsinize cells. Make sure cells have detached.
• Neutralize trypsin with HGM. Resuspend cells carefully but completely.
• Transfer cell suspension to 15 ml Falcon tubes.
Note:We generally freeze vials containing ~5*10^5 cells, so I count the cells before pelleting (in order to roughly determine the amount of freezing mix needed)
• Pellet cells in centrifuge (5-7’ 150 g at room temperature).
• Aspirate media and place pellet on ice.
• Add cold freezing media to pellet. Resuspend by pipetting up and down.
• Aliquot cell suspension into cryovials and transfer vials to Mr. Frosty.
• Place Mr. Frosty in -80°C freezer and store at least overnight.
• Transfer frozen cells to liquid N2 or cryobiology facility.
Thawing
• Prepare a culture flask coated with gelatin or collagen. I start a vial containing 500K cells into a T75 flask (~75 cm2 surface).
• Prepare 1 tube with 12 ml HGM and put at 37°C to warm.
• Thaw 1 vial of HUVECs from liquid N2 in water bath at 37°C.
• When the cell suspension is nearly but not completely thawed, take it out of the water bath together with the 12 ml HGM and spray vial and tube with 70% ethanol.
• Put into tissue culture hood and wipe off excess ethanol.
• Transfer contents of vial to the HGM.
• Transfer to the gelatin/collagen-coated flask.
• Put flasks in 37°C, 5% CO2 incubator. At end of day change the media to remove DMSO from freezing mix and the cells that failed to attached (not many).
There are 2 aspects..freezing and subsequent thawing.
To avoid osmotic shock it is better to have a volume of cells then slowly add 20% DMSO with gentle mixing rather than just drop the dmso/fbs directly at 10% on to the cells. The cells should then be cooled at about 1 degree per minute in an alcohol based cell freezer ( or an inflated polystyrene box with the samples loosely wrapped in tissue is ok. Then transferred to liquid nitrogen.
Thaw by shaking gently in a 37 water bath until the ice is almost gone ( this is important as the salt concentration is very high in the first part of the thawing process unless mixed) Gentle spin to remove the dmso and resuspend and hopefully it will work
good luck
paul
I am using HUVECs for several years now. I am using the following protocol for freezing of HUVECs: Cell pellet in cryo vials suspended in ice cold freezing medium (10% cell culture grade DMSO, 20% serum, 70% M199). Then cryo vial put in -20 0C for two hours which is followed by keeping in -80 0C for overnight. Next day the cryo vials put into liquid nitrogen for permanent storage.
I agree with Paul and Tapan. Also, check your water bath temperature with a calibrated thermometer that is immersed to the calibration line. A 1C difference ( induces apoptosis) is enough to kill primary cells while your lab mates have success with immortalized lines. Trypan blue staining is a quick measure of dead cells but will under estimate plating efficiency. After transfer to the T75, healthy cells will look like "pearls".
You should avoid spinning this cells. It is much better if cells are transfered from cryo tube to dish with fresh medium. They should stay in the incubator for 3-4 hours (cells would adhere), then medium should be changed to remove DMSO. In our hands it works.
This is the (detailed) protocol that we use in our lab on a daily basis. We use Mr. Frosty freezing containers (Thermo Scientific) to gently freeze cells in the -80°C freezers. Frozen cells are then transferred to liquid nitrogen for storage.
Freezing mix
• Heat-inactivated FCS (we use FCS from Hyclone) containing 10% DMSO (tissue culture grade, sterile).
• Keep sterile at 4°C.
Freezing
• Pre-cool Mr. Frosty freezing container at 4°C.
• Pre-cool freezing media on ice. Will need ~1 ml per vial.
• Equilibrate 50 ml tube with HUVEC growth media (HGM: in our case EGM-2 from Lonza supplemented with FCS to 18% final concentration FCS) to RT.
• Aspirate media, rinse 1x RT PBS, aspirate and then trypsinize cells. Make sure cells have detached.
• Neutralize trypsin with HGM. Resuspend cells carefully but completely.
• Transfer cell suspension to 15 ml Falcon tubes.
Note:We generally freeze vials containing ~5*10^5 cells, so I count the cells before pelleting (in order to roughly determine the amount of freezing mix needed)
• Pellet cells in centrifuge (5-7’ 150 g at room temperature).
• Aspirate media and place pellet on ice.
• Add cold freezing media to pellet. Resuspend by pipetting up and down.
• Aliquot cell suspension into cryovials and transfer vials to Mr. Frosty.
• Place Mr. Frosty in -80°C freezer and store at least overnight.
• Transfer frozen cells to liquid N2 or cryobiology facility.
Thawing
• Prepare a culture flask coated with gelatin or collagen. I start a vial containing 500K cells into a T75 flask (~75 cm2 surface).
• Prepare 1 tube with 12 ml HGM and put at 37°C to warm.
• Thaw 1 vial of HUVECs from liquid N2 in water bath at 37°C.
• When the cell suspension is nearly but not completely thawed, take it out of the water bath together with the 12 ml HGM and spray vial and tube with 70% ethanol.
• Put into tissue culture hood and wipe off excess ethanol.
• Transfer contents of vial to the HGM.
• Transfer to the gelatin/collagen-coated flask.
• Put flasks in 37°C, 5% CO2 incubator. At end of day change the media to remove DMSO from freezing mix and the cells that failed to attached (not many).
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