The freezing protocol is usually mentioned when you search your cell line on the database of where you bought your cells. For instance, if your cells are from Cellonex or ATCC, then you can search for the cell line on their database and it will give you all the information on it. This will include proper storage, maintenance etc

Hello,

I am working on Fibroblasts, guess, you can use a similar freezing protocol with HUVECs

Since you are using Endothelial Cell Medium (ECM), you need to prepare the freezing medium that contains:

1. 70% (700 µL of Endothelial Cell Medium)

2. 20% (200 µL of FBS)

3. 10% (100 µL of DMSO)

Note:

The volumes listed (700 µL media + 200 µL FBS + 100 µL DMSO, totaling 1 mL) are sufficient for preparing one cryovial. If you need to freeze multiple vials, adjust the total volume accordingly by scaling up the components proportionally.

Let's say you want to freeze 5 cryovials, so:

Media: 700 µL × 5 = 3.5 mL

FBS: 200 µL × 5 = 1.0 mL

DMSO: 100 µL × 5 = 0.5 mL

Total = 5 mL for 5 vials

Freezing Protocol:

1. Collect cells into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

2. Carefully remove the supernatant without disturbing the cell pellet.

3. Resuspend the cell pellet in cold freezing medium (previously prepared).

4. Aliquot 1 mL of the cell suspension into each labeled cryovial (include cell type, passage number, and date on the label).

5. Place the vials in a Mr Frost freezing container and transfer to a –80°C freezer for overnight freezing to ensure a gradual cooling rate.

6. For short-term experiments, cells can be kept at –80°C for up to 3 months. For long-term storage, transfer the vials to liquid nitrogen after 24 hours.

I hope this information is helpful. All the best!

Hello Inês Soares Marques

You may use 90% FBS + 10% DMSO (tissue culture grade, sterile) solution as a freezing medium for HUVECs because HUVECs are sensitive cells, and using a freezing medium with a higher concentration of FBS can help improve their survival and recovery after cryopreservation.

You may use the following protocol.

1. First and foremost, precool Mr. Frosty freezing container.

2. Prepare the freezing media (90% FBS+ 10% DMSO) as per the number of vials (1ml per vial) to be frozen, and place the freezing media on ice.

3. Trypsinize the cells at 80% confluency which you may have already seeded in flasks.

4. After neutralizing trypsin with complete media, transfer the cells carefully in a 15 ml centrifuge tube.

5. Take the cell count using hemocytometer. Generally, 0.5 – 1 x 10^6 cells are frozen per freezing vial.

6. Pellet the cells at 150-200g for 5-7 mins, aspirate the media and place the cell pellet on ice.

7. Add cold freezing media which you have already prepared in step 2 to the cell pellet and pipette up and down.

8. Aliquot the cell suspension into freezing cryovials (1ml containing 0.5-1 x 10^6 cells per cryovial ) and transfer the vials to Mr. Frosty freezing container which has already been precooled in step 1.

9. Transfer Mr. Frosty container to -80 degree C for an overnight stay.

10. The next day, transfer the freezing cryovials from Mr. Frosty container to liquid nitrogen.

Good Luck!