I have seen multiple sources suggesting that culturing Caco-2 cells in a flask or something like a 6-well plate for 14-21 days with media changes should cause them to form a differentiated monolayer. When I tried this in a 6 well plate, the cells hit confluence and developed a relatively tight, structured layer within a few days of seeding, however, the cells then just started to grow on top of each other and by 2 weeks were showing signs of massive overgrowth with lots of detached cells floating in the media.
Is there a trick to making this work such as the presence of and NEAA supplement or a specific seeding density? I'm starting to be concerned that my cells on transwell inserts may not be forming proper monolayers either despite high TEER values. I am using DMEM GlutaMAX media with 10% FBS and 1% Anti-Anti. I tried three different seeding densities - 2, 4, 8 x 10^4 cells/cm2. Media was replaced twice a week.
What I recollect is the temporally differentiated cells in monolayer on plastic formed domes or blisters. Overgrowth will also occur since these are cancer cell derived. This was simply a spontaneous event that did not require a special stimulus. There were instances in several labs where the cells lost their monolayer forming phenotype qualified by lower TER and multiple layering. To my knowledge, Why this occurred was never established and necessitated thawing lower passage cells.
I am trying to the same.
i want to test cytokine/chemokine secretion and am currently not interested in permeability. After testing a few methods, i now grow them at 5*10^5 per cm in a 6 well coated with PDL. it works great.
I use 7-day differentiation protocol (with adding MITO+) and validated by ZO-1 staining and microscopy imaging of villi formation
Hello Caco-2 cells spontaneously differentiate in plastic support when they reach confluency. Culture medium is DMEM-glutamax 4.5g/L glucose+ 1% NEAA+ 20% serum, at 10% CO2 with a daily change of culture medium.
Update: I seeded cells at 200k cells/cm2 and that generated a much more stable looking monolayer without overgrowth than cells seeded at 86k cells/cm2. Cells on a high passage number performed poorly and experienced greater loss of viability over a 3 week incubation period compared to low passage number cells. NEAA made no visible difference to cell morphology or monolayer formation.
NEAA is required for cell growth. In cell culture i always add NEAA during all the experiment (growing and differentiation).
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