Dear colleagues,
I wish to find out, if anyone has an idea on the optimal number of freez-thaw cycles for an immortalised HUVEC cell line.
I first thawed these cells from liquid nitrogen, cultured the cells for 6 passages in complete endothelial medium (Endopan 3). During this period they grew very well and looked normal.
Then I harvested and stored the cells at -80 degrees C in Mr. Frosty. After one month, I thawed them a second time and now they grow so slowly and are their morphology is not optimal. What could be the problem?
I thank you in advance for your ideas and contirbutions.
My intuition tells me that your freezing process may have killed the cells. Here are some brief words of wisdom to help you harvest and re-freeze cells:
1. After harvesting and counting, leave the cells suspended in their growth medium at twice the intended density (for aliquoting into cryovials) on ice for at least 20-30 min (this slows down their metabolic rate)
2. Slowly (ie. dropwise) add a 2X freezing medium to your cells (usually the freezing medium is 20 vol.% DMSO in your growth medium) - invert the tube to mix. It should take you several minutes to add the DMSO solution.
3. After diluting your cells to 1X in your freezing medium, start aliquoting your cells into cryovials, and once complete, transfer the cryovials to the Mr Frosty and store at -80C
4. DO NOT leave your cells in the -80C for more than a couple days - transfer them to liquid nitrogen as soon as possible. If you are storing your cells for more than a week in -80C, your cells have likely died.
5. Upon thawing, only leave your vial of cells in the water bath long enough to thaw most of the solution. A few small ice crystals should be leftover when you move the vial to your sterile culture hood and dilute in medium
6. Dilute the cells from the cryovial immediately in growth medium, and plate immediately with the DMSO still in the medium
7. The very next day after thaw, change the medium with fresh growth medium
These steps should drastically improve your viability after re-freezing and re-thawing.
Everything David has said is valid. I have frozen endothelial cells in 10% DMSO and 90% FBS at 1x10^6 cells per ml per cryovial. Endothelial cells don't tend to like prolonged passage, so their morphology is likely to change over time. When using HUVECs, primary cells is the best option and ideally primary HUVECs shouldn't be used past P4-5. Some attachment factors such as fibronectin and collagen coating on the culture flasks may activate the ECs and prolong their longevity, but use these with caution as they may influence specific pathways.
I agree with pretty much everything David and Xi write. But I would add that if using primary cells, magnitude of cell responses - in particular endothelial cell growth rates - are very much batch specific. Indeed I have seen distinct growth and survival differences from commercial endothelial cells from the same batch number but different cryovials delivered directly from the supplier and prepared immediately on arrival. As a result, I have never used immortalized HUVECS but can see the attraction of using them! You should note tho' that if you use immortalized HUVECs you may well be asked to confirm any findings in primary ECs anyway.
In my previous lab in Vascular Biology, we were taught that in the liquid nitrogen tank endothelial cells do better in the vapor phase above the liquid.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology
- Culture Cells