I need to treat HUVEC cells with different glucose concentrations for up to 14 days. How do I do this in a 6 well plate because the cells will reach confluence within a few days? Can I use trpysin and passage the cells from a plate and then re-seed? Would this not affect the duration of glucose treatment?
HUVEC show contact inhibition, they will grow up to confluency and can be kept in a 6-well for quite some time, a week or even two.If you would like to compare influences, it would be appropriate to check for influencing factors- passage number, conditions applied including CO2 and oxygen levels. It might be worth comparing different states: after reaching confluency and during the growth stage. Certainly, I support the views expressed already. Try to use an early passage number as starting the culture and possibly compare individual cultures from different umbilical veins that may show different patterns.
I would not recommend to use trypsin and certainly not to re-passage while running the reaction experiments.
Wishing you successful run of experiments. :-)
HUVEC will not be coming out upon reaching confluency so, I suppose you should not have a problem in working with the cells. It is also because HUVEC will behave in a different way depending on the cell confluency due to which most of the experiments are recommended to be done upon 80% confluency.
Hope this will help you.
HUVECS can be treated chronically with different glucose concentrations by sub-culturing for such long periods of time. Glucose treatment doesn't vary for such trypsinisations, provided you add the glucose to the medium before supplementing it to the cells.
HUVEC show contact inhibition, they will grow up to confluency and can be kept in a 6-well for quite some time, a week or even two.If you would like to compare influences, it would be appropriate to check for influencing factors- passage number, conditions applied including CO2 and oxygen levels. It might be worth comparing different states: after reaching confluency and during the growth stage. Certainly, I support the views expressed already. Try to use an early passage number as starting the culture and possibly compare individual cultures from different umbilical veins that may show different patterns.
I would not recommend to use trypsin and certainly not to re-passage while running the reaction experiments.
Wishing you successful run of experiments. :-)
A lot depends on what response you're testing. If you want them to behave somewhat like they would in a blood vessel, having them confluent would be an advantage, because the cell-cell contacts will be more "realistic". But if you're testing for the effect of glucose concentrations on maintaining growth in culture, successive passaging may be useful, as a test of resilience.
It depends on what you are trying to achieve as an endpoint of your study. If you are examining growth rate and are seeding at a certain cell number say 2x104 per plate in six replicates on the 6 well plate, and all other culture conditions being equal, the rate of growth to confluency will be effected by the glucose concentration if there is a dependency on the glucose for survival. If you only want to keep them in different concentrations of glucose for 14 days then once again seeding number should allow them to slowly grow to confluency at a similar rate if glucose is not dependent. Of course as stated above passage number will effect your results and the lower the passage number the better. Later passage numbers will often undergo senescence and hypertropathy if you stress them to far with a low seeding number.
If your question possibly is based on intent to continue a HUVEC culture by passaging/etc, keep in mind that HUVEC are not stable -- their phenotype drifts fairly rapidly with passaging, something that may or may not render them unsuitable for your purposes. So you'll want to carefully monitor the markers/functions/features that you are interested in as they (possibly) change with successive passages.
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