Cell growth density affects the structural and functional properties of Caco‐2 differentiated monolayer
Author(s)
Natoli, Manuela
Date Issued
March 16, 2010
Type
Doctoral Thesis
Abstract
The human intestinal Caco-2 cell line has been extensively used as a model of the
intestinal barrier. However, it is widely reported in literature that culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often lead to problems of
reproducibility making difficult to compare results. We developed a new cell-growth protocol in which Caco-2 cells were subcultured at 50% of confluence (Low Density, LD) instead of 90% of
confluence, as usually reported in literature (High Density, HD). Using this new protocol, Caco-2 cells retained a higher proliferation potential resulting in a cell population, which, reaching confluence was able to differentiate almost synchronously, forming a more homogeneous and
perfectly polarized cell monolayer, as compared to that obtained with the HD cells. This comparison has been done by analyzing the gene expression and the structural characteristics of the 21-days differentiated monolayers by microarrays hybridization and by confocal microscopy.
We then investigated if these differences could also modify the effects of toxicants on 21-days-differentiated LD and HD cells. We analyzed the 2 hours-acute toxicity of CuCl2 in terms of actin depolymerization and MT2A (metallothionein 2A) and HSPA1A (heat shock protein 70) genes induction. Copper treatment resulted in different levels of actin depolymerization and gene
expression induction in relationship with the culture protocol, the LD cells showing a more homogeneous and stronger response. Our results suggest that cell growth density could influence a number of morphological and physiological properties of differentiated Caco-2 cells and these
effects must be taken in account when these cells are used as intestinal model.
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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