I want to try a long term culture of Lens Epithelial cells, culturing them for more than two months.
Up until now i have been culturing the cells for 3 to 4 weeks in RPMI culture medium supplemented with 5% FCS and 1% antibiotic-antimitotic, changing the media every 2 to 3 days. And this has worked fine.
Is there any way to only have to change media once a week or once every 5 days or so?
Would doubling the volume of media available to the cells increase time between media changes? Is the lowering of pH due to the accumulation of metabolites in the medium the main limiting factor?
Hi Justin the amount of media/nutrients required is generally related to cell number/cell density. Depending on this you could probably reduce media changes to perhaps twice per week (Monday and Friday perhaps).
I would not recommend increasing the volume of media much beyond ~ 0.3 ml per cm2 of the container/flask. Doing so reduces the availability of the gas (O2) to the cells.
Flask manufacturers state a maximum media amount per flask in most of their product details for this reason (I have attached one from Corning I find useful).
Unfortunately unless you have a perfusion bioreactor regular feeding is essential.
Many cell lines (especially if they are confluent and/or in G0) can "tick" along with minimal feeding but having had no personal experience of lens epithelial cells (or the density your cells are at) I hesitate to recommend feeding them less than twice per week.
Sorry I can't be more help
Gary
You could lower your seeding density, or possibly change to a higher nutrient media like DMEM which has a higher glucose content to that of RPMI. If you do decide to change media, do a ratio change over slowly (75:25, 50:50, 25:75) to allow the cells to acclimate to the difference in nutrient content. Maybe do a trial run on cells you can afford to lose if the cells do not do well in the new media.
Don't use standard L-Glutamine for medium supplementation in your long term cultures, as it's unstable. Better use L-glutamine in dipeptide forms, such as alanyl-l-glutamine.
However, in my experience most cells grow fine with only two medium changes a week (like Monday and Thursday).
And don't increase the medium volume in your flasks. As Gary said, the gas supply will be worse.
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