During my internship, I used HUVEC from one single aliquote from Promocell. The aim was to activate cells by a TNF-alpha stimulation (used at 5ng/ml) in hypoxic conditions (0,1% O2) for 24h.
At the end of the incubation period, we performed a flow cytometry experiment to see the expression of ICAM-1 and VCAM-1, but with 5 experiments (one per week), we had alternate results ( 2 were activated but preceded and followed by non-activated ones ).
All the cells were cultured in the same conditions, same medium, and same TNF-a aliquote, they were all used at passage 5.
I don't understand why i found such variation, does anyone have an idea ?
In our experiments we use HUVECs in pooled version, not from one single donor. When we activated cells with TNF-a (5 ng/ml) after 6 h we observed huge ICAM-1 and VCAM-1 increase in every experiment we did. The only thing is that each experiment was started when cells where completely confluent. They should reach 100% of confluency at the same time from passage. In general endothelial cells need to be in contact as it is in real tissue. If you start experiment when cells are not completely confluent the response could be not repetitive and completely different mechanism could be activated.
Best regards,
Dorota@
It could be that the variation was due to a genetic drift of the HUVECs.
I assume that the cells were relative old at the time of the experiments if the HUVEC were used in passage 5? And 5 experiments mean that you worked with five separate HUVEC populations?
The longer primary cells are cultivated, the more they can change - mutations occur, expression profiles change and the cells are not the same as at the beginning of the cultivation. For example, if you thaw one vial of HUVEC, then split and cultivate then in two flasks (creating two populations), there is a good chance that you will see differences between those two populations - the more passages, the more differences, the more variations.
This is one reason, why experiments are usually done at low passage numbers before the cells change too much.
You checked that the cells did not yet reach senescence? These HUVEC are guaranteed to perform at least 15 population doublings (PD). But that does not mean you can passage them 15x.
A population doubling (PD) is a two-fold increase in the total number of cells during in vitro-culture. Calculating the number of PDs a culture has performed at a certain point gives an accurate measure of its age. In contrast, the term passage merely refers to the subculturing process, i.e. the detachment of the cells from a culture vessel (using eg. trypsin/EDTA or accutase) and replating them at a lower density to allow for further cell growth. The term passage does not take into account the different split ratios that can be applied. Different split ratios produce cultures with an identical number of passages but a varying number of doublings. Passage is therefore only a rough estimate of the actual age of a culture.
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