Dear Dr. Lael Werner

Several cell lines may be differentiated by adding synthetic or biological factors to the medium. However, Caco-2 cell line is rather different from others as it shows the ability to differentiate spontaneously on reaching confluence.

Towards confluence they start to polarize acquiring a characteristic apical brush border with microvilli. Tight junctions form between adjacent cells, and they express enzyme activities typical of enterocytes, namely, lactase, aminopeptidase N, sucrase-isomaltase and dipeptidylpeptidase IV.

Subculturing cells before they have reached confluence can help cells to form a more homogenous and polarized monolayer. One protocol suggests subculturing cells when they have reached 50% of confluence, in contrast to an existing recommendation of 80%. Using this protocol, Caco-2 cells retain a higher proliferation potential resulting in a cell population, which, on reaching confluence, is able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a high cell growing density.

You may want to refer to the article attached below for more information.

Article Cell Growing Density Affects the Structural and Functional P...

Regards,

Malcolm Nobre